Abstract:
A diagnostic tool for use in diagnosing diseases, the tool is a detector for detecting a presence of an array of markers being used to determine gene expression changes that are related to cellular immortalization, the presence of the markers being indicative of a specific disease and the markers and treatments found by the tool. A tool for interpreting results of a microarray, wherein the tool is a computer program for analyzing the results of microrarrays. A method of creating an array of markers for diagnosing the presence of disease by microarraying sera obtained from a patient to obtain molecular markers of disease and detecting markers that are present only in the sera of patients with a specific disease thereby detecting molecular markers being used to determine gene expression changes that are related to cellular immortalization and for use in diagnosing disease.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application is a Continuation-in-Part of PCT/US03/29624, which claims the benefit of priority under 35 U.S.C. Section 119(e) of U.S. Provisional Patent Application Nos. 60/412,228, filed Sep. 20, 2002 and 60/478,548, Filed Jun. 13, 2003, which is incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     1. Technical Field  
         [0003]     The present invention relates to molecular targets of cancer and aging. More specifically, the present invention relates to a microarray for use in determining molecular targets of cancer and aging.  
         [0004]     2. Description of the Related Art  
         [0005]     It is commonly known in the art that genetic mutations can be used for detecting cancer. For example, the tumorigenic process leading to colorectal carcinoma formation involves multiple genetic alterations (Fearon et al (1990) Cell 61, 759-767). Tumor suppressor genes such as p53, DCC and APC are frequently inactivated in colorectal carcinomas, typically by a combination of genetic deletion of one allele and point mutation of the second allele (Baker et al (1989) Science 244, 217-221; Fearon et al (1990) Science 247, 49-56; Nishisho et al (1991) Science 253, 665-669; and Groden et al (1991) Cell 66, 589-600). Recently, mutation of two mismatch repair genes that regulate genetic stability was associated with a form of familial colon cancer (Fishel et al (1993) Cell 75, 1027-1038; Leach et al (1993) Cell 75, 1215-1225; Papadopoulos et al (1994) Science 263, 1625-1629; and Bronner et al (1994) Nature 368, 258-261). Proto-oncogenes such as myc and ras are altered in colorectal carcinomas, with c-myc RNA being overexpressed in as many as 65% of carcinomas (Erisman et al (1985) Mol. Cell. Biol. 5, 1969-1976), and ras activation by point mutation occurring in as many as 50% of carcinomas (Bos et al (1987) Nature 327, 293-297; and Forrester et al (1987) Nature 327, 298-303). Other proto-oncogenes, such as myb and neu are activated with a much lower frequency (Alitalo et al (1984) Proc. Natl. Acad. Sci. USA 81, 4534-4538; and D&#39;Emilia et al (1989) Oncogene 4, 1233-1239). No common series of genetic alterations is found in all colorectal tumors, suggesting that a variety of such combinations can be able to generate these tumors.  
         [0006]     Increased tyrosine phosphorylation is a common element in signaling pathways that control cell proliferation. The deregulation of protein tyrosine kinases (PTKS) through overexpression or mutation has been recognized as an important step in cell transformation and tumorigenesis, and many oncogenes encode PTKs (Hunter (1989) in oncogenes and the Molecular Origins of Cancer, ed. Weinberg (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), pp. 147-173). Numerous studies have addressed the involvement of PTKs in human tumorigenesis. Activated PTKs associated with colorectal carcinoma include c-neu (amplification), trk (rearrangement), and c-src and c-yes (mechanism unknown) (D&#39;Emilia et al (1989), ibid; Martin-Zanca et al (1986) Nature 3, 743-748; Bolen et al (1987) Proc. Natl. Acad. Sci. USA 84, 2251-2255; Cartwright et al (1989) J. Clin. Invest. 83, 2025-2033; Cartwright et al (1990) Proc. Natl. Acad. Sci. USA 87, 558-562; Talamonti et al (1993) J. Clin. Invest. 91, 53-60; and Park et al (1993) Oncogene 8, 2627-2635).  
         [0007]     Obviously, protein tyrosine phosphatases (PTPs) are also intimately involved in regulating cellular phosphotyrosine levels. The growing family of PTPs consists of non-receptor and receptor-like enzymes (for review see Charbonneau et al (1992) Annu. Rev. Cell. Biol. 8, 463493; and Pot et al (1992) Biochim. Biophys. Acta 1136, 35-43). All share a conserved catalytic domain, which in the non-receptor PTPs is often associated with proximal or distal sequences containing regulatory elements directing protein-protein interaction, intracellular localization, or PTP stability. The receptor like PTPs usually contain two catalytic domains in their intracellular region, and in addition have a transmembrane region and heterogeneous extracellular regions. The extreme diversity of the extracellular region, compared to the relatively conserved intracellular portion of these enzymes, suggests that these PTPs are regulated by specific extracellular factors, few of which have been identified. Some PTPs can act in opposition to PTKs. For example, the nonreceptor PTP 1B and TC-PTP can reverse or block cell transformation induced by the oncogenic tyrosine kinases neu or v-fms, while another non-receptor PTP (known as 3HC134, CL100, HVH1, PAC-1, erp, or MKP-1) can reverse the PTK-mediated activation of a central signaling enzyme, MAP kinase (Brown-Shimer et al (1992) Cancer Res. 52, 478-482; Zander et al (1993) Oncogene 8, 1175-1182; Sun et al (1993) Cell 75, 487-493; and Ward et al (1994) Nature 367, 651-654). Conversely, other PTPs can act in conjunction with PTKs. Two receptor-like PTPs, PTPa and CD45, respectively activate the tyrosine kinases c-src or Ick and fyn while the non-receptor SH-PTP2 (PTP 1D, PTP-2C, Syp) positively transduces a mitogenic signal from the PDGF receptor tyrosine kinase to ras (WP 94/01119; Zheng et al (1992) Nature 359, 336-339; den Hertog et al (1993) EMUB J. 12, 3789-3798; Mustelin et al (1989) Proc. Natl. Acad. Sci. USA 86, 6302-6306; Ostergaard et al (1989) Proc. Natl. Acad. Sci. USA 86, 8959-8963; Cahir McFarland et al (1989) Proc. Natl. Acad. Sci. USA 90, 1402-1406; and Li et al (1994) Mol. Cell. Biol. 14, 509-517).  
         [0008]     Very few studies have examined alterations in PTP expression or activity that can be associated with tumorigenesis. As indicated above, two PTP-related mechanisms, either the inactivation or the overactivation of a PTP, could increase cellular phosphotyrosine levels and result in uncontrolled cell proliferation and tumorigenesis. In relation to PTP inactivation, it is of interest that the gene encoding receptor-like PTP7 is situated on a region of chromosome 3 that is often lost in renal and lung carcinomas, and that a PTPW allele is lost in some renal carcinoma and lung carcinoma cell lines (LaForgia et al (1991) Proc. Natl. Acad. Sci. USA 88, 5036-5040). As regards PTP overactivation, it has been shown that when PTPa is overexpressed in rat embryo fibroblasts, cell transformation occurs and the cells are tumorigenic in nude mice (WO 94/01119 and Zheng et al (1992), ibid). PTPα is a receptor-like enzyme with a short, unique extracellular domain and two tandem catalytic domains (WO 92/01050; Matthews et al (1990) Proc. Natl. Acad. Sci. USA 87, 4444-4448; Sap et al (1990) Proc. Natl. Acad. Sci. USA 87, 6112-6116; and Krueger et al (1990) EMBO J. 9, 3241-3252). Compared to many other receptor-like PTPs with a restricted and lineage-specific expression, PTPα is widely expressed (Sap et al (1990), ibid and Krueger et al (1990), ibid).  
         [0009]     Mutations, such as those disclosed above can be useful in detecting cancer. However, there have been few advancements that can repeatably be used in diagnosing cancer prior to the existence of a tumor. For example, breast cancer, which is by far the most common form of cancer in women, is the second leading cause of cancer death in humans. Despite many recent advances in diagnosing and treating breast cancer, the prevalence of this disease has been steadily rising at a rate of about 1% per year since 1940. Today, the likelihood that a women living in North America can develop breast cancer during her lifetime is one in eight.  
         [0010]     The current widespread use of mammography has resulted in improved detection of breast cancer. Nonetheless, the death rate due to breast cancer has remained unchanged at about 27 deaths per 100,000 women. All too often, breast cancer is discovered at a stage that is too far advanced, when therapeutic options and survival rates are severely limited. Accordingly, more sensitive and reliable methods are needed to detect small (less than 2 cm diameter), early stage, in situ carcinomas of the breast. Such methods should significantly improve breast cancer survival, as suggested by the successful employment of Papinicolou smears for early detection and treatment of cervical cancer.  
         [0011]     In addition to the problem of early detection, there remain serious problems in distinguishing between malignant and benign breast disease, in staging known breast cancers, and in differentiating between different types of breast cancers (e.g. estrogen dependent versus non-estrogen dependent tumors). Recent efforts to develop improved methods for breast cancer detection, staging and classification have focused on a promising array of so-called cancer “markers.” Cancer markers are typically proteins that are uniquely expressed (e.g. as a cell surface or secreted protein) by cancerous cells, or are expressed at measurably increased or decreased levels by cancerous cells compared to normal cells. Other cancer markers can include specific DNA or RNA sequences marking deleterious genetic changes or alterations in the patterns or levels of gene expression associated with particular forms of cancer.  
         [0012]     A large number and variety of breast cancer markers have been identified to date, and many of these have been shown to have important value for determining prognostic and/or treatment-related variables. Prognostic variables are those variables that serve to predict disease outcome, such as the likelihood or timing of relapse or survival. Treatment-related variables predict the likelihood of success or failure of a given therapeutic plan. Certain breast cancer markers clearly serve both functions. For example, estrogen receptor levels are predictive of relapse and survival for breast cancer patients, independent of treatment, and are also predictive of responsiveness to endocrine therapy. Pertschuk et al., Cancer 66: 1663-1670, 1990; Parl and Posey, Hum. Pathol. 19: 960-966, 1988; Kinsel et al., Cancer Res. 49: 1052-1056, 1989; Anderson and Poulson Cancer 65: 1901-1908, 1989.  
         [0013]     The utility of specific breast cancer markers for screening and diagnosis, staging and classification, monitoring and/or therapy purposes depends on the nature and activity of the marker in question. For general reviews of breast cancer markers, see Porter-Jordan et al., Hematol. Oncol. Clin. North Amer. 8: 73-100, 1994; and Greiner, Pharmaceutical Tech., May, 1993, pp. 2844. As reflected in these reviews, a primary focus for developing breast cancer markers has centered on the overlapping areas of tumorigenesis, tumor growth and cancer invasion. Tumorigenesis and tumor growth can be assessed using a variety of cell proliferation markers (for example Ki67, cyclin D1, and proliferating cell nuclear antigen (PCNA)), some of which can be important oncogenes as well. Tumor growth can also be evaluated using a variety of growth factor and hormone markers (for example estrogen, epidermal growth factor (EGF), erbB-2, transforming growth factor (TGF)a), which can be overexpressed, underexpressed or exhibit altered activity in cancer cells. By the same token, receptors of autocrine or exocrine growth factors and hormones (for example insulin growth factor (IGF) receptors, and EGF receptor) can also exhibit changes in expression or activity associated with tumor growth. Lastly, tumor growth is supported by angiogenesis involving the elaboration and growth of new blood vessels and the concomitant expression of angiogenic factors that can serve as markers for tumorigenesis and tumor growth.  
         [0014]     In addition to tumorigenic, proliferation, and growth markers, a number of markers have been identified that can serve as indicators of invasiveness and/or metastatic potential in a population of cancer cells. These markers generally reflect altered interactions between cancer cells and their surrounding microenvironment. For example, when cancer cells invade or metastasize, detectable changes can occur in the expression or activity of cell adhesion or motility factors, examples of which include the cancer markers Cathepsin D, plasminogen activators, collagenases and other factors. In addition, decreased expression or overexpression of several putative tumor “suppressor” genes (for example nm23, p53 and rb) has been directly associated with increased metastatic potential or deregulation of growth predictive of poor disease outcome.  
         [0015]     In summary, the evaluation of proliferation markers, oncogenes, growth factors and growth factor receptors, angiogenic factors, proteases, adhesion factors and tumor suppressor genes, among other cancer markers, can provide important information concerning the risk, presence, status or future behavior of cancer in a patient. Determining the presence or level of expression or activity of one or more of these cancer markers can aid in the differential diagnosis of patients with uncertain clinical abnormalities, for example by distinguishing malignant from benign abnormalities. Furthermore, in patients presenting with established malignancy, cancer markers can be useful to predict the risk of future relapse, or the likelihood of response in a particular patient to a selected therapeutic course. Even more specific information can be obtained by analyzing highly specific cancer markers, or combinations of markers, which can predict responsiveness of a patient to specific drugs or treatment options.  
         [0016]     Methods for detecting and measuring cancer markers have been recently revolutionized by the development of immunological assays, particularly by assays that utilize monoclonal antibody technology. Previously, many cancer markers could only be detected or measured using conventional biochemical assay methods, which generally require large test samples and are therefore unsuitable in most clinical applications. In contrast, modern immunoassay techniques can detect and measure cancer markers in relatively much smaller samples, particularly when monoclonal antibodies that specifically recognize a targeted marker protein are used. Accordingly, it is now routine to assay for the presence or absence, level, or activity of selected cancer markers by immunohistochemically staining tissue specimens obtained via conventional biopsy methods. Because of the highly sensitive nature of immunohistochemical staining, these methods have also been successfully employed to detect and measure cancer markers in smaller, needle biopsy specimens which require less invasive sample gathering procedures compared to conventional biopsy specimens. In addition, other immunological methods have been developed and are now well known in the art that allow for detection and measurement of cancer markers in non-cellular samples such as serum and other biological fluids from patients. The use of these alternative sample sources substantially reduces the morbidity and costs of assays compared to procedures employing conventional biopsy samples, which allows for application of cancer marker assays in early screening and low risk monitoring programs where invasive biopsy procedures are not indicated.  
         [0017]     For the purpose of cancer evaluation, the use of conventional or needle biopsy samples for cancer marker assays is often undesirable, because a primary goal of such assays is to detect the cancer before it progresses to a palpable or detectable tumor stage. Prior to this stage, biopsies are generally contraindicated, making early screening and low risk monitoring procedures employing such samples untenable. Therefore, there is general need in the art to obtain samples for cancer marker assays by less invasive means than biopsy, for example by serum withdrawal.  
         [0018]     Efforts to utilize serum samples for cancer marker assays have met with limited success, largely because the targeted markers are either not detectable in serum, or because telltale changes in the levels or activity of the markers cannot be monitored in serum. In addition, the presence of cancer markers in serum probably occurs at the time of micro-metastasis, making serum assays less useful for detecting pre-metastatic disease.  
         [0019]     Previous attempts to develop non-invasive breast cancer marker assays utilizing mammary fluid samples have included studies of mammary fluid obtained from patients presenting with spontaneous nipple discharge. In one of these studies, conducted by Inaji et al., Cancer 60: 3008-3013, 1987, levels of the breast cancer marker carcinoembryonic antigen (CEA) were measured using conventional, enzyme linked immunoassay (ELISA) and sandwich-type, monoclonal immunoassay methods. These methods successfully and reproducibly demonstrated that CEA levels in spontaneously discharged mammary fluid provide a sensitive indicator of nonpalpable breast cancer. In a subsequent study, also by Inaji et al., Jpn. J. Clin. Oncol. 19: 373-379, 1989, these results were expanded using a more sensitive, dry chemistry, dot-immunobinding assay for CEA determination. This latter study reported that elevated CEA levels occurred in 43% of patients tested with palpable breast tumors, and in 73% of patients tested with nonpalpable breast tumors. CEA levels in the discharged mammary fluid were highly correlated with intratumoral CEA levels, indicating that the level of CEA expression by breast cancer cells is closely reflected in the mammary fluid CEA content. Based on these results, the authors concluded that immunoassays for CEA in spontaneously discharged mammary fluid are useful for screening nonpalpable breast cancer.  
         [0020]     Although the evaluation of mammary fluid has been shown to be a useful method for screening nonpalpable breast cancer in women who experience spontaneous nipple discharge, the rarity of this condition renders the methods of Inaji et al, inapplicable to the majority of women who are candidates for early breast cancer screening. In addition, the first Inaji report cited above determined that certain patients suffering spontaneous nipple discharge secrete less than 10.mu.l of mammary fluid, which is a critically low level for the ELISA and sandwich immunoassays employed in that study. It is likely that other antibodies used to assay other cancer markers can exhibit even lower sensitivity than the anti-CEA antibodies used by Inaji and coworkers, and can therefore not be adaptable or sensitive enough to be employed even in dry chemical immunoassays of small samples of spontaneously discharged mammary fluid.  
         [0021]     In view of the above, an important need exists in the art for more widely applicable, non-invasive methods and materials to obtain biological samples for use in evaluating, diagnosing and managing breast and other diseases including cancer, particularly for screening early stage, nonpalpable tumors. A related need exists for methods and materials that utilize such readily obtained biological samples to evaluate, diagnose, and manage disease, particularly by detecting or measuring selected molecular cancer markers to provide highly specific, cancer prognostic and/or treatment-related information, and to diagnose and manage pre-cancerous conditions, cancer susceptibility, bacterial, and other infections, and other diseases.  
       SUMMARY OF THE INVENTION  
       [0022]     According to the present invention, there is provided a diagnostic tool for use in diagnosing diseases, the tool is a detector for detecting a presence of an array of markers indicative of a specific disease and the marker and treatments found therefrom. A tool for interpreting results of a microarray, wherein the tool is a computer program for analyzing the results of microrarrays. A method of creating an array of markers for diagnosing the presence of disease by microarraying sera obtained from a patient to obtain molecular markers of disease and detecting markers that are present only in the sera of patients with a specific disease thereby detecting molecular markers for use in diagnosing disease. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0023]     Other advantages of the present invention are readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:  
         [0024]      FIG. 1  is a photograph showing 5-aza-CdR mediated up-regulation of STAT1α;  
         [0025]      FIGS. 2A  and B are photographs showing the hierarchical clustering of gene expression using GeneSight software; and  
         [0026]      FIG. 3  is a photograph showing 5-aza-CdR mediated up-regulation of p16 INK4a  protein.  
         [0027]      FIG. 4  is a photograph showing the Western blot analysis of MDAH041 and MDAH087 cell lines, wherein UT: untreated; 5A: 5-aza-dC; 041-PC: precrisis MDAH041; 041-IM: immortal MDAH041; 087-PC: precrisis MDAH087; 087-N: MDAH087-N; 087-1: MDAH087-1; 087-10: MDAH087-10, and tubulin is a loading control;  
         [0028]      FIG. 5   a  is a photograph showing hierarchical clustering of gene expression data in MDAH041, MDAH087-N, MDAH087-1, and MDAH087-10, wherein each row represents a probe on the HGU95Av2 GeneChip®, each column represents the average comparisons of each cell line. 041-IM: immortal MDAH041; 087-N: MDAH087-N; 087-1: MDAH087-1; 087-10: MDAH087-10;  
         [0029]      FIG. 5   b  is a graph showing multidimensional scaling analysis of gene expression data in MDAH041, MDAH087-N, MDAH087-1, and MDAH087-10, wherein 5A: upregulated in 5-aza-dC-treated immortal cells versus untreated immortal cells; UT: Untreated, downregulated in immortal cells versus precrisis cells. 041-IM: immortal MDAH041; 087-N: MDAH087-N; 087-1: MDAH087-1; 087-10: MDAH087-10;  
         [0030]      FIG. 6A  through C are graphs depicting GoMiner analysis of differentially regulated genes in all four immortal LFS cell lines, wherein the genes, which were dysregulated (up- or downregulated) during immortalization and 5-aza-dC treatment in MDAH041, MDAH087-N, MDAH087-1, MDAH087-10 cells were analyzed by GoMiner according to biological process ( FIG. 6A ), cellular component ( FIG. 6B ) and molecular function ( FIG. 6C ). The first layer GO categories were plotted based on their −log 10 (p-value). IM: genes dysregulated during immortalization; 5A: genes dysregulated during 5-aza-dC treatment of immortal cells. p-values, which were smaller than 0.0001, were replaced with 0.0001 to get a viewable range of the plot. GO categories identified to be significant by corrected p-value were marked by *;  
         [0031]      FIG. 7  is a series of chromosome ideograms of genes differentially expressed genes in all four immortal LFS cell lines, fragile sites and imprinted genes, wherein the ideograms from left to right, for each chromosome, are reference ideogram of cytogenetic regions (R), ideogram of genes decreased during immortalization (D), ideogram of imprinted genes (I), and ideogram of genes increased after 5-aza-dC treatment (5A). The colored lines represent location of genes. Fragile sites are represented by a dot (F). Genes that are epigenetically regulated during immortalization are labeled on the ideograms;  
         [0032]      FIG. 8  is a series of chromosome ideograms depicting the localization of genes, in the four immortal LFS cell lines, with increased expression during immortalization  
         [0033]      FIG. 9  is a series of chromosome ideograms depicting the localization of genes, in the four immortal LFS cell lines, with decreased expression during immortalization  
         [0034]      FIG. 10  is a series of chromosome ideograms depicting the localization of genes, in the four immortal LFS cell lines, with increased expression after 5-aza-dC treatment  
         [0035]      FIG. 11  is a series of chromosome ideograms depicting the localization of genes, in the four immortal LFS cell lines, with decreased expression after 5-aza-dC treatment  
         [0036]      FIG. 12  is a series of chromosome ideograms depicting the localization of genes, in the four immortal LFS cell lines, with increased expression during immortalization and decreased expression after 5-aza-dC treatment  
         [0037]      FIG. 13  is a series of chromosome ideograms depicting the localization of genes, in the four immortal LFS cell lines, with decreased expression during immortalization and increased expression after 5-aza-dC treatment 
     
    
     DESCRIPTION OF THE INVENTION  
       [0038]     Generally, the present invention relates to a method of determining molecular targets of cancer and aging and the targets obtained by the same. The method includes analyzing the results obtained from a microarray that is used for determining the molecular targets of cancer and aging.  
         [0039]     The microarray of the present invention is any microarray that can be used to determine gene expression changes that are related to cellular immortalization. The gene expression changes that are determined as a result of the microarray are then compared to the gene expression changes due to variations in gene expression after inhibiting a fundamental pathway in the immortalization process. The genes expression changes relate to early events in the cellular progression to cancer both for molecular targets and diagnostic targets.  
         [0040]     More specifically, the pathway is affected by inhibiting a fundamental aspect of the pathway; for example, inhibition of DNA methylation in immortal fibroblast cells. The pathway can be a growth suppressor, a growth promotor, or is otherwise involved in cell growth or proliferation. The results of the comparison of the gene expression changes are compared to identify genes that are regulated in both conditions, thereby identifying genes that are molecular targets of cancer and aging.  
         [0041]     The use of microarray technology allows for the study of a complex interplay of genes and other genetic material, simultaneously. The pattern of genes expressed in a cell is characteristic of its state. Virtually all differences in cell state correlate with changes in mRNA levels of genes. Generally, microarray technology involves obtaining complementary genetic material to genetic material of interest and laying out the complementary genetic material in microscopic quantities on solid surfaces at defined positions. Genetic material from samples is then eluted over the surface and complementary genetic material binds thereto. The presence of bound genetic material then is detected by fluorescence following laser excitation.  
         [0042]     By “support or surface” as used herein, the term is intended to include, but is not limited to a solid phase, which is a porous or non-porous water insoluble material that can have any one of a number of shapes, such as strip, rod, particle, including beads and the like. Suitable materials are well known in the art and are described in, for example, Ullman, et al. U.S. Pat. No. 5,185,243, columns 10-11, Kum, et al., U.S. Pat. No. 4,868,104, column 6, lines 21-42 and Milburn, et al., U.S. Pat. No. 4,959,303, column 6, lines 14-31 that are incorporated herein by reference. Binding of ligands and receptors to the support or surface can be accomplished by well-known techniques, readily available in the literature. See, for example, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem. 245:3059 (1970). Whatever type of solid support is used, it must be treated so as to have bound to its surface either a receptor or ligand that directly or indirectly binds the antigen. Typical receptors include antibodies, intrinsic factor, specifically reactive chemical agents such as sulfhydryl groups that can react with a group on the antigen, and the like. For example, avidin or streptavidin can be covalently bound to spherical glass beads of 0.5-1.5 mm and used to capture a biotinylated antigen.  
         [0043]     The “molecular markers” that are isolated can be any marker known to those of skill in the art to be related to cancer or aging. The markers can be any detectable marker that is altered due to the present of cancer or the onset of aging. Examples of such markers include, but are not limited to, IFN pathway genes and molecular targets involved in immortalization.  
         [0044]     In the analysis there were identified several pathways with changes in gene expression, including the interferon signaling pathway, the cell cycle pathway, and genes for proteins in the cytoskeleton, that were differentially expressed after the immortalization in LFS cells. Fourteen genes were consistently epigenetically regulated during immortalization in all of the immortal cell lines studied, namely CREG, CYP1B1, IGFBPrP1, CLTB, KIAA1750, FLJ14675, OPTN, HPS5, HTATIP2, HSPA2, TNFAIP2, ALDH1A3, MAP1LC3B, and SERPINB2. A significant number of the epigenetically regulated genes, in each of the four immortal LFS cell lines, are in the IFN pathway. The involvement of the IFN pathway in cellular senescence and tumorigenesis is supported by the fact that a number of IFN induced proteins have tumor suppression activity when overexpressed in tumor cells. These proteins include double stranded RNA activated protein kinase (PKR), activated RNaseL, and the 200 gene family (Pitha 2000). Further, genes with expression that decreased during immortalization and increased after 5-aza-dC treatment, in common to all four immortal LFS cell lines, cluster on chromosome 4q12-q27, 6p22, 6p21.3, 7, 14,19 and X ( FIGS. 9 and 11 ).  
         [0045]     Immortalization is one of the necessary, multiple steps of tumorigenesis. Normal mammalian somatic cells can only divide a limited number of times in vitro. The maximum number of divisions is called the “Hayflick limit” (Hayflick L. et al., 1961). After that point the cells leave the cell cycle but remain metabolically active. This non-proliferative state is referred to as cellular senescence. Cells undergo a series of biochemical and morphological changes at senescence. Typical characteristics of senescing cells include large, flat morphology, a high frequency of nuclear abnormalities and positive staining for β-galactosidase activity specifically at pH 6.0. Senescence can be induced by a demethylation agent 5-aza-2′-deoxycytidine (5-aza-CdR) (Vogt M et. al, 1998). The counting mechanism for intrinsic replicative lifespan appears to be the shortening of telomeres with each cell division cycle (Counter, C. M. et al, 1992).  
         [0046]     Abnormal genetic changes or expression of viral oncoproteins in cells can prolong the division cycle beyond the Hayflick limit (Hayflick L. et al., 1961). The inactivation of p53 and pRb precedes the activation of telomere maintenance mechanism. The disruption of p16 INK4a  pathway creates a permissive environment for telomerase activation. After additional 20-30 population doublings, cells enter a state, which is referred to as crisis. At crisis, the cells continue to proliferate but have high rate of apoptosis. The expression of human telomerase reverse transcriptase (hTERT) is one of the telomere maintenance mechanisms that allow cells bypass senescence and expand the proliferative life span. The total cell number does not increase. After inactivation of p53 and pRb with DNA viral oncogenes, cells escape crisis and finally become immortalized at a low frequency (˜1 in 10 7 ).  
         [0047]     In addition to p53, pRb, p16 INK4a  (Vogt M et. al, 1998) and the genes required for telomere maintenance, some other genes can also involve in immortalization. The observation that not all cancers have mutated p53 suggests the upstream genes of p53 can prevent its normal function. Similarly, other genes involved in the pRb/p16 INK4a  pathway can substitute the abnormalities of these genes. They are also candidate tumor suppressor genes involved in immortalization (Bryan, T. M. et al., 1995, Kaul, S. C. et al, 1994).  
         [0048]     Mortalin is another important gene in cellular senescence and immortalization. The cytosolic mortalin is a marker of the mortal phenotype, however, the perinuclear mortalin can have a role in tumorigenesis (Kaul, S. C. et al, 1994, Wadhwa, R. et al., 1994).  
         [0049]     The greatest single risk factor for the development of cancer in mammals is aging. The incidence of cancer increases with age, beginning at about the mid-life span. In general, the rate at which cancer develops is proportional to the rate of aging. For example, mice develop cancer after about a year and a half of age roughly the midpoint in their life span, and humans develop cancer after 50 years, or half way through their life span. By contrast, other age-related diseases, such as Alzheimer&#39;s disease, are not believed to develop in short-lived mammals. Both cancer and other age related diseases are final results of a series of small, gradual changes at genetic level. Normal metabolism generates toxins as an inherent side effect. These toxins cause DNA damage, of which a small proportion is unrepaired by endogenous DNA repair mechanisms, and thus mutations accumulate. As DNA damage results in age-related degeneration, interventions must be designed to address molecular targets of aging. Somatic cells respond to these events by exiting the cell cycle and entering senescence, a metabolically active yet quiescent state. Bypassing senescence, commonly known as immortalization, has provided a relevant model for human aging at the cellular level. At the same time, bypassing cellular senescence is one of the necessary, multiple steps of tumorigenesis. Thus the phenomenon of immortalization is crucial to the understanding of both age related illnesses and cancer. By detecting the molecular targets involved in immortalization, one can determine proper targets of cancer prior to the existence of a tumor.  
         [0050]     Additionally, as disclosed in Esteller et al., the changes of 16 promoter hypermethylation regulated genes have been examined in over 600 primary tumor samples representing 15 major tumor types (Esteller et al (2001). Their results showed that although some of the gene changes are shared among different tumors, however, 70-90% tumor types do have a unique profile of three to four hypermethylation gene markers. In furtherance of the data disclosed in the Esteller et al. reference, the present invention provides that the promoter region hypermethylation is a molecular marker system for the early diagnosis of major forms of human cancer. Compared to genetic analysis, detection of promoter methylation offers many advantages: a) promoter methylation occurs over the same region within an individual gene, however, other DNA alterations such as mutations often vary over a wide region in the gene; b) promoter hypermethylation offers a positive signal against the background of normal DNA which is easier to detect comparing with the deletion mutation; c) the degree of transcription repression is dependent upon the density of methylation within the promoter region (Hsieh et al (1994); Vertino et al (1996); Graff et al (1997). Thus, the detection of methylation markers can be quantitative and qualitative with the aid of sensitive PCR strategies (Galm et al (2002); Herman, J. G. et al., 1996). Another key feature of methylation is its operational reversibility. Demethylation agents such as 5-azacytidine have already been used as chemotherapeutic agents. The identification of hypermethylation in gene promoters is not only a good molecular marker system for early tumor diagnosis, but also can be a desirable target for gene reactivation.  
         [0051]     Although IFN signaling pathways have been reported to be activated by the treatment of methylation inhibitor 5-aza-CdR in bladder and colon cancer cells, the IFN signaling pathway was not previously found to be activated with 5-aza-CdR in an immortal fibroblast preneoplastic cell line. The present invention provides that genes in IFN signaling pathway can be tumor suppressor genes, early genetic or epigenetic events involved in the progression of cells to immortalization and then cancer. The functional study on the biological function of IFN pathway genes in immortalization reveals the mechanism of how cancer cells escape the defense of IFN immune system. As functional genes i.e. candidate tumor suppressor genes in immortalization, these genes can serve as useful diagnostic markers in serum DNA assays or as therapeutic targets. The senescence initiating events leading to genomic instability and telomere stabilization are loss of checkpoint proteins such as p53, p21 CIP1/WAF1  and p16 INK4A . Gene profiling revealed 149 upregulated genes and 187 downregulated genes of which 14 were epigenetically downregulated in all four immortal LFS cell lines. In addition, several common pathways were involved in immortalization including the interferon pathway, genes involved in proliferation and cell cycle control, and the genes for cytoskeletal proteins.  
         [0052]     It is known that the immune system becomes less active during aging. The cellular response to interferon-gamma (IFN-gamma), the expression at the cell surface of the MHC class II gene IA complex product and the levels of IA-beta were decreased in aged macrophages (Herrero C et al, 2002). Moreover, the transcription of IFN regulated genes is impaired in aged macrophages. The impaired immune response associated with cellular senescence of immune cells. Indeed certain polymorphisms in IFN-gamma are associated with longevity (Lio 0 et al, 2002). The presence of the +874A allele, known to be associated with low IFN-production, allows extended longevity, possibly due to pro-inflammatory status during aging that might be detrimental for successful aging. The allele was significantly increased in female but not male centenarians seems indicating that a gender variable can be important in the biology of the aging process. It is clear that the IFN pathway is a factor in the aging process.  
         [0053]     The markers that are identified by the method of the present invention can then be used for treatment of disease. For example, in cancer, the molecular marker can be suppressed to prevent proliferation of cancerous cells using gene therapy techniques known to those of skill in the art. Alternatively, in aging, the marker can be enhanced to limit the number of cells that die as a normal result of the aging process using gene therapy techniques known to those of skill in the art.  
         [0054]     In order to determine which molecular markers are markers of cancer and aging, the microarrays must be analyzed. Preferably, the arrays are analyzed based either on fold change or via a noise sampling method (ANOVA). The fold change method is used to select the genes with at least a twofold change in expression. This is done using the Affymetrix Data Mining Tool (DMT), version 3,N-fold method (Affymetrix, Santa Clara, Calif., USA). For the control versus experiment comparisons, all possible pairings between the two controls and the two experiments are considered. ANOVA analysis (Kerr et al., 2000) can be used to isolate and eliminate the effects of within-slide and interslide variability and other sources of noise in the microarray. The effects of differential dye incorporation can also be eliminated by performing an exponential normalization (Houts, 2000) and/or a piece-wise linear normalization of the data obtained in the first round. The exponential normalization can be done by calculating the log ratio of all spots (excluding control spots or spots flagged for bad quality) and fitting an exponential decay to the log (Cy3/Cy5) vs. log (Cy5) curve. The curve fitted is of the form: 
 
 y=a+b   (−cx)  
 
         [0055]     where a, b and c are the parameters to be calculated during curve fitting. Once the curve is fitted, the values are normalized by subtracting the fitted log ratio from the observed log ratio.  
         [0056]     This normalization has been shown to obtain good results for cDNA microarrays but it relies on the hypothesis that the dye effect can be described by an exponential curve. The piece-wise linear normalization can be done by dividing the range of measured expression values into small intervals, calculating a curve of average expression values for each such interval and correcting that curve using piece-wise linear functions.  
         [0057]     All the gene expression data on HGU95Av2 were processed as previously described and used for the hierarchical clustering analysis implemented in GeneSight, version 3.2.6 (Biodiscovery, Los Angeles, Calif.). Euclidean distance was used for measuring similarities between two genes or samples, and complete linkage was used for clustering. For each of the four immortal LFS cell lines there were two comparisons, immortal cells versus precrisis cells, and 5-aza-dC treated immortal cells versus untreated immortal cells. Two-sided hierarchical analysis was carried out to determine the similarities of the four immortal LFS cell lines across the whole gene expression data.  
         [0058]     Multidimensional scaling is an alternative way to present the data in low dimension space. Multidimensional scaling analysis was performed using BRB-Array Tools version 3.2 beta to plot the data in three dimensions. The same comparisons and parameters used for hierarchical clustering were also used for multidimensional scaling analysis.  
         [0059]     Then, GoMiner (version 122) (Zeeberg et al. 2003) was used to annotate the gene expression data with GO categories. The entire HGU95Av2 GeneChip® probe set was the reference. Four experiment genes lists were analyzed: genes that were up- and downregulated during immortalization in all four immortal LFS cell lines (A and B in Table 7), and genes that were up- and downregulated after 5-aza-dC treatment in all four immortal LFS cell lines (C and D in Table 7). The probes from the lists were first converted to unique gene symbols using NetAffx, the Affymetrix online database (Build # 166) (Liu et al. 2003), and then the unique list of gene symbols were analyzed by GoMiner. The 8,487 unique gene symbols on the HGU95Av2 GeneChip® were linked to 6,020 GO categories. The one-sided Fisher&#39;s exact test p-values calculated by GoMiner were used to evaluate the statistical significance of changes for a GO category. The p-values for the first layer GO categories were converted to −log 10 (p-value) and graphed ( FIG. 6 ).  
         [0060]     Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al.,  Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, New York (1989), and in Ausubel et al.,  Current Protocols in Molecular Biology , John Wiley and Sons, Baltimore, Md. (1989) and in Perbal,  A Practical Guide to Molecular Cloning , John Wiley &amp; Sons, New York (1988), and in Watson et al.,  Recombinant DNA, Scientific American Books , New York and in Birren et al (eds)  Genome Analysis: A Laboratory Manual Series , Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out generally as in  PCR Protocols: A Guide To Methods And Applications , Academic Press, San Diego, Calif. (1990). In-situ (In-cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al, 1996, Blood 87:3822.)  
         [0061]     Standard methods in immunology known in the art and not specifically described are generally followed as in Stites et al.(eds), Basic and Clinical Immunology (8th Edition), Appleton &amp; Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W. H. Freeman and Co., New York (1980).  
         [0062]     Gene therapy, as used herein, refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype. The genetic material of interest encodes a product (e.g., protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. Alternatively, the genetic material of interest can encode a suicide gene. For a review, see, in general, the text “Gene Therapy” (Advances in Pharmacology 40, Academic Press, 1997).  
         [0063]     Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.  
         [0064]     In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, which is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in situ [Culver,  1998 ]. These genetically altered cells have been shown to express the transfected genetic material in situ.  
         [0065]     The gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. Often the 5′UTR and/or 3′UTR of the gene can be replaced by the 5′UTR and/or 3′UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5′UTR and/or 3′UTR of the actual gene to be transferred and only include the specific amino acid coding region.  
         [0066]     The expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription. Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non-translated DNA sequence that works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below.  
         [0067]     Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al.,  Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al.,  Current Protocols in Molecular Biology , John Wiley and Sons, Baltimore, Md. (1989), Chang et al.,  Somatic Gene Therapy , CRC Press, Ann Arbor, Mich. (1995), Vega et al.,  Gene Targeting , CRC Press, Ann Arbor, Mich. (1995),  Vectors: A Survey of Molecular Cloning Vectors and Their Uses , Butterworths, Boston Mass. (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. No. 4,866,042 for vectors involving the central nervous system and also U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.  
         [0068]     Introduction of nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.  
         [0069]     A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus-derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor that includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.  
         [0070]     Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur.  
         [0071]     Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.  
         [0072]     In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.  
         [0073]     As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. The vector to be used in the methods of the invention can depend on desired cell type to be targeted and can be known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used.  
         [0074]     Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles that are capable of undergoing further rounds of infection. The vector&#39;s genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector can depend upon the intended application. The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.  
         [0075]     The recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors can circulate until they recognize host cells with the appropriate target specificity for infection.  
         [0076]     An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.  
         [0077]     The above discussion provides a factual basis for the use of microarrays for detecting molecular markers of cancer and aging as disclosed above. The methods used with a utility of the present invention can be shown by the following non-limiting examples and accompanying figures.  
       EXAMPLES  
     Example 1  
       [0078]     Abrogating cellular senescence is a necessary step in the formation of a cancer cell. Promoter hypermethylation is an epigenetic mechanism of gene regulation known to silence gene expression in carcinogenesis. Treatment of spontaneously immortal Li-Fraumeni fibroblasts with 5-aza-2′-deoxycytidine (5AZA-dC), an inhibitor of DNA methyltransferase (DNMT), induces a senescence-like state. Microarrays containing 12,558 genes were used to determine the gene expression profile associated with cellular immortalization and also regulated by 5AZA-dC. Remarkably, among 85 genes with methylation-dependent downregulation (silencing) after immortalization, 39 (46%) are regulated during an interferon signaling known growth-suppressive pathway. The data included herein indicates that gene silencing can be associated with an early event in carcinogenesis, cellular immortalization.  
         [0079]     Immortalization is one of the necessary, multiple steps of tumorigenesis. Normal mammalian somatic cells can only divide a limited number of times in vitro. The maximum number of divisions is called the ‘Hayflick limit’ (Hayflick, 1976). This non-proliferative state is also referred to as replicative cellular senescence. Typical characteristics of senescing cells include a large, flat morphology, a high frequency of nuclear abnormalities, and positive staining for β-galactosidase activity specifically at pH 6.0. The counting mechanism for the intrinsic replicative lifespan appears to be the shortening of telomeres with each cell division cycle (Huschtscha and Holliday, 1983). The phenotype of senescence is a dominant trait, and the genes associated with it fall into four complementation groups (Pereira-Smith and Smith, 1983).  
         [0080]     Human cells can be immortalized through the transduction of viral and cellular oncogenes (Graham et al., 1977; Huschtscha and Holliday, 1983), various human oncogenes such as c-myc (Gutman and Wasylyk, 1991), or in some rare cases spontaneously (Bischoff et al., 1990; Rogan et al., 1995; Shay et al., 1995). These mechanisms of immortalization result in abrogation of p53 and pRB/p16 ink4 -mediated terminal proliferation arrest and the activation of a telomere maintenance mechanism (Rogan et al., 1995; Duncan et al., 2000). The activation of human telomerase reverse transcriptase (hTERT) expression is one of the telomere maintenance mechanisms that allow cells to bypass senescence. Certain immortalized human cell lines (Bryan et al., 1995) and some tumors (Bryan et al., 1997) maintain their telomeres in the absence of detectable telomerase activity by a mechanism, referred to as alternative lengthening of telomeres (ALT), that can involve telomere-telomere recombination (Dunham et al., 2000).  
         [0081]     Senescence can also be induced in immortal cells by a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine (5AZA-dC) (Vogt et al., 1998), implying that replicative senescence can result from epigenetic changes in gene expression (Herman and Baylin, 2000; Newell-Price et al., 2000; Baylin et al., 2001). Genes regulated by DNA methylation usually contain upstream regulatory regions and immediate downstream sequences enriched in CpG dinucleotides (CpG islands). Cytidine residues within CpG islands are methylated by DNMT that can recruit histone deacetylases resulting in the formation of condensed chromatin structures containing hypoacetylated histones. Hypomethylation of CpG islands in oncogenes and hypermethylation of tumor-suppressor genes are important regulatory mechanisms in tumor initiation and progression of cancer (Vogt et al., 1998; Baylin et al., 2001).  
         [0082]     Li-Fraumeni syndrome (LFS) is a familial cancer syndrome that is characterized by multiple primary tumors including soft-tissue sarcomas, osteosarcomas, breast carcinomas, brain tumors, leukemias, adrenal-cortical carcinomas, to a lesser extent melanoma and carcinomas of the lung, pancreas, and prostate. Heterozygous germlne p53 mutations were found in 75% of families having LFS (Malkin et al., 1990; Malkin, 1994). Fibroblast cell lines established from individuals with LFS develop changes in morphology, chromosomal abnormalities, and spontaneously form immortal cell lines (Hayflick, 1976; Bischoff et al., 1990; Malkin et al., 1990). Vogt et al. (1998) demonstrated that the treatment of immortal LFS fibroblasts with 5AZA-dC results in arrest of growth of the fibroblasts and development of a senescent phenotype. Repression of gene expression because of methylation-dependent silencing occurs upon cellular immortalization and a significant proportion of these genes are regulated in the interferon (IFN) pathway. Silencing of this growth-suppressive pathway can be an important early event in the development of cancer, specifically associated with immortalization.  
         [0000]     Materials and Methods  
         [0000]     Cell Culture and p53 Genotyping  
         [0083]     The MDAH041 (p53 frameshift mutation) cell line was derived from primary fibroblasts obtained by skin biopsy from patients with LFS. Characterization and immortalization of these cells was performed by Bischoff et al. (1990). All cells were grown in modified Eagles medium (MEM, Gibco BRL, MD, USA) with 10% fetal calf serum and antibiotics. The CRL1502 cell line was derived from primary fibroblasts obtained by skin biopsy from a normal donor (ATCC 1502, Rockville, Md., USA). The region containing the frameshift mutation in gene encoding p53 from LP preimmortal and HP immortal cells was sequenced to confirm the heterozygosity in LP preimmortal MDAH041 cells. Treatment of cells with 5AZA-dC Fibroblast cell cultures were seeded 3×10 5  per plate in MEM medium with 10% fetal calf serum and antibiotics. Cell cultures were treated with 1 μM 5AZA-dC on days 1, 3, and 5 each time with a full media change. After day 6, the cells were returned to regular medium without 5AZA-dC. Total RNA preparation was performed on day 8.  
         [0000]     RNA Isolation and the Affymetrix Microarray Assays  
         [0084]     The cells were grown to 80% confluence, the medium was changed, and after 16 hours the cells were washed with PBS, trypsinized, and pelleted at 300 g for 5 minutes. Total RNA was isolated using RNeasy kit (Qiagen Inc., Valencia, Calif., USA). 1.5×10 7  cells yielded 200 μg total RNA. The RNA targets (biotin-labelled RNA fragments) were synthesized from 5 μg of total RNA by first synthesizing double-stranded cDNA followed by standard Affymetrix protocols (Affymetrix, Santa Clara, Calif., USA).  
         [0000]     Quantitation of Gene Expression by Q-RT-PCR  
         [0085]     Total RNA (1 μg was reverse transcribed into cDNA using Superscript II (Life Technologies, Gaithersburg, Md., USA). All methods for reactions were performed as recommended by the manufacturer. The ABI 5700 Sequence Detection System was used for Q-RT-PCR. The protocols and analysis of data are identical to that of the ABI 7700 Sequence Detection System (ABISYBR). All methods for reactions and quantitation were performed as recommended by the manufacturer. An extensive explanation and derivation of the calculations involved can be found in the ABI User Bulletin× and also in the manual accompanying the SYBR Green PCR core kit. Primers used in Q-RT-PCR are shown in Table 11.  
         [0000]     Analysis of Microarray Data  
         [0086]     Microarray experiments were performed using the Affymetrix HG-U95A chip containing 12,558 probes. Two RNA preparations from immortal cells (HP) were compared with two RNA preparations from preimmortal cells (LP). In addition, two RNA preparations from immortal cells (HP) were compared with three total RNA preparations from immortal cells treated with 5AZA-dC using the HG-U95A chips.  
         [0087]     Two analysis methods were used to select differentially regulated genes: fold change and noise sampling method (ANOVA). The fold change method was used to select the genes with at least a twofold change in expression. This was done using the Affymetrix Data Mining Tool (DMT), version 3,N -fold method (Affymetrix, Santa Clara, Calif., USA). For the control versus experiment comparisons, all possible pairings between the two controls and the two experiments were considered.  
         [0088]     The noise sampling method is a variation of the ANOVA model proposed by Kerr and Churchill (Kerr et al., 2000; Draghici, 2002). The noise sampling method was implemented in GeneSight, version 3.2.21 (Biodiscovery, Los Angeles, Calif., USA). In order to apply the noise sampling method, the intensities obtained from each chip, were normalized by dividing by the mean intensity. Four ratios were formed by taking all possible combinations of experiments and controls. Genes differentially regulated with a 99.99% confidence (P ¼ 0.0001) were detected.  
         [0000]     CPG Island Analysis  
         [0089]     First, the genome sequence of each IFN-regulated RNA from UCSD Genome Browser (http:Hlgenome.ucsc.eduI) was retrieved. Then, the CpG islands were tested within an interval of 500 to 200 bp around the transcription starting site (TSS) using CpGPlot program (http://www.ebi.ac.uk/emboss/cpgplot/). The discrimination for CpG islands is based on the formal definition of CpG islands (Gardiner-Garden and Frommer, 1987)(length is over 200 bp, G+C content is greater than 50%, statistical expectation is greater than 0.6).  
         [0000]     Results  
         [0000]     Chances in Gene Expression After Immortalization  
         [0090]     Preimmortal (PD 11) and immortal (PD 212) fibroblast cells (MDAH041 cell line) from an LFS patient were employed to analyze the changes in gene expression during cellular immortalization. Total RNA was isolated from these cells and probes were synthesized for hybridization to microarrays, Affymetrix HGU95Av2 GeneChips. The genes were selected using two different methods: (i) the classical method of selecting the genes with at least a predetermined fold change and (ii) an ANOVA-based noise sampling selection method (Draghici, 2002). All the four possible pairings between preimmortal vs immortal cell gene expression comparisons were performed using independent cellular RNAs prepared from these cells. The fold change method was used to select the genes with twofold or greater change in gene expression. There were 169 upregulated and 450 down-regulated genes satisfying this condition (Table 1). The noise-sampling selection method is based on ANOVA (Kerr et al., 2000) and uses replicate measurements to estimate an empirical distribution of the noise. Given this distribution and a chosen confidence level, one can establish which genes are differentially regulated beyond the influence of the noise. The method identified 76 upregulated and 217 downregulated genes.  
         [0091]     The two methods are in some sense complementary. The noise-sampling method selects those genes that have reproducible changes higher than the noise threshold at some confidence level, whereas the N -fold method selects those genes that have a minimal fold change that can be confirmed with other assays such as quantitative real time PCR (Q-RT-PCR). The intersection of the subsets of genes reported as differentially regulated by both methods identified 59 upregulated genes and 192 downregulated genes after immortalization (Table 1). Using a representative set of the genes satisfying both conditions (for both downregulated and upregulated genes), the microarray data were confirmed using Q-RT .PCR (Table 2). Comparison of the levels of gene expression after immortalization obtained by using both microarray hybridization and Q-RT-PCR revealed outstanding accuracy of the data. Since Q-RT-PCR data can cover a larger range of expression levels, the data obtained using microarrays and Q-RT-.PCR differed quantitatively.  
         [0000]     Effect of 5AZA-dC Gene Expression in Immortal LFS Fibroblasts  
         [0092]     As was first shown by Fairweather et al. (1987), in vitro lifespan of normal human fibroblasts could be shortened by exposure of the cells to the demethylating agent 5AZA-dC. In agreement with this, Vogt et al. (1998) have shown that treatment of LFS immortal fibroblasts with 5AZA-dC results in growth arrest and senescence. Thus, there is a possibility that development of immortalization is related to methylation-induced silencing of gene expression. To address this issue, the immortal cells (MDAH041 high passage cell culture) were treated with 5AZA-dC to induce gene demethylation. Treated MDAH041 cells had flat morphology, contained lipofuscin granules, and showed senescence associated β-galactosidase activity at pH 6, typical for the senescent cells (Dimri et al., 1995). Total RNA was prepared from MDAH041, high-passage (HP) treated or untreated with 5AZA-dC,and used to prepare probes for the microarray hybridizations. Affymetrix HGU95Av2 GeneChips were again used and the data were analyzed as described above for the comparison of preimmortal and immortal MDAH041 cells. The comparison of treated and untreated HP cells identified 48 5AZA-dC upregulated and 190 5AZA-dC downregulated genes with at least a twofold change and 150 upregulated and 328 down-regulated genes selected by ANOVA (Table 1). There were 81 upregulated genes and only one downregulated gene that satisfied both conditions (P&lt;α and fold change &gt;2 (Table 1). A sampling of genes covering a range of gene expression changes was chosen and confirmed using Q-RT-PCR (Table 3).  
         [0093]     It was then determined whether changes in gene expression using Q-RT-PCR after 5AZA-dC treatment were specific to cells undergoing senescence by comparing gene expression changes induced by 5AZA-dC treatment in normal mortal human fibroblasts with those in the immortal MDAH041 cells. The expression levels of 15 of these genes were analyzed in preimmortal low-passage (LP) MDAH041 and normal mortal fibroblast cells (CRL-1502) untreated or treated with 5AZA-dC using Q-RT-PCR (Table 4). The vast majority of the 5AZA-dC-dependent changes in expression found in the immortal MDAH041 cells were not induced by 5AZA-dC treatment of the normal human fibroblasts or preimmortal MDAH041 LFS fibroblasts. The exception, IFN-inducible p27, is found in a known imprinted region on chromosome 14q32 and its induction by 5AZA-dC in all cells therefore was not surprising. In summary, while treatment with 5AZA-dC strongly induces expression of many genes silenced in immortal cells, the expression levels of the same genes were not significantly affected by 5AZA-dC treatment of mortal fibroblasts.  
         [0000]     Genes Downregulated After Immortalization and Silenced by Gene Methylation  
         [0094]     Since 5AZA-dC-induced gene expression results in the reversal of immortal phenotype and the induction of a senescent-like state, it was investigated whether inhibition of DNMT by 5AZA-dC upregulates genes repressed after immortalization. Table 5 shows the list of 85 genes selected by either or both selection methods as silenced after immortalization due to methylation. Interestingly, when the ‘reverse’ identification of genes was attempted (i.e. genes, both upregulated after immortalization but repressed by 5AZA-dC), no common genes were identified using the dual selection method approach (Table 1,comparison of A and C). In view of the fact that the numbers of genes identified in these comparisons (comparisons B and D (85 genes), and A and C (three genes)) were so vastly different, these suggested that methylation-dependent gene silencing is mechanistically significant to the process of immortalization. Microarray analysis of MDAH041 cells containing a tetracycline-modulated p53 gene revealed that none of these 85 genes were regulated by p53 in these cells. Analysis of the functional annotations of the genes downregulated in immortalization (Table 5), because of methylation-dependent silencing, revealed that a significant fraction, 39 out of 85 genes, are known to be regulated by the IFN pathway, with 19 of the 39 genes containing CpG islands identified using CpGPlot software (Table 6).  
         [0000]     Hierarchical Clustering  
         [0095]     The hierarchical map of the silenced gene expression set and two subsets of genes (identified by both software methods) that are repressed after immortalization by methylation-dependent silencing is shown in  FIGS. 2   a, b . In these figures, the height of each bridge between members of a cluster is proportional to the average squared distance of each leaf in the subtree from that subtree&#39;s centroid (or mean). These data indicate that the level of expression of the same set of genes that are downregulated during immortalization is also stimulated by 5AZA-dC-induced DNA demethylation. Interestingly, the approach showed that the total pattern of gene expression (12,558 genes) in preimmortal MDAH041 cells is similar to the 5AZA-dC-treated immortal MDAH041 cells as compared to the untreated immortal cells. In  FIG. 2   a , the set of 5 genes silenced by methylation show a pattern of low expression in the immortal fibroblasts (indicated by the green color) and higher expression in the preimmortal MDAH041 cells and in the 5AZA-dC-treated immortal cells (indicated by the red color).  FIG. 2   b  similarly shows the pattern of gene expression in the group of 30 genes selected by 99.99% confidence and a greater than twofold change in expression.  
         [0000]     Discussion  
         [0096]     The indefinite lifespan necessary for the formation of a cancer cell appears to be a complex genetic trait with four complementation groups of recessive genes (Pereira-Smith and Smith, 1983, 1988; Berube et al., 1998). Since treatment of spontaneously immortalized Li-Fraumeni cells, MDAH041, with the DNMT inhibitor, 5AZA-dC, results in a replicative senescent state (Baylin et al., 2001), epigenetic control of immortalization needed to be considered in these cells. Affymetrix microarrays were employed to profile gene expression changes associated with immortalization and determined which of those genes were also regulated by DNA demethylation. Genes downregulated after immortalization (493 genes) fit the pattern of recessive senescence genes predicted by the somatic cell genetics experiments (Pereira-Smith and Smith, 1988). Consistent with this hypothesis, it was reasoned that those in common with the 190 genes upregulated after the 5AZA-dC treatment would focus the gene set on those involved in replicative senescence. This gene set included a total of 85 genes from those available on the microarrays used. One of these genes is known to be maternally imprinted in the Prader-Willi Syndrome, NDN (Jay et al., 1997) (Table 5). The protein encoded by this gene, Necdin, is a growth suppressor expressed in postmitotic neurons of the brain (Nakada et al., 1998). The RNA is silenced during immortalization and activated by 5AZA-dC treatment of the immortal MDAH041 cells but not normal fibroblasts or preimmortal MDAH041 (Table 4). Interestingly, this gene was found to undergo loss of heterozygosity in the MDAH041 immortal cells.  
         [0097]     Downregulation in immortal MDAH041 cells of some genes (collagenase, cathepsin O, uPA) was observed that have been detected by others as upregulated genes during replicative senescence in dermal fibroblasts (Shelton et al., 1999). Downregulation of DOC1, IGFBP4 and IGFBP6 was also observed in immortal cells that is correlated with the published data before of Schwarze et al. (2002) who found upregulation of DOC1 and IGFBP3 in human prostate epithelial cells when passaged to senescence.  
         [0098]     Remarkably, 39 of these 85 genes were also known to be regulated in the IFN pathway and represent candidate regulatory genes in cellular immortalization. These data are in agreement with others who observed 5AZA-dC upregulation of IFN pathway genes in colon tumor cells (Karpf et al., 1999) and human bladder cancer cells (Liang et al., 2002). To calculate the significance of this observation, the UniGene clusters were used in order to eliminate overcounting genes with several accession numbers and/or Affymetrix probes. Currently, the 12,558 probes on the array correspond to 8628 Unigene clusters. Among these, there are 137 genes, or 0.015%, known to be IFN-regulated. Thus, a list of 85 random genes contains about 85 0.015% or approximately zero INF-regulated genes due to random chance. In fact, the list of 85 genes silenced in immortalization contained 39 IFN-regulated genes. The probability of this happening by chance is approximately 10 47 which shows that the silencing of the IFN-pathway genes is highly significant to the mechanism of cellular immortalization.  
         [0099]     Some IFN-regulated genes have previously been shown to be silenced by DNA methylation and reactivated by 5AZA-dC treatment (Liang et al., 2002). Consistent with this observation and the growth-inhibitory effect of IFNs, 5AZA-dC treatment has been shown to inhibit the growth of human tumor cell lines (Bender et al., 1998) and the data indicate that gene silencing can be an early event in cancer development. The IFN-regulated RNaseL gene is known to inhibit cell proliferation and induce apoptosis through the IFN-regulated (2′-5′) oligoadenylate synthetase pathway. RNaseL is a candidate tumor-suppressor gene that has been shown to be mutated in the germ line of hereditary prostate cancer patients (Carpten et al., 2002). This candidate tumor-suppressor gene, RNaseL, is activated by (2′-5′) oligoadenylate synthetase proteins and therefore it is noteworthy that in MDAH041 cells, three out of four of the isoforms of the (2′-5′) oligoadenylate synthetase are downregulated after immortalization because of methylation-dependent silencing (Table 6). In addition, IRF-1 has been shown to be a tumor-suppressor gene in human leukemias (Harada et al., 1993; Willman et al., 1993). The double-stranded RNA-activated protein kinase (PKR) has been shown to induce apoptosis, implying that its inactivation would be a procarcinogenic event (Jagus et al., 1999). The IFN-inducible proteins of the ‘HIN-200 gene family’ have been demonstrated to be growth inhibitory, have antitumor activity (Wen et al., 2001; Xin et al., 2001), and are able to bind to the Rb1 and p53 tumor-suppressor proteins (Choubey and Lengyel, 1995). One of the three members of this gene family, AIM2, is downregulated in MDAH041 cells and silenced by methylation (Table 6). AIM2 functions as a tumor suppressor for a melanoma cell line (DeYoung et al., 1997) and a T-cell tumor antigen in neuroecto-dermal tumors, as well as breast, ovarian, and colon carcinomas (Harada et al., 2001). The AIM2 gene contains a site of microsatellite instability (MSI) that results in gene inactivation in 47% of colorectal tumors analyzed with high MSI (Mori et al., 2001). Interestingly, p202, a member of the murine ‘200 gene family’, is a negative regulator of p53 whose gene expression is controlled by p53 as well (D&#39;Souza et al., 2001).  
         [0100]     MDAH041 LFS cells contain significant telomerase activity after immortalization (Gollahon et al., 1998). Although in microarray analysis, the hTERT gene for the protein of enzymatic subunit of telomerase was not significantly upregulated after immortalization of MDAH041 cells, 1.6-fold, using Q-RT .PCR that there was a significant increase in hTERT expression, 486-fold (Tables 2 and 7). This is consistent with the experience that genes with low basal expression levels are difficult to quantitate accurately using micro-arrays alone. 5AZA-dC treatment resulted in an additional 17-fold increase in hTERT RNA expression (Table 3). Interestingly, the promoter of the hTERT gene has been shown to be regulated by methylation at CpG islands (Dessain et al., 2000; Bechter et al., 2002). Using CpGPlot, an analysis was performed for the presence of CpG islands in the 39 interferon-regulated genes that were identified. In all, 19 of those genes contained CpG islands (Table 6). A subset of these 19 genes represent the primary inducers of cellular senescence and/or aging.  
         [0101]     p16 INK4a  is one of the tumor-suppressor genes whose expression is repressed by methylation, which permits cells to bypass early mortality checkpoints. Downregulation of p16 mRNA in immortal cells and upregulation by demethylation using RT .PCR was confirmed. When the level of protein expression was tested using Western blots, it was found that p16 INK4a  protein was much less abundant in immortal cells and upregulated approximately 500-fold by 5AZA-dC treatment. The 5AZA-dC-dependent upregulation of p16 INK4a  protein in immortal MDAH041 cells was observed by us and by Vogt et al. (1998), who demonstrated that retroviral transduction of a p16 INK4a  cDNA was able to induce senescence in MDAH041 cells. Although retroviral transduction of a p21 cDNA was also able to induce senescence in MDAH041 cells (Vogt et al., 1998), p21 protein levels were not regulated by 5AZA-dC treatment of immortal MDAH041 cells. It is noteworthy that p21 cip/waf  was also identified as sdi1 because of its high levels of expression in senescing mortal fibroblasts (Noda et al., 1994) and is regulated transcriptionally by DNMT (Young and Smith, 2001). p21 can also be regulated by STAT1 that is also a major transcriptional effector of the IFN pathway (Agrawal et al., 2002). The level of STAT1 protein is two-fold downregulated after immortalization and 4.7-fold upregulated in immortal cells by 5AZA-dC treatment. Therefore, STAT1 is silenced by methylation in immortal MDAH041 cells (Tables 5 and 6) and can be a key regulator of immortalization by controlling the interferon-regulated gene expression pathway and its growth-suppressive effectors. As these mechanisms become better understood, specific demethylation or deacetylation agents currently in preclinical evaluation and clinical trials in cancer patients can provide another approach to control cancer (Brown and Strathdee, 2002).  
       Example 2  
       [0102]     An indefinite lifespan or cellular immortalization is a necessary step in the formation of a cancer cell. Promoter hypermethylation is an important epigenetic mechanism of gene regulation in the development of cancer, cellular immortalization and aging. Oligonucleotide microarrays were used to discover the gene expression changes associated with cellular immortalization and compared those changes due to variations in gene expression after inhibiting DNA methylation in immortal fibroblast cells with 5-aza-2′-deoxycytidine. The goal was to identify candidate regulatory genes for immortalization as those regulated under both conditions. Among 84 such regulated genes, 31 genes were identified that are known to be involved in interferon-cytokine/JAK/STAT signaling, which are pathways known to be growth suppressive. These and other pathways of gene expression are thus highlighted as important molecular targets for intervention in cancer and aging.  
         [0000]     Cellular Immortalization  
         [0103]     Smith et al. in 1998 used cell fusion experiment to group &gt;40 immortal human cell lines into four complementation groups. Cell lines in the same complementation group generated hybrids with unlimited division potential. However, cell lines in different complementary group generated hybrids with a finite number of cell divisions (Pereira-Smith et al (1988). Based on this finding, later research used microcell-mediated chromosome transfer technique to identify involvement of mortality factor on chromosome 4 (MORF4) in cell senescence and immortalization (Leung et al (2001).  
         [0000]     DNA Methylation  
         [0104]     DNA Methylation as an epigenetic regulation in carcinogenesis gene function can be disrupted through either genetic alternations or epigenetic alternations. Genetic alternations include direct gene mutation or deletion. However, epigenetic alternations indicate the inheritance of aberrant states of gene expression following cell division. DNA methylation is one epigenetic mechanism that modifies the genome via covalent addition of a methyl group to the 5-position of cytosine ring in CpG dinucleotide (Holliday, (1990); Bird (1992); Boyes et al (1991). CpG dinucleotides usually cluster at the 5′-ends of regulatory region of genes and are referred to as CpG islands (Boyes et al (1991). DNA methylation in these CpG islands correlate with transcription silencing of the genes. The transcription repression can partly due to the affected ability of DNA-binding proteins to interact with their cognate cis elements (Jaenisch R. (1997). Methylation also plays a key role in genomic imprinting. The regulation of the imprinted gene expression is assumed to be a kind of competition between sense and antisense transcripts on both parental alleles. The methylation patterns of downstream region of the promoter, e.g. imprint control region (ICR) for Igf2 and differentially methylated region 2 (DMR2) for M6P-Igf2r determine the expression of antisense transcript or sense transcript of the imprinted allele (Barlow et al (1991); Counts et al (1996). The normal methylation status is very important for the maintenance of genome stability and abnormal methylation status can lead to carcinogenesis. Hypomethylation can lead to the aberrant expression of oncogenes (Ming et al (2000); Makos et al (1993) and regional hypermethylation can lead to genetic instability and transcription inhibition of tumor suppressor genes (Makos et al (1993); Magewu et al (1994). The methylated CpG sites in the p53 coding region act as hotspots for somatic mutations and account for 50% and 25% inactivating mutations in colon cancer and general cancers (Greenblatt et al (1994); Baylin et al (2001) as well as most germ line mutations in p53.  
         [0000]     Promoter Hypermethylation and Carcinogenesis  
         [0105]     Promoter hypermethylation has been indicated to be an early event in tumor progression (Wales et al (1995). The genes whose expression have been repressed by promoter hypermethylation have been suggested to be candidate tumor suppressor genes. Various techniques have been applied to search for epigenetically silenced genes in cancer, including searching in frequent LOH regions for promoter hypermethylation (Costello et al (2000);, restriction landmark genomic scanning (Toyota et al (1999), methylated CpG amplification-restriction digest analysis (Liang et al (2002) and microarray (Peris et al (1999). So far, promoter hypermethylation of numerous genes has been identified and their relation to carcinogenesis has been analyzed. This list includes p16 INK4a , p15 INK4b , p14 ARF , p73, APC, BRCA1, hMLH1, GSTP1, MGMT, COH1, TIMP3, DAPK, E-cadherin, LKB1, hSRBC etc. These genes play an important role in cellular pathways of DNA repair, cell cycle regulation, cell-cell recognition and apoptosis, which are important for regulation of tumor formation and aging. Wild type p16 INK4a  is a negative regulator of cell cycle. It can bind to cyclin-dependent kinase 4 (cdk4) and cyclin-dependent kinase 6 (cdk6) and prevent their phosphorylation of the retinoblastoma protein. The cell cycle progression through the G1 phase is thus blocked (Belinsky et al (1998). The promoter methylation of p16/NK4a has been studied in a wide range of tumor types (Foster et al (1998). The inactivation of p16/NK4a has been implicated in the immortalization process. (Loughran et al (1996); Brenner et al (1998); Kiyona et al (1998); Counts et al (1995) Besides the genes studied, a broad survey for more genes involved in carcinogenesis is ongoing. Since genetic and epigenetic regulations of gene function are cooperative in carcinogenesis (Baylin et al (2001), genes identified from promoter hypermethylation alone as a candidate tumor suppressor gene should be followed by intensive functional analysis for their biological importance (Malkin et al (1990).  
         [0000]     MDAH041 Cell Line  
         [0106]     MDAHO41 cells derived from patient with Li-Fraumeni syndrome were used. Li-Fraumeni syndrome is a rare familial dominant inherited cancer syndrome. Approximately 75% of LFS patients carry a germline mutation in the p53 gene (Malkin et al (1990). There is a high frequency of somatic mutation in the remaining wild type allele of p53, which leads to the spontaneous immortalization in LFS fibroblast. The MDAHO41 cell line has a point deletion in the p53 allele and the p53 protein is truncated. In precrisis MDAHO41 cells (population doubling &lt;43), the wild type p53 is present and the cells do not have detectable telomerase activity. In postcrisis MDAHO41 cells, the expression of p53 decreases, due to the loss of the wild type allele of p53 and telomerase activity can be detected (Gollahon et al (1998). It has been reported that the treatment of MDAHO41 cells with 5-aza-CdR results an arrest of growth of fibroblast and senescence-associated β-Galactosidase activity at pH 6 (Vogt M et. al, 1998). In the study, low passage MDAHO41 (precrisis) and high passage MDAHO41 (postcrisis) cells were used to study the changes in gene expression in the cellular progression to immortalization. The high passage MDAHO41 cells were then treated with 5-aza-CdR, trying to detect the genes upregulated by promoter dehypermethylation. The observation that cells with p53 germline mutations can spontaneously immortalize (Bischoff et al (1990); Bischoff et al (1991); and can be transformed into tumor cells by oncogenes.  
         [0000]     5-aza-2′-deoxycytidine Treatment of MDAHO41 Cells  
         [0107]     Treatment of immortal MDAH041 cells with 5-aza-2′-deoxycytidine results in a senescent-like state (Vogt M et. al, 1998). MDAH041 cells were cultured at 37° C. in 10% humidified CO 2  in DMEM (10% FBS, 500 units/ml penicillin, 100 μg/ml streptomycin. The cells were treated with 1 μM 5-aza-2′-deoxycytidine for 6 days with media changes on days 1,3, and 5.  
         [0000]     Immunoblotting of 16 INK4a  Protein After 5-aza-CdR Treatment  
         [0108]     The tumor suppressor p16 INK4a  protein is known to be regulated by DNA methylation at its promoter and to be able to induce senescence in immortal cells, (Vogt M et. al, 1998). Twenty μg of cell extract was boiled for 5 minutes in sample buffer, electrophoresed on a 15% SDS-polyacrylamide gel, and transferred to nitrocellulose. The blots were blocked with 5% nonfat dry milk and incubated with purified anti-human p16 INK4a  diluted 1:5,000 at 4° C. overnight. The anti-mouse IgG was incubated with the blot for 1 hour at room temperature. The signal was detected by enhanced chemiluminescence. SAOS2 cells and HT1080 cells served as positive and negative control for p16 INK4a , respectively. The expression of the p16 INK4a  protein was upregulated over 500 fold in the 5-aza-CdR-treated MDAH041 cells, as compared to the expression in the untreated immortal MDAH041 cells ( FIG. 3 ). This is consistent with previously published work that p16 INK4a  protein is upregulated by 5-aza-CdR-induced DNA demethylation in MDAH041 immortal cells (Vogt M et. al, 1998).  
         [0000]     Affymetrix Oligonucleotide Array Analysis of Gene Expression  
         [0109]     Affymetrix array was performed on low passage MDAHO41, 5aza-CdR treated and non-treated high passage MDAHO41 cells with three replicates of each in the lab. mRNA were reverse transcribed into cDNAs. DNA chips were performed followed the protocols from Affymetrix (Santa Clara, Calif.). The microarrays were scanned and processed.  
         [0000]     Data Analysis  
         [0110]     The expression profiles were analyzed with Data Mining Tools of Affymetrix. The expression level of the genes in 5-aza-CdR treated MDAHO41 cells were compared with those of untreated cells. Genes whose expression levels were up regulated &gt;2 fold in 5-aza-CdR treated cells were selected (Table 1). The gene expression levels in high passage MDAH041 cells were compared with those of low passage MDAH041 cells (Table 1). The genes whose expression level were down-regulated &gt;2 folds in high passage immortal cells were selected. The genes whose expression levels are low in untreated high passage, immortal MDAH041 cells but high after 5-aza-CdR treatment were candidate tumor (or growth) suppressor genes whose expression has been repressed by promoter hypermethylation in immortal cells. By intersecting the two groups of genes, 84 genes upregulated by demethylation and downregulation during immortalization were identified, Table 1. The differential expression of many of the genes was confirmed by quantitative RT-PCR, Table 2. After functional annotation of the 84 genes from GeneOntology, it was found that these 84 genes involved in a broad range of pathways including cell-cell signaling, transcription regulation, cellular proliferation, and cell adhesion. By examining these genes closer, it was found that ˜25% (n=31) genes are interferon inducible genes or genes involved in the interferon/cytokine/JAK/STAT signaling pathways, Table 3. The suggested that the impairment of interferon signaling pathway might be important in early development of cancer (through an immortalization-related mechanism) and/or can be involved in the process of aging. The statistical probability of this happening by chance to ˜10 −34  was calculated.  
                                                           TABLE 1                           Affymetrix Microarray Data: genes regulated by immortalization       and methylation            Accession #   Gene Name   IMMORT   5aza   Software                    L19686   Microphage migration inhibitory   −278.0   42.1   A/GS           factor (MIF)       X54489   Melanoma growth stimulatory activity   −146.3   64.6   A           (MGSA) (GRO-1)       M33882   Interferon-induced p78, Mx1   −99.3   202   A/GS       AI017574   Cysteine-rich heart protein   −85.0   8.8   A       U66711   Ly-6-related protein (9804) gene   −73.7   34.1   A/GS           (responsive to IFNs)       X82494   Fibulin-2   −70.1   21.8   A/GS       AF054825   VAMP45 (vesicle-associated   −69.1   9.1   A           membrane protein 5)       AL049946   Adlican   −60.2   17.6   A/GS       M33882   Interferon-induced p78, MxB   −52.1   122.1   A/GS*       M55153   Transglutaminase (TGase)   −50.9   144.6   A       AF037335   Carbonic anhydrase precursor (CA   −47.4   8.9   A           12)       L24564   Rad (Ras associated with diabetes)   −35.9   19.7   A/GS*       AA631972   Nk4 protein (natural killer cell   −35.0   20.2   A/GS           transcript 4)       U20982   Insulin-like growth factor binding   −33.3   3.8   A*           protein-4       AF039103   Tat-interacting protein TIP30   −30.3   8.9   A       J09309   Gamma-interferon-inducible protein   −27.8   31.2   A/GS           (IP-30)       AF053944   Aortic carboxypeptidase-like protein   −27.4   12.3   A/GS       AL080059   CDNA DKFZp564H142   −23.4   9.5   A       U88964   HEM45 (interferon-stimulated gene,   −21.7   50.2   A/GS           20-kd; ISG20)       U03688   Dioxin-inducible cytochrome P450   −20.6   6.9   A           (CYP1B1)       U59185   Putative monocarboxylate   −19.5   6.3   A           transporter       AB029000   KIAA 1077 protein Sulfatase FP   −18.6   10.9   A       U45878   Inhibitor of apoptosis protein 1   −18.4   13.1   A/GS       M28130   Interleukin 8   −15.5   92.7   A/GS       X04371   2-5A synthetase induced by   −15.4   76.5   A/GS*           interferon OAS-1       X02419   uPA gene (urokinase-plasminogen   −14.6   4.4   A           activator gene)       M13509   Skin collagenase MMP1   −14.4   4.8   A       AF026941   CIG5 (cytomegalovirus induces   −13.9   66.6   A           interferon-responsive)       AB025254   PCTAIRE 2 (pctaire protein kinase)   −13.7   19.3   A/GS       X67325   Interferon-stimulated gene p27   −13.0   482.0   A           mRNA       M36820   Cytokine (GRO-beta, GRO-2)   −12.9   29.6   A/GS       AF026939   CIG49 (cytomegalovirus induces   −12.8   70.2   A/GS           interferon-responsive)       M90657   Tumor antigen (L6)   −12.6   17.5   A/GS*       AF060228   Retinoic acid receptor responder 3   −12.2   7.1   A/GS       J04164   Interferon-inducible protein 9-27   −11.8   8.8   A       AI885852   Similar to gb: L 19779 HISTONE   −7.3   10.3   A/GS*           H2A.1       M36821   Cytokine (GRO-gamma)   −11.1   56.8   A       AL050162   TESTIN 3 testis derived transcript (3   −10.7   8.1   A           LIM domains)       U77643   K12 protein precursor (SECTM1)   −10.7   19.6   A/GS       D28137   BST-2 (bone marrow stroma cell   −9.9   38.7   A/GS           surface gene)       M17017   Beta-thromboglobin-like protein   −9.7   17.5   A/GS       AC004142   BAC clone RG118D07 from 7q31   −9.4   5.5   A       M24283   Major group rhinovirus receptor   −9.3   28.9   A           (HRV)       AL022723   HLA-F, gene for major   −8.9   29.4   A/GS*           histocompatibility complex class I F       U15932   Dual-specificity protein phosphatase   −8.9   12.6   A/GS       M24594   Interferon-inducible 56 Kd protein   −8.6   36.6   A/GS*       AB020315   Dickkopf-1 (hdkk-1)   −8.3   14.4   GS       J02931   Placental tissue factor (two forms)   −8.0   8.7   A       X86163   B2-bradykinin receptor, 3   −7.9   3.8   A       M13755   Interferon-induced 17-kDa/15-kDa   −7.6   17.0   A/GS*           protein       L20817   Tyrosine protein kinase (CAK) gene   −7.5   5.7   A       AJ225089   2-5 oligoadenylate synthetase 59 kDa   −7.4   40.0   A/GS           OAS-L       AF085692   Multidrug resistance-associated   −7.3   13.9   A           protein 3B       M26326   Keratin 18   −6.9   13.5   A       M22489   Bone morphogenetic protein 2A   −6.8   9.9   A       U37518   TNF-related apoptosis inducing   −6.7   42.2   A           ligand TRAIL       M92357   B94 protein (tumor necrosis factor-   −6.7   5.5   A           alpha-inducible)       X07523   Complement factor H   −6.6   6.8   A       AB018287   KIAA0744 protein   −6.5   8.6   A       U53831   Interferon regulatory factor 7B   −6.3   17.5   A/GS       X55110   Neurite outgrowth-promoting protein   −6.2   7.1   A       AL039458   Integral membrane glycoprotein LIG-   −6.1   5.2   A           1 (TM4SF1)       AL021977   Transcription Factor MAFF   −6.1   8.1   GS       M65292   Factor H homologue   −5.8   7.5   A       D29992   Placental protein 5 (PP5)   −5.7   29.3   A       AF070533   Optineurin-like protein   −5.7   4.2   A**       AF052135   Associated molecule with the SH3   −5.6   7.6   GS           domain of STAM       D28915   Microtubular protein 44   −5.5   17.8   A/GS       M62402   Insulin-like growth factor binding   −5.5   5.9   A/GS           protein 6       AF024714   Interferon-inducible protein AIM2   −5.3   21.6   A           (absent in melanoma)       M31165   Tumor necrosis factor-inducible   −5.2   10.3   A           (TSG-6)       U81607   Gravin   −5.1   12.2   GS       M30818   Interferon-inducible protein,   −5.0   42   A           myxovirus resistance, Mx2       M25915   Complement cytolysis inhibitor (CLI)   −4.9   5.1   A       AB013382   DUSP6 (dual specificity MAP kinase-   −4.7   4.6   A           phosphatase)       AB000115   mRNA expressed in osteoblast   −4.3   32.5   A/GS       D50919   Tripartite motif-containing protein 14,   −4.3   6.2   A           TRIM14       X58536   HLA class I locus C heavy chain   −4.1   5.7   A       AF010312   Pig 7   −4   9.4   GS       AI985272   Neuromedin B Precursor   −3.9   5.6   A       X57985   Genes for histones H2B.1 and H2A   −3.6   4.3   A       U07919   Aldehyde dehydrogenase 6   −3.5   4.1   A       AA883502   Ubiquitin-conjugating enzyme E2L6   −3.4   5.9   A           (UBE2L6)       U22970   Interferon-inducible peptide (6-16)   −3.3   10.2   GS       M87434   2-5 oligoadenylate synthetase 69/71 kDa   −2.7   19.6   A           OAS-2       M97935   Transcription factor ISGF-3 (Stat 1)   −1.9   7.6   A**                  
 
 Confirmation of Changes in Gene Expression 
 
         [0111]     Immortal (PO 212) and pre-immortal (PO 11) fibroblasts cells (MOAH041 cell line) were used to analyze the changes in gene expression during immortalization. Total RNA was isolated from these cells and used as a probe for hybridization on microarrays. Affymetrix HGU95Av2 GeneChips were used and the data were analyzed using Affymetrix Microarray Suite and Data Mining Tool software packages (Affymetrix). The microarray data were further confirmed using Quantitative Real Time-PCR (Q-RT-PCR) using a randomly selected set of these genes. Table 2 shows a comparison of the levels of gene expression during immortalization by using both microarray hybridization and Q-RT-PCR. In all cases, 16 down-regulated and 5 up-regulated genes chosen by bioinformatics methods, there is an excellent correlation of the data obtained using both techniques. Since Q-RT-PCR data is accurate over a larger range of expression levels, the data obtained using microarrays and Q-RT-PCR are quantitatively different.  
                                                                     TABLE 2                           Comparison of expression levels of genes differentially regulated       during immortalization* by Affymetrix microarray technology       and quantitative real-time PCR.                    Q-RT-PCR,       Gene   Microarray, fold change   fold change                    Down-regulated genes            MIF   277   65       MGSA   145   6700       Interferon-inducible   99.3   2700       protein p78       NDN   60   2790       CD24   45   7450       CYP1B1   20   45       (2-5′) oligoadenylate   19   160       synthetase E gene OAS1       CIG49   13   36       Interferon-inducible 56 kDA   8.6   108       Interferon-inducible   12   8       membrane protein 9-27       (IFITM1)       Dermatopontin   10   13       Interferon-regulatory   6   2683       factor 7B       MRP3   5   17       Interferon-induced 17-kDA/   4.5   43       15-kDA       GST4A   4   8       Signal Transducer and   1.9   8.5       Activator of Transcription       1, STAT1, 91 kD       AIM2   5.3   165       IP-30   27.8   12       P69/OAS-2   2.7   142       Interferon, alpha-inducible   13   70       p27            Up-regulated genes            WISP   8   20       SNF2A   6   3       ERCC2   5   4       RAGE3   7   10       HTERT   1.6   486                 *Fold change of gene expression level in the immortal cells (MDAHO41 high passage) relative to non-immortal cells (MDAHO41 low passage). Analysis of the genes involved in immortalization indicated that a large fraction of them are interferon (IFN) regulated, Table 3. Analysis of the chromosomal location of these IFN-regulated genes revealed that they are clustered in multiple loci around the human genome.             
 
         [0112]                                                                TABLE 3                           Affymetrix Microarray Data: CYTOKINE/JAK/STAT pathway genes       regulated by demethylation and immortalization            Gene   IMMORT   5AZA   CpG   Locus                     1. Interferon-induced 17-kDa/15-kDA   −4.2   13.9   +   1p36.33        2. Interferon-inducible peptide (6-16)   −3.3   10.2   −   1p36        3. mRNA expressed in osteoblast   −4.3   32.5   −   1p31        4. Microtubular protein p44 (IFI44)   −5.5   17.8   −   1p31.1        5. Interferon-inducible protein   −5.3   21.6   −   1q22          (Absent in Melanoma 2, AIM2)        6. Complement factor H   −6.6   6.8   −   1q32        7. CIG5 vipirin; similar to Inflammatory   −13.9   67.0   −   2p25.3          Response Protein 6        8. Signal Transducer Activator of   −1.9   7.6   +   2q32.2          Transcription 1 STAT1 91 kDa        9. TNF-related apoptosis inducing   −6.7   42.2   −   3q26          ligand, TRAIL       10. Cytokine (GRO-beta, GRO-2)   −12.9   29.6   +   4g12-13       11. Interleukin 8   −15.5   92.7   −   4q12-13       12. HLA class I locus C heavy chain   −4.1   5.7   +   6p21       13. uPA gene (urokinase-   −14.6   4.4   +   8p12          plasminogen activator gene)       14. Ly-6-related protein (9804) gene   −73.7   34.1   +   8q24.3       15. Tripartite motif-containing protein   −4.3   6.2   +   9q22-q31          14, TRIM14       16. CIG49 Interferon-induced protein   −12.8   70.2   −   10q24          with tetratricopepide repeats 4       17. Interferon-inducible 56 kDa   −8.6   36.6   −   10q25-q26          protein       18. Interferon-inducible membrane   −11.8   8.8   −   11p15.5          protein 9-27 (IFITM1)       19. Interferon regulatory factor 7B   −6.3   17.5   +   11p15.5       20. (2-5′) oligoadenylate synthetase   −15.4   76.5   −   12q24.1          p46/p42 E gene OAS-1       21. (2-5′) oligoadenylate synthetase   −7.4   40.0   −   12q24.2          59 kDa isoform OAS-L       22. (2-5′) oligoadenylate synthetase   −2.7   19.6   −   12q24.2          69/71 kDa isoform OAS-2       23. Interferon, alpha-inducible   −13.0   482.0   −   14q32          protein 27       24. HEM45, ISG-20   −21.7   50.2   −   15q26       25. NK4 protein (natural killer cell   −35.0   20.2   −   16p13.3          transcript 4)       26. Insulin-like growth factor binding   −33.3   3.8   −   17q12-q21          protein-4       27. Gamma-interferon-inducible   −27.8   31.2   +   19p13.1          protein (IP-30)       28. BST-2 (bone marrow stroma cell   −9.9   38.7   −   19p13.2          surface gene)       29. Major group rhinovirus receptor (HRV)   −9.3   28.9   +   19p13.3          ICAM       30. Interferon-inducible protein p78, Mx2   −99.3   202   +   21q22.3       31. Interferon-inducible protein Mx1   −5   42   −   21q22.3                 (Data was processed in Affymetrix Data Mining Tool. Triplicates were averaged.)            5aza: Up-regulation in 5-aza-CdR treated HP MDAHO41 cells vs. untreated HP MDAHO41 cells            041HP: down-regulation in HP MDAHO41 cells VS. LP MDAHO41 cells               
 Interferons 
 
         [0113]     Interferons are a group of pleiotropic cytokines. Human interferons can be divided into two major classes, type-I (IFN alpha, beta, omega) and type-II (IFN gamma). Although they have common antiviral, antiproliferative and immunomodulatory activities (Platanias (1995); Platanias (1999), their physical and immunochemical properties are different (Platanias (1995). Interferons are generally inducible proteins, type-I IFNs are expressed in a various type of cells induced by viral infection. Type-II IFN is produced by activated T lymphocytes and natural killer cells. The diverse biological functions of interferons are realized by the expression of interferon inducible genes after the cells receive the signals from interferons. Type-I IFN receptor (IFNR) and type-II IFN receptor (IFNGR) are different and both type-I IFN and type-II IFN can induce several signaling pathways (Imada et al (2000). Jak-Stat pathway is one major pathway, which can be induced in both type-I and type-II IFNs. Upon the binding of interferon with its receptor, Jaks, receptor associated tyrosine kinase, are activated. Stats can then be recruited to the receptors via their SH2 domain and tyrosine phosphorylated by Jaks. Activated Stats can form homodimers or heterodimers, and then translocate to the nucleus to activate the expression of target genes that have proper promoter regulatory elements (Leonard et al (1998); Uddin et al (1996). Pathways involved in type-I interferon signaling also include insulin receptor substrate (IRS)/PI-3′-kinase pathway and pathways involving adaptor proteins of the Crk-family (CrkL and Crkll) or vav proto-oncogene product. For type II interferon stimulated pathways, besides Jaks, some other tyrosine kinases, Fyn (src-family) and Pyk-2 can also be activated. (Takaoka et al (1999); Pitha (2000). IFNs have shown their antiviral effects on several virally induced carcinomas and their influence in cell metabolism, growth and differentiation has suggested their importance in inhibiting tumorigenesis. A number of IFNs induced genes have tumor suppression activities when over expressed in uninfected cells, e.g. double stranded RNA activated protein kinase (PKR), activated RNAseL, and the proteins of the 200 gene family (Karpf et al (1999). Some recent studies in examining the promoter methylation in bladder cancer cells and colon adenocarcinoma cells also showed the activation of IFN signaling pathways after the treatment of 5-aza-CdR to cancer cells. The suggested IFN signaling pathway was found to be a potential tumor-suppressive pathway (Peris et al (1999; Agrawal et al (2002). The experimental results first revealed that IFN signaling pathways can be disrupted in immortalization. Based on the current knowledge of IFN signaling pathway and the present data, the promoter hypermethylation regulation of IFN signaling pathways appears to play a significant role in immortalization and identification of immortalization genes in IFN signaling pathways.  
         [0000]     STAT 1  
         [0114]     Signal transducers and activators of transcription 1 (STAT1) is one of the seven identified Stat proteins play an important role in cytokine signaling transduction. STAT1 is involved in both type-I and type-IIIFN signaling pathways. ( FIGS. 1, 3 ) It forms homodimer or heterodimer with other Stat proteins to activates the genes who have IFN-stimulated response elements (ISRE) or IFN-gamma activated sequences (GAS). Although STAT1α can be induced by several kinds of cytokines and is involved in diverse signaling pathways, the predominant role for STAT1α a is suggested to be growth inhibition (Uddin et al (1996). The antiproliferative function of STAT1α is revealed by its induction of the CDK inhibitor p21 WAF1  (Chin et al (1997), caspase 1 (Xu et al (1998), Fas and FasL (Kaplan et al (1998), which leads to cell cycle arrest and apoptosis. The deficiency of STAT1α can thus confer a selective advantage to tumor cells. In the study of STAT1α knockout mice, mice lacking STAT1α develops spontaneous and chemically induced tumors more rapidly and with more rapid frequency comparing with their wild-type littermates (Huang et al (2000). The regulation of STAT1α by promoter hypermethylation in tumor cells has been implicated in the study of colon cancer and bladder cancer cells (Peris et al (1999; Agrawal et al (2002). The negative regulatory effects of STAT1α in angiogenesis, tumorigenesis and metastasis have also been demonstrated in a transfection study in mouse fibrosarcoma (Altman et al (2001). These data combined with the findings suggest STAT1α to be a tumor suppressor gene involved in immortalization with the implication that IFN pathway genes are regulated by promoter hypermethylation. At a functional level, STAT1α could be a promising transcriptional regulator immortalization and cancer. The regulation of STAT1α at the mRNA level was confirmed by quantitative RT-PCR (Table 4 and at the protein level,  FIG. 3 ). The genes regulated by demethylation were also tested by quantitative RT-PCR and their up regulation was confirmed, Table 4.  
                                                         TABLE 4                           Confirmation of expression levels of genes identified by Affymetrix       microarray technology as differentially upregulated during Saza-CdR       induced DNA demethylation* using Quantitative Real-Time PCR.                    Microarray, fold   Q-RT-PCR,           Gene   change   fold change                        Up-regulated genes                Interferon-inducible p78   202   478           (2-5′) OAS1   92   4379           CIG49   70   204           MGAS   65   839           MIF   42   128           Interferon-inducible 56 kDa   36.6   1807           Interferon regulatory factor   17.5   20031           7B           CYP1B1   7   77           MRP3   14   54           Interferon-induced 17/15-kDA   14   228           Interferon-inducible   9   278           membrane protein 9-27           (IFITM1)           IP-30   31.2   7           Signal Transducer and   7.6   158           Activator of Transcription 1,           STAT1, 91 kD           Interferon, alpha-inducible   482   1320           p27           P69/OAS-2   19.6   231           AIM2   21.6   686                         *Fold change of gene expression in the immortal cells treated with 5aza-CdR relative to untreated cells.             
 
         [0115]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                   
               
               
                 Comparison of expression level of genes differentially 
               
               
                 regulated in immortal** and normal* cells after 
               
               
                 5azaCdR-induced DNA demethylation. 
               
             
          
           
               
                   
                   
                 MDAH041HP vs. 
               
               
                   
                 1502 vs. 
                 MDAH0411HP 
               
               
                 Gene Name 
                 1502 5aza* 
                 5aza** 
               
               
                   
               
             
          
           
               
                 STAT 1, 91 kD 
                 1.8↑ 
                 158↑ 
               
               
                 Interferon-inducible protein p78 
                 1.8↑ 
                 480↑ 
               
               
                 MIF 
                 1.7↓ 
                 130↑ 
               
               
                 MGSF 
                 3.2↓ 
                 800↑ 
               
               
                 NDN 
                 1.4↓ 
                 10↑ 
               
               
                 Interferon-inducible 56 kDa 
                 1.2↓ 
                 1807↑ 
               
               
                 protein 
               
               
                 Interferon-inducible membrane 
                 1.8↓ 
                 278↑ 
               
               
                 protein 9-27 (IFITM1) 
               
               
                 Interferon-induced 17-kDa/15-kDA 
                 1.8↑ 
                 443↑ 
               
               
                 (2-5′) oligoadenylate synthetase 
                 2↑ 
                 1072↑ 
               
               
                 E gene OAS1 
               
               
                 CIG49 
                 1.1↑ 
                 204↑ 
               
               
                 Interferon-regulatory factor 7B 
                 1.5↑ 
                 20031↑ 
               
               
                   
               
               
                   *Fold change of gene expression level in a non-immortal normal skin fibroblast (NSF) cell line 1502 before and after treatment with 5azaCdR.    
               
               
                   **Fold change of gene expression level in an immortal fibroblast cell line (041, high passage) before and after treatment with 5azaCdR.    
               
               
                   # ↑ and ↓ indicate increase and decrease in gene expression, respectively.    
               
             
          
         
       
     
         [0116]     To determine the specificity and significance of these findings, the expression levels of 11 genes in normal fibroblast cells (strain 1502) with 5-aza-CdR treated or untreated using Q-RT-PCR Table 5 were analyzed. Treatment of nonimmortal cells with 5Aza-dC does not result in an induction of an senescence-like state in the cells. When the expression levels of 11 of these genes were analyzed in normal fibroblast cells (strain 1502) 5Aza-dC treated or untreated using Q-RT-PCR (Table 5) no 5Aza-dC dependent changes in expression were observed. None of these genes were significantly altered in their expression after the 5-aza-CdR-treatment.  
         [0117]     In summary. while 5Aza-dC-treatment strongly induces expression of many genes in Immortal cells. expression of the same genes is not significantly altered after the 5Aza-dC-treatment of normal fibroblasts. Therefore the immortal-specific gene expression changes observed in immortal MDAHO41 cells also regulated by treatment with 5Aza-dC has identified gene targets of cellular immortalization that were silenced by methylation.  
       Example 3  
       [0118]     The genes listed in Table 8 were increased (decreased) across four independently immortalized cell lines: MDAH041, MDAH087-N, MDAH087-1 and MDAH087-10. All three variants are derived from an original cell line. Each variant has different germlne p53 mutations, however all lose their wild type p53 upon immortalization. If a gene increased (decreased) across less then 4/4 of the cell lines, the gene is not present in these lists.  
         [0119]     Several situations could exist: 1. Genes decreased after treatment with 5-aza-deoxycidine (5-aza-dC); 2. Genes increased after treatment with 5-aza-dC; 3. Genes decreased during immortalization; 4. Genes increased during immortalization; and 5. Intersection of the genes that decreased during immortalization and increased after treatment with 5-aza-dC (Intersection of lists 1 and 4). For 1 and 2, 5-aza-dC treated immortalized cells were compared to untreated immortalized cells. For 3 and 4, immortalized cells were compared to pre-crisis cells. MDAH041 immortal cells were compared to MDAH041 pre-crisis cells. MDAH087-N, MDAH087-1 and MDAH087-10 were compared to MDAH087 pre-crisis cells.  
         [0120]     The Affymetrix probe ID for a probe. A probe is a sequence that is unique to 1 gene. Note, there are sometimes multiple probes for 1 gene. The microarry chip used was HG-U95Av2.  
         [0121]     There were multiple microarray chips, representing independent experiments, for each cell line. First we determined the genes that increased (decreased), during immortalization, across all chip comparisons for an individual cell line. Similarly, we determined the genes that increased (decreased), after treatment with 5-aza-dC, across all chip comparisons for an individual cell line. We used the list of genes generated for the individual cell lines to determine genes that were in common across all four cell lines. The results are shown in Table 8.  
                                                                   p53 sequence analysis of           LFS patients&#39; fibroblasts            Cell Line   Codon   Mutation Type                    MDAH087   248 CGG/TGG   Arg to Trp                   MDAH172   175 CGC/CAC   Arg to His               MDAH174   175 CGC/CAC   Arg to His               MAT170-1   133 ATG/ACG   Met to Thr               MAT170-3   133 ATG/ACG   Met to Thr               MAT120-1   N.D.   wt by Western Blot               MDAH041   184 GAT A/GAA   Frameshift stop after               60 amino acids                 ND = not determined             
 
         [0122]    
       
         
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
               
               
                   
                 NUMBER OF GENES 
               
             
          
           
               
                   
                 MDAH087-N 
                   
               
               
                   
                 MDAH087-1 
                 Magic Bullets 
               
             
          
           
               
                   
                 MDAH041 
                 MDAH087-N 
                 MDAH087-1 
                 MDAH087-10 
                 MDAH087-10 
                 Total 
                   
               
               
                   
                 Total Probe 
                 Total Probe 
                 Total Probe 
                 Total Probe 
                 Total Probe 
                 Probe 
                 Unique 
               
               
                 Gene Set 
                 IDs 
                 IDs 
                 IDs 
                 IDs 
                 IDs 
                 IDs 
                 Unignes 
               
               
                   
               
             
          
           
               
                 A. IM vs PC 
                 440 
                 576 
                 796 
                 332 
                 136 
                 26 
                 26 
               
               
                 upregulated* 
               
               
                 B. IM vs PC 
                 625 
                 486 
                 613 
                 467 
                 221 
                 85 
                 80 
               
               
                 downregulated* 
               
               
                 C. IM 5azaCdR 
                 420 
                 311 
                 266 
                 134 
                 40 
                 6 
                 6 
               
               
                 vs IM untreated, 
               
               
                 downregulated** 
               
               
                 D. IM 5azaCdR 
                 547 
                 447 
                 329 
                 306 
                 125 
                 85 
                 76 
               
               
                 vs IM untreated 
               
               
                 upregulated** 
               
               
                 Genes in Sets 
                 119 
                 52 
                 44 
                 33 
                 8 
                 4 
                 3 
               
               
                 B and D# 
               
               
                 Genes in Sets 
                 30 
                 101 
                 72 
                 24 
                 3 
                 0 
                 0 
               
               
                 A and C# 
               
             
          
           
               
                 P: Present 
                 Downregulated 
                 Downregulated 
                 Downregulated 
                 Downregulated 
                 Downregulated 
                 Downregulated 
               
               
                 M: Marginal 
                 Genes: 
                 Genes: 
                 Genes: 
                 Genes: 
                 Genes: 
                 Genes: 
               
               
                 A: Absent 
                 Call: P M A 
                 Call: P M A 
                 Call: P M A 
                 Call: P M A 
                 Call: P M A 
                 Call: P M A 
               
               
                 I: Increase 
                 Change: D MD 
                 Change: D MD 
                 Change: D MD 
                 Change: D MD 
                 Change: D MD 
                 Change: D MD 
               
               
                 MI: Marginal 
                 Upregulated Genes: 
                 Upregulated 
                 Upregulated 
                 Upregulated 
                 Upregulated 
                 Upregulated 
               
               
                 Increase 
                 Call: P M 
                 Genes: 
                 Genes: 
                 Genes: 
                 Genes: 
                 Genes: 
               
               
                 D: Decrease 
                 Change: I MI 
                 Call: P M 
                 Call: P M 
                 Call: P M 
                 Call: P M 
                 Call: P M 
               
               
                 MD: Marginal 
                   
                 Change: I MI 
                 Change: I MI 
                 Change: I MI 
                 Change: I MI 
                 Change: I MI 
               
               
                 Decrease 
               
               
                   
               
               
                   Fold change: No fold Percent comparisons: 100% Total number of comparisons in ( )    
               
               
                   Notes:    
               
               
                     1 Low passage/Pre-crisis chips used: PC2, PC6    
               
               
                   High passage/Immortalized chips used: AE1, AE2, OE2    
               
               
                   5-aza-dC treated immortal chips used: AE1, AE2, AE4    
               
             
          
         
       
     
         [0123]    
       
         
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
               
             
             
               
                 (B-D)Intersection_magic_bullets 
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                 PC vs HP 
                 041 5-aza 
                 PC vs IM 
                   
               
               
                   
                   
                   
                   
                   
                 041 HP Ave 
                 Ave Signal 
                 N-UT Ave 
               
               
                 Probe ID 
                 Unigene 
                 Locus ID 
                 Symbol 
                 Chromosome 
                 Signal Log 
                 Log 
                 Signal Log 
               
               
                   
               
             
          
           
               
                 36686_at 
                 Hs.75748 
                 220 
                 ALDH1A3 
                 15q26.3 
                 −1.42 
                 −2.67 
                 1.50 
                 2.83 
                 −1.18 
                 −2.26 
                   
               
               
                 40071_at 
                 Hs.154654 
                 1545 
                 CYP1B1 
                 2p21 
                 −3.47 
                 −11.04 
                 1.91 
                 3.75 
                 −3.03 
                 −8.16 
               
               
                 859_at 
                 Hs.154654 
                 1545 
                 CYP1B1 
                 2p21 
                 −3.81 
                 −14.04 
                 3.03 
                 8.17 
                 −3.06 
                 −8.36 
               
               
                 32730_at 
                 Hs.173094 
                 85453 
                 KIAA1750 
                 8q22.1 
                 −4.80 
                 −27.76 
                 1.53 
                 2.88 
                 −4.23 
                 −18.61 
               
               
                   
                   
                   
                   
                   
                 Log(2) 
               
               
                   
               
             
          
           
               
                 (B-D)Intersection_magic_bullets 
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                 10-5aza 
                   
               
               
                   
                 N-5aza Ave 
                 1-UT Ave 
                 1-5aza Ave 
                 10-UT Ave 
                 Ave Signal 
               
               
                 Probe ID 
                 Signal Log 
                 Signal Log 
                 Signal Log 
                 Signal Log 
                 Log 
               
               
                   
               
             
          
           
               
                 36686_at 
                 1.70 
                 3.24 
                 −3.00 
                 −8.00 
                 1.19 
                 2.29 
                 −2.15 
                 −4.42 
                 1.84 
                 3.83 
                   
               
               
                 40071_at 
                 2.95 
                 7.73 
                 −1.85 
                 −3.60 
                 2.06 
                 4.16 
                 −2.39 
                 −5.25 
                 2.23 
                 4.70 
               
               
                 659_at 
                 3.00 
                 7.98 
                 −2.06 
                 −4.17 
                 2.29 
                 4.69 
                 −2.36 
                 −5.13 
                 2.23 
                 4.69 
               
               
                 32730_at 
                 2.35 
                 5.10 
                 −1.90 
                 −3.72 
                 1.71 
                 3.28 
                 −3.93 
                 −15.24 
                 3.18 
                 9.09 
               
               
                   
               
             
          
           
               
                 (A) HP_I_Annotated_Averages 
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 041 5-aza 
                   
                   
               
               
                   
                   
                   
                   
                   
                 041 HP Ave 
                 Ave Fold 
                 N-UT Ave 
               
               
                 Probe ID 
                 Unigene 
                 Locus ID 
                 Symbol 
                 Chromosome 
                 Fold Change 
                 Change 
                 Fold Change 
               
               
                   
               
             
          
           
               
                 32331_at 
                 Hs.274691 
                 205 
                 AK3 
                 1p31.3 
                 1.34 
                 2.52 
                 −0.02 
                 −1.02 
                 2.03 
                 3.11 
                   
               
               
                 39230_at 
                 Hs.226307 
                 9582 
                 APOBEC38 
                 22q13.1-q13.2 
                 0.92 
                 1.89 
                 −1.27 
                 −2.40 
                 1.46 
                 2.86 
               
               
                 38201_at 
                 Hs.317432 
                 586 
                 BCAT1 
                 12pter-q12 
                 3.08 
                 8.47 
                 0.66 
                 1.58 
                 1.51 
                 4.52 
               
               
                 32238_at 
                 Hs.193163 
                 274 
                 BIN1 
                 2q14 
                 1.72 
                 3.29 
                 0.64 
                 1.56 
                 0.66 
                 3.23 
               
               
                 35615_at 
                 Hs.30736 
                 23246 
                 BOP1 
                 6q24.3 
                 1.47 
                 2.77 
                 0.23 
                 1.18 
                 1.07 
                 2.08 
               
               
                 1942_s_at 
                 Hs.95577 
                 1019 
                 CDK4 
                 12q14 
                 0.53 
                 1.44 
                 −0.49 
                 −1.41 
                 0.96 
                 2.26 
               
               
                 37931_at 
                 Hs.85004 
                 1059 
                 CENPB 
                 20p13 
                 0.96 
                 1.95 
                 0.16 
                 1.12 
                 0.99 
                 2.23 
               
               
                 39231_at 
                 Hs.22670 
                 1105 
                 CHD1 
                 5q15-q21 
                 0.87 
                 1.83 
                 −0.03 
                 −1.02 
                 1.10 
                 1.77 
               
               
                 33650_at 
                 Hs.65234 
                 55681 
                 DDX27 
                 20q13.13 
                 1.18 
                 2.26 
                 0.21 
                 1.15 
                 0.68 
                 2.21 
               
               
                 1537_at 
                 Hs.77432 
                 1958 
                 EGFR 
                 7p12 
                 2.23 
                 4.69 
                 1.10 
                 2.14 
                 3.59 
                 2.76 
               
               
                 40845_at 
                 Hs.256583 
                 3609 
                 ILF3 
                 19p13.2 
                 1.44 
                 2.70 
                 0.19 
                 1.14 
                 1.42 
                 2.08 
               
               
                 36624_at 
                 Hs.75432 
                 3615 
                 IMPDH2 
                 3p21.2 
                 0.81 
                 1.53 
                 −0.59 
                 −1.50 
                 0.71 
                 4.72 
               
               
                 39926_at 
                 Hs.37501 
                 4090 
                 MADH5 
                 5q31 
                 1.06 
                 2.08 
                 0.48 
                 1.39 
                 1.05 
                 2.01 
               
               
                 673_at 
                 Hs.172665 
                 4522 
                 MTHFD1 
                 14q24 
                 1.55 
                 2.93 
                 −0.56 
                 −1.48 
                 1.01 
                 2.07 
               
               
                 1973_s_at 
                 Hs.79070 
                 4609 
                 MYC 
                 8q24.12-q24.13 
                 1.76 
                 3.36 
                 0.42 
                 1.33 
                 2.24 
                 1.63 
               
               
                 1979_s_at 
                 Hs.15243 
                 4839 
                 NOL1 
                 12p13 
                 1.03 
                 2.04 
                 0.85 
                 1.81 
                 1.06 
                 2.88 
               
               
                 35705_at 
                 Hs.37288 
                 9975 
                 NR1D2 
                 3p24.1 
                 1.44 
                 2.71 
                 1.17 
                 2.24 
                 1.46 
                 12.06 
               
               
                 36125_s_at 
                 Hs.74111 
                 22913 
                 RALY 
                 20q11.21-q11.23 
                 0.93 
                 1.90 
                 0.17 
                 1.12 
                 1.14 
                 1.84 
               
               
                 39731_at 
                 Hs.146381 
                 27316 
                 RBMX 
                 Xq26 
                 0.54 
                 1.45 
                 −0.94 
                 −1.92 
                 0.82 
                 2.14 
               
               
                 41363_at 
                 Hs.102456 
                 8487 
                 SIP1 
                 14q13 
                 1.23 
                 2.34 
                 0.36 
                 1.28 
                 1.16 
                 1.99 
               
               
                 38455_at 
                 Hs.83753 
                 6628 
                 SNRP8 
                 20p13 
                 0.74 
                 1.67 
                 −0.44 
                 −1.35 
                 1.18 
                 1.94 
               
               
                 34851_at 
                 Hs.250822 
                 6790 
                 STK6 
                 20q13.2-q13.3 
                 0.54 
                 1.46 
                 −1.53 
                 −2.88 
                 1.06 
                 2.10 
               
               
                 318_at 
                 Hs.75307 
                 7052 
                 TGM2 
                 20q12 
                 1.46 
                 2.74 
                 0.20 
                 1.15 
                 1.69 
                 1.58 
               
               
                 910_at 
                 Hs.105097 
                 7083 
                 TK1 
                 17q23.2-q25.3 
                 1.37 
                 2.58 
                 −0.96 
                 −1.94 
                 2.18 
                 2.84 
               
               
                 1581_s_at 
                 Hs.75248 
                 7155 
                 TOP2B 
                 3p24 
                 1.77 
                 3.40 
                 0.32 
                 1.25 
                 1.52 
                 2.74 
               
               
                 40792_s_at 
                 Hs.367689 
                 7204 
                 TRIO 
                 5p15.1-p14 
                 1.37 
                 2.58 
                 1.04 
                 2.05 
                 1.64 
                 4.08 
               
               
                   
               
             
          
           
               
                 (A) HP_I_Annotated_Averages 
                   
               
             
          
           
               
                   
                 N-5aza Ave 
                 1-UT Ave 
                 1-5aza Ave 
                 10-UT Ave 
                 10-5aza Ave 
                   
               
               
                 Probe ID 
                 Fold Change 
                 Fold Change 
                 Fold Change 
                 Fold Change 
                 Fold Change 
               
               
                   
               
             
          
           
               
                 32331_at 
                 −0.54 
                 −1.46 
                 1.42 
                 2.68 
                 −1.03 
                 −2.04 
                 1.84 
                 3.57 
                 −0.87 
                 −1.82 
                   
               
               
                 39230_at 
                 −1.03 
                 −2.05 
                 1.55 
                 2.92 
                 0.17 
                 1.12 
                 1.42 
                 2.67 
                 0.92 
                 1.89 
               
               
                 38201_at 
                 0.46 
                 1.37 
                 1.71 
                 3.28 
                 0.65 
                 1.80 
                 1.96 
                 3.89 
                 0.16 
                 1.12 
               
               
                 32238_at 
                 0.44 
                 1.36 
                 0.89 
                 1.86 
                 −0.17 
                 −1.12 
                 0.88 
                 1.84 
                 0.13 
                 1.09 
               
               
                 35615_at 
                 0.36 
                 1.28 
                 1.31 
                 2.48 
                 −0.24 
                 −1.18 
                 1.12 
                 2.17 
                 −0.03 
                 −1.02 
               
               
                 1942_s_at 
                 −0.54 
                 −1.46 
                 1.25 
                 2.37 
                 −0.33 
                 −1.25 
                 1.07 
                 2.09 
                 −0.16 
                 −1.12 
               
               
                 37931_at 
                 −0.47 
                 −1.38 
                 0.92 
                 1.69 
                 −0.47 
                 −1.38 
                 1.02 
                 2.03 
                 −0.38 
                 −1.30 
               
               
                 39231_at 
                 −0.37 
                 −1.29 
                 0.97 
                 1.95 
                 0.07 
                 1.05 
                 0.86 
                 1.82 
                 0.30 
                 1.23 
               
               
                 33650_at 
                 0.30 
                 1.23 
                 0.79 
                 1.73 
                 0.23 
                 1.18 
                 1.12 
                 2.18 
                 −0.28 
                 −1.22 
               
               
                 1537_at 
                 −0.18 
                 −1.14 
                 3.12 
                 8.67 
                 0.39 
                 1.31 
                 2.96 
                 7.77 
                 0.85 
                 1.81 
               
               
                 40845_at 
                 −0.24 
                 −1.16 
                 1.08 
                 2.12 
                 −0.09 
                 −1.07 
                 1.37 
                 2.59 
                 0.31 
                 1.24 
               
               
                 36624_at 
                 −0.28 
                 −1.21 
                 0.95 
                 1.93 
                 −0.22 
                 −1.16 
                 0.79 
                 1.73 
                 −0.15 
                 −1. 
               
               
                 39926_at 
                 0.20 
                 1.15 
                 0.92 
                 1.89 
                 0.42 
                 1.34 
                 1.35 
                 2.55 
                 0.27 
                 1 
               
               
                 673_at 
                 −0.03 
                 −1.02 
                 1.93 
                 3.81 
                 −0.43 
                 −1.35 
                 1.07 
                 2.10 
                 −0.06 
                 −1.04 
               
               
                 1973_s_at 
                 −0.25 
                 −1.19 
                 1.34 
                 2.52 
                 −0.15 
                 −1.11 
                 1.20 
                 2.30 
                 0.15 
                 1.11 
               
               
                 1979_s_at 
                 1.02 
                 2.02 
                 1.90 
                 3.73 
                 0.37 
                 1.29 
                 1.27 
                 2.41 
                 0.80 
                 1.74 
               
               
                 35705_at 
                 −0.17 
                 −1.13 
                 1.33 
                 2.51 
                 0.02 
                 1.02 
                 1.47 
                 2.77 
                 0.05 
                 1.04 
               
               
                 36125_s_at 
                 −0.02 
                 −1.01 
                 0.82 
                 1.77 
                 0.18 
                 1.13 
                 1.04 
                 2.05 
                 0.36 
                 1.28 
               
               
                 39731_at 
                 −0.55 
                 −1.46 
                 1.01 
                 2.01 
                 −0.62 
                 −1.54 
                 0.77 
                 1.70 
                 −0.39 
                 1.31 
               
               
                 41363_at 
                 0.50 
                 1.41 
                 1.64 
                 3.11 
                 −0.06 
                 −1.05 
                 0.87 
                 1.83 
                 0.51 
                 1.42 
               
               
                 38455_at 
                 0.07 
                 1.05 
                 1.03 
                 2.01 
                 0.28 
                 1.21 
                 1.03 
                 2.04 
                 0.52 
                 1.43 
               
               
                 34851_at 
                 −1.12 
                 −2.18 
                 1.13 
                 2.18 
                 −0.04 
                 −1.03 
                 1.22 
                 2.33 
                 0.02 
                 1.01 
               
               
                 318_at 
                 −0.36 
                 −1.28 
                 2.44 
                 5.44 
                 −0.28 
                 −1.21 
                 1.40 
                 2.63 
                 0.51 
                 1.43 
               
               
                 910_at 
                 −1.30 
                 −2.48 
                 1.73 
                 3.31 
                 0.11 
                 1.08 
                 1.87 
                 3.19 
                 0.28 
                 1.22 
               
               
                 1581_s_at 
                 −0.21 
                 −1.16 
                 1.09 
                 2.13 
                 −0.42 
                 −1.33 
                 1.31 
                 2.48 
                 −0.22 
                 −1.17 
               
               
                 40792_s_at 
                 −0.05 
                 −1.03 
                 1.48 
                 2.79 
                 −0.12 
                 −1.09 
                 1.46 
                 2.75 
                 0.29 
                 1.23 
               
               
                   
               
             
          
           
               
                 (B) HP_D_Annotated_Averages 
                   
               
             
          
           
               
                   
                   
                   
                   
                 041 HP Ave 
                 041 5-aza 
                 N-UT Ave 
                   
               
               
                   
                   
                   
                   
                 Fold 
                 Ave Fold 
                 Fold 
               
               
                 Probe ID 
                 Unigene 
                 Locus ID 
                 Symbol 
                 Change 
                 Change 
                 Change 
               
               
                   
               
             
          
           
               
                 32755_at 
                 Hs.195851 
                 59 
                 ACTA2 
                 −3.97 
                 −15.65 
                 0.04 
                 1.03 
                 −2.56 
                 −5.91 
                   
               
               
                 39063_at 
                 Hs.118127 
                 70 
                 ACTC 
                 −3.08 
                 −8.43 
                 0.10 
                 1.07 
                 −6.50 
                 −90.30 
               
               
                 36686_at 
                 Hs.75746 
                 220 
                 ALDH1A3 
                 −1.42 
                 −2.67 
                 1.50 
                 2.83 
                 −1.18 
                 −2.26 
               
               
                 32527_at 
                 Hs.74120 
                 10974 
                 APM2 
                 −6.79 
                 −110.66 
                 1.36 
                 2.57 
                 −5.38 
                 −41.50 
               
               
                 39043_at 
                 Hs.433506 
                 10095 
                 ARPC18 
                 −0.87 
                 −1.83 
                 1.08 
                 2.12 
                 −1.52 
                 −2.86 
               
               
                 41776_at 
                 Hs.279910 
                 475 
                 ATOX1 
                 −0.72 
                 −1.64 
                 0.32 
                 1.25 
                 −0.99 
                 −1.98 
               
               
                 36497_at 
                 Hs.57548 
                 113146 
                 C14orf78 
                 −0.96 
                 −1.94 
                 −0.60 
                 −1.52 
                 −2.56 
                 −5.90 
               
               
                 37112_at 
                 Hs.101359 
                 9750 
                 C6orf32 
                 −5.23 
                 −37.57 
                 2.80 
                 6.97 
                 −2.57 
                 −5.93 
               
               
                 41207_at 
                 Hs.18075 
                 23733 
                 C9orf3 
                 −1.43 
                 −2.69 
                 1.92 
                 3.78 
                 −2.60 
                 −6.05 
               
               
                 38418_at 
                 Hs.82932 
                 595 
                 CCND1 
                 −1.84 
                 −3.57 
                 1.14 
                 2.20 
                 −1.04 
                 −2.06 
               
               
                 2020_at 
                 Hs.82932 
                 595 
                 CCND1 
                 −1.90 
                 −3.73 
                 1.19 
                 2.27 
                 −1.01 
                 −2.01 
               
               
                 39351_at 
                 Hs.278573 
                 966 
                 CD59 
                 −0.91 
                 −1.88 
                 −0.31 
                 −1.24 
                 −0.84 
                 −1.79 
               
               
                 41138_at 
                 Hs.433387 
                 4267 
                 CD99 
                 −1.49 
                 −2.80 
                 0.58 
                 1.49 
                 −1.44 
                 −2.70 
               
               
                 32363_at 
                 Hs.194687 
                 9023 
                 CH25H 
                 −4.42 
                 −21.46 
                 1.03 
                 2.04 
                 −4.17 
                 −18.04 
               
               
                 40698_at 
                 Hs.85201 
                 9976 
                 CLECSF2 
                 −3.97 
                 −15.71 
                 −0.44 
                 −1.36 
                 −2.64 
                 −6.23 
               
               
                 34203_at 
                 Hs.21223 
                 1264 
                 CNN1 
                 −3.25 
                 −9.51 
                 0.85 
                 1.80 
                 −3.58 
                 −11.93 
               
               
                 39031_at 
                 Hs.421621 
                 1346 
                 COX7A1 
                 −4.76 
                 −27.00 
                 2.70 
                 6.51 
                 −7.34 
                 −161.83 
               
               
                 32242_at 
                 Hs.408767 
                 1410 
                 CRYAB 
                 −3.89 
                 −14.79 
                 0.69 
                 1.61 
                 −3.468 
                 −10.99 
               
               
                 32243_g_at 
                 Hs.408767 
                 1410 
                 CRYAB 
                 −3.66 
                 −12.63 
                 −0.27 
                 −1.21 
                 −3.51 
                 −11.42 
               
               
                 859_at 
                 Hs.154654 
                 1545 
                 CYP1B1 
                 −3.81 
                 −14.04 
                 3.03 
                 8.17 
                 −3.06 
                 −8.36 
               
               
                 40071_at 
                 Hs.154654 
                 1545 
                 CYP1B1 
                 −3.47 
                 −11.04 
                 1.91 
                 3.75 
                 −3.03 
                 −8.16 
               
               
                 39140_at 
                 Hs.95665 
                 54505 
                 DDXx 
                 −0.72 
                 −1.65 
                 −0.26 
                 −1.20 
                 −1.03 
                 −2.04 
               
               
                 33337_at 
                 Hs.185973 
                 8560 
                 DEGS 
                 −0.53 
                 −1.44 
                 −0.38 
                 −1.30 
                 −1.35 
                 −2.55 
               
               
                 37000_at 
                 Hs.76285 
                 25874 
                 DKFZP564B167 
                 −0.66 
                 −1.58 
                 −0.17 
                 −1.13 
                 −1.46 
                 −2.74 
               
               
                 36861_at 
                 Hs.72157 
                 25878 
                 DKFZp56411922 
                 −3.65 
                 −12.54 
                 2.50 
                 5.68 
                 −4.03 
                 −16.30 
               
               
                 35977_at 
                 Hs.40499 
                 22943 
                 DKK1 
                 −2.97 
                 −7.82 
                 1.62 
                 3.07 
                 −0.85 
                 −1.80 
               
               
                 36133_at 
                 Hs.349499 
                 1832 
                 DSP 
                 −2.44 
                 −5.42 
                 0.03 
                 1.02 
                 −6.45 
                 −87.53 
               
               
                 37600_at 
                 Hs.81071 
                 1893 
                 ECM1 
                 −2.00 
                 −4.00 
                 0.07 
                 1.05 
                 −1.30 
                 −2.46 
               
               
                 39098_at 
                 Hs.9295 
                 2006 
                 ELN 
                 −2.45 
                 −5.46 
                 1.12 
                 2.17 
                 −2.75 
                 −6.71 
               
               
                 39861_at 
                 Hs.119257 
                 2017 
                 EMS1 
                 −1.34 
                 −2.53 
                 −0.50 
                 −1.41 
                 −1.12 
                 −2.17 
               
               
                 41385_at 
                 Hs.103839 
                 23136 
                 EPB41L3 
                 −7.02 
                 −129.64 
                 −0.90 
                 −1.87 
                 −4.64 
                 −24.85 
               
               
                 32148_at 
                 Hs.183738 
                 10160 
                 FARP1 
                 −1.16 
                 −2.24 
                 −0.45 
                 −1.36 
                 −2.03 
                 −4.09 
               
               
                 39038_at 
                 Hs.11494 
                 10516 
                 FBLN5 
                 −2.11 
                 −4.31 
                 −0.17 
                 −1.13 
                 −1.86 
                 −3.63 
               
               
                 37743_at 
                 Hs.79226 
                 9638 
                 FEZ1 
                 −4.46 
                 −22.01 
                 0.22 
                 1.16 
                 −3.52 
                 −11.48 
               
               
                 38651_at 
                 Hs.103419 
                 9637 
                 FEZ2 
                 −0.63 
                 −1.55 
                 −0.15 
                 −1.11 
                 −0.51 
                 −1.42 
               
               
                 40468_at 
                 Hs.301763 
                 23048 
                 FNBP1 
                 −0.69 
                 −1.61 
                 −0.38 
                 −1.30 
                 −0.73 
                 −1.65 
               
               
                 35785_at 
                 Hs.336429 
                 23710 
                 GABARAPL1 
                 −3.77 
                 −13.64 
                 1.69 
                 3.22 
                 −1.05 
                 −2.08 
               
               
                 905_at 
                 Hs.3764 
                 2987 
                 GUK1 
                 −0.56 
                 −1.47 
                 0.57 
                 1.49 
                 −0.71 
                 −1.63 
               
               
                 38824_at 
                 Hs.90753 
                 10553 
                 HTATIP2 
                 −4.15 
                 −17.69 
                 2.49 
                 5.61 
                 −2.73 
                 −6.61 
               
               
                 39781_at 
                 Hs.1516 
                 3487 
                 IGFBP4 
                 −4.31 
                 −19.86 
                 1.48 
                 2.80 
                 −1.69 
                 −3.22 
               
               
                 38636_at 
                 Hs.102171 
                 3671 
                 ISLR 
                 −2.04 
                 −4.12 
                 0.58 
                 1.49 
                 −2.52 
                 −5.72 
               
               
                 35318_at 
                 Hs.5737 
                 9917 
                 KIAA0475 
                 −0.72 
                 −1.65 
                 −1.00 
                 −2.00 
                 −0.91 
                 −1.87 
               
               
                 36453_at 
                 Hs.5333 
                 9920 
                 KIAA0711 
                 −4.33 
                 −20.04 
                 0.33 
                 1.26 
                 −4.11 
                 −17.29 
               
               
                 41585_at 
                 Hs.49500 
                 23231 
                 KIAA0746 
                 −3.82 
                 −14.09 
                 −1.65 
                 −3.13 
                 −2.66 
                 −6.31 
               
               
                 32730_at 
                 Hs.173094 
                 85453 
                 KIAA1750 
                 −4.80 
                 −27.76 
                 1.53 
                 2.88 
                 −4.23 
                 −18.81 
               
               
                 38972_at 
                 Hs.109438 
                 115207 
                 LOC115207 
                 −2.63 
                 −6.18 
                 0.51 
                 1.42 
                 −2.43 
                 −5.37 
               
               
                 35917_at 
                 Hs.194301 
                 4130 
                 MAP1A 
                 −1.49 
                 −2.82 
                 −0.68 
                 −1.60 
                 −1.71 
                 −3.28 
               
               
                 34403_at 
                 Hs.3745 
                 4240 
                 MFGE8 
                 −4.02 
                 −16.26 
                 −0.18 
                 −1.14 
                 −1.55 
                 −2.92 
               
               
                 33447_at 
                 Hs.180224 
                 10627 
                 MLCB 
                 −1.23 
                 −2.34 
                 1.28 
                 2.42 
                 −2.71 
                 −6.55 
               
               
                 36073_at 
                 Hs.50130 
                 4692 
                 NDN 
                 −7.57 
                 −190.46 
                 −0.25 
                 −1.19 
                 −1.92 
                 −3.79 
               
               
                 38750_at 
                 Hs.8546 
                 4854 
                 NOTCH3 
                 −2.62 
                 −6.13 
                 −0.19 
                 −1.14 
                 −2.99 
                 −7.92 
               
               
                 41742_s_at 
                 Hs.278898 
                 10133 
                 OPTN 
                 −1.57 
                 −2.98 
                 1.38 
                 2.59 
                 −0.86 
                 −1.82 
               
               
                 32260_at 
                 Hs.194673 
                 8682 
                 PEA15 
                 −0.72 
                 −1.64 
                 −0.64 
                 −1.55 
                 −1.66 
                 −3.15 
               
               
                 40434_at 
                 Hs.18426 
                 5420 
                 PODXL 
                 −4.54 
                 −23.18 
                 0.10 
                 1.07 
                 −3.59 
                 −12.03 
               
               
                 35841_at 
                 Hs.441072 
                 5441 
                 POLR2L 
                 −0.65 
                 −1.57 
                 −0.70 
                 −1.62 
                 −1.25 
                 −2.38 
               
               
                 503_at 
                 Hs.441072 
                 5441 
                 POLR2L 
                 −0.71 
                 −1.64 
                 −0.64 
                 −1.56 
                 −1.13 
                 −2.19 
               
               
                 34797_at 
                 Hs.406043 
                 8611 
                 PPAP2A 
                 −1.41 
                 −2.65 
                 0.16 
                 1.12 
                 −1.47 
                 −2.77 
               
               
                 39366_at 
                 Hs.303090 
                 5507 
                 PPP1R3C 
                 −1.89 
                 −3.70 
                 −0.31 
                 −1.24 
                 −0.85 
                 −1.80 
               
               
                 36533_at 
                 Hs.302085 
                 5740 
                 PTG1S 
                 −4.35 
                 −20.35 
                 −0.07 
                 −1.05 
                 −4.01 
                 −16.09 
               
               
                 39244_at 
                 Hs.119007 
                 5867 
                 RAB4A 
                 −5.16 
                 −35.63 
                 1.13 
                 2.19 
                 −3.54 
                 −11.65 
               
               
                 38264_at 
                 Hs.90875 
                 5877 
                 RABIF 
                 −2.22 
                 −4.66 
                 0.12 
                 1.08 
                 −0.86 
                 −1.82 
               
               
                 38331_at 
                 Hs.96038 
                 6016 
                 RIT1 
                 −1.84 
                 −3.58 
                 0.64 
                 1.55 
                 −0.74 
                 −1.67 
               
               
                 32827_at 
                 Hs.206097 
                 22800 
                 RRAS2 
                 −0.77 
                 −1.70 
                 −0.49 
                 −1.41 
                 −1.16 
                 −2.23 
               
               
                 39338_at 
                 Hs.400250 
                 6281 
                 S100A10 
                 −1.23 
                 −2.35 
                 −0.26 
                 −1.20 
                 −1.88 
                 −3.69 
               
               
                 38138_at 
                 Hs.417004 
                 6282 
                 S100A11 
                 −0.60 
                 −1.52 
                 0.45 
                 1.36 
                 −1.70 
                 −3.26 
               
               
                 38087_s_at 
                 Hs.81256 
                 6275 
                 S100A4 
                 −2.06 
                 −4.18 
                 −0.14 
                 −1.10 
                 −2.13 
                 −4.39 
               
               
                 39775_at 
                 Hs.151242 
                 710 
                 SERPING1 
                 −2.46 
                 −5.51 
                 −0.06 
                 −1.04 
                 −1.53 
                 −2.89 
               
               
                 34993_at 
                 Hs.151899 
                 6444 
                 SGCD 
                 −2.52 
                 −5.74 
                 −1.10 
                 −2.14 
                 −2.29 
                 −4.88 
               
               
                 39260_at 
                 Hs.351306 
                 9122 
                 SLC16A4 
                 −5.48 
                 −44.53 
                 3.33 
                 10.03 
                 −2.37 
                 −5.16 
               
               
                 32574_at 
                 Hs.77813 
                 6609 
                 SMPD1 
                 −1.08 
                 −2.11 
                 0.42 
                 1.34 
                 −1.10 
                 −2.14 
               
               
                 1686_g_at 
                 Hs.296169 
                 10638 
                 SPHAR 
                 −5.03 
                 −32.56 
                 0.07 
                 1.05 
                 −3.64 
                 −12.47 
               
               
                 40419_at 
                 Hs.160483 
                 2040 
                 STOM 
                 −1.63 
                 −3.10 
                 0.52 
                 1.43 
                 −2.51 
                 −5.70 
               
               
                 35832_at 
                 Hs.70823 
                 23213 
                 SULF1 
                 −4.97 
                 −31.31 
                 2.02 
                 4.05 
                 −8.08 
                 −270.60 
               
               
                 36931_at 
                 Hs.433399 
                 6876 
                 TAGLN 
                 −1.15 
                 −2.21 
                 0.84 
                 1.79 
                 −1.38 
                 −2.59 
               
               
                 1596_g_at 
                 Hs.89640 
                 7010 
                 TEK 
                 −2.97 
                 −7.82 
                 −0.58 
                 −1.49 
                 −5.02 
                 −32.37 
               
               
                 37643_at 
                 Hs.82359 
                 355 
                 TNFRSF6 
                 −2.27 
                 −4.82 
                 0.40 
                 1.32 
                 −2.64 
                 −6.21 
               
               
                 1441_s_at 
                 Hs.82359 
                 355 
                 TNFRSF6 
                 −4.46 
                 −22.06 
                 2.09 
                 4.25 
                 −2.96 
                 −7.80 
               
               
                 32313_at 
                 Hs.300772 
                 7169 
                 TPM2 
                 −1.16 
                 −2.23 
                 −0.42 
                 −1.34 
                 −1.64 
                 −3.12 
               
               
                 32314_g_at 
                 Hs.300772 
                 7169 
                 TPM2 
                 −0.72 
                 −1.65 
                 −0.01 
                 −1.01 
                 −1.30 
                 −2.47 
               
               
                 39331_at 
                 Hs.336780 
                 7280 
                 TUB8 
                 −0.44 
                 −1.36 
                 −0.51 
                 −1.43 
                 −1.97 
                 −3.90 
               
               
                 32533_s_at 
                 Hs.74669 
                 10791 
                 VAMP5 
                 −2.84 
                 −7.18 
                 0.86 
                 1.81 
                 −0.85 
                 −1.80 
               
               
                 40147_at 
                 Hs.157236 
                 10493 
                 VAT1 
                 −0.68 
                 −1.60 
                 −0.29 
                 −1.22 
                 −0.48 
                 −1.39 
               
               
                 36170_at 
                 Hs.7486 
                 23474 
                 YF13H12 
                 −1.10 
                 −2.14 
                 0.53 
                 1.44 
                 −1.30 
                 −2.46 
               
               
                 39170_at 
                 Hs.99768 
                   
                   
                 −1.10 
                 −2.15 
                 −0.55 
                 −1.46 
                 −1.07 
                 −2.10 
               
               
                 39162_at 
                 Hs.356224 
                   
                   
                 −0.70 
                 −1.62 
                 −1.23 
                 −2.35 
                 −0.77 
                 −1.71 
               
               
                   
               
             
          
           
               
                 (B) HP_D_Annotated_Averages 
                   
               
             
          
           
               
                   
                 N-5aza Ave 
                 1-UT Ave 
                 1-5aza Ave 
                 10-UT Ave 
                 10-5aza 
                   
               
               
                   
                 Fold 
                 Fold 
                 Fold 
                 Fold 
                 Ave Fold 
               
               
                 Probe ID 
                 Change 
                 Change 
                 Change 
                 Change 
                 Change 
               
               
                   
               
             
          
           
               
                 32755_at 
                 −0.73 
                 −1.66 
                 −2.04 
                 −4.11 
                 −1.55 
                 −2.92 
                 −1.34 
                 −2.53 
                 −1.71 
                 −3.28 
                   
               
               
                 39063_at 
                 0.43 
                 1.35 
                 −7.45 
                 −174.85 
                 1.22 
                 2.33 
                 −7.32 
                 −159.79 
                 −0.29 
                 1.22 
               
               
                 36686_at 
                 1.70 
                 3.24 
                 −3.00 
                 −8.00 
                 1.19 
                 2.29 
                 −2.15 
                 −4.42 
                 1.94 
                 3.83 
               
               
                 32527_at 
                 0.04 
                 1.03 
                 −5.17 
                 −35.92 
                 2.93 
                 7.60 
                 −2.72 
                 −6.60 
                 0.25 
                 1.19 
               
               
                 39043_at 
                 0.10 
                 1.07 
                 −0.99 
                 −1.99 
                 0.24 
                 1.18 
                 −1.02 
                 −2.02 
                 0.21 
                 1.16 
               
               
                 41776_at 
                 0.78 
                 1.72 
                 −1.06 
                 −2.09 
                 0.20 
                 1.15 
                 −1.24 
                 −2.36 
                 0.42 
                 1.34 
               
               
                 36497_at 
                 0.24 
                 1.18 
                 −1.35 
                 −2.54 
                 −0.87 
                 −1.82 
                 −3.25 
                 −9.50 
                 −0.33 
                 −1.26 
               
               
                 37112_at 
                 1.05 
                 2.07 
                 −4.93 
                 −30.45 
                 2.04 
                 4.12 
                 −3.49 
                 −11.21 
                 −0.37 
                 −1.29 
               
               
                 41207_at 
                 1.20 
                 2.30 
                 −0.99 
                 −1.99 
                 0.88 
                 1.83 
                 −1.11 
                 −2.16 
                 0.81 
                 1.75 
               
               
                 38418_at 
                 0.04 
                 1.03 
                 −1.13 
                 −2.19 
                 0.10 
                 1.07 
                 −1.34 
                 −2.53 
                 0.51 
                 1.43 
               
               
                 2020_at 
                 0.19 
                 1.14 
                 −1.10 
                 −2.14 
                 0.15 
                 1.11 
                 −1.24 
                 −2.35 
                 0.55 
                 1.47 
               
               
                 39351_at 
                 −0.37 
                 −1.29 
                 −0.99 
                 −1.99 
                 −0.49 
                 −1.41 
                 −1.32 
                 −2.50 
                 −0.60 
                 −1.51 
               
               
                 41138_at 
                 −0.48 
                 −1.39 
                 −1.51 
                 −2.85 
                 −0.17 
                 −1.12 
                 −0.98 
                 −1.97 
                 −0.05 
                 −1.03 
               
               
                 32363_at 
                 0.98 
                 1.97 
                 −2.49 
                 −5.63 
                 −0.43 
                 −1.35 
                 −3.53 
                 −11.58 
                 0.51 
                 1.43 
               
               
                 40698_at 
                 0.18 
                 1.14 
                 −1.21 
                 −2.31 
                 −0.10 
                 −1.07 
                 −3.09 
                 −8.50 
                 0.22 
                 1.16 
               
               
                 34203_at 
                 −0.33 
                 −1.25 
                 −3.42 
                 −10.67 
                 0.70 
                 1.63 
                 −3.33 
                 −10.06 
                 1.22 
                 2.33 
               
               
                 39031_at 
                 3.84 
                 14.35 
                 −6.97 
                 −125.51 
                 2.88 
                 7.34 
                 −7.36 
                 −163.71 
                 2.79 
                 6.93 
               
               
                 32242_at 
                 −0.28 
                 −1.21 
                 −2.96 
                 −7.75 
                 −0.63 
                 −1.55 
                 −5.49 
                 −44.79 
                 −0.73 
                 −1.66 
               
               
                 32243_g_at 
                 −0.01 
                 −1.01 
                 −2.70 
                 −6.51 
                 −0.73 
                 −1.66 
                 −5.49 
                 −44.79 
                 −0.25 
                 −1.19 
               
               
                 859_at 
                 3.00 
                 7.98 
                 −2.06 
                 −4.17 
                 2.29 
                 4.69 
                 −2.36 
                 −5.13 
                 2.23 
                 4.69 
               
               
                 40071_at 
                 2.95 
                 7.73 
                 −1.85 
                 −3.60 
                 2.06 
                 4.16 
                 −2.39 
                 −5.25 
                 2.23 
                 4.70 
               
               
                 39140_at 
                 0.47 
                 1.39 
                 −0.87 
                 −1.83 
                 −0.33 
                 −1.26 
                 −0.81 
                 −1.76 
                 −0.18 
                 −1.14 
               
               
                 33337_at 
                 −0.04 
                 −1.03 
                 −0.95 
                 −1.93 
                 −0.22 
                 −1.17 
                 −0.95 
                 −1.93 
                 −0.31 
                 −1.24 
               
               
                 37000_at 
                 0.49 
                 1.40 
                 −1.20 
                 −2.29 
                 0.35 
                 1.27 
                 −1.45 
                 −2.73 
                 0.18 
                 1.13 
               
               
                 36861_at 
                 0.34 
                 1.27 
                 −3.44 
                 −10.88 
                 0.54 
                 1.46 
                 −2.88 
                 −7.36 
                 0.90 
                 1.87 
               
               
                 35977_at 
                 −0.33 
                 −1.26 
                 −2.17 
                 −4.48 
                 0.30 
                 1.23 
                 −1.79 
                 −3.45 
                 0.07 
                 1.05 
               
               
                 36133_at 
                 1.20 
                 2.30 
                 −3.38 
                 −10.42 
                 −0.73 
                 −1.66 
                 −3.37 
                 −10.34 
                 0.51 
                 1.43 
               
               
                 37600_at 
                 −0.16 
                 −1.11 
                 −1.96 
                 −3.89 
                 −0.19 
                 −1.14 
                 −1.81 
                 −3.51 
                 0.23 
                 1.17 
               
               
                 39098_at 
                 −0.11 
                 −1.08 
                 −3.63 
                 −12.39 
                 −0.81 
                 −1.75 
                 −4.59 
                 −24.11 
                 0.59 
                 1.51 
               
               
                 39861_at 
                 −0.03 
                 −1.02 
                 −1.71 
                 −3.28 
                 0.10 
                 1.07 
                 −1.25 
                 −2.38 
                 −0.27 
                 −1.20 
               
               
                 41385_at 
                 −0.54 
                 −1.46 
                 −1.56 
                 −2.95 
                 −0.03 
                 −1.02 
                 −5.51 
                 −45.68 
                 3.42 
                 10.71 
               
               
                 32148_at 
                 −0.07 
                 −1.05 
                 −3.71 
                 −13.12 
                 0.35 
                 1.28 
                 −2.17 
                 −4.49 
                 0.01 
                 1.01 
               
               
                 39038_at 
                 −1.14 
                 −2.21 
                 −0.92 
                 −1.89 
                 −1.94 
                 −3.85 
                 −1.90 
                 −3.72 
                 −1.13 
                 −2.18 
               
               
                 37743_at 
                 −0.61 
                 −1.53 
                 −3.96 
                 −15.58 
                 0.91 
                 1.88 
                 −2.19 
                 −4.57 
                 0.01 
                 1.01 
               
               
                 38651_at 
                 −0.28 
                 −1.21 
                 −0.95 
                 −1.93 
                 −0.01 
                 −1.01 
                 −1.26 
                 −2.39 
                 −0.30 
                 −1.23 
               
               
                 40468_at 
                 0.25 
                 1.19 
                 −1.20 
                 −2.29 
                 0.02 
                 1.01 
                 −1.30 
                 −2.47 
                 −0.03 
                 −1.02 
               
               
                 35785_at 
                 0.67 
                 1.59 
                 −2.22 
                 −4.67 
                 1.01 
                 2.01 
                 −1.65 
                 −3.15 
                 0.32 
                 1.25 
               
               
                 905_at 
                 0.19 
                 1.14 
                 −0.63 
                 −1.55 
                 −0.15 
                 −1.11 
                 −0.67 
                 −1.59 
                 0.10 
                 1.07 
               
               
                 38824_at 
                 1.90 
                 3.74 
                 −1.51 
                 −2.85 
                 1.03 
                 2.05 
                 −2.47 
                 −5.52 
                 1.28 
                 2.42 
               
               
                 39781_at 
                 0.79 
                 1.73 
                 −1.31 
                 −2.48 
                 0.16 
                 1.12 
                 −2.35 
                 −5.08 
                 0.66 
                 1.58 
               
               
                 38636_at 
                 −0.06 
                 −1.04 
                 −2.63 
                 −6.18 
                 0.12 
                 1.09 
                 −2.16 
                 −4.46 
                 0.16 
                 1.12 
               
               
                 35318_at 
                 −0.89 
                 −1.85 
                 −1.02 
                 −2.02 
                 −0.56 
                 −1.48 
                 −1.78 
                 −3.42 
                 0.06 
                 1.04 
               
               
                 36453_at 
                 0.08 
                 1.06 
                 −4.76 
                 −27.13 
                 0.04 
                 1.03 
                 −3.33 
                 −10.04 
                 −0.55 
                 −1.46 
               
               
                 41585_at 
                 0.16 
                 1.12 
                 −2.20 
                 −4.61 
                 −0.01 
                 −1.01 
                 −2.61 
                 −6.09 
                 −0.42 
                 −1.34 
               
               
                 32730_at 
                 2.35 
                 5.10 
                 −1.90 
                 −3.72 
                 1.71 
                 3.28 
                 −3.93 
                 −15.24 
                 3.18 
                 9.09 
               
               
                 38972_at 
                 0.40 
                 1.32 
                 −2.50 
                 −5.67 
                 0.00 
                 1.00 
                 −1.24 
                 −2.36 
                 −0.47 
                 −1.39 
               
               
                 35917_at 
                 −0.86 
                 −1.82 
                 −0.88 
                 −1.84 
                 −1.03 
                 −2.05 
                 −1.11 
                 −2.16 
                 −1.89 
                 −3.71 
               
               
                 34403_at 
                 −0.13 
                 −1.09 
                 −3.59 
                 −12.01 
                 0.50 
                 1.42 
                 −3.21 
                 −9.23 
                 0.40 
                 1.32 
               
               
                 33447_at 
                 1.12 
                 2.17 
                 −1.90 
                 −3.72 
                 0.89 
                 1.86 
                 −1.26 
                 −2.39 
                 0.08 
                 1.05 
               
               
                 36073_at 
                 −0.59 
                 −1.51 
                 −4.20 
                 −18.40 
                 2.45 
                 5.46 
                 −4.35 
                 −20.44 
                 3.44 
                 10.84 
               
               
                 38750_at 
                 −1.80 
                 −3.48 
                 −3.76 
                 −13.50 
                 0.39 
                 1.31 
                 −5.28 
                 −38.76 
                 0.07 
                 1.05 
               
               
                 41742_s_at 
                 0.83 
                 1.77 
                 −1.85 
                 −3.60 
                 0.70 
                 1.63 
                 −1.09 
                 −2.13 
                 0.34 
                 1.26 
               
               
                 32260_at 
                 −0.15 
                 −1.11 
                 −1.34 
                 −2.53 
                 −0.35 
                 −1.27 
                 −1.65 
                 −3.13 
                 −0.14 
                 −1.11 
               
               
                 40434_at 
                 0.98 
                 1.97 
                 −2.27 
                 −4.82 
                 0.84 
                 1.79 
                 −5.38 
                 −41.74 
                 1.74 
                 3.34 
               
               
                 35841_at 
                 −0.05 
                 −1.03 
                 −1.13 
                 −2.18 
                 −0.31 
                 −1.24 
                 −1.70 
                 −3.25 
                 −0.19 
                 −1.14 
               
               
                 503_at 
                 0.00 
                 1.00 
                 −0.98 
                 −1.97 
                 −0.31 
                 −1.24 
                 −1.41 
                 −2.65 
                 −0.19 
                 −1.14 
               
               
                 34797_at 
                 0.33 
                 1.26 
                 −1.82 
                 −3.52 
                 −0.28 
                 −1.22 
                 −1.57 
                 −2.97 
                 −0.48 
                 −1.39 
               
               
                 39366_at 
                 −0.73 
                 −1.66 
                 −2.24 
                 −4.72 
                 −0.13 
                 −1.09 
                 −2.64 
                 −6.23 
                 −0.77 
                 −1.70 
               
               
                 36533_at 
                 2.47 
                 5.55 
                 −2.77 
                 −8.84 
                 1.43 
                 2.70 
                 −4.54 
                 −23.21 
                 −0.24 
                 −1.18 
               
               
                 39244_at 
                 2.12 
                 4.34 
                 −1.51 
                 −2.84 
                 0.43 
                 1.34 
                 −3.00 
                 −7.98 
                 1.96 
                 3.90 
               
               
                 38264_at 
                 0.14 
                 1.10 
                 −1.19 
                 −2.28 
                 0.30 
                 1.23 
                 −0.92 
                 −1.89 
                 −0.06 
                 −1.04 
               
               
                 38331_at 
                 0.66 
                 1.58 
                 −0.61 
                 −1.52 
                 0.02 
                 1.01 
                 −1.13 
                 −2.19 
                 0.17 
                 1.12 
               
               
                 32827_at 
                 −0.39 
                 −1.31 
                 −1.40 
                 −2.65 
                 −0.73 
                 −1.66 
                 −1.63 
                 −3.10 
                 −0.23 
                 −1.18 
               
               
                 39338_at 
                 0.52 
                 1.43 
                 −0.86 
                 −1.81 
                 −0.13 
                 −1.09 
                 −1.34 
                 −2.54 
                 0.26 
                 1.19 
               
               
                 38138_at 
                 0.32 
                 1.25 
                 −0.78 
                 −1.72 
                 −0.17 
                 −1.12 
                 −1.18 
                 −2.27 
                 0.02 
                 1.01 
               
               
                 38087_s_at 
                 0.56 
                 1.48 
                 −1.34 
                 −2.53 
                 −1.46 
                 −2.75 
                 −3.98 
                 −15.73 
                 1.05 
                 2.06 
               
               
                 39775_at 
                 −0.34 
                 −1.26 
                 −1.17 
                 −2.24 
                 −0.44 
                 −1.36 
                 −1.29 
                 −2.44 
                 −0.28 
                 −1.21 
               
               
                 34993_at 
                 −0.83 
                 −1.77 
                 −2.17 
                 −4.51 
                 −2.56 
                 −5.88 
                 −1.32 
                 −2.50 
                 −2.62 
                 −6.13 
               
               
                 39260_at 
                 2.46 
                 5.52 
                 −3.71 
                 −13.07 
                 1.81 
                 3.50 
                 −2.98 
                 −7.91 
                 1.02 
                 2.03 
               
               
                 32574_at 
                 −0.28 
                 −1.21 
                 −1.45 
                 −2.72 
                 −0.48 
                 −1.39 
                 −1.30 
                 −2.48 
                 −0.46 
                 −1.37 
               
               
                 1686_g_at 
                 1.57 
                 2.97 
                 −1.80 
                 −3.49 
                 0.00 
                 1.00 
                 −1.40 
                 −2.63 
                 0.08 
                 1.05 
               
               
                 40419_at 
                 1.18 
                 2.27 
                 −2.33 
                 −5.02 
                 0.52 
                 1.43 
                 −1.06 
                 −2.08 
                 0.38 
                 1.30 
               
               
                 35832_at 
                 −0.04 
                 −1.03 
                 −7.66 
                 −202.25 
                 0.44 
                 1.36 
                 −5.21 
                 −36.97 
                 −1.85 
                 −3.60 
               
               
                 36931_at 
                 −0.38 
                 −1.30 
                 −2.88 
                 −7.34 
                 −1.16 
                 −2.24 
                 −1.72 
                 −3.29 
                 −1.51 
                 −2.85 
               
               
                 1596_g_at 
                 3.97 
                 15.63 
                 −4.60 
                 −24.17 
                 −1.11 
                 −2.15 
                 −4.01 
                 −16.07 
                 −0.85 
                 −1.80 
               
               
                 37643_at 
                 0.55 
                 1.46 
                 −1.91 
                 −3.75 
                 −0.15 
                 −1.11 
                 −1.34 
                 −2.53 
                 −0.19 
                 −1.14 
               
               
                 1441_s_at 
                 0.31 
                 1.24 
                 −1.79 
                 −3.47 
                 0.16 
                 1.11 
                 −1.86 
                 −3.62 
                 0.66 
                 1.58 
               
               
                 32313_at 
                 −1.13 
                 −2.20 
                 −1.04 
                 −2.06 
                 −0.82 
                 −1.76 
                 −0.74 
                 −1.67 
                 −0.93 
                 −1.90 
               
               
                 32314_g_at 
                 −0.89 
                 −1.85 
                 −0.80 
                 −1.74 
                 −0.62 
                 −1.53 
                 −0.51 
                 −1.42 
                 −0.83 
                 −1.78 
               
               
                 39331_at 
                 1.26 
                 2.40 
                 −0.92 
                 −1.90 
                 0.65 
                 1.57 
                 −0.86 
                 −1.82 
                 0.31 
                 1.24 
               
               
                 32533_s_at 
                 0.01 
                 1.00 
                 −2.06 
                 −4.16 
                 −0.15 
                 −1.11 
                 −1.10 
                 −2.14 
                 −0.66 
                 −1.58 
               
               
                 40147_at 
                 −0.02 
                 −1.02 
                 −0.90 
                 −1.87 
                 0.17 
                 1.12 
                 −1.02 
                 −2.02 
                 0.24 
                 1.18 
               
               
                 36170_at 
                 0.02 
                 1.01 
                 −1.58 
                 −3.00 
                 0.20 
                 1.15 
                 −1.07 
                 −2.10 
                 −0.16 
                 −1.12 
               
               
                 39170_at 
                 −0.73 
                 −1.66 
                 −1.99 
                 −3.98 
                 −0.36 
                 −1.28 
                 −1.21 
                 −2.32 
                 −0.79 
                 −1.73 
               
               
                 39162_at 
                 0.03 
                 1.02 
                 −0.70 
                 −1.62 
                 −0.27 
                 −1.20 
                 −0.68 
                 −1.61 
                 −0.70 
                 −1.62 
               
               
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                 041 HP Ave 
                 041 5-aza 
                 N-UT Ave 
               
               
                   
                   
                   
                   
                   
                 Fold 
                 Ave Fold 
                 Fold 
               
               
                 Probe ID 
                 Unigene 
                 Locus ID 
                 Symbol 
                 Chromosome 
                 Change 
                 Change 
                 Change 
               
               
                   
               
             
          
           
               
                 (C) SA_D_Annotated_Averages 
               
             
          
           
               
                 37858_at 
                 Hs.8769 
                 83804 
                 BCMP1 
                 Xp11.0 
                 −0.82 
                 −1.77 
                 −0.88 
                 −1.87 
                 −0.83 
                 −1.55 
               
               
                 32643_at 
                 Hs.1681 
                 2532 
                 GBE1 
                 3p12.3 
                 −0.34 
                 −1.30 
                 −0.92 
                 −1.89 
                 0.33 
                 1.17 
               
               
                 38348_at 
                 Hs.235887 
                 3275 
                 HRMTIL1 
                 21q22.3 
                 0.03 
                 1.02 
                 −0.71 
                 −1.83 
                 0.52 
                 1.43 
               
               
                 38065_at 
                 Hs.4980 
                 9079 
                 LDB2 
                 4p18 
                 0.02 
                 1.76 
                 −0.85 
                 −1.83 
                 1.06 
                 2.08 
               
               
                 34283_at 
                 Hs.22907 
                 703824 
                 LOC783824 
                 18p13.12 
                 0.15 
                 1.11 
                 −1.28 
                 −2.42 
                 −0.37 
                 −1.30 
               
               
                 34357_at 
                 Hs.3343 
                 78227 
                 PNGOH 
                 1p12 
                 0.32 
                 1.25 
                 −0.84 
                 −1.79 
                 0.51 
                 1.47 
               
             
          
           
               
                 (D) SA_t_Annotated_Averages 
               
             
          
           
               
                 36589_at 
                 Hs.75313 
                 231 
                 AKR181 
                 7q35 
                 −0.42 
                 −1.34 
                 1.07 
                 2.10 
                 −0.20 
                 −1.15 
               
               
                 36688_at 
                 Hs.75746 
                 220 
                 ALDH1A3 
                 15q28.3 
                 −1.42 
                 −2.67 
                 1.50 
                 2.63 
                 −1.18 
                 −2.26 
               
               
                 39158_at 
                 Hs.9734 
                 22809 
                 ATFS 
                 19q13.3 
                 0.13 
                 1.10 
                 2.10 
                 4.30 
                 0.06 
                 1.04 
               
               
                 1717_s_at 
                 Hs.127799 
                 330 
                 BIRC3 
                 11q22 
                 −2.79 
                 −8.92 
                 2.70 
                 6.51 
                 0.67 
                 1.59 
               
               
                 40385_at 
                 Hs.75498 
                 8384 
                 CCL20 
                 2q33-q37 
                 1.84 
                 3.58 
                 4.29 
                 19.58 
                 −1.22 
                 −2.33 
               
               
                 1274_s_at 
                 Hs.423615 
                 997 
                 CDC34 
                 19p13.3 
                 0.79 
                 1.73 
                 0.74 
                 1.67 
                 0.12 
                 1.09 
               
               
                 1211_s_at 
                 Hs.155568 
                 8738 
                 CRADD 
                 12q21.33-q23 
                 0.92 
                 1.89 
                 1.47 
                 2.77 
                 0.88 
                 1.84 
               
               
                 33637_g_at 
                 Hs.167379 
                 1485 
                 CTAG1 
                 Xq28 
                 0.73 
                 1.85 
                 2.97 
                 7.82 
                 0.69 
                 1.82 
               
               
                 33838_at 
                 Hs.167379 
                 1485 
                 CTAG1 
                 Xq28 
                 0.23 
                 1.17 
                 4.34 
                 20.28 
                 0.34 
                 1.27 
               
               
                 37187_at 
                 Hs.73765 
                 2920 
                 CXCL2 
                 4q21 
                 −2.75 
                 −5.73 
                 3.70 
                 13.01 
                 2.49 
                 3.82 
               
               
                 34022_at 
                 Hs.89690 
                 2921 
                 CXC13 
                 4q21 
                 −1.33 
                 −2.51 
                 3.84 
                 14.27 
                 0.79 
                 1.72 
               
               
                 33410_at 
                 Hs.164021 
                 8372 
                 CXCL6 
                 4q21 
                 −6.21 
                 −73.94 
                 3.34 
                 10.10 
                 5.54 
                 46.53 
               
               
                 859_at 
                 Hs.154634 
                 1545 
                 CYP1B1 
                 2p21 
                 −3.81 
                 −14.04 
                 3.03 
                 8.17 
                 −3.06 
                 −8.36 
               
               
                 40071_at 
                 Hs.154634 
                 1545 
                 CYP1B1 
                 2p21 
                 −3.47 
                 −11.04 
                 1.81 
                 3.75 
                 −3.03 
                 −6.16 
               
               
                 33972_s_at 
                 Hs.73078 
                 1618 
                 DAZL 
                 3p24.3 
                 0.51 
                 1.42 
                 6.35 
                 81.28 
                 −1.02 
                 −2.02 
               
               
                 33871_f_at 
                 Hs.73078 
                 1818 
                 DAZL 
                 3p24.3 
                 −0.34 
                 −1.27 
                 8.08 
                 67.57 
                 0.89 
                 1.55 
               
               
                 529_at 
                 Hs.2128 
                 1847 
                 DUSP5 
                 10q25 
                 −1.97 
                 −3.80 
                 2.20 
                 4.58 
                 2.32 
                 4.98 
               
               
                 41193_at 
                 Hs.180393 
                 1848 
                 DUSP6 
                 12q22-q23 
                 −2.08 
                 −4.21 
                 1.96 
                 3.90 
                 1.74 
                 3.34 
               
               
                 38326_at 
                 Hs.432132 
                 50486 
                 G0S2 
                 1q32.2-q41 
                 −0.86 
                 −1.82 
                 2.72 
                 6.58 
                 0.14 
                 1.10 
               
               
                 1107_s_at 
                 Hs.432233 
                 9636 
                 G1P2 
                 1p38.33 
                 −1.88 
                 −3.62 
                 4.03 
                 16.32 
                 1.10 
                 2.14 
               
               
                 31960_f_at 
                 Hs.367724 
                 2574 
                 GAGE2 
                 Xp11.4-p11.2 
                 2.68 
                 6.42 
                 7.52 
                 182.91 
                 0.46 
                 1.38 
               
               
                 33671_f_at 
                 Hs.183199 
                 2576 
                 GAGE4 
                 Xp11.4-p11.2 
                 2.31 
                 4.97 
                 7.85 
                 229.92 
                 0.50 
                 1.41 
               
               
                 37085_f_at 
                 Hs.378444 
                 2577 
                 GAGE5 
                 Xp11.4-p11.2 
                 1.41 
                 2.65 
                 7.44 
                 173.63 
                 −0.42 
                 −1.33 
               
               
                 31498_f_at 
                 Hs.272484 
                 2578 
                 GAGE6 
                 Xp11.4-p11.2 
                 1.19 
                 2.27 
                 8.70 
                 104.21 
                 1.80 
                 3.49 
               
               
                 33680_f_at 
                 Hs.278806 
                 2579 
                 GAGE7 
                 Xp11.2-p11.2 
                 1.42 
                 2.57 
                 6.94 
                 122.93 
                 −0.72 
                 −1.65 
               
               
                 31954_f_at 
                 Hs.251677 
                 28748 
                 GAGE78 
                 Xp11.4-p11.2 
                 1.98 
                 3.94 
                 7.38 
                 166.18 
                 0.32 
                 1.24 
               
               
                 31595_s_at 
                 Hs.76057 
                 2582 
                 GALE 
                 1p36-p35 
                 −1.23 
                 −2.35 
                 1.06 
                 2.09 
                 0.45 
                 1.38 
               
               
                 37944_at 
                 Hs.86724 
                 2543 
                 GCH1 
                 14q22.1-q22.2 
                 −0.93 
                 −1.91 
                 4.33 
                 20.04 
                 0.71 
                 1.64 
               
               
                 34311_at 
                 Hs.28988 
                 2745 
                 GLRX 
                 5q14 
                 −2.03 
                 −4.07 
                 1.37 
                 2.58 
                 −0.39 
                 −1.31 
               
               
                 37483_at 
                 Hs.116753 
                 9734 
                 HDAC9 
                 7p21p15 
                 −1.88 
                 −3.87 
                 2.47 
                 5.54 
                 0.28 
                 1.21 
               
               
                 37018_at 
                 Hs.7644 
                 3006 
                 HIST1H1C 
                 6p21.3 
                 −0.75 
                 −1.68 
                 1.18 
                 2.27 
                 0.78 
                 1.71 
               
               
                 32980_f_at 
                 Hs.356901 
                 8347 
                 HIST1H2BC 
                 6p21.3 
                 0.01 
                 1.00 
                 1.55 
                 2.93 
                 0.72 
                 1.64 
               
               
                 31522_f_at 
                 Hs.182137 
                 8343 
                 HIST1H2BF 
                 6p21.3 
                 0.06 
                 1.04 
                 1.72 
                 3.29 
                 0.73 
                 1.65 
               
               
                 31524_f_at 
                 Hs.182140 
                 8346 
                 HIST1H2BI 
                 6p21.3 
                 0.18 
                 1.12 
                 1.80 
                 3.47 
                 0.69 
                 1.61 
               
               
                 153_f_at 
                 Hs.285735 
                 8970 
                 HIST1H2BJ 
                 6p21.33 
                 −0.89 
                 −1.81 
                 1.68 
                 3.19 
                 0.28 
                 1.22 
               
               
                 36347_f_at 
                 Hs.154576 
                 8341 
                 HIST1H2BN 
                 6p22-p21.3 
                 −0.14 
                 −1.10 
                 2.04 
                 4.11 
                 1.02 
                 2.02 
               
               
                 34984_at 
                 Hs.143042 
                 8351 
                 HIST1H3D 
                 6p21.3 
                 −0.59 
                 −1.50 
                 2.33 
                 5.03 
                 1.87 
                 3.65 
               
               
                 288_at 
                 Hs.417332 
                 8337 
                 HIST2H2AA 
                 1q21.2 
                 −1.45 
                 −2.73 
                 2.50 
                 5.66 
                 0.12 
                 1.09 
               
               
                 32609_at 
                 Hs.417332 
                 8337 
                 HIST2H2AA 
                 1q21.2 
                 −2.51 
                 −5.69 
                 2.55 
                 5.66 
                 −0.30 
                 −1.23 
               
               
                 1016_s_at 
                 Hs.25954 
                 3598 
                 IL13RA2 
                 Xq13.1-q28 
                 −2.43 
                 −5.40 
                 2.01 
                 4.03 
                 1.69 
                 3.23 
               
               
                 39402_at 
                 Hs.128256 
                 3533 
                 IL18 
                 2q14 
                 −0.32 
                 −1.25 
                 3.66 
                 12.66 
                 3.12 
                 8.66 
               
               
                 1520_s_at 
                 Hs.128256 
                 3553 
                 IL18 
                 2q14 
                 −0.38 
                 −1.30 
                 4.58 
                 23.92 
                 2.95 
                 7.71 
               
               
                 38299_at 
                 Hs.93913 
                 3569 
                 IL5 
                 7p21 
                 0.83 
                 1.78 
                 5.80 
                 48.39 
                 1.55 
                 2.93 
               
               
                 35372_r_at 
                 Hs.624 
                 3576 
                 IL8 
                 4q13-q21 
                 −2.24 
                 −4.73 
                 3.42 
                 10.72 
                 2.18 
                 4.56 
               
               
                 33304_at 
                 Hs.183487 
                 3669 
                 ISG20 
                 15q26 
                 −1.51 
                 −2.85 
                 3.62 
                 12.28 
                 2.12 
                 4.35 
               
               
                 41481_at 
                 Hs.27198 
                 3673 
                 ITGA2 
                 5q23-q31 
                 −3.00 
                 −7.97 
                 3.94 
                 15.30 
                 1.45 
                 2.77 
               
               
                 41179_at 
                 Hs.179946 
                 22838 
                 KIAA1100 
                 5q35.3 
                 −0.18 
                 1.14 
                 0.85 
                 1.60 
                 1.04 
                 2.05 
               
               
                 32730_at 
                 Hs.173094 
                 85453 
                 KIAA1750 
                 8q22.1 
                 −4.80 
                 −27.76 
                 1.53 
                 2.88 
                 −4.23 
                 −18.81 
               
               
                 35768_at 
                 Hs.406013 
                 3875 
                 KRT18 
                 12q13 
                 −2.03 
                 −4.08 
                 4.05 
                 16.58 
                 −1.99 
                 −3.97 
               
               
                 36288_at 
                 Hs.32952 
                 3887 
                 KRTHB1 
                 12q13 
                 0.53 
                 1.45 
                 5.32 
                 39.85 
                 0.41 
                 1.33 
               
               
                 36929_at 
                 Hs.75317 
                 3914 
                 LAMB3 
                 1q32 
                 0.23 
                 1.18 
                 2.06 
                 4.18 
                 1.87 
                 3.65 
               
               
                 37754_at 
                 Hs.79339 
                 3959 
                 LGAL93BP 
                 17q25 
                 −0.22 
                 −1.17 
                 4.25 
                 19.05 
                 −0.35 
                 −1.28 
               
               
                 38062_at 
                 Hs.49587 
                 9404 
                 LPXN 
                 11q12.1 
                 −0.79 
                 −1.73 
                 0.83 
                 1.54 
                 −0.22 
                 −1.18 
               
               
                 36711_at 
                 Hs.51305 
                 23764 
                 MAFF 
                 22q13.1 
                 −1.62 
                 −3.06 
                 1.99 
                 3.98 
                 0.33 
                 1.26 
               
               
                 32428_f_at 
                 Hs.72879 
                 4100 
                 MAGEA1 
                 Xq28 
                 1.84 
                 3.58 
                 3.66 
                 12.87 
                 0.18 
                 1.12 
               
               
                 36302_f_at 
                 Hs.37107 
                 4103 
                 MAGEA4 
                 Xq28 
                 1.42 
                 2.67 
                 4.73 
                 28.57 
                 0.06 
                 1.04 
               
               
                 35097_at 
                 Hs.113824 
                 4113 
                 MAGEB2 
                 Xq21.3 
                 4.59 
                 24.03 
                 5.06 
                 33.40 
                 −1.15 
                 −2.22 
               
               
                 39370_at 
                 Hs.121849 
                 81631 
                 MAP1LC3B 
                 16q24.2 
                 −0.66 
                 −1.58 
                 0.73 
                 1.68 
                 −1.31 
                 −2.47 
               
               
                 38428_at 
                 Hs.83169 
                 4312 
                 MUP1 
                 11q22.3 
                 −3.26 
                 −9.58 
                 2.18 
                 4.52 
                 −0.09 
                 −1.06 
               
               
                 35138_at 
                 Hs.25010 
                 55918 
                 NXT2 
                 Xq22.3 
                 0.43 
                 1.34 
                 1.35 
                 2.55 
                 0.80 
                 1.74 
               
               
                 33649_at 
                 Hs.239138 
                 10113 
                 PBEF 
                 7q22.1 
                 0.02 
                 1.02 
                 2.32 
                 4.99 
                 1.29 
                 2.44 
               
               
                 1890_at 
                 Hs.296639 
                 9518 
                 PLAB 
                 19p13.1-13.2 
                 0.30 
                 1.23 
                 1.29 
                 2.45 
                 −0.93 
                 −1.91 
               
               
                 37310_at 
                 Hs.77274 
                 3328 
                 PLAU 
                 10q24 
                 −4.17 
                 −17.94 
                 1.99 
                 3.97 
                 0.27 
                 1.20 
               
               
                 41048_at 
                 Hs.96 
                 5366 
                 PMAIP1 
                 18q21.31 
                 −1.16 
                 −2.23 
                 2.10 
                 4.30 
                 0.80 
                 1.74 
               
               
                 38886_at 
                 Hs.1050 
                 9267 
                 PSCD1 
                 17q25 
                 −0.71 
                 −1.63 
                 1.16 
                 2.23 
                 0.11 
                 1.08 
               
               
                 41184_s_at 
                 Hs.180062 
                 5696 
                 PSMB8 
                 6p21.3 
                 −0.64 
                 −1.55 
                 1.82 
                 3.52 
                 1.43 
                 2.69 
               
               
                 34304_s_at 
                 Hs.20491 
                 6303 
                 SAT 
                 Xp22.1 
                 −0.62 
                 −1.54 
                 1.20 
                 2.31 
                 −0.02 
                 −1.02 
               
               
                 35488_at 
                 Hs.19312 
                 6817 
                 SNAPC1 
                 14q22 
                 0.32 
                 1.25 
                 1.21 
                 2.31 
                 0.02 
                 1.02 
               
               
                 40898_at 
                 Hs.182248 
                 8878 
                 SO5TM1 
                 5q35 
                 −0.70 
                 −1.62 
                 1.07 
                 2.10 
                 0.02 
                 1.01 
               
               
                 36409_f_at 
                 Hs.289105 
                 6757 
                 SSX2 
                 Xp11.23-p11.7 
                 0.68 
                 1.60 
                 5.07 
                 33.87 
                 0.08 
                 1.06 
               
               
                 33855_f_at 
                 Hs.178749 
                 10214 
                 SSX3 
                 Xp11.23 
                 0.97 
                 1.96 
                 1.89 
                 3.71 
                 1.10 
                 2.15 
               
               
                 35950_at 
                 Hs.278632 
                 6759 
                 SSX4 
                 Xp11.23 
                 0.43 
                 1.35 
                 1.54 
                 2.91 
                 0.41 
                 1.33 
               
               
                 32134_at 
                 Hs.165986 
                 26138 
                 TES 
                 7q31.2 
                 −4.91 
                 −30.13 
                 2.89 
                 7.40 
                 −4.06 
                 −16.70 
               
               
                 37388_at 
                 Hs.205944 
                 7980 
                 TFP12 
                 7q22 
                 −1.40 
                 −2.64 
                 4.30 
                 19.72 
                 1.57 
                 2.97 
               
               
                 231_at 
                 Hs.75307 
                 7052 
                 TGM2 
                 20q12 
                 −1.93 
                 −3.80 
                 3.20 
                 9.18 
                 1.46 
                 2.74 
               
               
                 38404_s_at 
                 Hs.75307 
                 7052 
                 TGM2 
                 20q12 
                 −6.23 
                 −75.15 
                 7.14 
                 140.88 
                 1.59 
                 3.00 
               
               
                 1693_s_at 
                 Hs.5831 
                 7076 
                 TIMP1 
                 Xp11.3-p11.23 
                 −0.39 
                 −1.31 
                 0.93 
                 1.90 
                 −1.25 
                 −2.37 
               
               
                 595_at 
                 Hs.211600 
                 7128 
                 TNFA1P3 
                 6q23 
                 −1.61 
                 −3.05 
                 1.07 
                 2.09 
                 −0.33 
                 −1.25 
               
               
                 34892_at 
                 Hs.31233 
                 8793 
                 TNFRSF108 
                 8p22-p21 
                 −0.07 
                 −1.05 
                 0.93 
                 1.91 
                 −1.07 
                 −2.09 
               
               
                 40090_g_at 
                 Hs.155020 
                 114049 
                 WBSCR22 
                   
                 0.30 
                 1.23 
                 1.40 
                 2.64 
                 −0.10 
                 −1.07 
               
               
                 1173_s_at 
                   
                   
                   
                   
                 −0.37 
                 −1.29 
                 1.54 
                 2.91 
                 −0.04 
                 −1.03 
               
               
                 39525_at 
                 Hs.351597 
                   
                   
                   
                 0.368 
                 1.28 
                 1.35 
                 2.55 
                 −0.70 
                 −1.62 
               
               
                 189_at 
                   
                   
                   
                   
                 −0.72 
                 −1.65 
                 1.79 
                 3.45 
                 −0.27 
                 −1.21 
               
               
                 39420_at 
                 Hs.408544 
                   
                   
                   
                 −0.71 
                 −1.83 
                 1.09 
                 2.12 
                 −0.16 
                 −1.11 
               
               
                 126_s_at 
                   
                   
                   
                   
                 0.02 
                 1.01 
                 3.04 
                 8.23 
                 −1.10 
                 −2.14 
               
               
                   
               
             
          
           
               
                   
                   
                 N-5aza Ave 
                 1-UT Ave 
                 1-5aza Ave 
                 10-UT Ave 
                 10-5aza 
                   
               
               
                   
                   
                 Fold 
                 Fold 
                 Fold 
                 Fold 
                 Ave Fold 
               
               
                   
                 Probe ID 
                 Change 
                 Change 
                 Change 
                 Change 
                 Change 
               
               
                   
                   
               
             
          
           
               
                   
                 (C) SA_D_Annotated_Averages 
               
             
          
           
               
                   
                 37858_at 
                 −1.54 
                 −2.90 
                 −2.12 
                 −4.34 
                 −1.24 
                 −2.37 
                 −1.89 
                 −3.23 
                 −1.40 
                 −2.84 
               
               
                   
                 32643_at 
                 −0.89 
                 −1.85 
                 −0.37 
                 −1.38 
                 −0.78 
                 −1.89 
                 0.15 
                 1.11 
                 −0.812 
                 −1.77 
               
               
                   
                 38348_at 
                 −0.73 
                 −1.86 
                 −0.79 
                 −1.88 
                 −0.70 
                 −1.02 
                 −0.20 
                 −1.15 
                 −0.76 
                 −1.70 
               
               
                   
                 38065_at 
                 −1.50 
                 −3.83 
                 3.01 
                 4.01 
                 −1.85 
                 −3.68 
                 1.02 
                 3.03 
                 −0.87 
                 −1.83 
               
               
                   
                 34283_at 
                 −1.30 
                 −4.92 
                 −0.32 
                 −1.25 
                 −1.79 
                 −3.48 
                 0.29 
                 1.32 
                 −3.16 
                 −4.34 
               
               
                   
                 34357_at 
                 −0.83 
                 −1.90 
                 0.49 
                 1.40 
                 −0.59 
                 −1.51 
                 0.57 
                 1.43 
                 −0.93 
                 −1.80 
               
             
          
           
               
                   
                 (D) SA_t_Annotated_Averages 
               
             
          
           
               
                   
                 36589_at 
                 0.62 
                 1.54 
                 0.48 
                 1.39 
                 0.41 
                 1.33 
                 −0.17 
                 −1.13 
                 0.55 
                 1.47 
               
               
                   
                 36688_at 
                 1.70 
                 3.24 
                 −3.00 
                 −8.00 
                 1.19 
                 2.29 
                 −2.15 
                 −4.42 
                 1.94 
                 3.83 
               
               
                   
                 39158_at 
                 2.43 
                 5.37 
                 0.29 
                 1.22 
                 2.43 
                 5.41 
                 0.80 
                 1.74 
                 1.98 
                 3.94 
               
               
                   
                 1717_s_at 
                 3.22 
                 9.34 
                 0.43 
                 1.34 
                 3.21 
                 9.27 
                 0.48 
                 1.39 
                 2.73 
                 6.61 
               
               
                   
                 40385_at 
                 6.57 
                 117.24 
                 −1.20 
                 −2.30 
                 7.09 
                 136.13 
                 −1.09 
                 −2.12 
                 6.15 
                 70.79 
               
               
                   
                 1274_s_at 
                 0.85 
                 1.80 
                 −0.28 
                 −1.21 
                 1.03 
                 2.04 
                 0.32 
                 1.24 
                 0.95 
                 1.94 
               
               
                   
                 1211_s_at 
                 1.64 
                 3.12 
                 −0.54 
                 −1.46 
                 1.91 
                 3.72 
                 0.24 
                 1.18 
                 1.81 
                 3.05 
               
               
                   
                 33637_g_at 
                 3.39 
                 10.48 
                 1.75 
                 3.37 
                 1.81 
                 3.51 
                 1.10 
                 2.14 
                 2.45 
                 5.46 
               
               
                   
                 33838_at 
                 4.86 
                 29.13 
                 −0.02 
                 −1.02 
                 4.24 
                 18.84 
                 0.79 
                 1.73 
                 3.48 
                 11.03 
               
               
                   
                 37187_at 
                 3.08 
                 8.43 
                 3.46 
                 11.00 
                 3.27 
                 9.82 
                 2.98 
                 7.76 
                 2.85 
                 7.19 
               
               
                   
                 34022_at 
                 2.48 
                 5.58 
                 1.59 
                 3.02 
                 4.72 
                 18.58 
                 1.14 
                 2.20 
                 2.96 
                 7.78 
               
               
                   
                 33410_at 
                 1.44 
                 2.77 
                 8.97 
                 125.51 
                 2.83 
                 7.60 
                 5.50 
                 45.38 
                 1.72 
                 3.29 
               
               
                   
                 859_at 
                 3.00 
                 7.98 
                 −2.06 
                 −4.17 
                 2.29 
                 4.68 
                 −2.36 
                 −5.13 
                 2.23 
                 4.69 
               
               
                   
                 40071_at 
                 2.95 
                 7.73 
                 −1.85 
                 −3.60 
                 2.06 
                 4.16 
                 −2.38 
                 −5.25 
                 2.23 
                 4.70 
               
               
                   
                 33972_s_at 
                 6.26 
                 76.65 
                 −0.28 
                 −1.21 
                 5.31 
                 39.70 
                 0.05 
                 1.03 
                 5.68 
                 51.35 
               
               
                   
                 33871_f_at 
                 5.64 
                 49.90 
                 −0.50 
                 −1.41 
                 5.92 
                 60.45 
                 0.43 
                 1.35 
                 5.71 
                 52.47 
               
               
                   
                 529_at 
                 1.20 
                 2.30 
                 1.10 
                 2.14 
                 1.79 
                 3.47 
                 0.38 
                 1.29 
                 1.41 
                 2.68 
               
               
                   
                 41193_at 
                 1.54 
                 2.92 
                 2.01 
                 4.02 
                 1.49 
                 2.82 
                 0.67 
                 1.59 
                 2.30 
                 4.91 
               
               
                   
                 38326_at 
                 3.41 
                 10.52 
                 3.75 
                 13.45 
                 1.42 
                 2.68 
                 1.39 
                 2.62 
                 2.90 
                 7.47 
               
               
                   
                 1107_s_at 
                 2.27 
                 4.81 
                 −1.05 
                 −2.07 
                 0.97 
                 1.96 
                 −1.01 
                 −2.02 
                 1.79 
                 3.45 
               
               
                   
                 31960_f_at 
                 7.78 
                 220.30 
                 7.84 
                 199.24 
                 1.38 
                 2.61 
                 0.95 
                 1.93 
                 7.03 
                 130.79 
               
               
                   
                 33671_f_at 
                 7.40 
                 185.38 
                 8.23 
                 300.59 
                 1.40 
                 2.65 
                 −0.01 
                 −1.01 
                 7.94 
                 245.00 
               
               
                   
                 37085_f_at 
                 7.58 
                 191.34 
                 7.30 
                 157.04 
                 1.37 
                 2.58 
                 1.03 
                 2.03 
                 6.28 
                 77.59 
               
               
                   
                 31498_f_at 
                 6.44 
                 88.76 
                 8.04 
                 263.81 
                 1.40 
                 2.65 
                 1.36 
                 2.57 
                 7.56 
                 168.83 
               
               
                   
                 33680_f_at 
                 7.44 
                 173.24 
                 7.03 
                 130.84 
                 1.40 
                 2.64 
                 0.16 
                 1.11 
                 6.79 
                 110.32 
               
               
                   
                 31954_f_at 
                 8.35 
                 326.79 
                 8.76 
                 439.59 
                 1.44 
                 2.72 
                 0.67 
                 1.53 
                 7.47 
                 176.88 
               
               
                   
                 31595_s_at 
                 1.27 
                 2.41 
                 0.64 
                 1.56 
                 1.40 
                 2.65 
                 −0.54 
                 −1.45 
                 2.42 
                 5.37 
               
               
                   
                 37944_at 
                 2.53 
                 5.79 
                 0.74 
                 1.87 
                 2.80 
                 8.97 
                 1.37 
                 2.58 
                 2.11 
                 4.30 
               
               
                   
                 34311_at 
                 1.29 
                 2.44 
                 −1.96 
                 −3.90 
                 1.72 
                 3.30 
                 −1.71 
                 −3.27 
                 1.33 
                 2.52 
               
               
                   
                 37483_at 
                 0.88 
                 1.81 
                 0.24 
                 1.18 
                 1.47 
                 2.78 
                 0.43 
                 1.34 
                 1.27 
                 2.41 
               
               
                   
                 37018_at 
                 1.95 
                 3.85 
                 2.43 
                 5.39 
                 1.83 
                 3.56 
                 0.30 
                 1.23 
                 2.38 
                 5.26 
               
               
                   
                 32980_f_at 
                 1.48 
                 2.79 
                 1.48 
                 2.75 
                 1.33 
                 2.52 
                 0.11 
                 1.08 
                 1.28 
                 2.39 
               
               
                   
                 31522_f_at 
                 1.85 
                 3.59 
                 1.73 
                 3.31 
                 1.48 
                 2.78 
                 −0.17 
                 −1.13 
                 1.68 
                 3.21 
               
               
                   
                 31524_f_at 
                 1.46 
                 2.75 
                 1.85 
                 3.61 
                 1.05 
                 2.07 
                 0.26 
                 1.20 
                 1.11 
                 2.15 
               
               
                   
                 153_f_at 
                 1.77 
                 3.42 
                 1.76 
                 3.39 
                 1.59 
                 3.02 
                 −0.82 
                 −1.77 
                 2.58 
                 5.98 
               
               
                   
                 36347_f_at 
                 1.74 
                 3.35 
                 2.28 
                 4.85 
                 1.20 
                 2.30 
                 0.06 
                 1.04 
                 2.03 
                 4.10 
               
               
                   
                 34984_at 
                 2.86 
                 7.28 
                 3.41 
                 10.59 
                 2.40 
                 5.29 
                 −0.43 
                 −1.35 
                 4.20 
                 18.42 
               
               
                   
                 288_at 
                 2.90 
                 7.49 
                 0.68 
                 1.60 
                 2.18 
                 4.52 
                 −1.02 
                 −2.03 
                 2.23 
                 4.71 
               
               
                   
                 32609_at 
                 3.25 
                 9.51 
                 0.30 
                 1.23 
                 2.46 
                 5.52 
                 −1.85 
                 −3.81 
                 2.78 
                 6.68 
               
               
                   
                 1016_s_at 
                 3.40 
                 10.59 
                 2.34 
                 5.07 
                 4.06 
                 16.71 
                 0.74 
                 1.87 
                 3.78 
                 13.71 
               
               
                   
                 39402_at 
                 2.04 
                 4.11 
                 0.96 
                 1.94 
                 3.87 
                 14.61 
                 2.06 
                 4.18 
                 2.25 
                 4.75 
               
               
                   
                 1520_s_at 
                 2.41 
                 5.33 
                 0.81 
                 1.52 
                 4.68 
                 25.81 
                 1.53 
                 2.68 
                 3.20 
                 9.20 
               
               
                   
                 38299_at 
                 3.46 
                 10.99 
                 0.52 
                 1.44 
                 3.87 
                 14.63 
                 3.01 
                 8.05 
                 1.71 
                 3.27 
               
               
                   
                 35372_r_at 
                 2.61 
                 5.11 
                 1.65 
                 3.13 
                 4.01 
                 16.09 
                 2.80 
                 6.98 
                 2.77 
                 6.80 
               
               
                   
                 33304_at 
                 2.23 
                 4.68 
                 1.11 
                 2.16 
                 1.17 
                 2.26 
                 1.13 
                 2.19 
                 1.32 
                 2.49 
               
               
                   
                 41481_at 
                 2.33 
                 5.04 
                 2.37 
                 5.13 
                 1.57 
                 2.97 
                 −0.06 
                 −1.04 
                 3.74 
                 13.33 
               
               
                   
                 41179_at 
                 0.81 
                 1.76 
                 0.01 
                 1.01 
                 1.04 
                 2.06 
                 −0.07 
                 −1.05 
                 1.38 
                 2.57 
               
               
                   
                 32730_at 
                 2.35 
                 5.10 
                 −1.90 
                 −3.72 
                 1.71 
                 3.28 
                 −3.93 
                 −15.24 
                 3.18 
                 9.09 
               
               
                   
                 35768_at 
                 4.61 
                 24.40 
                 0.30 
                 1.23 
                 1.96 
                 3.90 
                 −0.20 
                 −1.15 
                 2.48 
                 5.58 
               
               
                   
                 36288_at 
                 6.27 
                 77.11 
                 −0.01 
                 −1.00 
                 5.03 
                 32.87 
                 1.20 
                 2.29 
                 5.51 
                 45.57 
               
               
                   
                 36929_at 
                 1.33 
                 2.52 
                 1.35 
                 2.55 
                 1.90 
                 3.74 
                 1.42 
                 2.87 
                 2.50 
                 3.87 
               
               
                   
                 37754_at 
                 2.71 
                 6.56 
                 0.25 
                 1.19 
                 1.02 
                 2.03 
                 −1.28 
                 −2.42 
                 2.84 
                 6.23 
               
               
                   
                 38062_at 
                 0.79 
                 1.73 
                 −0.28 
                 −1.21 
                 1.18 
                 2.28 
                 0.25 
                 1.19 
                 0.92 
                 1.89 
               
               
                   
                 36711_at 
                 1.38 
                 2.57 
                 −0.64 
                 −1.55 
                 2.23 
                 4.70 
                 −0.54 
                 −1.48 
                 2.14 
                 4.42 
               
               
                   
                 32428_f_at 
                 4.56 
                 23.62 
                 3.86 
                 14.54 
                 1.39 
                 2.82 
                 0.45 
                 1.36 
                 4.54 
                 23.19 
               
               
                   
                 36302_f_at 
                 5.71 
                 52.51 
                 1.53 
                 2.59 
                 3.69 
                 12.91 
                 0.18 
                 1.13 
                 4.57 
                 23.77 
               
               
                   
                 35097_at 
                 8.95 
                 124.02 
                 2.80 
                 8.95 
                 3.39 
                 10.45 
                 −0.79 
                 −1.73 
                 5.62 
                 49.18 
               
               
                   
                 39370_at 
                 1.32 
                 2.50 
                 −1.01 
                 −2.01 
                 1.65 
                 3.13 
                 −0.43 
                 −1.35 
                 0.06 
                 1.82 
               
               
                   
                 38428_at 
                 2.26 
                 4.80 
                 −0.10 
                 −1.07 
                 3.16 
                 6.91 
                 −0.95 
                 −1.93 
                 2.65 
                 6.27 
               
               
                   
                 35138_at 
                 1.45 
                 2.72 
                 1.49 
                 2.81 
                 1.73 
                 3.32 
                 0.28 
                 1.21 
                 1.40 
                 7.64 
               
               
                   
                 33649_at 
                 1.21 
                 2.32 
                 1.33 
                 2.51 
                 2.17 
                 4.49 
                 1.68 
                 3.21 
                 1.85 
                 3.61 
               
               
                   
                 1890_at 
                 1.81 
                 3.51 
                 −0.77 
                 −1.70 
                 2.50 
                 5.67 
                 −0.57 
                 −1.49 
                 2.22 
                 4.66 
               
               
                   
                 37310_at 
                 2.21 
                 4.61 
                 0.36 
                 1.28 
                 2.41 
                 5.30 
                 −0.35 
                 −1.27 
                 2.53 
                 5.79 
               
               
                   
                 41048_at 
                 1.87 
                 3.17 
                 1.32 
                 2.50 
                 1.55 
                 2.93 
                 1.18 
                 2.26 
                 2.26 
                 4.79 
               
               
                   
                 38885_at 
                 0.88 
                 1.84 
                 −0.45 
                 −1.38 
                 1.09 
                 2.12 
                 0.15 
                 1.12 
                 0.98 
                 1.97 
               
               
                   
                 41184_s_at 
                 0.55 
                 1.58 
                 0.21 
                 1.15 
                 0.77 
                 1.71 
                 0.07 
                 1.05 
                 0.82 
                 1.76 
               
               
                   
                 34304_s_at 
                 0.95 
                 1.93 
                 0.18 
                 1.12 
                 1.29 
                 2.45 
                 0.43 
                 1.34 
                 0.95 
                 1.93 
               
               
                   
                 35488_at 
                 1.28 
                 2.44 
                 0.02 
                 1.02 
                 1.58 
                 3.00 
                 −0.40 
                 −1.32 
                 1.90 
                 3.74 
               
               
                   
                 40898_at 
                 1.08 
                 2.12 
                 −0.36 
                 −1.28 
                 1.54 
                 2.90 
                 −0.10 
                 −1.07 
                 0.94 
                 1.92 
               
               
                   
                 36409_f_at 
                 6.34 
                 81.07 
                 4.74 
                 28.89 
                 7.96 
                 7.79 
                 −0.14 
                 −1.10 
                 8.15 
                 283.83 
               
               
                   
                 33855_f_at 
                 1.36 
                 2.56 
                 2.33 
                 5.04 
                 2.54 
                 5.82 
                 0.23 
                 1.17 
                 4.85 
                 25.18 
               
               
                   
                 35950_at 
                 2.10 
                 4.30 
                 2.99 
                 7.94 
                 1.75 
                 3.36 
                 0.49 
                 1.40 
                 3.41 
                 10.66 
               
               
                   
                 32134_at 
                 2.36 
                 5.13 
                 −0.58 
                 −1.49 
                 0.94 
                 1.92 
                 −0.64 
                 −1.56 
                 0.85 
                 1.81 
               
               
                   
                 37388_at 
                 3.07 
                 8.39 
                 1.50 
                 2.83 
                 3.43 
                 10.81 
                 −0.26 
                 −1.19 
                 5.14 
                 35.32 
               
               
                   
                 231_at 
                 2.03 
                 4.07 
                 1.72 
                 3.29 
                 1.83 
                 3.57 
                 −0.23 
                 −1.17 
                 3.60 
                 12.11 
               
               
                   
                 38404_s_at 
                 2.84 
                 6.22 
                 1.82 
                 3.53 
                 2.24 
                 4.72 
                 −0.10 
                 −1.07 
                 3.71 
                 13.05 
               
               
                   
                 1693_s_at 
                 1.06 
                 2.09 
                 −0.67 
                 −1.59 
                 0.77 
                 1.71 
                 −0.74 
                 −1.68 
                 0.57 
                 1.48 
               
               
                   
                 595_at 
                 1.49 
                 2.81 
                 −0.97 
                 −1.95 
                 2.00 
                 3.99 
                 −0.04 
                 −1.03 
                 1.41 
                 2.66 
               
               
                   
                 34892_at 
                 1.09 
                 2.12 
                 −0.56 
                 −1.47 
                 1.09 
                 2.13 
                 −0.70 
                 −1.62 
                 1.26 
                 2.39 
               
               
                   
                 40090_g_at 
                 1.03 
                 2.05 
                 0.99 
                 1.98 
                 0.89 
                 1.62 
                 0.50 
                 1.41 
                 1.15 
                 2.21 
               
               
                   
                 1173_s_at 
                 1.13 
                 2.19 
                 0.37 
                 1.29 
                 1.22 
                 2.34 
                 0.46 
                 1.37 
                 0.86 
                 1.82 
               
               
                   
                 39525_at 
                 1.14 
                 2.21 
                 −0.72 
                 −1.65 
                 1.17 
                 2.25 
                 −0.50 
                 −1.41 
                 0.91 
                 1.88 
               
               
                   
                 189_at 
                 1.04 
                 2.06 
                 0.34 
                 1.27 
                 0.87 
                 1.83 
                 0.42 
                 1.34 
                 1.03 
                 2.05 
               
               
                   
                 39420_at 
                 1.04 
                 2.08 
                 −0.84 
                 −1.79 
                 2.20 
                 4.59 
                 −0.03 
                 −1.02 
                 1.23 
                 2.35 
               
               
                   
                 126_s_at 
                 6.33 
                 80.33 
                 3.47 
                 11.08 
                 3.78 
                 13.78 
                 0.61 
                 1.75 
                 7.81 
                 195.21 
               
               
                   
                   
               
             
          
         
       
     
       Example 4  
       [0000]     Materials adn Methods  
         [0000]     Cell Culture and Genotyping  
         [0124]     The cell lines MDAH041 (p53 frameshift mutation) and MDAH087 (p53 missense point mutation) were derived from primary fibroblasts by skin biopsy from a female and male patient, respectively, with LFS (Bischoff et al. 1990). Four independent, spontaneously immortalized LFS cell lines were developed: one immortal cell line from MDAH041, and three independent immortal cell lines derived from MDAH087 (MDAH087-1, MDAH087-10 and MDAH087-N) (Gollahon et al. 1998). All the cells were cultured at 37° C. in 10% humidified CO 2  in Modified Eagles Medium (Gibco BRL, MD, USA) with 10% fetal bovine serum and 500 units/ml penicillin, 100 μg/ml streptomycin. The appropriate regions in the gene p53 containing the mutation were sequenced in the precrisis and immortal cell lines to confirm heterozygosity in the precrisis cell lines, and loss of the remaining wild-type p53 mutation in the immortal cell lines.  
         [0000]     5-aza-dC Treatment, RNA Isolation and the Affymetrix Microarray Assays  
         [0125]     Precrisis and immortal MDAH041 and MDAH87 fibroblasts were treated with 5-aza-dC as described (Kulaeva et al. 2003). Total RNA was extracted using the QIAGEN RNeasy Kit (QIAGEN, Inc., Valencia, Calif.). cRNA preparation and oligonucleotide analysis was performed in accordance with Affymetrix protocols. cRNA was hybridized to Affymetrix HGU95Av2 arrays (Affymetrix, Santa Clara, Calif., USA), which contains 12,625 probes.  
         [0000]     Analysis of Microarray Data  
         [0126]     Microarray experiments on MDAH087 were performed using the Affymetrix HGU95Av2 GeneChip® containing 12,625 probes. Three RNA preparations from MDAH087-N, MDAH087-1 and from MDAH087-10 were each compared with two RNA preparations from MDAH087-PC cells, individually. Three RNA preparations from 5-aza-dC treated MDAH087-N, MDAH087-1 and MDAH087-10 were each compared with RNA preparations from the corresponding untreated immortal MDAH087 cells separately. All the pairings of the comparisons were considered. Microarray data on MDAH041-PC, MDAH041 immortal and MDAH041 5-aza-dC treated cells was used in the microarray analysis performed in this study. Affymetrix DMT version 5 (Affymetrix, Santa Clara, Calif., USA) was used to select genes with increased expression. Probes with a detection call of present or marginal α1=0.00 and α2=0.06), and had a change call of increase or marginal increase (Change p-value, γ1=0.0025 and γ2=0.003) in all four immortal LFS cell lines, irrespective of fold change, in &gt;65% of the chip comparisons, were identified as increased. Genes with decreased expression were similarly detected, but decreased probes with a detection call of absent were also included.  
         [0000]     Identification of CpG Islands in Genes  
         [0127]     To detect CpG islands in the promoter region of genes, there were identified −1000 bp to +500 bp, and &#39; 1 500 bp to +200 bp of the transcription start site using UCSC Golden Path, http://qenome.ucsc.edu. MethPrimer (http:H/mail.ucsf.edu/˜urolab/methprimer/index1.html) was then used to identify CpG islands. The program analyzes windows of 100 base pairs with each subsequent window shifting 1 base pair over from the prior window. To determine if there is a CpG island for each window the program calculates the observed ratio of C plus G to CpG, and minimum average percentage G plus C; the default values were used for these parameters, &gt;0.6 and &gt;50, respectively.  
         [0000]     Quantization of Gene Expression bv Q-RT-PCR  
         [0128]     Two μg total RNA was reverse transcribed into cDNA using Superscript II (Invitrogen, Carlsbad, Calif.). Q-RT-PCR was performed using the SYBR Green PCR Detection Kit (PE Biosystems, Warrington, United Kingdom) and run on the ABI 5700 Sequence Detection System (Applied Biosystems, Foster City, Calif.). Primer Express Program (Applied Biosystems, Foster City, Calif.) was used to design primers for Q-RT PCR (Table 11). The relative fold change, 2 −ΔΔc   T,  where, ΔΔC T =(C T Gene of interest −C T GAPDH ) experiment −(C T Gene of interest −C T GAPDH ) control ), of the transcript of interest was determined by comparing it to the reference gene transcript, GAPDH (Schmittgen et al. 2000). If the relative fold change was between 0 and 1, then the fold change was calculated by dividing −1 by the relative fold change. Fold changes of replicates were averaged.  
         [0000]     Western Blot Analysis  
         [0129]     Total cellular protein was harvested from untreated and 5-aza-dC treated LFS cells. Extracts were prepared using PBS-TDS (10 mM Na 2 HPO 4 , 154 mM NaCl, 12 mM cholic acid, sodium salt, 3.5 mM SDS, 31 mM sodium azide, 1 mM sodium fluoride, 1% Triton X-100) and 1% protease inhibitor cocktail (Sigma, St. Louis, Mo.). Lysates were incubated on ice for 30 minutes followed by centrifugation at 10,000×g. Protein was quantitated using the Bradford Reagent (Sigma, St. Louis, Mo.). Equal amounts of protein were electrophoresed in an appropriate percentage SDS-polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were incubated with antibodies as indicated. Antibodies to the following molecules were used: p21 CIP1/WAF1  (Upstate Biotechnologies, Lake Placid, N.Y.), p16 INK4a  (PharMingen, San Diego, Calif.), α-Tubulin (Sigma, St. Louis, Mo.), and p53, STAT1α, IGFBP3, IGFBP4 and IGFBPrP1 were from Santa Cruz (Santa Cruz, Calif.). The western blots were then incubated with a horseradish peroxidase-conjugated secondary and developed using SuperSignal West Pico (Pierce, Rockford, Ill.). As a control, parallel western blots were probed with α-Tubulin.  
         [0000]     Clustering Analysis  
         [0130]     All the gene expression data on HGU95Av2 were processed as previously described and used for the hierarchical clustering analysis implemented in GeneSight, version 3.2.6 (Biodiscovery, Los Angeles, Calif.). Euclidean distance was used for measuring similarities between two genes or samples, and complete linkage was used for clustering. For each of the four immortal LFS cell lines there were two comparisons, immortal cells versus precrisis cells, and 5-aza-dC treated immortal cells versus untreated immortal cells. Two-sided hierarchical analysis was carried out to determine the similarities of the four immortal LFS cell lines across the whole gene expression data.  
         [0131]     Multidimensional scaling is an alternative way to present the data in low dimension space. Multidimensional scaling analysis was performed using BRB-ArrayTools version 3.2 beta to plot the data in three dimensions. The same comparisons and parameters used for hierarchical clustering were also used for multidimensional scaling analysis.  
         [0000]     Gene Ontology Analysis  
         [0132]     GoMiner (version 122) (Zeeberg et al. 2003) was used to annotate the gene expression data with GO categories. The entire HGU95Av2 GeneChip® probe set was the reference. Four experiment genes lists were analyzed: genes that were up- and downregulated during immortalization in all four immortal LFS cell lines (A and B in Table 7), and genes that were up- and downregulated after 5-aza-dC treatment in all four immortal LFS cell lines (C and D in Table 7). The probes from the lists were first converted to unique gene symbols using NetAffx, the Affymetrix online database (Build # 166) (Liu et al. 2003), and then the unique list of gene symbols were analyzed by GoMiner. The 8,487 unique gene symbols on the HGU95Av2 GeneChip® were linked to 6,020 GO categories. The one-sided Fisher&#39;s exact test p-values calculated by GoMiner were used to evaluate the statistical significance of changes for a GO category. The p-values for the first layer GO categories were converted to −log 10 (p-value) and graphed ( FIG. 6 ).  
         [0133]     When large numbers of trials are tested, a multiple comparisons problem occurs because there is an accumulation of type I error for an individual test on the level of the whole experiment. False discovery rate (FDR) is one of the least conservative correction methods and allows for tests of variables with some dependencies (Benjamini 1995; Benjamini 2001). FDR was used to correct for type I error for an individual test to achieve an acceptable type I error at the level of the whole experiment. The corrected p-values were calculated as P×i/R (P is the original p-value calculated from GoMiner; i is the index for increasing-sorted p-value; R is the total GO categories) for each GO category.  
         [0000]     Chromosome Ideogram  
         [0134]     The chromosome region and cytogenetic location for the genes was obtained using NetAffx annotation file for HGU95Av2, which used NCBI genome version 34. For those genes that did not have a chromosome region or cytogenetic location associated with them, and genes for which there was a discrepancy in the chromosome region and cytogenetic region, chromosome information was obtained using NCBI and GeneLoc. A modified version of colored Chromosomes.pl (Böhringer S 2002) was then used to generate the chromosome ideograms.  
         [0000]     Comparison of Data with Interferon, Imprinting Genes and p53 Regulated Genes  
         [0135]     Common gene lists (Table 7) were searched for IFN regulated genes, p53 regulated genes and for imprinted genes. The list of 1,061 IFN regulated genes was prepared from the IFN stimulated gene database of IFN-α and IFN-β inducible genes (http://www.lerner.ccf.orq/labs/williams/der.html) (Der et al. 1998) and from the IFN regulated genes identified by Dr. Leaman, University of Toledo. The imprinted genes list is derived from imprinted genes lists at the websites http://www.geneimprint.com and http://cancer.otago.ac.nz/IGC/Web/home.html. A list of 512 p53 regulated genes was derived from microarray data of the MDAH041 cell line stably expressing the tetracycline inducible p53 gene. Three preparations of RNA from MDAH041 were harvested at 0, 7, 24 and 72 hours after induction of p53. cRNA preparation and microarray assays were performed as described above. Each of the post p53 induction time points was compared to the 0 time point. Genes that increased or decreased upon expression of p53 were selected using Affymetrix DMT version 5. The p53 regulated gene list is comprised of genes that either increased or decreased at one of the time points following induction of p53, across 65% of the comparisons.  
         [0136]     To determine in a particular list if the probability that the number of IFN or p53 is statistically significant, 1 minus cumulative distribution function (cdf) was employed (http://edpsych.ed.sc.edu/seaman/edrm712/questions/onesample.htm) (Draghici et al. 2003).  
         [0000]     Results  
         [0137]     Previously the gene expression changes during immortalization of the telomerase positive LFS immortal cell line MDAH041 and the role of methylation-dependent gene silencing in that process were analyzed (Kulaeva et al. 2003). To further examine the significance of the role of the IFN pathway and potentially identify other mechanisms commonly abrogated during immortalization, the study was expanded to include three independent LFS cell lines derived from the fibroblasts of a second LFS patient, MDAH087. The three MDAH087 telomerase negative, ALT cell lines (Bischoff et al. 1990; Gollahon et al. 1998), MDAH087-N, MDAH087-1 and MDAH087-10, in addition to the telomerase positive cell line MDAH041, were used in a systematic analysis of the gene expression changes during immortalization and after 5-aza-dC treatment.  
         [0000]     p53, p16 INK4a  and p21 CIP1/WAF1  Status in the Immortal Cell Lines  
         [0138]     To demonstrate that the immortal cell lines MDAH041, MDAH087-N, MDAH087-1 and MDAH087-10 resulted from independent immortalization events and were not replicates of a single cell line, a limited genotyping of key genes known to change during this process was performed. MDAH041 has a p53 germline mutation in exon 5, and MDAH087 has a p53 germline mutation in exon 7. Spontaneous immortalization of MDAH087 occurs in mechanistically distinct fashion among the three immortal variants. Initially, MDAH087 cells have one wild-type and one mutated p53 allele; the mutant p53 allele has a missense mutation (CGG (Arg)→TGG (Trp)) in exon 7, codon 248 (Malkin et al. 1990; Yin et al. 1992). The p53 gene was sequenced in the cell lines used in this study to ensure that precrisis MDAH087 (MDAH087-PC) was heterozygous for p53, and that the three immortal MDAH087 cell lines have lost their wild-type p53. Sequencing confirmed MDAH087-PC was heterozygous for p53. Although MDAH087-N and MDAH087-1 cell lines exhibited loss of heterozygosity (LOH) on chromosome 17 at the p53 gene locus with the loss of the wild copy p53 allele, the MDAH087-10 cell line retained both alleles. Sequencing of cDNA from MDAH087-10 cells revealed that the wild-type p53 allele was altered by a somatically acquired point mutation, resulting in P152G substitution, exon 5. This deleterious mutation has been identified in tumors and is listed in the International Agency for Research on Cancer (IARC) TP53 Mutation Database (http://www.iarc.fr/P53/), mutation identification numbers 1015, 1337, 2976 and 18119. P152G substitution was not found in MDAH087-PC, MDAH087-N or MDAH087-1.  
         [0139]     The p53 mutation in MDAH041 causes a premature stop codon, thus in the MDAH041 immortal cells there is no detectable p53 by western blot analysis ( FIG. 4 ). The p53 mutation in MDAH087 has a missense mutation in the DNA-binding domain. The mutant p53 protein found in MDAH087 is readily detected by western blot analysis due to its longer half-life as compared to the wild-type p53 present in normal fibroblasts ( FIG. 4 ).  
         [0140]     A second difference among the four immortal LFS cell lines is the protein expression pattern of the cyclin-dependent kinase inhibitors, p16 INK4a  and p21 CIP1/WAF1 . In precrisis MDAH041 (MDAH041-PC) cells there is little to no protein expression of p16 INK4a  or p21 CIP1/WAF1 . Immortal MDAH041 cells also do not express either p16 INK4a  or p21 CIP1/WAF1 , but protein expression of both was induced upon treatment with 5-aza-dC. MDAH087-PC cells express both p16 INK4a  and p21 CIP1/WAF1 , but their expression is lost from the immortal MDAH087 cell lines. Treatment of MDAH087-N cells with the DNA methyltransferase inhibitor 5-aza-dC induced protein expression of p16 INK4a , but not of p21 CIP1/WAF1  (Vogt et al. 1998) ( FIG. 4 ). In contrast to MDAH087-N, 5-aza-dC induced expression of p21 CIP1/WAF1  in MDAH087-1 and MDAH087-10, but not expression of p16 INK4a  ( FIG. 4 ). These data demonstrate that the four immortal LFS cell lines used in this study contain a different complement of genetic and epigenetic changes, indicating they are independent immortalizations. Analysis of additional genetic or epigenetic events could reveal critical mechanisms of interest to the process of cellular immortalization.  
         [0000]     Microarray Profiling of Gene Expression in Immortal LFS Fibroblasts  
         [0141]     Total RNA was prepared from each of the LFS cell lines early in their lifespans and as immortal cell lines. Probes were synthesized and hybridized to the Affymetrix HGU95Av2 GeneChip®. The immortal MDAH041 cell line was compared with the MDAH041-PC cell line and the immortal MDAH087 cell lines, MDAH087-N, MDAH087-1 and MDAH087-10, were each individually compared with the MDAH087-PC cell line. All the possible pairings (6 comparisons per cell line) between precrisis versus immortal cell lines were analyzed. In previous studies of the MDAH041 cell line, genes were selected that had at least a 2-fold change in gene expression on the microarrays. The same criteria were used to identify genes that changed and were common to all four immortal LFS cell lines. The Affymetrix Data Mining Tool (DMT) version 5 was used to select genes whose expression increased or decreased, without specification of fold change, in greater than 65% of the chip comparisons for an individual immortal cell line. The number of genes differentially expressed during immortalization, for the each of four LFS immortal cell lines, is shown in Table 7. Less stringent criteria than the 2-fold change criteria was used to identify 897 genes with upregulated expression and 1,120 genes with downregulated expression changes in MDAH041. In the three immortal MDAH087 cell lines, the number of genes with increased or decreased expression ranged from 785 to 1,267. Using this approach there were found 149 upregulated and 187 downregulated genes common to all four immortal LFS cell lines. In general, 98% of the changes in gene expression were greater than 1.5-fold, and 67% were greater than 2-fold. Of the 149 upregulated and 187 downregulated genes there were a statistically significant number of p53-regulated genes (29 upregulated genes, p-value=7.48×10 −9 , 23 downregulated genes, p-value=0.0005) (Table 8). In addition there were four known imprinted genes, PHLDA2, CD81, MEG3 and NDN (Table 8), among the 187 downregulated genes, further indicating that methylation-dependent silencing is important to the mechanism of cellular immortalization.  
         [0142]     The DNA methyltransferase inhibitor 5-aza-dC induces growth arrest and senescence in LFS immortal fibroblasts (Vogt et al. 1998). Treatment of immortal MDAH041 cells with 5-aza-dC induced significant changes in gene expression (Kulaeva et al. 2003). 5-aza-dC treated immortal LFS cells have a senescence-like morphology and exhibit senescence-associated β-galactosidase activity. To further investigate the role of DNA methylation regulated gene expression during immortalization the study was expanded to include the MDAH087 cell lines (MDAH087-N, MDAH087-1, and MDAH087-10). 5-aza-dC treatment of immortal MDAH087 cell lines resulted in a flat senescence-like morphology and the activation of the senescence-associated β-galactosidase activity.  
         [0143]     Total RNA, prepared from 5-aza-dC-treated immortal MDAH041 and MDAH087 cell lines, was used to prepare probes for hybridization to the Affymetrix HGU95Av2 GeneChip®. All the possible pairings (6 comparisons for MDAH041; 9 comparisons for each of the MDAH087 cell lines) of treated and untreated immortal cells were analyzed with Affymetrix DMT version 5. To identify genes that increased or decreased after 5-aza-dC treatment, the same criteria as was used for identifying gene expression changes in immortal cells was used; genes were selected whose expression increased or decreased after 5-aza-dC treatment, without specification of fold change, in greater than 65% of the chip comparisons for an individual immortal cell line. In MDAH041 cells, the number of genes with upregulated and downregulated expression is 877 and 803, respectively (Table 7). This is in comparison to the 190 genes with upregulated expression and the 48 genes with downregulated gene expression in a previous study in which a criterion of 2-fold change in expression was used (Kulaeva et al. 2003). For the immortal MDAH087 cell lines, the number of genes with increased or decreased expression ranged from 408 to 772 (Table 7). When compared the four spontaneously immortal LFS cell lines, 185 upregulated and 46 downregulated genes common to all four immortal LFS cell lines were identified (Table 7). None of the 185 upregulated or the 46 downregulated genes were known imprinted genes. However there were a statistically significant number of p53-regulated genes whose expression was methylation dependent (7 upregulated genes p-value=0.017, 26 downregulated genes p-value=2.46×10 −5 ) (Table 8).  
         [0000]     Genes Downregulated After Immortalization by Gene Methylation  
         [0144]     In the study of the MDAH041 cell line (Kulaeva et al. 2003), 85 genes were repressed during immortalization and upregulated after treatment of these cells with 5-aza-dC. This indicated the significance of methylation-dependent gene silencing to the process of immortalization. In this analysis 14 genes, common to all four immortal LFS cell lines, were identified whose expression decreased during immortalization and increased after treatment with 5-aza-dC (Table 7, Genes in sets B and D). These 14 epigenetically regulated genes are statistically significant when compared to the 4 genes that decreased during immortalization and decreased after 5-aza-dC using an exact binomial test, p-value=0.034. Average fold change in expression of these fourteen genes are listed in Table 9. Among the 14 genes, 11 (78%) of them have computational CpG islands in their promoter regions (1000 bp upstream and 500 bp downstream of the transcriptional start site). This suggested that these 11 genes are repressed by promoter hypermethylation during immortalization. To determine the significance of that observation, 16 genes whose expression decreased in all four immortal LFS cell lines were tested for computational CpG islands, but was not affected by treatment with 5-aza-dC, and 60 randomly chosen genes from the HGU95Av2 microarray GeneChip®. Fifteen out of the 16 downregulated methylation insensitive genes (94%) and 49 out of the 60 randomly chosen genes (82%) had computational CpG islands. Focusing the search region to 500 bp upstream and 200 bp downstream of the transcriptional start site reduced only the number of computational CpG islands in the random gene set by 2 (47/60; 78%) and had no effect on the presence of computational CpG islands in the 14 methylation sensitive downregulated genes or the 16 methylation insensitive downregulated genes. The percentage of genes that were identified as having a CpG island in their promoter is approximately the same as the percent that is generally found in the human genome (Antequera 2003). It is evident that the presence of a computational CpG island is not a dependable measure that a gene is actually regulated by promoter methylation.  
         [0145]     There were only 2 genes identified that were upregulated during immortalization and decreased after 5-aza-dC treatment in immortal cells, cell division cycle 25B (CDC25B) and LIM domain-binding 2 (LDB2) (Table 7, Genes in sets A and C). This evidence supports the observation (Kulaeva et al. 2003) that methylation-dependent gene silencing is more frequently associated with the spontaneous immortalization of LFS cell lines.  
         [0000]     Epigenetic Control of Interferon Regulated Genes in the LFS Cell Lines  
         [0146]     In a study, 39 of the 85 genes identified in MDAH041 as epigenetically repressed during immortalization were known to be regulated in the IFN signaling pathway (Kulaeva et al. 2003). The probability of that occurring by chance was 8.31×10 −47 , and thus it was concluded that the IFN-pathway genes play a significant mechanistic role in the acquisition of cellular immortalization. This probability was calculated based on having 137 IFN-regulated genes out of 8,628 unique genes on the HGU95Av2 GeneChip®. Based on more recent information there are 1,061 IFN-regulated genes and there are 8,903 unique genes on the HGU95Av2 GeneChip®. Using this updated information it was determined that in MDAH041 cells the number of IFN-regulated genes that are epigenetically regulated during immortalization remained statistically significant (p-value=2×10 −15 ). In the individual MDAH087 immortal cell lines, the number of IFN-regulated genes that decreased during immortalization and increased after 5-aza-dC treatment was also significant (MDAH087-N, p-value=3×10 −6 ; MDAH087-1, p-value=1.3×10 −7 ; MDAH087-10 p-value=1.2×10 −6 ). However, the set of epigenetically silenced IFN genes found in common to all four immortal LFS cell lines was not significant (p-value=0.51). Therefore, it was concluded that although the interferon pathway appears to play a significant role in the cellular immortalization of LFS cell lines, there are differences among the cell lines in the specific genes of this pathway that are dysregulated.  
         [0147]     To further evaluate the IFN pathway in MDAH041 the expression of the IFN signaling pathway gene, STAT1α, was determined by western blot analysis ( FIG. 4 ). Consistent with STAT1α transcript expression (Table 10), protein levels of STAT1α decreased in immortal MDAH041 cells and increased in response to treatment of immortal MDAH041 cell with 5-aza-dC. Changes in STAT1α protein ( FIG. 4 ) and mRNA (Table 10) expression were also analyzed in MDAH087 cells. As was seen in MDAH041, STAT1α gene expression decreased during immortalization and increased after 5-aza-dC treatment in MDAH087 cells. Interestingly, in contrast to Q-RT-PCR and microarray data, there was no difference in protein expression levels of STAT1α between MDAH087-PC and the immortal MDAH087 cell lines, nor was there found a difference in levels between untreated and 5-aza-dC treated samples ( FIG. 4 ). Furthermore after stimulation of LFS cells with either VSV viral infection (Balachandran et al. 2000) or the double-strand RNA analog poly (I:C), which mimics viral infection, it was found that the cell lines were differentially sensitive to these treatments. While poly (I:C) treatment induced STAT1α and IFNβ gene expression in MDAH041-PC, MDAH087-PC, immortal MDAH041 and MDAH087-N, there was no induction of STAT1α and IFN β in either MDAH087-1 or MDAH087-10. Because different sets of IFN regulated genes were dysregulated in each of the four immortal LFS cell lines, it was concluded that while the specific mechanism of inactivation of the IFN pathway varies from cell line to cell line, abrogation of this significant pathway is necessary, albeit not sufficient, for immortalization to occur.  
         [0000]     Confirmation of Microarray Gene Expression Data by Q-RT-PCR Analysis  
         [0148]     Q-RT-PCR was used to confirm the gene expression changes observed in the microarray data (Table 10). The expression of nine representative genes was examined, CREG, IGFBPrP1, CLTB, KIAA1750, OPTN, HSPA2, TNFAIP2, ALDH1A3 and SERPINB2, among the fourteen genes that were identified as decreased during immortalization and increased after 5-aza-dC treatment in all four immortal LFS cell lines. Three of the genes, CLTB, HSPA2 and OPTN, in the list of fourteen genes that were epigenetically regulated during immortalization, had multiple probes on the Affymetrix HGU95Av2 GeneChip®. Although all of the replicate probes on the chip for these genes were not identified as decreased during immortalization and increased after 5-aza-dC treatment in all four immortal LFS cell lines, Q-RT-PCR did in fact confirm that these genes were in fact epigenetically regulated during immortalization. In addition, as IGF-binding proteins (IGFBP) are known to be involved in cellular immortalization, two additional IGFBP genes, IGFBP3 and IGFBP4, were also analyzed by Q-RT-PCR; both of these genes decreased during immortalization among all four immortal LFS cell lines. Overall 96% (Table 10, 92 out of 96 comparisons) of the changes in gene expression observed in the microarrays were confirmed by Q-RT-PCR. Microarray and Q-RT-PCR fold-changes were considered in accordance when either both had significant fold changes in the same direction, or neither had a significant fold-change. The cutoff for significant fold-change for microarray was ±1.3-fold, and for Q-RT-PCR was ±1.8-fold. In 3 of the 4 instances when there was a discrepancy between microarray and Q-RT-PCR gene expression changes, the microarray fold-change was low, less than 1.1-fold, or the Q-RT-PCR fold-change was low, less than 0.61-fold. In one instance, IGFBP3 gene expression changes in MDAH087-10 after 5-aza-dC treatment, the microarray fold-change was significant (−2.66), but the Q-RT-PCR fold-change was not significant (−1.4), thus microarray and Q-RT-PCR fold-changes in concordance were not considered. These few discrepancies between microarray and Q-RT-PCR fold-changes were isolated instances that only occurred in 2 of the 12 genes analyzed, HSPA2 and IGFBP3. Furthermore, for HSPA2 there was only one such discrepancy. Thus microarray is a reliable method to measure expression changes.  
         [0000]     Confirmation of Gene Expression Data by Western Blot Analysis  
         [0149]     Western blot analysis was employed to determine if protein expression data correlated with changes in gene expression ( FIG. 4 ). Consistent with microarray and Q-RT-PCR data, IGFBP3 protein levels decreased during immortalization in MDAH041, MDAH087-N, MDAH087-1 and MDAH087-10 cell lines, but only increased in response to 5-aza-dC in one LFS cell line, MDAH041. There was a decrease in expression of IGFBP4 in all four immortal LFS cell lines. IGFBP4 runs as a doublet, the higher band likely represents the glycosylated form of this protein (Carr et al. 1994). Interestingly, the glycoslyated form of IGFBP4 is more prevalent in the immortal cell lines than in the precrisis cell lines ( FIG. 4 ). After 5-aza-dC treatment there was a slight induction of IGFBP4 in MDAH041, MDAH087-N and MDAH087-1, but no induction in MDAH087-10. This is in contrast to gene expression data (Table 10), where IGFBP4 was not induced in MDAH087-1 cells and was induced in MDAH087-10 cell following 5-aza-dC treatment. Also inconsistent with microarray and Q-RT-PCR data, there was little to no induction of either IGFBP4 or IGFBPrP1 protein ( FIG. 4 ) in MDAH041 following treatment with 5-aza-dC. Q-RT-PCR confirmed the microarray data by an independent method demonstrating that the microarray gene expression data is accurate. The rare instances when there was a difference between Q-RT-PCR and microarray data with western blot data suggests that posttranslational mechanisms, not reflected in the gene expression data, control the protein expression from these genes during immortalization.  
         [0000]     Global Analysis of Gene Expression by Hierarchical Clustering  
         [0150]     MDAH041, MDHAO87-N, MDAH087-1 and MDAH087-10 are independent immortalizations of human skin fibroblasts with apparent similarities in the mechanisms by which they became immortal. To globally assess these mechanistic similarities the entire gene expression data set was analyzed using hierarchical clustering ( FIG. 5   a ). The immortal versus precrisis cells expression datasets cluster distinctively from the 5-aza-dC versus untreated immortal cell expression datasets. The expression patterns revealed by the hierarchical cluster map show that the two processes, immortalization and demethylation, have reciprocal changes in gene regulation. These data support the observation that the treatment of immortal MDAH041 and MDAH087 cell lines with 5-aza-dC can reverse the immortal phenotype, and cause the cells to senescence (Kulaeva et al. 2003; Vogt et al. 1998).  
         [0151]     To determine if the profiles of any two pairs of cell lines more closely resembled one another than to the other pairs of cell lines, the number of genes common between all possible pairings of the immortal cell lines was examined. By this analysis there were no pairs of immortal cell lines that were more alike. However, upon examination of the expression data using hierarchical clustering it was found that the three immortal MDAH087 cell lines are more similar to one another than MDAH041 is with any one of them. It was not surprising that MDAH041 and MDAH087 immortal cell lines have differences in genes expression patterns, as these cell lines seem to be in different immortalization complementation groups (Gollahon et al. 1998), and MDAH041 is telomerase positive while MDAH087 telomeres stabilize via ALT. Among the three MDAH087 cell lines, MDAH087-N and MDAH087-10 were more closely related to one another than the other possible pairings of the MDAH087 cell lines. MDAH041 clustered separately from the three MDAH087 cell lines. While immortal MDAH087-N and MDAH087-10 were the closest of the possible immortal cell pairs, after 5-aza-dC treatment MDAH087-N had a gene expression pattern that was more similar to MDAH087-1 than to MDAH087-1 0.  
         [0152]     Upon examination of the 5-aza-dC cluster diagram, only a small subset of genes was regulated by 5-aza-dC in common to all four immortal LFS cell lines. Yet, within any immortal cell line, there are hundreds of genes whose expression increases following treatment with 5-aza-dC. This suggests that among the four immortal LFS cells lines, while there are similar pathways involved in bypassing senescence, and in induction of senescence with 5-aza-dC, different sets of genes are regulated in each of the immortal LFS cell lines. Despite individual differences in the genes dysregulated during immortalization among the four immortal LFS cell lines, methylation-dependent gene silencing is necessary for LFS cells to bypass senescence and become immortal.  
         [0153]     Multidimensional scaling, like hierarchical clustering analysis is based on evaluating the similarity distance of the expression data and is used to reveal the relationship between the samples; however in multidimensional scaling the samples are plotted in a three-dimensional space ( FIG. 5   b ) and thus it provides another approach to visualizing the data. The three-dimensional models allow a more straightforward visualization of the similarities among the sample pairs than hierarchical clustering diagrams. The distance of each sample pair in the three-dimensional space represents their Euclidean distance. The four colored balls, which represent each of the immortal cell lines, were relatively far from the balls that represented the 5-aza-dC treated immortal cell lines ( FIG. 5   b ). Among the four immortal LFS cell lines, the MDAH041 cell line is set apart from the three MDAH087 cell lines, reflecting that MDAH041 has a different set of genes that are differentially expressed during immortalization than the MDAH087 immortal cells lines. Of the MDAH087 immortal cell lines, MDAH087-N and MDAH087-10 were closer to each other in the immortalization comparisons; however in the 5-aza-dC comparisons, MDAH087-N and MDAH087-1 expression patterns were closer to each other. Overall the relationship among the four immortal LFS cell lines determined using multidimensional clustering analysis agrees with the results from the hierarchical clustering analysis.  
         [0000]     Identification of Pathways Associated with Cellular Immortalization  
         [0154]     To identify mechanisms, in addition to the IFN-regulated pathway, critical to cellular immortalization, the biological, cellular and molecular characteristics of the genes differentially expressed during immortalization and after 5-aza-dC treatment (Table 7) were studied using the Gene Ontology (GO) software program, GoMiner. GoMiner links genes with GO categories, allowing one to identify the biological process, cellular component and molecular function associated with the genes in the lists (Zeeberg et al. 2003). The p-value for each GO category was calculated as an evaluation of the significance of the number of gene changes associated with each GO category. The categories that had five or more genes that changed, or had an uncorrected p-value &lt;0.01 are reported in Table 14. Subcategories (Table 14, categories in bold), of the three primary GO categories biological processes, cellular components and molecular function, were graphed, using the −log 10 (p-value) ( FIG. 6 ) to facilitate evaluation of which functional categories were significant during immortalization and or 5-aza-dC treatment. Sublayers of the significant GO categories were then explored in finer detail to determine more specifically which GO functional categories accounted for the changes. To avoid misinterpretations resulting from failure to correct for multiple comparisons in GoMiner, the p-values were recalculated using False Discovery Rate (FDR) ( FIG. 6 , denoted by asterisks) for each GO category. Possibly due to the small number (&lt;200) of dysregulated genes in the lists, there were only a few GO categories that achieved significance after correction by FDR.  
         [0155]     In the primary GO category biological processes, cell adhesion (GO:0007155) and cell motility (GO:0006928) are among the subcategories ( FIG. 6   a ; Table 14a) that had a significant number of genes that were downregulated during immortalization. Among the categories with a significant number of genes upregulated during immortalization were cell proliferation (GO:0008283), metabolism (GO:0008152), and cell organization and biogenesis (GO:0016043). After correction by FDR, only cell proliferation (GO:0008283) remained significant. Within the cell proliferation (GO:0008283) category most of the changes occurred in cell cycle (GO:0007049), cytokinesis (GO:0000910) and regulation of cell proliferation (GO:0042172), but only cell cycle (GO:0007049) remained significant after correction by FDR. In these categories, MYC, E2F transcription factor 4 (E2F4), cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 3 (CDKN3) were among the genes identified as dysregulated during immortalization. Identification of these GO categories and genes, supports the observation that dysregulation of the cell cycle, through the retinoblastoma signaling pathway, and dysregulation of cyclin-dependent kinase inhibitors, are required for immortalization of fibroblasts (Kiyono et al. 1998; Tsutsui et al. 2002). Through classification of genes in GO categories known genes in the cell cycle pathway that are involved in immortalization were identified, thus this data indicated that one can identify other pathways that are involved in immortalization using this approach.  
         [0156]     After treatment with 5-aza-dC, the categories in the primary GO category biological process with a significant number of genes with changes in gene expression included cell death (GO:0008219), cell proliferation (GO:0008283), response to stress (GO:0006950), and cell organization and biogenesis (GO:0016043). Genes in these categories include cell division cycle 34 (CDC34), cyclin-dependent kinase inhibitor 2C (CDNK2C) and fibroblast growth factor 2 (FGF2). During immortalization, a significant number of genes in the cell proliferation category increased in expression, and after 5-aza-dC treatment a significant number of genes in the cell proliferation category decreased in expression. Interestingly, in the cell proliferation category (GO:0008283), among the 35 genes (Table 15) whose expression was upregulated during immortalization and the 11 genes whose expression was downregulated following demethylation, only one gene was found in both gene sets, CDC25B.  
         [0157]     Of the GO categories identified for the genes differentially expressed after 5-aza-dC treatment, the only GO category that remained significant after correction by FDR was response to wounding (GO:009611), a subcategory of response to extracellular stimulus (GO:0009991). This is consistent with the finding that the IFN pathway is important in immortalization (Kulaeva et al. 2003), as several of the genes identified in the wounding category (GO:009611) are interferon and/or cytokine regulated genes (Table 16).  
         [0158]     In the primary GO category cellular component ( FIG. 6B ), genes downregulated during immortalization were primarily in the GO categories extracellular (GO:0005576), cytoplasm (GO:0005737) and a subcategory of cytoplasm, cytoskeleton (GO:0005856), while the genes with upregulated expression were in the GO categories chromosome (GO:0005694) and nucleus (GO:0005634), and subcategories of cytoplasm (GO:0005737) and mitochondrion (GO:0005739). However only the GO categories nucleus (GO:0005634) and cytoskeleton (GO:0005856) remained significant after correction by FDR. The cytoskeleton (GO:0005856) category is consistent with the morphological changes that occur as cells senesce; typically, as cells senesce they become very large and flat. Thus, one would predict that changes in cytoskeletal genes would contribute to the processes of immortalization and senescence. The genes that were upregulated after treatment with 5-aza-dC were in GO categories extracellular (GO:0005576), nucleus (GO:0005634), and chromosome (GO:0005694). Only chromosome (GO:0005694) remained significant after correction by FDR. There were no subcategories of cellular component with a significant number of genes that decreased after 5-aza-dC treatment.  
         [0159]     In the primary GO category molecular function ( FIG. 6C ), genes that were upregulated during immortalization were in the subcategory catalytic activity (GO:0003824) and those downregulated during immortalization were in the subcategories cell adhesion molecule activity (GO:0005194) and structural molecule activity (GO:0005198). Although none of these categories retained significance after correction by FDR, the identification of structural molecule activity (GO:0005198) in molecular function is consistent with cytoskeleton genes (GO:0005856) being identified as significant in the cellular component. Sixteen of the 24 genes from structural molecular activity genes (GO:0005198), and 1 of the 9 genes in the cell adhesion molecular activity (GO:0005194), overlap with the genes in the cytoskeletal category (GO:0005856) (Table 17). Additionally the cytoskeletal protein, gelsolin, is epigenetically regulated in MDAH041 immortal cells. There were two GO categories examined that were both significantly downregulated during immortalization and upregulated during demethylation: a subcategory of biological process, regulation of cell proliferation (GO:0042127, p-value IM_Down &lt;0.02; p-value 5A_Up &lt;0.05) and a subcategory of cellular component, extracellular (GO:0005576, p-value IM_Down &lt;0.005; p-value 5A_Up &lt;0.01). The genes downregulated during immortalization did not overlap with the genes upregulated during demethylation in the regulation of cell proliferation category (GO:0008283), except for the gene IGFBPrP1. This finding indicates that the arrest of cell proliferation (GO:0008283) associated with 5-aza-dC-treatment results in changes in a different set of genes from those altered to permit cells to maintain their proliferative capacity during immortalization.  
         [0000]     Regional Control of Gene Expression  
         [0160]     To determine whether epigenetic regulation of gene expression is controlled in a regional fashion on certain chromosomes during immortalization, chromosome ideograms were annotated to indicate areas of altered gene regulation ( FIGS. 7-13 ). Genes with an increase in expression during immortalization were found on chromosome 3p, 12, 14q, 17q21, 17q23, 19p13.3, 19q13, 20 and 22 ( FIG. 8 ). Of interest, there are no genes on chromosome 3 that decreased in expression during immortalization in common to all four immortal LFS cell lines. However, there are genes on chromosome 3 that decrease in expression during immortalization that are in common to the three MDAH087 immortal cell lines. This is intriguing because introduction of normal chromosome 3 into a renal cell carcinoma cell line and an ovarian carcinoma cell line induces senescence (Horikawa et al. 1998; Rimessi et al. 1994; Tanaka et al. 1998); induction of senescence was attributed to a gene on chromosome 3p14.2-p21.1 decreasing telomerase activity (Horikawa et al. 1998; Tanaka et al. 1998). The reason that there may not be any genes decreased on chromosome 3 that are in common to MDAH041 and the three MDAH087 immortal cell lines may be a reflection of how the telomeres are stabilized in these cell lines during immortalization, MDAH041 by increased telomerase activity and MDAH087 by ALT. Thus, in order for MDAH041 to bypass senescence and become immortal, genes on chromosome 3 that naturally inhibit telomerase activity are selected against. As MDAH087 immortal cell lines stabilize telomeres by ALT, there is no selective pressure against genes that inhibit telomerase activity. Therefore one or more of the chromosome 3 genes that are specifically downregulated in MDAH041 cells may be a critical negative regulator of telomerase that is lost when these cells become immortal. When the four immortal LFS cell lines were individually analyzed, in each of the cell lines, 19q13 is a region that had a large number of genes with increased expression. In all three immortal MDAH087 cell lines, both chromosomes 17 and 22 had a cluster of genes that increased during immortalization. In MDAH041, chromosome region 20q had a cluster of genes that increased during immortalization, which was not observed in the MDAH087 immortal cell lines.  
         [0161]     Decreased gene expression was found during immortalization in all four immortal LFS cell lines on multiple chromosomes including 1q21, 1q32, 1q41, 6q, 9q34, 10, 11p15, 11q23, 13q, 14q32 and 15 ( FIG. 9 ). In the analysis of the individual cell lines, in each of the cell lines, 10q is a region with a group of genes whose expression decreases during immortalization. In MDAH041 and MDAH087-N there is a group of genes with decreased expression during immortalization at 9q22 and 9q32. In MDAH041, MDAH087-N and MDAH087-1 there are clusters of genes at 6q and 11q that decrease during immortalization. In all cell lines except MDAH087-1 there are clusters of genes at 9q34 and 14q32 that have a decrease in expression during immortalization. Also of interest are chromosomes 4, 6 and 13. There is only one gene on chromosome 4 with increased expression during immortalization, but there are three genes on chromosome 4p and five genes on chromosome 4q with decreased expression in all four immortal LFS cell lines during immortalization. On chromosome 6p there are both genes with increased expression and with decreased expression during immortalization, but on chromosome 6q there are only genes with a decrease in expression during immortalization. Common to all four immortal LFS cell lines there are four genes located on chromosome 13 with a decrease in expression, but no genes on this chromosome with an increase in expression after immortalization. Of note, these genes are located in the region of chromosome 13q22 to 13q32 and are not located near RB, which is at chromosome 13q14.2. There is LOH along the q arm of chromosome 13, including the region where RB is located, in MDAH041, MDAH087-1 and MDAH087-10, but RB protein expression is unaffected. Thus, the decrease in expression of the four genes is a consequence of combination of mechanisms, such as LOH in combination with methylation or gene mutations. Genes with expression that decreased during immortalization and increased after 5-aza-dC treatment, in common to all four immortal LFS cell lines, cluster on chromosome 4q12-q27, 6p22, 6p21.3, 7,14, 19 and X ( FIGS. 9 and 11 ).  
         [0000]     Discussion  
         [0162]     Tumors evolve from normal cells due to a series of genetic and epigenetic changes that result in phenotypic alterations found in cancer cells. One of the earliest identifiable phenotypes is that of escaping cellular senescence, immortalization, which provides the proliferative capacity necessary for a tumor to develop. A number of genetic factors have been shown to play a role in the acquisition of the immortal phenotype including changes in tumor suppressor genes such as p53 and p16, oncogenes such as c-myc, and the upregulation of the enzyme telomerase. Telomerase provides protection of telomeres whose erosion results in a reduction in the cells proliferative capacity. Such genetic changes will provide molecular targets for intervention at the earliest stages of cancer development. LFS cells spontaneously immortalize in cell culture without the aid of chemical mutagens or transforming viruses, and as such provide a useful model system to study cellular immortalization. The goal was to confirm the role of IFN genes in the process of immortalization using three independent immortal cell lines derived from a second LFS cell line, MDAH087. In the analysis there were identified several pathways with changes in gene expression, including the interferon signaling pathway, the cell cycle pathway, and genes for proteins in the cytoskeleton, that were differentially expressed after the immortalization in LFS cells. Fourteen genes were consistently epigenetically regulated during immortalization in all of the immortal cell lines studied.  
         [0163]     Hierarchical clustering and multidimensional analysis was used to determine the relationships between the four immortal LFS cell lines, and to identify genes that were similarly regulated in the four immortal LFS cell lines. Both approaches indicated that the three immortal MDAH087-derived cell lines, although independently immortalized, were more closely related to one another than MDAH041 was to any of these cell lines. As expected, the gene expression patterns were closely related, but not identical among the three MDAH087 immortal cell lines.  
         [0164]     Data from another study suggested that genes with a similar expression pattern may be functionally related (Allocco et al. 2004). Although the gene expression patterns were not identical in the four immortal LFS cell lines, the Gene Ontology Pathways in which the genes are classified were similar. Thus certain pathways, but not necessarily particular genes, must be abrogated or enhanced in order for a cell to become immortal. The mechanisms by which a particular pathway is disrupted varied among cell lines. A significant number of the epigenetically regulated genes, in each of the four immortal LFS cell lines, are in the IFN pathway. The involvement of the IFN pathway in cellular senescence and tumorigenesis is supported by the fact that a number of IFN induced proteins have tumor suppression activity when overexpressed in tumor cells. These proteins include double stranded RNA activated protein kinase (PKR), activated RNaseL, and the 200 gene family (Pitha 2000). Recent studies examining the promoter methylation in bladder cancer cells and colon cells also showed the activation of IFN signaling pathways after the treatment of cancer cells with 5-aza-dC implying that their promoters are silenced by DNA methylation (Karpf et al. 1999; Liang et al. 2002). The data were the first to demonstrate that IFN signaling pathways were silenced by methylation in an early step of cancer development, immortalization (Kulaeva et al. 2003). These results support the hypothesis that genes in the IFN signaling pathways may act as growth suppressors in the progression of cells to immortalization. Recently, the interferon inducible gene, IFI 16, was shown to contribute to senescence in prostate epithelial and fibroblast cells (Xin et al. 2003; Xin et al. 2004).  
         [0165]     By categorizing the genes in gene ontology categories, it was determined that genes coding for regulatory proteins of the cell cycle and/or structural proteins of the cytoskeleton were differentially expressed during immortalization. Identification of the cell cycle as a significant pathway is consistent with studies that found that the cell cycle is dysregulated during immortalization (Vogt et al. 1998; Yin et al. 1992). In the cell cycle pathway, in addition to the well-known cell cycle genes, RB and p16 INK4 , that are involved in cellular immortalization, other cell cycle regulators were found among the fourteen genes epigenetically regulated in all four immortal LFS cell lines. These include CREG and SERPINB2, which are mechanistically involved with the RB protein, contribute to cellular immortalization and CREG which causes a delay in G1/S transition when overexpressed in NTERA-2 cells (Di Bacco and Gill 2003).  
         [0166]     Two of the fourteen epigenetically regulated genes, MAP1LC3B and HPS5 are associated with the cytoskeleton. The cytoskeletal protein CRP1, which happens to be regulated by IFN, decreases during immortalization in all four immortal LFS cell lines, further supporting the involvement of the IFN pathway and cytoskeletal proteins in immortalization.  
         [0167]     The fourteen epigenetically regulated genes that are common to all four immortal LFS cell lines do not have a significantly higher percent of genes with CpG islands when compared to another set of genes that were similarly downregulated in immortal cells but not regulated by 5-aza-dC. In addition, the size of the CpG island(s) was not found within the epigenetically regulated genes correlated with their being epigenetically regulated. Of these fourteen genes, there are twelve genes that have a known function, nine of which, IGFBPrP1, ALDH1A3, SERPINB2, also known as PAI-2, CREG, TNFAIP2, HTATIP2, CYP1B1, HPS5 and MAP1LC3B, have been associated with tumorigenesis, senescence, CpG methylation or the cytoskeleton, and based on microarray analysis and literature (Antalis et al. 1998; Der et al. 1998); D. Leaman, personal communication), three are regulated by IFN, ALDH1A3, OPTN and SERPINB2.  
         [0168]     Four of the fourteen epigenetically regulated genes may regulate the cell cycle by being functionally associated with p53 or RB. IGFBPrP1, and ALDH1A3 are regulated by p53, and as previously discussed SERPINB2 and CREG associate in cells with RB. In addition, CDC25B, which is increased in expression after immortalization and decreased in expression after 5-aza-dC treatment, is also regulated by p53. Consistent with the findings that SERPINB2 decreases during immortalization and increases after 5-aza-dC treatment, it is overexpressed in skin cells when they senesce (West et al. 1996) and SERPINB2 decreases 25-fold after the transformation of RHEK-1 cells (Yang et al. 1999). In addition, using the CGAP Virtual Northern blot of EST libraries, SERPINB2 is expressed in normal skin but not in cancerous skin. In keeping with the hypothesis that the IFN pathway plays a key role in cellular immortalization, SERPINB2 was found to protect cells from alpha virus infection through the induction of IFN-stimulated gene factor 3 (ISGF3) and through the induction of a low-level interferon-alpha/beta production (Antalis et al. 1998). Interestingly, like CYP1B1, another of the fourteen epigenetically regulated genes, SERPINB2 is regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Jana et al. 2000) through ligand-mediated activation of for the aryl-hydrocarbon receptor (AhR) (Bock 1994). This suggests that AhR and the genes regulated by it may play a role in regulating senescence. This hypothesis is corroborated by Ray and Swanson who found AhR protein levels increase during senescence of keratinocytes (Ray and Swanson 2004). In addition, TCDD causes transcriptional silencing in part through promoter methylation and under these conditions AhR is involved in inhibiting senescence of primary human epidermal keratinocytes (Ray and Swanson 2004).  
         [0169]     HTATIP2 and TNFAIP2 are among the fourteen genes epigenetically regulated in all four immortal cell lines. HTATIP2 is a putative tumor suppressor gene that promotes apoptosis and inhibits angiogenesis (Ito et al. 2003). The loss of HTATIP2 increases fibroblast transformation and ectopic expression of HTATIP2 leads to growth suppression. Furthermore HTATIP2-null mice are more susceptible to tumor development, including hepatocellular carcinomas (Ito et al. 2003). TNFAIP2 is cytokine/retinoic acid-inducible (Rusiniak et al. 2000). As retinoids are able to induce senescence-like growth arrest in tumor cells (Lotan and Nicolson 1977; Ma et al. 2003; Roninson and Dokmanovic 2003; Rusiniak et al. 2000), the findings may indicate that induction of senescence by retinoids is at least partially through the induction of TNFAIP2.  
         [0170]     Of the fourteen genes decreased during immortalization and increased after 5-aza-dC, of particular interest was IGFBPrP1. IGFBPrP1 is a member of the insulin-like growth factor-binding protein (IGFBP) superfamily consisting of six, IGFBPs and nine, IGFBP-related proteins (IGFBPrP). In addition to IGFBPrP1, other members of the IGFBP family of genes, including IGFBP3, IGFBP4 and IGFBPrP5 are silenced in the four immortal LFS cell lines. IGFBP3, IGFPB4, IGFBPrP1 and IGFBPrP5 all have CpG islands in their promoters and are potentially silenced during immortalization by methylation of these CpG islands. The IGFBPs and IGFBPrPs are involved in cell proliferation, differentiation, and apoptosis. IGFBPs bind to insulin-like growth factors (IGF-I and IGF-2), and function as their carrier, prolong their half-life, modulate their availability and prevent them from binding IGF-I and IGF-11 receptors (IGF-IR and IGF-IIR) (Hwa et al. 1999; Rajaram et al. 1997). IGFs are able to protect cells from apoptosis, act as mitogens and are necessary for the establishment and maintenance of the transformed phenotype (Benini et al. 2001). A mutation in codon 248 of p53, such as that found in all the MDAH087 cell lines, causes stimulation of the IGF-I-R (Girnita et al. 2000; Werner et al. 1996). In the microarray analysis IGF-I-R increases in all four immortal LFS cell lines, possibly a result of the loss of wild-type p53. The loss of IGFBP3 and IGFBPrP1 may be a consequence of the increase in lifespan due to the loss of p53, which also leads to genomic instability and immortalization.  
         [0171]     There are many reports of poor prognosis and increased risk of cancer when IGFBPs and IGFPBrPs are dysregulated. Breast cancer patients with low levels of IGFBPrP1 had a poor prognosis (Landberg et al. 2001). There is an inverse correlation of IGFBP3 with risk and prognosis of prostate, breast, lung, and colorectal cancer (Monzavi and Cohen 2002; Oh et al. 1995; Yu and Rohan 2000). Furthermore it has been shown to inhibit cell proliferation in breast and prostate cancer cells (Hwa et al. 1999; Oh et al. 1993; Oh et al. 1995; Rajaram et al. 1997). In one study 75% of the human hepatocellular carcinomas analyzed had a reduction in IGFBP3 expression and 33% of the hepatocellular carcinomas contained hypermethylation in the IGFBP3 promoter (Hanafusa et al. 2002). Treating hypermethylated hepatocellular carcinoma cell lines with 5-aza-dC re-established the expression of IGFBP3. Thus IGFBP3 may be an epigenetically regulated gene capable of inducing cellular senescence. Increasing levels of IGFBP3 and/or the other IGFBPs may decrease the levels of free IGFs leading to decreased binding of IGF to IGF-IR and IGF-IIR, thereby inhibiting cell growth and proliferation, and/or promoting apoptosis. Because the lifespan of these cells is insufficient to survive at a distant site, it is less likely that they will become immortal and acquire the ability to metastasize. Schwarze et. al. used cDNA microarrays to identify genes in human prostate epithelial cells upregulated in senescence and repressed during immortalization, and found that IGFBP3 was decreased in immortal cells and upregulated in senescent cells (Schwarze et al. 2002). Also consistent with the gene expression analysis of immortal LFS cells, IGFBP3 increases during senescence of human oral keratinocytes (Kang et al. 2003). Similarly, IGFBPrP1 was found to be overexpressed during senescence of human mammary epithelial cells and human prostate epithelial cells (Lopez-Bermejo et al. 2000; Swisshelm et al. 1995). Expression of IGFBPrP1 in MCF-7 breast cancer cells induces a senescence-like state (Wilson et al. 2002). Methylation of IGFBPrP1 corresponds to a decrease in its expression during hepatocarcinogenesis (Komatsu et al. 2000). To test the hypothesis that a decrease in IGFBP genes permits immortalization and subsequently can lead to tumorigenesis, the CGAP Virtual Northern blot of EST libraries were queried and found that IGFBP is expressed in normal liver tissue but not in cancerous liver tissue. Thus the epigenetic regulation of IGFBP3 and IGFBPrP1 is consistent with the findings supporting the involvement of IGFBP genes in senescence.  
         [0172]     HSPA2, (HSP70 isoform 2) one of the fourteen epigenetically regulated genes, is hypermethylated in breast cancer, but can be reactivated upon treatment with a demethylating agent (Shi et al. 2002). Using the CGAP Virtual Northern blot of EST libraries, it was found that HSP70-2 is expressed in normal testicular tissue but not in testicular cancer tissue. Interestingly, HSP70-2 disruption in the mouse genome results in male meiosis defects and infertility (Dix et al. 1996).  
         [0173]     By microarray and Q-RT-PCR analysis, CYP1B1 was found to be epigenetically regulated in all four immortal LFS cell lines. Consistent with the finding, another lab found that CYP1B1 expression is enhanced during senescence human oral keratinocytes (Kang et al. 2003). Contradictory to these results, CYP1B1 was found to decrease with differentiation of mouse embryo fibroblasts (MEFs), and increase in MEFs that escaped senescence (Alexander et al. 1997). However, it was concluded that while the CYP1B1 mRNA may be altered in expression during immortalization, its function in this process is unlikely to be significant.  
         [0174]     There were two genes, CDC25B and LDB2, which increased during immortalization and decreased with 5-aza-dC treatment. Consistent with the findings, CDC25B message is overexpressed in human tumors, including pancreatic, prostate head and neck, and in some cancer cell lines (Gasparotto et al. 1997; Guo et al. 2004; Ngan et al. 2003; Ullmannova et al. 2003). In agreement with the finding that CDC25B decreases after induction of senescence with 5-aza-dC, treatment of cells with another differentiation agent, butyric acid, resulted in a decrease in CDC25B expression (Ullmannova et al. 2003).which has epigenetic effects by inhibiting histone deacetylases,  
         [0175]     There are few imprinted genes that are found alone on chromosomes, as they frequently occur in clusters (Verona et al. 2003). Thus if an epigenetically regulated gene localizes to such an imprinted cluster region, it is possible that it may be part of a previously unidentified imprinted gene. In the data genes were found that decrease during immortalization that are known imprinted genes as well as genes that fall in imprinting regions and may be putative imprinted genes. CD81 (11p15.5), MEG3 (14q32) and NDN (15q11.2-q12) are known imprinted genes that decrease in all four immortal LFS cell lines during immortalization. Genes that have a decrease in expression during immortalization and are located near known imprinted genes (putative novel imprinting genes), include CD59 (11p13), DKFZp564J0323 (11p13-11qter), SMPD1 (11p15.4-p15.1), PHLDA2 (11p15.5), CRYAB (11q22.q23.1), IGSF4 (11q23.2), TAGLN (11q23.2), CD63 (12q12-q13), LUM (12q21.3-q22), TNFAIP2 (14q32), FBLN5 (14q32.1), KNS2 (14q32.3) and C14orf78 (14q32.33). Of particular interest is TNFAIP2, which is one of the fourteen genes that decreases during immortalization and increases after 5-aza-dC treatment in all four immortal LFS cell lines.  
         [0176]     In summary, gene expression analysis was performed using microarrays to identify pathways critical to the process of cellular immortalization. The senescence initiating events leading to genomic instability and telomere stabilization are loss of checkpoint proteins such as p53, p21 CIP1/WAF1 , and p16 INK4a . Gene profiling revealed 149 upregulated genes and 187 downregulated genes of which 14 were epigenetically downregulated in all four immortal LFS cell lines. In addition, several common pathways were involved in immortalization including the interferon pathway, genes involved in proliferation and cell cycle control, and the genes for cytoskeletal proteins.  
         [0177]     Throughout this application, various publications, including United States patents, are referenced by author and year, and patents, by number. Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.  
         [0178]     The invention has been described in an illustrative manner, and it is to be understood that the terminology that has been used is intended to be in the nature of words of description rather than of limitation.  
         [0179]     Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the described invention, the invention can be practiced otherwise than as specifically described.  
                                                                                                                                     TABLE 7                           Summary of differentially regulated genes in MDAH041 and MDAH087 immortal cell lines.                                Common to 4           MDAH041   MDAH087-N   MDAH087-1   MDAH087-10   IM Cell Lines            Comparison   Probe   Gene   Probe   Gene   Probe   Gene   Probe   Gene   Probe   Gene                    A. IM vs PC upregulated   1120   897   1276   1038   1544   1267   979   801   192   149       B. IM vs PC downregulated   1270   1120   894   785   1093   954   921   807   207   187       C. 5-aza-dC vs IM downregulated   928   803   869   717   816   713   484   408   49   46       D. 5-aza-dC vs IM upregulated   1063   877   936   772   894   730   875   724   226   185       Genes in Sets B and D   304   263   172   152   169   147   133   121   15   14       Genes in Sets A and C   175   159   355   284   262   233   109   90   2   2                 Data was analyzed using Affymetrix DMT version 5;            PC: precrisis cells;            IM: immortal cells;            5-aza-dC: 5-aza-dC-treated immortal cells.            Probe: Probe ID from Affymetrix HGU95Av2 arrays.            Gene: Unigene number based on Unigene build #166.             
 
         [0180]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                   
               
               
                 Categorization of the genes regulated in all four immortal LFS cell lines 
               
             
          
           
               
                   
                 Common 
                   
                 p53 
                 Im- 
               
               
                   
                 to 4 
                 IFN 
                 regu- 
                 print- 
               
               
                   
                 IM Cell 
                 regulated 
                 lated 
                 ed 
               
               
                 Comparison 
                 Lines 
                 gene 
                 gene 
                 gene 
               
               
                   
               
             
          
           
               
                 A. IM vs PC upregulated 
                 149 
                 43 
                 29 
                 0 
               
               
                 B. IM vs PC downregulated 
                 187 
                 40 
                 23 
                 4 
               
               
                 C. 5-aza-dC vs IM downregulated 
                 47 
                 10 
                 7 
                 0 
               
               
                 D. 5-aza-dC vs IM upregulated 
                 185 
                 56 
                 26 
                 0 
               
               
                 Genes in Sets B and D 
                 14 
                 2 
                 1 
                 0 
               
               
                 Genes in Sets A and C 
                 2 
                 1 
                 1 
                 0 
               
               
                   
               
               
                   Data was analyzed using Affymetrix DMT version 5;    
               
               
                   PC: precrisis cells;    
               
               
                   IM: immortal cells;    
               
               
                   5-aza-dC: 5-aza-dC-treated immortal cells.    
               
               
                   Probe: Probe ID from Affymetrix HGU95Av2 arrays.    
               
               
                   Gene: Unigene number based on Unigene build #166    
               
             
          
         
       
     
         [0181]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                   
               
               
                 Genes differentially regulated after immortalization and 
               
               
                 demethylation in MDAH041 and MDAH087 cells 
               
             
          
           
               
                   
                 MDAH041 
                 MDAH087-N 
                 MDAH087-1 
                 MDAH087-10 
                   
               
             
          
           
               
                 Gene 
                 LocusLink ID 
                 IM 
                 5A 
                 IM 
                 5A 
                 IM 
                 5A 
                 IM 
                 5A 
                 CpG 
                 LOCUS 
               
               
                   
               
             
          
           
               
                 CREG 
                 8804 
                 −1.6 
                 2.2 
                 −1.5 
                 1.6 
                 −9.3 
                 4.1 
                 −3.3 
                 2.6 
                 + 
                  1q24 
               
               
                 CYP1B1 
                 1545 
                 −14.0 
                 8.2 
                 −8.4 
                 8.0 
                 −4.2 
                 4.9 
                 −5.1 
                 4.7 
                 + 
                  2p21 
               
               
                 IGFBPrP1 
                 3490 
                 −2.0 
                 2.5 
                 −4.5 
                 3.0 
                 −10.1 
                 1.9 
                 −4.9 
                 1.7 
                 + 
                  4q12 
               
               
                 CLTB 
                 1212 
                 −1.3 
                 1.5 
                 −1.9 
                 2.4 
                 −4.0 
                 2.1 
                 −2.4 
                 1.8 
                 + 
                  5q35 
               
               
                 KIAA1750 
                 85453 
                 −27.8 
                 2.9 
                 −18.8 
                 5.1 
                 −3.7 
                 3.3 
                 −15.2 
                 9.1 
                 + 
                  8q22.1 
               
               
                 FLJ14675 
                 84909 
                 −2.7 
                 3.8 
                 −6.0 
                 2.3 
                 −2.0 
                 1.8 
                 −2.2 
                 1.7 
                 + 
                  9q22 
               
               
                 OPTN 
                 10133 
                 −3.0 
                 2.6 
                 −1.8 
                 1.8 
                 −3.6 
                 1.6 
                 −2.1 
                 1.3 
                 + 
                 10p14 
               
               
                 HPS5 
                 11234 
                 −1.6 
                 1.6 
                 −1.6 
                 2.2 
                 −1.7 
                 1.7 
                 −2.4 
                 1.5 
                 + 
                 11p14 
               
               
                 HTATIP2 
                 10553 
                 −17.7 
                 5.6 
                 −6.6 
                 3.7 
                 −2.9 
                 2.0 
                 −5.5 
                 2.4 
                 + 
                 11p15.1 
               
               
                 HSPA2 
                 3306 
                 −2.0 
                 1.6 
                 −12.4 
                 10.9 
                 −5.2 
                 5.3 
                 −5.2 
                 6.4 
                 − 
                 14q24.1 
               
               
                 TNFAIP2 
                 7127 
                 −6.0 
                 5.4 
                 −9.3 
                 5.4 
                 −4.3 
                 3.1 
                 −3.7 
                 2.1 
                 − 
                 14q32 
               
               
                 ALDH1A3 
                 220 
                 −2.7 
                 2.8 
                 −2.3 
                 3.2 
                 −8.0 
                 2.3 
                 −4.4 
                 3.8 
                 + 
                 15q26.3 
               
               
                 MAP1LC3B 
                 81631 
                 −1.6 
                 1.7 
                 −2.5 
                 2.5 
                 −2.0 
                 3.1 
                 −1.3 
                 1.8 
                 + 
                 16q24.2 
               
               
                 SERPINB2 
                 5055 
                 −1.3 
                 4.2 
                 −3.0 
                 10.7 
                 −2.6 
                 8.8 
                 −4.3 
                 7.0 
                 − 
                 18q21.3 
               
               
                   
               
               
                   Fold change of gene expression level were processed in Affymetrix DMT, version 5.    
               
               
                   Fold changes for genes with multiple probes were averaged;    
               
               
                   5A: upregulation in 5-aza-dC-treated immortal cells versus untreated immortal cells;    
               
               
                   IM: down-regulation in immortal cells versus precrisis cells;    
               
             
          
         
       
     
         [0182]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                   
               
               
                 Comparison of changes in expression in microarrays and by Q-RT-PCR analysis 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 MDAH041 
                 MDAH087-N 
               
             
          
           
               
                 Gene 
                 IM vs PC 
                 IM vs 5-aza-dC 
                 IM vs PC 
                 IM vs 5-aza-dC 
               
             
          
           
               
                 Symbol 
                 LocusLinkID 
                 MA 
                 QP 
                 MA 
                 QP 
                 MA 
                 QP 
                 MA 
                 QP 
               
               
                   
               
               
                 ALDH1A3 
                 220 
                 −2.7 
                 −13.94 
                 2.8 
                 7.84 
                 −2.3 
                 −4.31 
                 3.2 
                 11.51 
               
               
                 CLTB 
                 1212 
                 −1.3 
                 −7.51 
                 1.5 
                 2.04 
                 −1.9 
                 −6.11 
                 2.4 
                 7.62 
               
               
                 CREG 
                 8804 
                 −1.65 
                 −8.50 
                 2.21 
                 2.88 
                 −1.54 
                 −2.75 
                 1.55 
                 4.80 
               
               
                 HSPA2 
                 3306 
                 −2.0 
                 0.60 
                 1.6 
                 2.74 
                 −12.4 
                 −144.35 
                 10.9 
                 46.6 
               
               
                 IGFBPrP1 
                 3490 
                 −2.00 
                 −8.80 
                 2.51 
                 12.27 
                 −4.47 
                 −27.15 
                 3.02 
                 5.34 
               
               
                 KIAA1750 
                 85453 
                 −27.76 
                 −3.78 
                 2.88 
                 3.86 
                 −18.81 
                 −403.99 
                 5.10 
                 28.43 
               
               
                 OPTN 
                 10133 
                 −3.0 
                 −15.30 
                 2.6 
                 9.21 
                 −1.8 
                 −4.68 
                 1.8 
                 6.36 
               
               
                 SERPINB2 
                 5055 
                 −1.3 
                 −42.08 
                 4.2 
                 12.90 
                 −3.0 
                 −6.52 
                 10.7 
                 66.72 
               
               
                 TNFAIP2 
                 7127 
                 −5.97 
                 −18.90 
                 5.41 
                 6.10 
                 −9.34 
                 −32.01 
                 5.41 
                 39.56 
               
               
                 STAT1α 
                 6772 
                 −1.57 
                 −16.21 
                 2.92 
                 119.85 
                 −1.3 
                 −4.39 
                 1.99 
                 9.05 
               
               
                 IGFBP3 
                 3486 
                 −2.24 
                 0.24 
                 2.48 
                 8.29 
                 −6.25 
                 −111.11 
                 −1.03 
                 2.82 
               
               
                 IGFBP4 
                 3487 
                 −21.66 
                 −50.58 
                 3.72 
                 7.00 
                 −2.89 
                 −24.21 
                 1.84 
                 4.82 
               
               
                   
               
             
          
           
               
                   
                 MDAH087-1 
                 MDAH087-10 
               
             
          
           
               
                 Gene 
                 IM vs PC 
                 IM vs 5-aza-dC 
                 IM vs PC 
                 IM vs 5-aza-dC 
               
             
          
           
               
                 Symbol 
                 LocusLinkID 
                 MA 
                 QP 
                 MA 
                 QP 
                 MA 
                 QP 
                 MA 
                 QP 
               
               
                   
               
               
                 ALDH1A3 
                 220 
                 −8.0 
                 −42.13 
                 2.3 
                 69.28 
                 −4.4 
                 −10.42 
                 3.8 
                 9.71 
               
               
                 CLTB 
                 1212 
                 −4.0 
                 −3.66 
                 2.1 
                 3.08 
                 −2.4 
                 −5.4 
                 1.8 
                 7.5 
               
               
                 CREG 
                 8804 
                 −9.32 
                 −8.34 
                 4.10 
                 4.93 
                 −3.28 
                 −3.11 
                 2.56 
                 4.49 
               
               
                 HSPA2 
                 3306 
                 −5.2 
                 −7.21 
                 5.3 
                 24.43 
                 −5.2 
                 −17.7 
                 6.4 
                 3.65 
               
               
                 IGFBPrP1 
                 3490 
                 −10.15 
                 −52.02 
                 1.93 
                 1.93 
                 −4.86 
                 −41.85 
                 1.69 
                 4.10 
               
               
                 KIAA1750 
                 85453 
                 −3.72 
                 −10.83 
                 3.28 
                 5.23 
                 −15.24 
                 −30.42 
                 9.09 
                 31.41 
               
               
                 OPTN 
                 10133 
                 −3.6 
                 −8.65 
                 1.6 
                 30.54 
                 −2.1 
                 −1553.76 
                 1.3 
                 431.43 
               
               
                 SERPINB2 
                 5055 
                 −2.6 
                 −5.19 
                 8.8 
                 22.63 
                 −4.3 
                 −12.17 
                 7.0 
                 58.24 
               
               
                 TNFAIP2 
                 7127 
                 −4.34 
                 −8.44 
                 3.08 
                 7.28 
                 −3.69 
                 −5.14 
                 2.14 
                 3.72 
               
               
                 STAT1α 
                 6772 
                 −1.51 
                 −3.94 
                 1.05 
                 1.56 
                 −2.49 
                 −15.23 
                 1.42 
                 3.23 
               
               
                 IGFBP3 
                 3486 
                 −5.76 
                 −46.68 
                 −1.58 
                 −2.61 
                 −6.80 
                 −83.13 
                 −2.66 
                 −1.40 
               
               
                 IGFBP4 
                 3487 
                 −2.15 
                 −10.59 
                 1.08 
                 −0.03 
                 −4.37 
                 −34.80 
                 1.9 
                 3.72 
               
               
                   
               
               
                   Average fold change of gene expression after immortalization (IM versus PC) and after treatment with 5-aza-dC (IM versus 5-aza-dC)    
               
               
                   MA: fold change microarray;    
               
               
                   QP: fold change Q-RT-PCR    
               
             
          
         
       
     
         [0183]    
       
         
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                   
               
               
                 Primers used in Q-RT-PCR 
                   
               
             
          
           
               
                 Gene 
                 Forward prime 1   
                 Reverse primer 1   
               
               
                   
               
             
          
           
               
                 ALDH1A3 
                 GCCAGGGTCTTTGTGGATTG 
                 AGCTCTCTGGGCTATTGATTCTG 
                   
               
               
                   
                   
                 T 
               
               
                   
               
               
                 CLTB 
                 AACAACCGGATCGCTGACA 
                 CCTCCTTGGATTCCTTCACG 
               
               
                   
               
               
                 CREG 
                 CAGCTTCAGCCAGGGACAAA 
                 GGGCAGTTGAGGAAGCCTTAG 
               
               
                   
               
               
                 GAPDH 
                 ATCAAGAAGGTGGTGAAGCAG 
                 TGTCGCTGTTGAAGTCAGAGG 
               
               
                   
               
               
                 HSPA2 
                 ACCGAAACCAGATGGCAGAG 
                 GGACCACCTTGGTAAAGTTTGCT 
               
               
                   
               
               
                 IGFBP3 
                 AACTGTGGCCATGACTGAGGA 
                 CTCCCTGAGCCTGACTTTGC 
               
               
                   
               
               
                 IGFBP4 
                 ACCCACTCCCAAAGCTCAGA 
                 TGCCAGCCAACCAAGCA 
               
               
                   
               
               
                 IGFBPrP1 
                 GCCATGCATCCAATTCCC 
                 TCGGCACCTTCACCTTTTTT 
               
               
                   
               
               
                 KIAA1750 
                 TATGGTCAACCTGGTTTCATCTGT 
                 CTCCCAAAGTAGTCACGGTTGC 
               
               
                   
               
               
                 OPTN 
                 GAGAAGGCTCTGGCTTCCAA 
                 GAGCCCTGAGGATGGTCATG 
               
               
                   
               
               
                 SERPINB2 
                 GGACGGGCCAATTTCTCAG 
                 CTTCAGTGCCCTCCTCATTCA 
               
               
                   
               
               
                 TNFAIP2 
                 TAGCCTCCTAAAGTGCTGGGAT 
                 TCTCTGGGTAGGCGCAATGT 
               
               
                   
               
               
                     1 All primers have a 5′-3′ orientation    
               
             
          
         
       
     
         [0184]     Table 12 Summary of differentially regulated genes/probes in MDAH041 and MDAH087 cells during immortalization.  
                                                                   TABLE 12a                           192 probes upregulated IM vs PC (see Table 1A)            Symbol   Affymetrix HGU95Av2 Probe ID   LocusLink   Average Singal Log 2     Fold Change   Locus                    AK3   32331_at   205   1.66   3.15   1p31.3       ALDH6A1   32676_at   4329   1.30   2.46   14q24.3       ANAPC7   37171_at   51434   0.83   1.78   12q13.12       APEX1   2025_s_at   328   0.83   1.78   14q11.2-q12       APOBEC3B   39230_at   9582   1.33   2.52   22q13.1-q13.2       ARF6   37984_s_at   382   0.91   1.88   14q21.3       ARHGEF2   40100_at   9181   0.72   1.65   1q21-q22       ASPH   37528_at   444   1.16   2.23   8q12.1       ATP2B1   37661_at   490   1.59   3.01   12q21-q23       ATP5S   40027_at   27109   1.09   2.13   14q22.1       BAX   2065_s_at   581   0.79   1.73   19q13.3-q13.4       BCAT1   38201_at   586   2.06   4.18   12pter-q12       BCAT2   41111_at   587   0.77   1.70   19q13       BIN1   132238_at   274   1.04   2.05   2q14       BOP1   35615_at   23246   1.24   2.37   8q24.3       BSG   36162_at   682   1.00   2.00   19p13.3       BTBD2   35155_at   55643   1.81   3.50   19p13.3       C6orf69   35001_at   222658   1.13   2.19   6p21.31       C6orf69   35002_g_at   222658   2.17   4.51   6p21.31       CDC20   38414_at   991   1.06   2.09   1p34.1       CDC25B   1347_at   994   0.64   1.56   20p13       CDKN3   1599_at   1033   1.05   2.07   14q22       CELSR3   40020_at   1951   1.00   2.00   3p24.1-p21.2       CENPB   37931_at   1059   0.98   1.97   20p13       CHC1   37927_at   1104   1.04   2.05   1p36.1       CHD1   39231_at   1105   0.95   1.93   5q15-q21       CKS2   40690_at   1164   1.02   2.02   9q22       COX11   34723_at   1353   1.17   2.25   17q22       CSNK1G2   446_at   1455   1.33   2.51   19p13.3       CSNK2A1   40258_at   1457   0.89   1.86   20p13       CSNK2A1   594_s_at   1457   0.96   1.95   20p13       CSNK2B   32843_s_at   1460   1.00   1.99   6p21-p12       D10S170   37162_at   8030   0.83   1.78   10q21       DDX27   33650_at   55661   0.99   1.99   20q13.13       DDX3X   826_at   1654   1.06   2.09   Xp11.3-p11.23       DKFZP564J157   35745_f_at   54458   0.93   1.90   12q12       DKFZP564J157   35746_r_at   54458   0.76   1.70   12q12       DKFZP564O243   41018_at   25864   0.80   1.74   3p21.1       DLG7   37231_at   9787   1.02   2.03   14q22.2       DPYD   38220_at   1806   1.10   2.14   1p22       E2-EPF   893_at   27338   1.41   2.65   19q13.43       E2F4   1703_g_at   1874   1.24   2.36   16q21-q22       EGFR   1537_at   1956   2.97   7.86   7p12       EMP1   1321_s_at   2012   2.58   5.96   12p12.3       ERF   38996_at   2077   1.12   2.17   19q13       EWSR1   423_at   2130   1.11   2.17   22q12.2       FBL   39173_at   2091   0.91   1.88   19q13.1       FNTB   37488_at   2342   0.89   1.86   14q23-q24       FUS   39180_at   2521   1.00   2.00   16p11.2       FZD2   36799_at   2535   1.01   2.01   17q21.1       FZD2   628_at   2535   1.16   2.23   17q21.1       GAPD   AFFX-HUMGAPDH/M33197_5_at   2597   0.42   1.34   12p13       GG2-1   33243_at   25816   1.17   2.25   5q23.1       GNA13   1139_at   10672   1.61   3.05   17q24.3       GNS   36262_at   2799   1.31   2.48   12q14       GNS   36263_g_at   2799   2.04   4.12   12q14       GUSB   33308_at   2990   0.60   1.51   7q21.11       H1F0   33386_at   3005   0.91   1.88   22q13.1       H1FX   318_at   8971   1.75   3.36   3q21.3       H1FX   319_g_at   8971   1.06   2.09   3q21.3       H2AFY   36576_at   9555   0.67   1.59   5q31.3-q32       HAN11   38171_at   10238   0.76   1.69   17q24.2       HAN11   41591_at   10238   0.90   1.86   17q24.2       HDAC1   476_s_at   3065   0.92   1.90   1p34       IDH3B   40110_at   3420   0.79   1.73   20p13       IER3   1237_at   8870   1.46   2.75   6p21.3       IGF1R   34718_at   3480   0.86   1.81   15q25-q26       ILF3   40845_at   3609   1.33   2.51   19p13.2       ILF3   40846_g_at   3609   0.91   1.88   19p13.2       IMPDH2   36624_at   3615   0.77   1.70   3p21.2       KCNG1   37498_at   3755   0.98   1.97   20q13       KCNMA1   40737_at   3778   0.84   1.79   10q22-q23       KIAA0186   39677_at   9837   1.24   2.36   20P11.21       KIAA0528   35252_at   9847   1.04   2.05   12p12.2       KIAA0863   37837_at   22850   1.43   2.70   18q23       KPNB2   40463_at   3842   0.82   1.76   5q13.2       LDB2   36065_at   9079   1.23   2.34   4p16       LMNB1   37985_at   4001   1.35   2.54   5q23.3-q31.1       LMNB2   36987_at   84823   1.03   2.05   19p13.3       MADH5   1013_at   4090   0.96   1.94   5q31       MADH5   39926_at   4090   1.09   2.13   5q31       MAP1B   39531_at   4131   1.47   2.76   5q13       MAP1B   41373_s_at   4131   1.97   3.92   5q13       MAPK1   976_s_at   5594   0.82   1.77   22q11.2       MAPKAPK2   1439_s_at   9261   2.18   4.54   1q32       MAT2A   32571_at   4144   0.88   1.84   2p11.2       MAX   1981_s_at   4149   1.54   2.91   14q23       MAZ   1764_s_at   4150   2.07   4.20   16p11.2       MAZ   32553_at   4150   0.59   1.51   16p11.2       MET   35684_at   4233   1.15   2.23   7q31       MRPS12   33214_at   6183   0.78   1.72   19q13.1-q13.2       MSC   35992_at   9242   1.87   3.64   8q21       MTHFD1   673_at   4522   1.39   2.62   14q24       MTHFD1   674_g_at   4522   1.01   2.01   14q24       MYC   1827_s_at   4609   1.14   2.20   8q24.12-q24.13       MYC   1973_s_at   4609   1.63   3.10   8q24.12-q24.13       MYC   37724_at   4609   1.31   2.48   8q24.12-q24.13       MYO10   35362_at   4651   0.80   1.74   5p15.1-p14.3       NDUFV1   37329_at   4723   0.79   1.73   11q13       NOL1   1979_s_at   4839   1.31   2.49   12p13       NOL5A   34882_at   10528   0.82   1.77   20p13       NR1D2   35705_at   9975   1.43   2.69   3p24.1       NR1H2   518_at   7376   0.93   1.91   19q13.3-19q13.3       NRAS   1539_at   4893   1.38   2.60   1P13.2       NRP1   36836_at   8829   1.54   2.91   10p12       OSBP   41313_at   5007   0.92   1.89   11q12-q13       PAI-RBP1   40440_at   26135   0.97   1.96   1p31-p22       PDK1   36386_at   5163   1.61   3.06   2q31.1       PES1   41869_at   23481   0.87   1.82   22q12.1       PGK1   31488_s_at   5230   1.29   2.44   Xq13       PITPNB   353_at   23760   1.51   2.86   22q12.1       PLD3   36151_at   23646   1.04   2.06   19q13.2       PLK   37228_at   5347   1.08   2.12   16p12.3       PLTP   40081_at   5360   1.06   2.09   20q12-q13.1       PPP2R1A   922_at   5518   0.63   1.54   19q13.41       PPP4C   382_at   5531   0.59   1.50   16p12-16p11       PPP5C   391_at   5536   1.18   2.26   19q13.3       PPP5C   392_g_at   5536   1.72   3.30   19q13.3       PPT1   34774_at   5538   1.10   2.14   1p32       PRIM1   798_at   5557   1.75   3.35   12q13       PRIM2A   122_at   5558   0.90   1.86   6p12-p11.1       PRKCA   32304_at   5578   1.05   2.07   17q22-q23.2       PRKDC   2012_s_at   5591   1.20   2.30   8q11       PROSC   40545_at   11212   1.09   2.13   8p11.2       PRRX1   40126_at   5396   1.13   2.19   1q24       PTMS   40580_r_at   5763   1.01   2.02   12p13       PTPN11   1870_at   5781   1.77   3.41   12q24       PTRF   36369_at   284119   1.95   3.87   17q21.31       RAB5A   600_at   5868   0.94   1.92   3p24-p22       RAD21   38114_at   5885   0.77   1.70   8q24       RAF1   1917_at   5894   0.85   1.80   3p25       RALY   36125_s_at   22913   0.98   1.97   20q11.21-q11.23       RBMX   39731_at   27316   0.78   1.72   Xq26       REA   37364_at   11331   0.75   1.68   12p13       RELA   1045_s_at   5970   1.84   3.59   11q13       RFC5   38863_at   5985   1.16   2.23   12q24.2-q24.3       RRP4   32974_at   23404   1.17   2.24   9q34       SCG2   36924_r_at   7857   2.44   5.43   2q35-q36       SFRS3   351_f_at   6428   1.05   2.08   6p21       SIP1   41363_at   8487   1.22   2.34   14q13       SLC25A1   38998_g_at   6576   0.62   1.53   22q11.21       SMC2L1   37502_at   10592   1.18   2.27   9q31.2       SNRPB   38455_at   6628   0.99   1.99   20p13       SNRPB   38456_s_at   6628   0.54   1.46   20p13       SQLE   35839_at   6713   0.85   1.80   8q24.1       STK6   34851_at   6790   0.99   1.98   20q13.2-q13.3       TCOF1   40596_at   6949   0.83   1.77   5q32-q33.1       TFRC   AFFX-HUMTFRR/M11507_3_at   7037   1.60   3.03   3q26.2-qter       TFRC   AFFX-HUMTFRR/M11507_5_at   7037   2.00   4.00   3q26.2-qter       TFRC   AFFX-HUMTFRR/M11507_M_at   7037   2.22   4.66   3q26.2-qter       TMPO   32682_at   7112   2.20   4.59   12q22       TMPO   32683_at   7112   1.26   2.39   12q22       TOMM40   35620_at   10452   1.36   2.57   19q13       TOP1   1710_s_at   7150   1.80   3.48   20q12-q13.1       TOP2B   1581_s_at   7155   1.42   2.68   3p24       TPX2   39109_at   22974   0.85   1.80   20q11.2       TRIO   40581_at   7204   1.48   2.80   5p15.1-p14       TRIO   40792_s_at   7204   1.49   2.80   5p15.1-p14       TULP3   31943_g_at   7289   1.13   2.19   12p13.3       TXNDC   34768_at   81542   0.90   1.86   14q22.1       UBE2C   1651_at   11065   1.08   2.11   20q13.12       UBE2M   33781_s_at   9040   0.88   1.84   19q13.43       UBTF   38795_s_at   7343   1.10   2.14   17q21.3       VEGF   1953_at   7422   1.08   2.11   6p12       VEGF   36100_at   7422   1.04   2.06   6p12       VEGF   36101_s_at   7422   3.52   11.49   6p12       WNT5A   1669_at   7474   1.09   2.13   3p21-p14       WNT5A   31862_at   7474   1.04   2.05   3p21-p14       —   1196_at   —   1.49   2.80   —       —   1258_s_at   —   1.04   2.05   —       —   1609_g_at   —   1.34   2.54   —       —   1635_at   —   0.92   1.89   —       —   1750_at   —   0.97   1.96   —       —   1753_s_at   —   1.44   2.70   —       —   1812_s_at   —   1.16   2.24   —       —   1942_s_at   —   0.95   1.93   —       —   2049_s_at   —   2.38   5.19   —       —   31510_s_at   —   0.57   1.49   —       —   33761_s_at   376645   1.22   2.32   1q21.2       —   34069_s_at   —   0.70   1.62   —       —   34374_g_at   —   0.73   1.65   —       —   38022_s_at   —   2.89   7.40   —       —   39470_at   —   1.76   3.38   —       —   39537_at   —   2.40   5.28   —       —   39560_at   —   2.23   4.69   —       —   39694_at   —   0.94   1.92   —       —   40608_at   376135   2.39   5.25   12q15       —   434_at   —   3.70   13.02   —       —   625_at   —   1.29   2.45   —       —   910_at   —   1.74   3.33   —       —   919_at   —   1.32   2.50   —       —   953_g_at   —   1.23   2.34   —                  
 
         [0185]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 12b 
               
             
             
               
                   
               
               
                   
               
               
                 207 genes/probes downregulated IM vs PC (see Table 1B) 
               
             
          
           
               
                   
                 Affymetrix HGU95Av2 
                   
                 Average 
                 Fold 
                   
               
               
                 Symbol 
                 Probe ID 
                 LocusLink 
                 Signal Log 2   
                 Change 
                 Locus 
               
               
                   
               
             
          
           
               
                 ABAT 
                 33446_at 
                 18 
                 −1.92 
                 −3.77 
                 16p13.2 
               
               
                 ACTA2 
                 32755_at 
                 59 
                 −2.48 
                 −5.57 
                 10q23.3 
               
               
                 ACTC 
                 39063_at 
                 70 
                 −6.09 
                 −67.90 
                 15q11-q14 
               
               
                 ACTR1A 
                 40052_at 
                 10121 
                 −0.58 
                 −1.50 
                 10q24.33 
               
               
                 ADD1 
                 32145_at 
                 118 
                 −0.48 
                 −1.40 
                 4p16.3 
               
               
                 ALDH1A3 
                 36686_at 
                 220 
                 −1.94 
                 −3.82 
                 15q26.3 
               
               
                 ANXA11 
                 36637_at 
                 311 
                 −0.97 
                 −1.96 
                 10q23 
               
               
                 APM2 
                 32527_at 
                 10974 
                 −5.01 
                 −32.31 
                 10q23.31 
               
               
                 ARPC1B 
                 39043_at 
                 10095 
                 −1.10 
                 −2.14 
                 7q22.1 
               
               
                 ASS 
                 40541_at 
                 445 
                 −1.76 
                 −3.38 
                 9q34.1 
               
               
                 ATOX1 
                 41776_at 
                 475 
                 −1.00 
                 −2.00 
                 5q32 
               
               
                 ATP2B4 
                 40913_at 
                 493 
                 −0.88 
                 −1.84 
                 1q25-q32 
               
               
                 ATP6V0E 
                 33875_at 
                 8992 
                 −1.09 
                 −2.13 
                 5q35.2 
               
               
                 BPAG1 
                 32780_at 
                 667 
                 −0.58 
                 −1.50 
                 6p12-p11 
               
               
                 C14orf78 
                 36497_at 
                 113146 
                 −2.03 
                 −4.08 
                 14q32.33 
               
               
                 C21orf80 
                 34287_at 
                 23275 
                 −0.74 
                 −1.67 
                 21q22.3 
               
               
                 C6orf109 
                 38697_at 
                 25844 
                 −0.77 
                 −1.70 
                 6p21.1 
               
               
                 C6orf32 
                 37112_at 
                 9750 
                 −4.05 
                 −16.61 
                 6p22.3-p21.32 
               
               
                 CAP2 
                 33405_at 
                 10486 
                 −1.34 
                 −2.53 
                 6p22.3 
               
               
                 CAPG 
                 38391_at 
                 822 
                 −2.50 
                 −5.65 
                 2cen-q24 
               
               
                 CCND1 
                 2020_at 
                 595 
                 −1.31 
                 −2.48 
                 11q13 
               
               
                 CCND1 
                 38418_at 
                 595 
                 −1.34 
                 −2.53 
                 11q13 
               
               
                 CD59 
                 39351_at 
                 966 
                 −1.01 
                 −2.02 
                 11p13 
               
               
                 CD63 
                 37003_at 
                 967 
                 −0.76 
                 −1.69 
                 12q12-q13 
               
               
                 CD81 
                 35282_r_at 
                 975 
                 −1.11 
                 −2.16 
                 11p15.5 
               
               
                 CD97 
                 35625_at 
                 976 
                 −0.70 
                 −1.63 
                 19p13 
               
               
                 CD99 
                 41138_at 
                 4267 
                 −1.35 
                 −2.55 
                 Xp22.32 
               
               
                 CDKN1A 
                 2031_s_at 
                 1026 
                 −2.02 
                 −4.04 
                 6p21.2 
               
               
                 CH25H 
                 32363_at 
                 9023 
                 −3.66 
                 −12.60 
                 10q23 
               
               
                 CLECSF2 
                 40698_at 
                 9976 
                 −2.73 
                 −6.62 
                 12p13-p12 
               
               
                 CLIC1 
                 36131_at 
                 1192 
                 −0.62 
                 −1.54 
                 6p22.1-p21.2 
               
               
                 CLTB 
                 32523_at 
                 1212 
                 −1.16 
                 −2.24 
                 4q2-q3 
               
               
                 CNN1 
                 34203_at 
                 1264 
                 −3.39 
                 −10.50 
                 19p13.2-p13.1 
               
               
                 COL4A1 
                 39333_at 
                 1282 
                 −2.82 
                 −7.04 
                 13q34 
               
               
                 COL4A2 
                 36659_at 
                 1284 
                 −1.93 
                 −3.82 
                 13q34 
               
               
                 COX7A1 
                 39031_at 
                 1346 
                 −6.61 
                 −97.34 
                 19q13.1 
               
               
                 CREG 
                 35311_at 
                 8804 
                 −1.57 
                 −2.97 
                 1q24 
               
               
                 CRIP1 
                 33232_at 
                 1396 
                 −2.63 
                 −6.18 
                 7q11.23 
               
               
                 CRYAB 
                 32242_at 
                 1410 
                 −3.95 
                 −15.41 
                 11q22.3-q23.1 
               
               
                 CRYAB 
                 32243_g_at 
                 1410 
                 −3.84 
                 −14.32 
                 11q22.3-q23.1 
               
               
                 CSRP1 
                 38700_at 
                 1465 
                 −1.14 
                 −2.21 
                 1q32 
               
               
                 CXCL12 
                 32666_at 
                 6387 
                 −3.40 
                 −10.56 
                 10q11.1 
               
               
                 CYB5R1 
                 35329_at 
                 51706 
                 −1.08 
                 −2.11 
                 1p36.13-q41 
               
               
                 CYBA 
                 35807_at 
                 1535 
                 −1.58 
                 −2.99 
                 16q24 
               
               
                 CYP1B1 
                 40071_at 
                 1545 
                 −2.68 
                 −6.42 
                 2p21 
               
               
                 CYP1B1 
                 859_at 
                 1545 
                 −2.82 
                 −7.08 
                 2p21 
               
               
                 DEGS 
                 33337_at 
                 8560 
                 −0.94 
                 −1.92 
                 1q42.12 
               
               
                 DHX29 
                 39140_at 
                 54505 
                 −0.86 
                 −1.81 
                 5q11.2 
               
               
                 DIA1 
                 36668_at 
                 1727 
                 −0.69 
                 −1.62 
                 22q13.31-qter 
               
               
                 DKFZP564B167 
                 37000_at 
                 25874 
                 −1.19 
                 −2.28 
                 1q24 
               
               
                 DKFZp564I1922 
                 36861_at 
                 25878 
                 −3.50 
                 −11.31 
                 Xp22.33 
               
               
                 DKK1 
                 35977_at 
                 22943 
                 −1.94 
                 −3.84 
                 10q11.2 
               
               
                 DNASE1L1 
                 37214_g_at 
                 1774 
                 −1.55 
                 −2.92 
                 Xq28 
               
               
                 DSP 
                 36133_at 
                 1832 
                 −3.91 
                 −15.04 
                 6p24 
               
               
                 DUSP14 
                 38272_at 
                 11072 
                 −1.05 
                 −2.07 
                 17q12 
               
               
                 ECM1 
                 37600_at 
                 1893 
                 −1.77 
                 −3.40 
                 1q21 
               
               
                 ELN 
                 39098_at 
                 2006 
                 −3.36 
                 −10.23 
                 7q11.23 
               
               
                 EMS1 
                 39861_at 
                 2017 
                 −1.35 
                 −2.56 
                 11q13 
               
               
                 ENG 
                 32562_at 
                 2022 
                 −0.93 
                 −1.90 
                 9q33-q34.1 
               
               
                 EPB41L3 
                 41385_at 
                 23136 
                 −4.68 
                 −25.66 
                 18p11.32 
               
               
                 ETHE1 
                 36170_at 
                 23474 
                 −1.26 
                 −2.40 
                 19q13.32 
               
               
                 FAM20B 
                 35318_at 
                 9917 
                 −1.10 
                 −2.15 
                 1p36.13-q41 
               
               
                 FAM8A1 
                 38318_at 
                 51439 
                 −1.11 
                 −2.16 
                 6p22-p23 
               
               
                 FARP1 
                 32148_at 
                 10160 
                 −2.27 
                 −4.82 
                 13q32.2-q32.3 
               
               
                 FBLN5 
                 39038_at 
                 10516 
                 −1.70 
                 −3.24 
                 14q32.1 
               
               
                 FBXO9 
                 38990_at 
                 26268 
                 −0.80 
                 −1.74 
                 6p12.3-p11.2 
               
               
                 FEZ1 
                 37743_at 
                 9638 
                 −3.53 
                 −11.58 
                 11q24.2 
               
               
                 FEZ2 
                 38651_at 
                 9637 
                 −0.84 
                 −1.79 
                 2p21 
               
               
                 FGF7 
                 1466_s_at 
                 2252 
                 −2.03 
                 −4.09 
                 15q15-q21.1 
               
               
                 FKBP9 
                 38761_s_at 
                 11328 
                 −1.20 
                 −2.30 
                 7p11.1 
               
               
                 FLJ10055 
                 33193_at 
                 55062 
                 −0.84 
                 −1.78 
                 17q24.3 
               
               
                 FLJ10849 
                 35181_at 
                 55752 
                 −1.27 
                 −2.40 
                 4q21.22 
               
               
                 FLJ14675 
                 41207_at 
                 84909 
                 −1.53 
                 −2.89 
                 9q22.33 
               
               
                 FLJ31737 
                 41013_at 
                 196740 
                 −1.20 
                 −2.30 
                 10q11.23 
               
               
                 FNBP1 
                 40468_at 
                 23048 
                 −0.98 
                 −1.97 
                 9q34 
               
               
                 GABARAPL1 
                 35785_at 
                 23710 
                 −2.18 
                 −4.52 
                 12p13.31 
               
               
                 GAF1 
                 33882_at 
                 26056 
                 −0.57 
                 −1.48 
                 2p13-p12 
               
               
                 GOLGA3 
                 34861_at 
                 2802 
                 −0.80 
                 −1.74 
                 12q24.33 
               
               
                 GSN 
                 32612_at 
                 2934 
                 −1.27 
                 −2.42 
                 9q33 
               
               
                 GUK1 
                 905_at 
                 2987 
                 −0.64 
                 −1.56 
                 1q32-q41 
               
               
                 HOXD4 
                 38294_at 
                 3233 
                 −1.04 
                 −2.05 
                 2q31.1 
               
               
                 HPS5 
                 35223_at 
                 11234 
                 −0.81 
                 −1.76 
                 11p14 
               
               
                 HSPA2 
                 36925_at 
                 3306 
                 −2.34 
                 −5.07 
                 14q24.1 
               
               
                 HTATIP2 
                 38824_at 
                 10553 
                 −2.71 
                 −6.55 
                 11p15.1 
               
               
                 IGFBP3 
                 37319_at 
                 3486 
                 −2.27 
                 −4.84 
                 7p13-p12 
               
               
                 IGFBP4 
                 1737_s_at 
                 3487 
                 −2.30 
                 −4.93 
                 17q12-q21.1 
               
               
                 IGFBP4 
                 39781_at 
                 3487 
                 −2.41 
                 −5.33 
                 17q12-q21.1 
               
               
                 IGFBP7 
                 2062_at 
                 3490 
                 −2.20 
                 −4.58 
                 4q12 
               
               
                 IGSF4 
                 35829_at 
                 23705 
                 −2.39 
                 −5.24 
                 11q23.2 
               
               
                 ISLR 
                 38636_at 
                 3671 
                 −2.34 
                 −5.05 
                 15q23-q24 
               
               
                 ITGA1 
                 37484_at 
                 3672 
                 −1.90 
                 −3.72 
                 5q11.2 
               
               
                 ITGA7 
                 36892_at 
                 3679 
                 −2.00 
                 −3.99 
                 12q13 
               
               
                 KCTD12 
                 38972_at 
                 115207 
                 −2.20 
                 −4.59 
                 13q22.1 
               
               
                 KIAA0033 
                 41129_at 
                 23027 
                 −0.92 
                 −1.90 
                 11p15.3 
               
               
                 KIAA0367 
                 33442_at 
                 23273 
                 −2.16 
                 −4.47 
                 9q21.31 
               
               
                 KIAA0711 
                 36453_at 
                 9920 
                 −4.13 
                 −17.53 
                 8p23.3 
               
               
                 KIAA0746 
                 41585_at 
                 23231 
                 −2.82 
                 −7.07 
                 4p15.31 
               
               
                 KIAA1026 
                 39615_at 
                 23254 
                 −1.36 
                 −2.57 
                 1p36.13 
               
               
                 KIAA1128 
                 37617_at 
                 54462 
                 −0.81 
                 −1.75 
                 10q23.2 
               
               
                 KIAA1279 
                 40831_at 
                 26128 
                 −0.82 
                 −1.77 
                 10q22.1 
               
               
                 KIAA1750 
                 32730_at 
                 85453 
                 −3.71 
                 −13.12 
                 8q22.1 
               
               
                 KNS2 
                 39057_at 
                 3831 
                 −0.69 
                 −1.62 
                 14q32.3 
               
               
                 LITAF 
                 37025_at 
                 9516 
                 −1.87 
                 −3.66 
                 16p13.3-p12 
               
               
                 LOC92689 
                 38643_at 
                 92689 
                 −0.61 
                 −1.53 
                 4p14 
               
               
                 LRP10 
                 34409_at 
                 26020 
                 −0.80 
                 −1.74 
                 14q11.2 
               
               
                 LUM 
                 38038_at 
                 4060 
                 −1.59 
                 −3.02 
                 12q21.3-q22 
               
               
                 MAP1A 
                 35917_at 
                 4130 
                 −1.30 
                 −2.46 
                 15q13-qter 
               
               
                 MAP1B 
                 38396_at 
                 4131 
                 −0.65 
                 −1.57 
                 5q13 
               
               
                 MAP1LC3B 
                 39370_at 
                 81631 
                 −0.85 
                 −1.80 
                 16q24.2 
               
               
                 MAP2K3 
                 1622_at 
                 5606 
                 −1.32 
                 −2.50 
                 17q11.2 
               
               
                 ME1 
                 31824_at 
                 4199 
                 −1.38 
                 −2.61 
                 6q12 
               
               
                 MEG3 
                 39026_r_at 
                 55384 
                 −0.98 
                 −1.97 
                 14q32 
               
               
                 MFGE8 
                 34403_at 
                 4240 
                 −3.09 
                 −8.52 
                 15q26 
               
               
                 MGMT 
                 2052_g_at 
                 4255 
                 −1.78 
                 −3.44 
                 10q26 
               
               
                 MRCL3 
                 33447_at 
                 10627 
                 −1.77 
                 −3.42 
                 18p11.31 
               
               
                 M-RIP 
                 38730_at 
                 23164 
                 −0.71 
                 −1.64 
                 17p11.2 
               
               
                 MYL9 
                 39145_at 
                 10398 
                 −1.49 
                 −2.81 
                 20q11.23 
               
               
                 NDN 
                 36073_at 
                 4692 
                 −4.51 
                 −22.82 
                 15q11.2-q12 
               
               
                 NID 
                 35366_at 
                 4811 
                 −1.25 
                 −2.38 
                 1q43 
               
               
                 NME4 
                 39089_at 
                 4833 
                 −0.85 
                 −1.80 
                 16p13.3 
               
               
                 NOTCH3 
                 38750_at 
                 4854 
                 −3.66 
                 −12.62 
                 19p13.2-p13.1 
               
               
                 NQO1 
                 38066_at 
                 1728 
                 −1.71 
                 −3.27 
                 16q22.1 
               
               
                 NT5C2 
                 31794_at 
                 22978 
                 −1.13 
                 −2.18 
                 10q24.33 
               
               
                 OPTN 
                 41742_s_at 
                 10133 
                 −1.34 
                 −2.53 
                 10p14 
               
               
                 OPTN 
                 41743_i_at 
                 10133 
                 −1.19 
                 −2.28 
                 10p14 
               
               
                 OPTN 
                 41744_at 
                 10133 
                 −1.95 
                 −3.85 
                 10p14 
               
               
                 P4HA2 
                 34390_at 
                 8974 
                 −1.17 
                 −2.25 
                 5q31 
               
               
                 PBP 
                 32611_at 
                 5037 
                 −0.80 
                 −1.74 
                 12q24.23 
               
               
                 PCBD 
                 34352_at 
                 5092 
                 −1.38 
                 −2.60 
                 10q22 
               
               
                 PCMT1 
                 37736_at 
                 5110 
                 −0.63 
                 −1.55 
                 6q24-q25 
               
               
                 PEA15 
                 32260_at 
                 8682 
                 −1.34 
                 −2.53 
                 1q21.1 
               
               
                 PHLDA2 
                 31888_s_at 
                 7262 
                 −0.67 
                 −1.59 
                 11p15.5 
               
               
                 PKIG 
                 34376_at 
                 11142 
                 −0.64 
                 −1.56 
                 20q12-q13.1 
               
               
                 PODXL 
                 40434_at 
                 5420 
                 −3.94 
                 −15.39 
                 7q32-q33 
               
               
                 POLR2L 
                 35841_at 
                 5441 
                 −1.18 
                 −2.27 
                 11p15 
               
               
                 POLR2L 
                 503_at 
                 5441 
                 −1.06 
                 −2.08 
                 11p15 
               
               
                 PON2 
                 40504_at 
                 5445 
                 −1.05 
                 −2.07 
                 7q21.3 
               
               
                 PPAP2A 
                 34797_at 
                 8611 
                 −1.57 
                 −2.96 
                 5q11 
               
               
                 PPP1R3C 
                 39366_at 
                 5507 
                 −1.90 
                 −3.74 
                 10q23-q24 
               
               
                 PPP2CB 
                 924_s_at 
                 5516 
                 −0.43 
                 −1.35 
                 8p12-p11.2 
               
               
                 PRDX2 
                 39729_at 
                 7001 
                 −1.86 
                 −3.64 
                 19p13.2 
               
               
                 PRSS11 
                 718_at 
                 5654 
                 −1.33 
                 −2.51 
                 10q26.3 
               
               
                 PRSS11 
                 719_g_at 
                 5654 
                 −1.34 
                 −2.54 
                 10q26.3 
               
               
                 PSMD1 
                 1314_at 
                 5707 
                 −0.65 
                 −1.57 
                 2q37.1 
               
               
                 PTGES 
                 38131_at 
                 9536 
                 −1.79 
                 −3.46 
                 9q34.3 
               
               
                 PTGIS 
                 36533_at 
                 5740 
                 −3.92 
                 −15.10 
                 20q13.11-q13.13 
               
               
                 QKI 
                 39760_at 
                 9444 
                 −1.26 
                 −2.39 
                 6q26-27 
               
               
                 RAB4A 
                 39244_at 
                 5867 
                 −3.30 
                 −9.85 
                 1q42-q43 
               
               
                 RABIF 
                 38264_at 
                 5877 
                 −1.30 
                 −2.46 
                 1q32-q41 
               
               
                 RECK 
                 35234_at 
                 8434 
                 −1.67 
                 −3.18 
                 9p13-p12 
               
               
                 RIT1 
                 38331_at 
                 6016 
                 −1.08 
                 −2.11 
                 1q22 
               
               
                 RRAS 
                 38338_at 
                 6237 
                 −0.58 
                 −1.50 
                 19q13.3-qter 
               
               
                 RRAS2 
                 32827_at 
                 22800 
                 −1.24 
                 −2.36 
                 11p15.2 
               
               
                 S100A10 
                 39338_at 
                 6281 
                 −1.33 
                 −2.51 
                 1q21 
               
               
                 S100A11 
                 38138_at 
                 6282 
                 −1.07 
                 −2.09 
                 1q21 
               
               
                 S100A4 
                 38087_s_at 
                 6275 
                 −2.38 
                 −5.20 
                 1q21 
               
               
                 SDFR1 
                 35747_at 
                 27020 
                 −0.62 
                 −1.54 
                 15q22 
               
               
                 SERPINB2 
                 37185_at 
                 5055 
                 −1.37 
                 −2.58 
                 18q21.3 
               
               
                 SERPINE2 
                 41246_at 
                 5270 
                 −1.28 
                 −2.43 
                 2q33-q35 
               
               
                 SERPING1 
                 39775_at 
                 710 
                 −1.61 
                 −3.06 
                 11q12-q13.1 
               
               
                 SGCD 
                 34991_at 
                 6444 
                 −2.00 
                 −4.00 
                 5q33-q34 
               
               
                 SGCD 
                 34993_at 
                 6444 
                 −2.07 
                 −4.21 
                 5q33-q34 
               
               
                 SGCD 
                 41378_at 
                 6444 
                 −2.59 
                 −6.00 
                 5q33-q34 
               
               
                 SHC1 
                 38118_at 
                 6464 
                 −0.64 
                 −1.56 
                 1q21 
               
               
                 SLC16A4 
                 39260_at 
                 9122 
                 −3.63 
                 −12.42 
                 1p13.2 
               
               
                 SLC20A2 
                 36956_at 
                 6575 
                 −0.98 
                 −1.97 
                 8p12-q21 
               
               
                 SLC4A4 
                 35285_at 
                 8671 
                 −1.25 
                 −2.37 
                 4q21 
               
               
                 SMPD1 
                 32574_at 
                 6609 
                 −1.23 
                 −2.35 
                 11p15.4-p15.1 
               
               
                 SPTAN1 
                 33833_at 
                 6709 
                 −0.72 
                 −1.64 
                 9q33-q34 
               
               
                 STAT1 
                 32859_at 
                 6772 
                 −1.57 
                 −2.98 
                 2q32.2 
               
               
                 STOM 
                 40419_at 
                 2040 
                 −1.88 
                 −3.69 
                 9q34.1 
               
               
                 STX12 
                 38685_at 
                 23673 
                 −1.12 
                 −2.17 
                 1p35-p34.1 
               
               
                 STX6 
                 41663_at 
                 10228 
                 −0.77 
                 −1.71 
                 1q25.1 
               
               
                 STX7 
                 38774_at 
                 8417 
                 −0.84 
                 −1.79 
                 6q23.1 
               
               
                 SULF1 
                 35832_at 
                 23213 
                 −6.48 
                 −89.21 
                 8q13.2 
               
               
                 TAGLN 
                 36931_at 
                 6876 
                 −1.78 
                 −3.43 
                 11q23.2 
               
               
                 TEK 
                 1596_g_at 
                 7010 
                 −4.15 
                 −17.71 
                 9p21 
               
               
                 TM4SF10 
                 37958_at 
                 83604 
                 −1.32 
                 −2.49 
                 Xp11.4 
               
               
                 TM7SF1 
                 32083_at 
                 7107 
                 −2.82 
                 −7.08 
                 1q42-q43 
               
               
                 TNFAIP2 
                 38631_at 
                 7127 
                 −2.45 
                 −5.47 
                 14q32 
               
               
                 TNFRSF6 
                 1441_s_at 
                 355 
                 −2.77 
                 −6.82 
                 10q24.1 
               
               
                 TNFRSF6 
                 37643_at 
                 355 
                 −2.04 
                 −4.11 
                 10q24.1 
               
               
                 TPM2 
                 32313_at 
                 7169 
                 −1.14 
                 −2.21 
                 9p13.2-p13.1 
               
               
                 TPM2 
                 32314_g_at 
                 7169 
                 −0.83 
                 −1.78 
                 9p13.2-p13.1 
               
               
                 TRIM22 
                 36825_at 
                 10346 
                 −4.06 
                 −16.67 
                 11p15 
               
               
                 TRIP-Br2 
                 37312_at 
                 9792 
                 −0.54 
                 −1.46 
                 2p15 
               
               
                 TUBB 
                 39331_at 
                 7280 
                 −1.05 
                 −2.07 
                 6p25 
               
               
                 UROS 
                 36652_at 
                 7390 
                 −0.71 
                 −1.64 
                 10q25.2-q26.3 
               
               
                 VAMP3 
                 35783_at 
                 9341 
                 −0.47 
                 −1.39 
                 1p36.23 
               
               
                 VAMP5 
                 32533_s_at 
                 10791 
                 −1.71 
                 −3.27 
                 2p11.2 
               
               
                 VAT1 
                 40147_at 
                 10493 
                 −0.77 
                 −1.70 
                 17q21 
               
               
                 VEGFC 
                 159_at 
                 7424 
                 −0.99 
                 −1.98 
                 4q34.1-q34.3 
               
               
                 VIL2 
                 40103_at 
                 7430 
                 −1.19 
                 −2.29 
                 6q25.2-q26 
               
               
                 ZFHX1B 
                 35681_r_at 
                 9839 
                 −1.11 
                 −2.16 
                 2q22 
               
               
                 — 
                 1586_at 
                 — 
                 −2.26 
                 −4.78 
                 — 
               
               
                 — 
                 1685_at 
                 — 
                 −1.47 
                 −2.77 
                 — 
               
               
                 — 
                 1686_g_at 
                 — 
                 −2.97 
                 −7.81 
                 — 
               
               
                 — 
                 296_at 
                 — 
                 −1.09 
                 −2.14 
                 — 
               
               
                 — 
                 297_g_at 
                 — 
                 −1.07 
                 −2.10 
                 — 
               
               
                 — 
                 36867_at 
                 — 
                 −2.03 
                 −4.09 
                 — 
               
               
                 — 
                 38351_at 
                 — 
                 −4.44 
                 −21.64 
                 — 
               
               
                 — 
                 39162_at 
                 — 
                 −0.71 
                 −1.64 
                 — 
               
               
                 — 
                 39170_at 
                 — 
                 −1.35 
                 −2.54 
                 — 
               
               
                 — 
                 39750_at 
                 — 
                 −1.46 
                 −2.76 
                 — 
               
               
                 — 
                 631_g_at 
                 — 
                 −0.88 
                 −1.84 
                 — 
               
               
                 — 
                 AFFX-BioC-3_at 
                 — 
                 −2.22 
                 −4.67 
                 — 
               
               
                 — 
                 AFFX-CreX-5_at 
                 — 
                 −3.49 
                 −11.26 
                 — 
               
               
                   
               
             
          
         
       
     
         [0186]     Table 13. Summary of differentially regulated genes/probes after demethylation in immortal MDAH041 and MDAH087 cells.  
                                                                   TABLE 13a                           49 probes downregulated 5-aza-dC vs IM (see Table 1C)                Affymetrix       Average   Fold           Symbol   HGU95Av2 Probe ID   LocusLink   Signal Log 2     Change   Locus                    AARS   36185_at   16   −0.87   −1.83   16q22       AD-017   33126_at   55830   −0.63   −1.55   3p21.31       ARHA   37309_at   387   −0.42   −1.34   3p21.3       ARHGEF6   37543_at   9459   −0.91   −1.88   Xq26       AXL   38433_at   558   −0.75   −1.68   19q13.1       C5orf13   39710_at   9315   −1.56   −2.94   5q22.2       CCNF   35907_at   899   −1.04   −2.06   16p13.3       CDC25B   1347_at   994   −0.98   −1.97   20p13       CDKN2C   36053_at   1031   −1.27   −2.41   1p32       CPN2   34778_at   1370   −0.91   −1.88   8p23-p22       DHCR24   36658_at   1718   −0.68   −1.60   1p33-p31.1       EVI2A   36313_at   2123   −1.20   −2.29   17q11.2       F2R   41700_at   2149   −0.79   −1.73   5q13       GBE1   32643_at   2632   −0.85   −1.80   3p12.3       GCN1L1   36603_at   10985   −0.56   −1.47   12q24.2       HRMT1L1   39348_at   3275   −0.73   −1.65   21q22.3       ICT1   40758_at   3396   −0.87   −1.82   17q25.2       INSIG1   35303_at   3638   −1.15   −2.21   7q36       KIAA0372   40517_at   9652   −0.91   −1.88   5q15       KIAA1049   41268_g_at   22980   −0.87   −1.82   16q24.3       LDB2   36065_at   9079   −1.30   −2.47   4p16       MAP1A   35917_at   4130   −1.12   −2.17   15q13-qter       MRPL23   34358_at   6150   −0.78   −1.72   11p15.5-p15.4       MRPL9   41514_s_at   65005   −0.61   −1.52   1       MYST4   35203_at   23522   −0.73   −1.65   10q22.2       NAG   31896_at   51594   −0.72   −1.64   2p24       NDUFS4   38695_at   4724   −0.70   −1.62   5q11.1       PABPC4   40506_s_at   8761   −0.60   −1.51   1p32-p36       PDGFRA   1731_at   5156   −1.49   −2.81   4q11-q13       PGAM1   41221_at   5223   −0.59   −1.51   10q25.3       POLD2   1470_at   5425   −0.80   −1.74   7p13       PPP2R5C   40784_at   5527   −0.75   −1.68   14q32       RAD23B   1874_at   5887   −0.68   −1.60   9q31.2       RECK   35234_at   8434   −1.93   −3.81   9p13-p12       RECK   35236_g_at   8434   −1.32   −2.49   9p13-p12       RME8   39403_at   23317   −0.82   −1.76   3q22.1       RNASE4   32664_at   6038   −1.44   −2.71   14q11.1       SLC35E2   41243_at   9906   −0.70   −1.63   1p36.33       SMARCD3   32565_at   6604   −1.24   −2.37   7q35-q36       TCFL5   35614_at   10732   −0.98   −1.98   20q13.3-qter       TM4SF10   37958_at   83604   −1.29   −2.45   Xp11.4       TUBGCP3   38353_at   10426   −1.05   −2.06   13q34       WDR18   35983_at   57418   −0.67   −1.59   19p13.3       —   33451_s_at   —   −0.88   −1.84   —       —   34283_at   —   −1.89   −3.70   —       —   34367_at   —   −0.82   −1.77   —       —   38440_s_at   —   −0.73   −1.65   —       —   39750_at   —   −0.94   −1.92   —       —   40567_at   —   −0.84   −1.79   —                  
 
         [0187]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 13b 
               
               
                   
               
               
                   
               
               
                 226 probes upregulated 5-aza-dC vs IM (see Table 1D) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 ADFP 
                 34378_at 
                 123 
                 1.06 
                 2.09 
                 9p22.1 
               
               
                 AKR1B1 
                 36589_at 
                 231 
                 0.66 
                 1.58 
                 7q35 
               
               
                 ALDH1A3 
                 36686_at 
                 220 
                 1.58 
                 2.99 
                 15q26.3 
               
               
                 ATF3 
                 287_at 
                 467 
                 1.40 
                 2.64 
                 1q32.3 
               
               
                 ATF5 
                 39158_at 
                 22809 
                 2.24 
                 4.71 
                 19q13.3 
               
               
                 ATP2B1 
                 37661_at 
                 490 
                 0.76 
                 1.69 
                 12q21-q23 
               
               
                 ATP6V1H 
                 33741_at 
                 51606 
                 0.58 
                 1.50 
                 8p22-q22.3 
               
               
                 BAZ1A 
                 37971_at 
                 11177 
                 0.68 
                 1.61 
                 14q12-q13 
               
               
                 BCAP31 
                 41724_at 
                 10134 
                 0.62 
                 1.54 
                 Xq28 
               
               
                 BHLHB2 
                 40790_at 
                 8553 
                 0.85 
                 1.81 
                 3p26 
               
               
                 BIRC3 
                 1717_s_at 
                 330 
                 2.97 
                 7.81 
                 11q22 
               
               
                 BTG3 
                 37218_at 
                 10950 
                 0.81 
                 1.75 
                 21q21.1-q21.2 
               
               
                 C20orf18 
                 32203_at 
                 10616 
                 0.76 
                 1.70 
                 20p13 
               
               
                 C6orf9 
                 39049_at 
                 63940 
                 1.73 
                 3.32 
                 6p21.3 
               
               
                 C20orf18 
                 32203_at 
                 10616 
                 0.76 
                 1.70 
                 20p13 
               
               
                 C6orf9 
                 39049_at 
                 63940 
                 1.73 
                 3.32 
                 6p21.3 
               
               
                 CASP7 
                 38281_at 
                 840 
                 0.77 
                 1.70 
                 10q25 
               
               
                 CASP8 
                 33774_at 
                 841 
                 0.95 
                 1.94 
                 2q33-q34 
               
               
                 CCL20 
                 40385_at 
                 6364 
                 6.10 
                 68.57 
                 2q33-q37 
               
               
                 CDC34 
                 1274_s_at 
                 997 
                 0.89 
                 1.85 
                 19p13.3 
               
               
                 CLTB 
                 32523_at 
                 1212 
                 0.94 
                 1.92 
                 4q2-q3 
               
               
                 CLU 
                 36780_at 
                 1191 
                 1.15 
                 2.21 
                 8p21-p12 
               
               
                 CRADD 
                 1211_s_at 
                 8738 
                 1.66 
                 3.16 
                 12q21.33-q23.1 
               
               
                 CREG 
                 35311_at 
                 8804 
                 1.29 
                 2.45 
                 1q24 
               
               
                 CREM 
                 32067_at 
                 1390 
                 0.81 
                 1.75 
                 10p11.21 
               
               
                 CTAG1 
                 33636_at 
                 1485 
                 4.23 
                 18.72 
                 Xq28 
               
               
                 CTAG1 
                 33637_g_at 
                 1485 
                 2.65 
                 6.29 
                 Xq28 
               
               
                 CXCL2 
                 37187_at 
                 2920 
                 3.22 
                 9.33 
                 4q21 
               
               
                 CXCL3 
                 34022_at 
                 2921 
                 3.37 
                 10.36 
                 4q21 
               
               
                 CXCL6 
                 35410_at 
                 6372 
                 2.36 
                 5.12 
                 4q21 
               
               
                 CYP1B1 
                 859_at 
                 1545 
                 2.64 
                 6.22 
                 2p21 
               
               
                 CYP1B1 
                 40071_at 
                 1545 
                 2.29 
                 4.88 
                 2p21 
               
               
                 D6S2654E 
                 34957_at 
                 26240 
                 1.60 
                 3.02 
                 6p25-pter 
               
               
                 DAZL 
                 33972_r_at 
                 1618 
                 5.90 
                 59.74 
                 3p24.3 
               
               
                 DAZL 
                 33971_f_at 
                 1618 
                 5.84 
                 57.19 
                 3p24.3 
               
               
                 DDR1 
                 36643_at 
                 780 
                 0.89 
                 1.86 
                 6p21.3 
               
               
                 DKFZP564M182 
                 37161_at 
                 26156 
                 0.91 
                 1.88 
                 16p13.13 
               
               
                 DNAJA1 
                 39118_at 
                 3301 
                 0.87 
                 1.83 
                 9p13-p12 
               
               
                 DNAJA1 
                 276_at 
                 3301 
                 0.78 
                 1.72 
                 9p13-p12 
               
               
                 DOCK4 
                 41620_at 
                 9732 
                 1.11 
                 2.16 
                 7q31.1 
               
               
                 DUSP1 
                 1005_at 
                 1843 
                 1.22 
                 2.33 
                 5q34 
               
               
                 DUSP11 
                 39727_at 
                 8446 
                 0.33 
                 1.26 
                 2p13.1 
               
               
                 DUSP5 
                 529_at 
                 1847 
                 1.65 
                 3.14 
                 10q25 
               
               
                 DUSP6 
                 41193_at 
                 1848 
                 1.82 
                 3.54 
                 12q22-q23 
               
               
                 EEF1A2 
                 35174_i_at 
                 1917 
                 1.62 
                 3.08 
                 20q13.3 
               
               
                 ELL2 
                 40606_at 
                 22936 
                 0.79 
                 1.72 
                 5q15 
               
               
                 ETV6 
                 38491_at 
                 2120 
                 0.79 
                 1.73 
                 12p13 
               
               
                 F2RL1 
                 38247_at 
                 2150 
                 2.89 
                 7.43 
                 5q13 
               
               
                 F3 
                 36543_at 
                 2152 
                 1.08 
                 2.12 
                 1p22-p21 
               
               
                 FGF2 
                 1828_s_at 
                 2247 
                 0.90 
                 1.87 
                 4q26-q27 
               
               
                 FLJ10097 
                 40916_at 
                 56271 
                 0.89 
                 1.85 
                 Xq22.1-q22 
               
               
                 FLJ14675 
                 41207_at 
                 84909 
                 1.20 
                 2.30 
                 9q22.33 
               
               
                 FLJ23027 
                 33915_at 
                 84193 
                 0.49 
                 1.41 
                 14q32.32 
               
               
                 FMR1 
                 37994_at 
                 2332 
                 0.62 
                 1.53 
                 Xq27.3 
               
               
                 FUS 
                 39420_at 
                 2521 
                 1.39 
                 2.62 
                 16p11.2 
               
               
                 G0S2 
                 38326_at 
                 50486 
                 2.61 
                 6.11 
                 1q32.2-q41 
               
               
                 G1P2 
                 1107_s_at 
                 9636 
                 2.26 
                 4.80 
                 1p36.33 
               
               
                 GAGE1 
                 31497_at 
                 2543 
                 1.92 
                 3.79 
                 Xp11.4-p11 
               
               
                 GAGE5 
                 31954_f_at 
                 2577 
                 6.16 
                 71.51 
                 Xp11.4-p11 
               
               
                 GAGE5 
                 33671_f_at 
                 2577 
                 6.15 
                 70.77 
                 Xp11.4-p11 
               
               
                 GAGE5 
                 31960_f_at 
                 2577 
                 5.93 
                 60.89 
                 Xp11.4-p11 
               
               
                 GAGE5 
                 37065_f_at 
                 2577 
                 5.67 
                 50.77 
                 Xp11.4-p11 
               
               
                 GAGE5 
                 33680_f_at 
                 2577 
                 5.64 
                 49.89 
                 Xp11.4-p11 
               
               
                 GAGE5 
                 31498_f_at 
                 2577 
                 5.53 
                 46.10 
                 Xp11.4-p11 
               
               
                 GALE 
                 31598_s_at 
                 2582 
                 1.54 
                 2.91 
                 1p36-p35 
               
               
                 GCH1 
                 37944_at 
                 2643 
                 2.94 
                 7.68 
                 14q22.1-q22.2 
               
               
                 GCLC 
                 31850_at 
                 2729 
                 0.89 
                 1.86 
                 6p12 
               
               
                 GFPT1 
                 32626_at 
                 2673 
                 0.61 
                 1.53 
                 2p13 
               
               
                 GLRX 
                 34311_at 
                 2745 
                 1.43 
                 2.69 
                 5q14 
               
               
                 HCLS1 
                 31820_at 
                 3059 
                 2.80 
                 6.94 
                 3q13 
               
               
                 HDAC9 
                 37483_at 
                 9734 
                 1.52 
                 2.86 
                 7p21.1 
               
               
                 HES1 
                 37393_at 
                 3280 
                 1.91 
                 3.77 
                 3q28-q29 
               
               
                 HIST1H1C 
                 37018_at 
                 3006 
                 1.84 
                 3.58 
                 6p21.3 
               
               
                 HIST1H2AC 
                 34308_at 
                 8334 
                 1.14 
                 2.20 
                 6p21.3 
               
               
                 HIST1H2AG 
                 284_at 
                 8969 
                 2.84 
                 7.17 
                 6p22.1 
               
               
                 HIST1H2AG 
                 285_g_at 
                 8969 
                 2.35 
                 5.09 
                 6p22.1 
               
               
                 HIST1H2BK 
                 32819_at 
                 85236 
                 1.05 
                 2.07 
                 6p21.33 
               
               
                 HIST1H2BL 
                 35576_f_at 
                 8340 
                 1.65 
                 3.13 
                 6p22-p21.3 
               
               
                 HIST1H2BN 
                 36347_f_at 
                 8341 
                 1.75 
                 3.37 
                 6p22-p21.3 
               
               
                 HIST1H3D 
                 34964_at 
                 8351 
                 2.95 
                 7.73 
                 6p21.3 
               
               
                 HIST2H2AA 
                 32609_at 
                 8337 
                 2.76 
                 6.78 
                 1q21.3 
               
               
                 HIST2H2AA 
                 286_at 
                 8337 
                 2.45 
                 5.48 
                 1q21.3 
               
               
                 HIST2H2BE 
                 33352_at 
                 8349 
                 1.84 
                 3.57 
                 1q21-q23 
               
               
                 HLA-C 
                 37383_f_at 
                 3107 
                 1.36 
                 2.56 
                 6p21.3 
               
               
                 HPS5 
                 35223_at 
                 11234 
                 0.80 
                 1.74 
                 11p14 
               
               
                 HSPA2 
                 36925_at 
                 3306 
                 2.30 
                 4.92 
                 14q24.1 
               
               
                 HTATIP2 
                 38824_at 
                 10553 
                 1.68 
                 3.19 
                 11p15.1 
               
               
                 ICAM1 
                 32640_at 
                 3383 
                 4.15 
                 17.76 
                 19p13.3-p13.2 
               
               
                 IER3 
                 1237_at 
                 8870 
                 0.91 
                 1.88 
                 6p21.3 
               
               
                 IFIT4 
                 38584_at 
                 3437 
                 2.79 
                 6.93 
                 10q24 
               
               
                 IFRD1 
                 32901_s_at 
                 3475 
                 0.93 
                 1.91 
                 7q22-q31 
               
               
                 IGFBP7 
                 2062_at 
                 3490 
                 1.16 
                 2.23 
                 4q12 
               
               
                 IL11 
                 35464_at 
                 3589 
                 1.44 
                 2.71 
                 19q13.3-q13.4 
               
               
                 IL13RA2 
                 1016_s_at 
                 3598 
                 3.31 
                 9.94 
                 Xq13.1-q28 
               
               
                 IL1B 
                 39402_at 
                 3553 
                 2.95 
                 7.75 
                 2q14 
               
               
                 IL6 
                 38299_at 
                 3569 
                 3.66 
                 12.63 
                 7p21 
               
               
                 IL8 
                 35372_r_at 
                 3576 
                 3.20 
                 9.20 
                 4q13-q21 
               
               
                 INHBA 
                 40357_at 
                 3624 
                 1.08 
                 2.11 
                 7p15-p13 
               
               
                 INPP5F 
                 36089_at 
                 22876 
                 1.52 
                 2.86 
                 10q26.13 
               
               
                 ISG20 
                 33304_at 
                 3669 
                 2.08 
                 4.24 
                 15q26 
               
               
                 ITGA2 
                 41481_at 
                 3673 
                 2.89 
                 7.43 
                 5q23-q31 
               
               
                 KIAA0247 
                 38393_at 
                 9766 
                 1.03 
                 2.04 
                 14q24.1 
               
               
                 KIAA0690 
                 36520_at 
                 23223 
                 1.38 
                 2.61 
                 10q24.2 
               
               
                 KIAA1111 
                 41399_at 
                 23133 
                 0.82 
                 1.76 
                 Xp11.22 
               
               
                 KIAA1750 
                 32730_at 
                 85453 
                 2.19 
                 4.58 
                 8q22.1 
               
               
                 KRT18 
                 35766_at 
                 3875 
                 3.28 
                 9.69 
                 12q13 
               
               
                 KRT7 
                 41294_at 
                 3855 
                 2.37 
                 5.17 
                 12q12-q13 
               
               
                 KRTHB1 
                 36288_at 
                 3887 
                 5.53 
                 46.25 
                 12q13 
               
               
                 LAMB3 
                 36929_at 
                 3914 
                 1.95 
                 3.86 
                 1q32 
               
               
                 LAMP2 
                 38403_at 
                 3920 
                 0.64 
                 1.56 
                 Xq24 
               
               
                 LGALS3BP 
                 37754_at 
                 3959 
                 2.66 
                 6.31 
                 17q25 
               
               
                 LOC56902 
                 33720_at 
                 56902 
                 0.73 
                 1.66 
                 2p13.3 
               
               
                 LPXN 
                 36062_at 
                 9404 
                 0.88 
                 1.84 
                 11q12.1 
               
               
                 LRIG1 
                 34800_at 
                 26018 
                 1.25 
                 2.38 
                 3p14 
               
               
                 LRRN4 
                 37796_at 
                 4034 
                 1.42 
                 2.67 
                 7q22 
               
               
                 MADH3 
                 1454_at 
                 4088 
                 0.73 
                 1.66 
                 15q21-q22 
               
               
                 MAFF 
                 36711_at 
                 23764 
                 1.93 
                 3.82 
                 22q13.1 
               
               
                 MAGEA2 
                 33518_f_at 
                 4101 
                 3.45 
                 10.95 
                 Xq28 
               
               
                 MAGEA4 
                 36302_f_at 
                 4103 
                 4.68 
                 25.58 
                 Xq28 
               
               
                 MAGEA5 
                 34575_f_at 
                 4104 
                 2.85 
                 7.21 
                 Xq28 
               
               
                 MAGEB2 
                 35097_at 
                 4113 
                 5.26 
                 38.20 
                 Xp21.3 
               
               
                 MAGEC1 
                 34932_at 
                 9947 
                 2.68 
                 6.42 
                 Xq26 
               
               
                 MAP1LC3B 
                 39370_at 
                 81631 
                 1.14 
                 2.20 
                 16q24.2 
               
               
                 MAP2K3 
                 2075_s_at 
                 5606 
                 0.60 
                 1.51 
                 17q11.2 
               
               
                 MAX 
                 1981_s_at 
                 4149 
                 0.93 
                 1.91 
                 14q23 
               
               
                 MICB 
                 35937_at 
                 4277 
                 0.91 
                 1.88 
                 6p21.3 
               
               
                 MLLT2 
                 39037_at 
                 4299 
                 0.67 
                 1.59 
                 4q21 
               
               
                 MMP1 
                 38428_at 
                 4312 
                 2.56 
                 5.90 
                 11q22.3 
               
               
                 MMP13 
                 39632_at 
                 4322 
                 4.05 
                 16.52 
                 11q22.3 
               
               
                 MPG 
                 37768_at 
                 4350 
                 0.75 
                 1.69 
                 16p13.3 
               
               
                 MYD88 
                 38369_at 
                 4615 
                 0.96 
                 1.95 
                 3p22 
               
               
                 NAB1 
                 38692_at 
                 4664 
                 0.91 
                 1.88 
                 2q32.3-q33 
               
               
                 NFKB1 
                 1377_at 
                 4790 
                 0.92 
                 1.89 
                 4q24 
               
               
                 NFKB1 
                 1378_g_at 
                 4790 
                 0.87 
                 1.83 
                 4q24 
               
               
                 NFKB1 
                 38438_at 
                 4790 
                 0.61 
                 1.53 
                 4q24 
               
               
                 NFKB2 
                 544_at 
                 4791 
                 1.44 
                 2.70 
                 10q24 
               
               
                 NFKB2 
                 40362_at 
                 4791 
                 1.29 
                 2.44 
                 10q24 
               
               
                 NFKBIA 
                 1461_at 
                 4792 
                 1.02 
                 2.02 
                 14q13 
               
               
                 NFKBIE 
                 38276_at 
                 4794 
                 1.29 
                 2.44 
                 6p21.1 
               
               
                 NMB 
                 40686_at 
                 4828 
                 1.47 
                 2.78 
                 15q22-qter 
               
               
                 NMI 
                 36472_at 
                 9111 
                 0.92 
                 1.90 
                 2p24.3-q21 
               
               
                 NOL1 
                 1979_s_at 
                 4839 
                 0.76 
                 1.69 
                 12p13 
               
               
                 NXT2 
                 35136_at 
                 55916 
                 1.48 
                 2.79 
                 Xq23 
               
               
                 OPTN 
                 41742_s_at 
                 10133 
                 0.81 
                 1.75 
                 10p14 
               
               
                 PALM2 
                 35985_at 
                 114299 
                 0.61 
                 1.53 
                 9q31-q33 
               
               
                 PBEF 
                 33849_at 
                 10135 
                 1.89 
                 3.70 
                 7q22.2 
               
               
                 PDAP2 
                 38115_at 
                 11334 
                 0.52 
                 1.43 
                 3p21.3 
               
               
                 PISD 
                 38090_at 
                 23761 
                 0.72 
                 1.64 
                 22q12.2 
               
               
                 PLAB 
                 1890_at 
                 9518 
                 1.96 
                 3.88 
                 19p13.1-13 
               
               
                 PLAT 
                 33452_at 
                 5327 
                 1.15 
                 2.23 
                 8p12 
               
               
                 PLAU 
                 37310_at 
                 5328 
                 2.28 
                 4.87 
                 10q24 
               
               
                 PLEKHB2 
                 39525_at 
                 55041 
                 1.14 
                 2.21 
                 2q21.2 
               
               
                 PMAIP1 
                 41048_at 
                 5366 
                 1.89 
                 3.72 
                 18q21.32 
               
               
                 PSCD1 
                 38666_at 
                 9267 
                 1.03 
                 2.04 
                 17q25 
               
               
                 PSMB8 
                 41184_s_at 
                 5696 
                 1.02 
                 2.02 
                 6p21.3 
               
               
                 PTPRR 
                 1658_g_at 
                 5801 
                 0.84 
                 1.79 
                 12q15 
               
               
                 PVRL2 
                 32156_at 
                 5819 
                 0.77 
                 1.71 
                 19q13.2-q13.4 
               
               
                 RAB9A 
                 39628_at 
                 9367 
                 0.52 
                 1.44 
                 Xp22.2 
               
               
                 RBBP4 
                 1318_at 
                 5928 
                 0.83 
                 1.77 
                 1p34.3 
               
               
                 RBP1 
                 38634_at 
                 5947 
                 2.33 
                 5.02 
                 3q23 
               
               
                 RELB 
                 570_at 
                 5971 
                 1.40 
                 2.65 
                 19q13.32 
               
               
                 RNF44 
                 41179_at 
                 22838 
                 1.02 
                 2.02 
                 5q35.3 
               
               
                 RRAD 
                 1776_at 
                 6236 
                 3.81 
                 14.05 
                 16q22 
               
               
                 RRAD 
                 39528_at 
                 6236 
                 3.50 
                 11.31 
                 16q22 
               
               
                 S100A2 
                 2027_at 
                 6273 
                 0.87 
                 1.82 
                 1q21 
               
               
                 SAA1 
                 33272_at 
                 6288 
                 2.74 
                 6.66 
                 11p15.1 
               
               
                 SAT 
                 34304_s_at 
                 6303 
                 1.10 
                 2.14 
                 Xp22.1 
               
               
                 SATB2 
                 41708_at 
                 23314 
                 0.58 
                 1.50 
                 2q33 
               
               
                 SCG2 
                 36924_r_at 
                 7857 
                 1.55 
                 2.93 
                 2q35-q36 
               
               
                 SERPINB2 
                 37185_at 
                 5055 
                 2.85 
                 7.23 
                 18q21.3 
               
               
                 SERPINB8 
                 36312_at 
                 5271 
                 1.23 
                 2.34 
                 18q21.3 
               
               
                 SFN 
                 33323_r_at 
                 2810 
                 1.52 
                 2.87 
                 1p35.3 
               
               
                 SFN 
                 33322_i_at 
                 2810 
                 1.26 
                 2.39 
                 1p35.3 
               
               
                 SLC39A8 
                 40456_at 
                 64116 
                 1.12 
                 2.18 
                 4q22-q24 
               
               
                 SMOX 
                 1649_at 
                 54498 
                 1.08 
                 2.12 
                 20p13 
               
               
                 SMOX 
                 1650_g_at 
                 54498 
                 1.06 
                 2.09 
                 20p13 
               
               
                 SNAPC1 
                 35488_at 
                 6617 
                 1.50 
                 2.82 
                 14q22 
               
               
                 SOD2 
                 34666_at 
                 6648 
                 1.53 
                 2.90 
                 6q25.3 
               
               
                 SQSTM1 
                 40898_at 
                 8878 
                 1.16 
                 2.23 
                 5q35 
               
               
                 SSX2 
                 36409_f_at 
                 6757 
                 5.63 
                 49.57 
                 Xp11.23-p11.22 
               
               
                 SSX3 
                 33655_f_at 
                 10214 
                 2.61 
                 6.11 
                 Xp11.23 
               
               
                 SSX4 
                 35950_at 
                 6759 
                 2.20 
                 4.60 
                 Xp11.23 
               
               
                 STC1 
                 41354_at 
                 6781 
                 1.13 
                 2.19 
                 8p21-p11.2 
               
               
                 TAP1 
                 40153_at 
                 6890 
                 1.48 
                 2.79 
                 6p21.3 
               
               
                 TAX1BP1 
                 35279_at 
                 8887 
                 0.48 
                 1.39 
                 7p15 
               
               
                 TERF2IP 
                 38982_at 
                 54386 
                 1.05 
                 2.07 
                 16q23.1 
               
               
                 TES 
                 32134_at 
                 26136 
                 1.76 
                 3.39 
                 7q31.2 
               
               
                 TFPI2 
                 37388_at 
                 7980 
                 3.99 
                 15.85 
                 7q22 
               
               
                 TGM2 
                 38404_at 
                 7052 
                 3.93 
                 15.24 
                 20q12 
               
               
                 TGM2 
                 231_at 
                 7052 
                 2.66 
                 6.34 
                 20q12 
               
               
                 TKTL1 
                 37120_at 
                 8277 
                 6.35 
                 81.66 
                 Xq28 
               
               
                 TNFAIP2 
                 38631_at 
                 7127 
                 1.90 
                 3.73 
                 14q32 
               
               
                 TNFAIP3 
                 595_at 
                 7128 
                 1.49 
                 2.81 
                 6q23 
               
               
                 TNFRSF10B 
                 34892_at 
                 8795 
                 1.09 
                 2.13 
                 8p22-p21 
               
               
                 TNIP1 
                 38970_s_at 
                 10318 
                 0.84 
                 1.79 
                 5q32-q33.1 
               
               
                 TOP1 
                 1710_s_at 
                 7150 
                 0.83 
                 1.78 
                 20q12-q13. 
               
               
                 TRAF1 
                 849_g_at 
                 7185 
                 1.69 
                 3.22 
                 9q33-q34 
               
               
                 TSNAX 
                 41051_at 
                 7257 
                 0.52 
                 1.44 
                 1q42.1 
               
               
                 TXNRD1 
                 39425_at 
                 7296 
                 0.59 
                 1.50 
                 12q23-q24. 
               
               
                 UPP1 
                 37351_at 
                 7378 
                 1.70 
                 3.25 
                 7p12.3 
               
               
                 WBSCR22 
                 40090_at 
                 114049 
                 1.07 
                 2.10 
               
               
                 ZNF267 
                 34544_at 
                 10308 
                 1.05 
                 2.07 
                 16p11.2 
               
               
                 — 
                 1173_g_at 
                 — 
                 1.19 
                 2.28 
                 — 
               
               
                 — 
                 126_s_at 
                 — 
                 5.19 
                 36.52 
                 — 
               
               
                 — 
                 1369_s_at 
                 — 
                 3.84 
                 14.33 
                 — 
               
               
                 — 
                 1520_s_at 
                 — 
                 3.72 
                 13.16 
                 — 
               
               
                 — 
                 153_f_at 
                 — 
                 1.91 
                 3.75 
                 — 
               
               
                 — 
                 1693_s_at 
                 — 
                 0.83 
                 1.78 
                 — 
               
               
                 — 
                 1842_at 
                 — 
                 1.42 
                 2.67 
                 — 
               
               
                 — 
                 189_s_at 
                 — 
                 1.18 
                 2.27 
                 — 
               
               
                 — 
                 291_s_at 
                 — 
                 3.14 
                 8.79 
                 — 
               
               
                 — 
                 31480_f_at 
                 — 
                 3.22 
                 9.30 
                 — 
               
               
                 — 
                 31522_f_at 
                 — 
                 1.68 
                 3.21 
                 — 
               
               
                 — 
                 31523_f_at 
                 — 
                 1.34 
                 2.53 
                 — 
               
               
                 — 
                 31524_f_at 
                 — 
                 1.35 
                 2.55 
                 — 
               
               
                 — 
                 31528_f_at 
                 — 
                 1.12 
                 2.17 
                 — 
               
               
                 — 
                 31633_g_at 
                 — 
                 0.71 
                 1.64 
                 — 
               
               
                 — 
                 31953_f_at 
                 — 
                 3.11 
                 8.66 
                 — 
               
               
                 — 
                 32426_f_at 
                 — 
                 3.54 
                 11.61 
                 — 
               
               
                 — 
                 32980_f_at 
                 — 
                 1.41 
                 2.65 
                 — 
               
               
                 — 
                 330_s_at 
                 — 
                 1.42 
                 2.68 
                 — 
               
               
                 — 
                 33761_s_at 
                 376645 
                 0.85 
                 1.80 
                 1q21.2 
               
               
                 — 
                 34577_at 
                 — 
                 1.43 
                 2.70 
                 — 
               
               
                 — 
                 35994_at 
                 — 
                 0.77 
                 1.70 
                 — 
               
               
                 — 
                 36757_at 
                 — 
                 2.58 
                 5.98 
                 — 
               
               
                 — 
                 408_at 
                 — 
                 4.31 
                 19.85 
                 — 
               
               
                 — 
                 645_at 
                 — 
                 3.70 
                 12.98 
                 — 
               
               
                 — 
                 669_s_at 
                 — 
                 1.07 
                 2.10 
                 — 
               
               
                   
               
             
          
         
       
     
         [0188]    
       
         
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 14 
               
             
             
               
                   
                   
               
               
                   
                   
               
               
                   
                 Immortal 
                 5-aza-dC 
               
             
          
           
               
                 Term 
                 GO ID 
                 Total 
                 Down 
                 P 
                 P* 
                 Up 
                 P 
                 P* 
                 Down 
                 P 
                 P* 
                 Up 
                 P 
                 P* 
               
               
                   
               
             
          
           
               
                 A. Biological process 
               
             
          
           
               
                 
                   Cell adhesion 
                 
                 0007155 
                 361 
                 13 
                 0.03 
                 1.00 
                 4 
                 0.93 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 7 
                 0.74 
                 1.00 
               
               
                 cell—cell adhesion 
                 0016337 
                 124 
                 3 
                 0.46 
                 1.00 
                 2 
                 0.71 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.95 
                 1.00 
               
               
                 
                   Cell-cell signaling 
                 
                 0007267 
                 365 
                 6 
                 0.75 
                 1.00 
                 4 
                 0.94 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 11 
                 0.23 
                 1.00 
               
               
                 
                   Signal transduction 
                 
                 0007165 
                 1321 
                 25 
                 0.67 
                 1.00 
                 25 
                 0.63 
                 1.00 
                 6 
                 0.56 
                 1.00 
                 33 
                 0.35 
                 1.00 
               
               
                 cell surface receptor linked 
                 0007166 
                 588 
                 11 
                 0.65 
                 1.00 
                 17 
                 0.07 
                 1.00 
                 2 
                 0.76 
                 1.00 
                 14 
                 0.51 
                 1.00 
               
               
                 signal transduction 
               
               
                 intracellular signaling cascade 
                 0007242 
                 444 
                 7 
                 0.80 
                 1.00 
                 8 
                 0.66 
                 1.00 
                 2 
                 0.60 
                 1.00 
                 10 
                 0.59 
                 1.00 
               
               
                 
                   Cell death 
                 
                 0008219 
                 272 
                 6 
                 0.47 
                 1.00 
                 9 
                 0.09 
                 1.00 
                 2 
                 0.35 
                 1.00 
                 15 
                 0.00 
                 1.00 
               
               
                 anti-apoptosis 
                 0006916 
                 59 
                 3 
                 0.11 
                 1.00 
                 4 
                 0.03 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 5 
                 0.01 
                 1.00 
               
               
                 apoptotic program 
                 0008632 
                 26 
                 1 
                 0.41 
                 1.00 
                 1 
                 0.41 
                 1.00 
                 1 
                 0.11 
                 1.00 
                 3 
                 0.02 
                 1.00 
               
               
                 induction of apoptosis 
                 0006917 
                 83 
                 2 
                 0.50 
                 1.00 
                 3 
                 0.23 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 3 
                 0.30 
                 1.00 
               
               
                 induction of programmed cell 
                 0012502 
                 83 
                 2 
                 0.50 
                 1.00 
                 3 
                 0.23 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 3 
                 0.30 
                 1.00 
               
               
                 death 
               
               
                 
                   Cell differentiation 
                 
                 0030154 
                 106 
                 2 
                 0.63 
                 1.00 
                 2 
                 0.62 
                 1.00 
                 1 
                 0.38 
                 1.00 
                 5 
                 0.10 
                 1.00 
               
               
                 lymphocyte differentiation 
                 0030098 
                 22 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 3 
                 0.01 
                 1.00 
               
               
                 
                   Cell organization and 
                 
                 0016043 
                 282 
                 5 
                 0.68 
                 1.00 
                 10 
                 0.05 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 12 
                 0.03 
                 1.00 
               
               
                 
                   biogenesis 
                 
               
               
                 cytoplasm organization and 
                 0007028 
                 166 
                 5 
                 0.24 
                 1.00 
                 4 
                 0.42 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.98 
                 1.00 
               
               
                 biogenesis 
               
               
                 nuclear organization and 
                 0006997 
                 109 
                 0 
                 1.00 
                 1.00 
                 6 
                 0.02 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 11 
                 0.00 
                 1.00 
               
               
                 biogenesis 
               
               
                 
                   Cell proliferation 
                 
                 0008283 
                 667 
                 18 
                 0.12 
                 1.00 
                 37 
                 0.00 
                 0.00 
                 11 
                 0.00 
                 1.00 
                 18 
                 0.29 
                 1.00 
               
               
                 cell cycle 
                 0007049 
                 442 
                 12 
                 0.18 
                 1.00 
                 32 
                 0.00 
                 0.00 
                 7 
                 0.00 
                 1.00 
                 14 
                 0.15 
                 1.00 
               
               
                 regulation of cell proliferation 
                 0042127 
                 175 
                 8 
                 0.02 
                 1.00 
                 7 
                 0.06 
                 1.00 
                 3 
                 0.04 
                 1.00 
                 8 
                 0.05 
                 1.00 
               
               
                 cytokinesis 
                 0000910 
                 64 
                 1 
                 0.73 
                 1.00 
                 5 
                 0.01 
                 0.57 
                 2 
                 0.03 
                 1.00 
                 0 
                 1.00 
                 1.00 
               
               
                 
                   Transport 
                 
                 0006810 
                 873 
                 18 
                 0.49 
                 1.00 
                 13 
                 0.90 
                 1.00 
                 1 
                 0.99 
                 1.00 
                 12 
                 0.99 
                 1.00 
               
               
                 intracellular transport 
                 0046907 
                 258 
                 6 
                 0.42 
                 1.00 
                 3 
                 0.89 
                 1.00 
                 1 
                 0.69 
                 1.00 
                 6 
                 0.56 
                 1.00 
               
               
                 ion transport 
                 0006811 
                 320 
                 7 
                 0.47 
                 1.00 
                 7 
                 0.45 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 2 
                 1.00 
                 1.00 
               
               
                 protein transport 
                 0015031 
                 223 
                 5 
                 0.47 
                 1.00 
                 2 
                 0.94 
                 1.00 
                 1 
                 0.64 
                 1.00 
                 7 
                 0.26 
                 1.00 
               
               
                 vesicle-mediated transport 
                 0016192 
                 167 
                 7 
                 0.05 
                 1.00 
                 3 
                 0.65 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 2 
                 0.91 
                 1.00 
               
               
                 
                   Cell motility 
                 
                 0006928 
                 222 
                 10 
                 0.01 
                 1.00 
                 3 
                 0.82 
                 1.00 
                 1 
                 0.64 
                 1.00 
                 1 
                 1.00 
                 1.00 
               
               
                 cell migration 
                 0016477 
                 39 
                 5 
                 0.00 
                 1.00 
                 1 
                 0.54 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.60 
                 1.00 
               
               
                 
                   Metabolism 
                 
                 0008152 
                 3192 
                 44 
                 1.00 
                 1.00 
                 78 
                 0.00 
                 1.00 
                 18 
                 0.11 
                 1.00 
                 78 
                 0.29 
                 1.00 
               
               
                 alcohol metabolism 
                 0006066 
                 151 
                 2 
                 0.81 
                 1.00 
                 4 
                 0.35 
                 1.00 
                 2 
                 0.15 
                 1.00 
                 4 
                 0.47 
                 1.00 
               
               
                 amine metabolism 
                 0009308 
                 175 
                 2 
                 0.87 
                 1.00 
                 6 
                 0.13 
                 1.00 
                 1 
                 0.55 
                 1.00 
                 3 
                 0.78 
                 1.00 
               
               
                 amino acid and derivative 
                 0006519 
                 149 
                 2 
                 0.81 
                 1.00 
                 4 
                 0.34 
                 1.00 
                 1 
                 0.49 
                 1.00 
                 3 
                 0.68 
                 1.00 
               
               
                 metabolism 
               
               
                 biosynthesis 
                 0009058 
                 525 
                 7 
                 0.91 
                 1.00 
                 9 
                 0.72 
                 1.00 
                 6 
                 0.03 
                 1.00 
                 7 
                 0.97 
                 1.00 
               
               
                 carbohydrate metabolism 
                 0005975 
                 228 
                 3 
                 0.84 
                 1.00 
                 6 
                 0.30 
                 1.00 
                 2 
                 0.27 
                 1.00 
                 4 
                 0.78 
                 1.00 
               
               
                 catabolism 
                 0009056 
                 450 
                 4 
                 0.98 
                 1.00 
                 8 
                 0.67 
                 1.00 
                 2 
                 0.61 
                 1.00 
                 9 
                 0.73 
                 1.00 
               
               
                 electron transport 
                 0006118 
                 150 
                 6 
                 0.08 
                 1.00 
                 4 
                 0.34 
                 1.00 
                 2 
                 0.14 
                 1.00 
                 4 
                 0.46 
                 1.00 
               
               
                 energy pathways 
                 0006091 
                 158 
                 3 
                 0.62 
                 1.00 
                 4 
                 0.38 
                 1.00 
                 2 
                 0.16 
                 1.00 
                 1 
                 0.98 
                 1.00 
               
               
                 lipid metabolism 
                 0006629 
                 299 
                 6 
                 0.56 
                 1.00 
                 5 
                 0.71 
                 1.00 
                 1 
                 0.75 
                 1.00 
                 4 
                 0.92 
                 1.00 
               
               
                 nucleobase, nucleoside, 
                 0006139 
                 1351 
                 12 
                 1.00 
                 1.00 
                 41 
                 0.00 
                 1.00 
                 6 
                 0.59 
                 1.00 
                 41 
                 0.03 
                 1.00 
               
               
                 nucleotide and nucleic acid 
               
               
                 metabolism 
               
               
                 organic acid metabolism 
                 0006082 
                 226 
                 5 
                 0.48 
                 1.00 
                 5 
                 0.46 
                 1.00 
                 1 
                 0.64 
                 1.00 
                 3 
                 0.90 
                 1.00 
               
               
                 phosphorus metabolism 
                 0006793 
                 423 
                 6 
                 0.86 
                 1.00 
                 17 
                 0.00 
                 1.00 
                 5 
                 0.04 
                 1.00 
                 9 
                 0.66 
                 1.00 
               
               
                 protein metabolism 
                 0019538 
                 1170 
                 16 
                 0.97 
                 1.00 
                 25 
                 0.37 
                 1.00 
                 9 
                 0.06 
                 1.00 
                 27 
                 0.55 
                 1.00 
               
               
                 regulation of metabolism 
                 0019222 
                 32 
                 2 
                 0.13 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.53 
                 1.00 
               
               
                 
                   Response to endogenous 
                 
                 0009719 
                 134 
                 1 
                 0.94 
                 1.00 
                 4 
                 0.27 
                 1.00 
                 1 
                 0.46 
                 1.00 
                 2 
                 0.82 
                 1.00 
               
               
                 
                   stimulus 
                 
               
               
                 response to DNA damage 
                 0006974 
                 133 
                 1 
                 0.93 
                 1.00 
                 4 
                 0.27 
                 1.00 
                 1 
                 0.45 
                 1.00 
                 2 
                 0.82 
                 1.00 
               
               
                 stimulus 
               
               
                 response to external stimulus 
                 0009605 
                 813 
                 18 
                 0.37 
                 1.00 
                 6 
                 1.00 
                 1.00 
                 2 
                 0.90 
                 1.00 
                 30 
                 0.01 
                 1.00 
               
               
                 response to abiotic stimulus 
                 0009628 
                 224 
                 2 
                 0.94 
                 1.90 
                 4 
                 0.65 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 8 
                 0.15 
                 1.00 
               
               
                 response to biotic stimulus 
                 0009607 
                 552 
                 13 
                 0.32 
                 1.00 
                 1 
                 1.00 
                 1.00 
                 1 
                 0.93 
                 1.00 
                 26 
                 0.00 
                 1.00 
               
               
                 
                   Response to extracellular 
                 
                 0009991 
                 10 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.21 
                 1.00 
               
               
                 
                   stimulus 
                 
               
               
                 response to wounding 
                 0009611 
                 173 
                 3 
                 0.68 
                 1.00 
                 1 
                 0.97 
                 1.00 
                 1 
                 0.55 
                 1.00 
                 14 
                 0.00 
                 0.00 
               
               
                 taxis 
                 0042330 
                 77 
                 1 
                 0.79 
                 1.00 
                 1 
                 0.79 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 7 
                 0.00 
                 1.00 
               
               
                 
                   Response to stress 
                 
                 0006950 
                 501 
                 12 
                 0.31 
                 1.00 
                 7 
                 0.88 
                 1.00 
                 3 
                 0.39 
                 1.00 
                 21 
                 0.01 
                 1.00 
               
               
                 response to DNA damage 
                 0006974 
                 133 
                 1 
                 0.93 
                 1.00 
                 4 
                 0.27 
                 1.00 
                 1 
                 0.45 
                 1.00 
                 2 
                 0.82 
                 1.00 
               
               
                 stimulus 
               
               
                 response to 
                 0009613 
                 287 
                 8 
                 0.22 
                 1.00 
                 1 
                 1.00 
                 1.00 
                 1 
                 0.73 
                 1.00 
                 17 
                 0.00 
                 1.00 
               
               
                 pest/pathogen/parasite 
               
               
                 
                   Bbiological 
                   — 
                   process unknown 
                 
                 0000004 
                 232 
                 3 
                 0.85 
                 1.00 
                 4 
                 0.68 
                 1.00 
                 2 
                 0.28 
                 1.00 
                 6 
                 0.46 
                 1.00 
               
             
          
           
               
                 B. Cellular component 
               
             
          
           
               
                 
                   Extracellular 
                 
                 0005576 
                 586 
                 22 
                 0.00 
                 1.00 
                 4 
                 1.00 
                 1.00 
                 1 
                 0.94 
                 1.00 
                 23 
                 0.01 
                 1.00 
               
               
                 extracellular matrix 
                 0005578 
                 181 
                 11 
                 0.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 4 
                 0.61 
                 1.00 
               
               
                 extracellular space 
                 0005615 
                 228 
                 7 
                 0.17 
                 1.00 
                 1 
                 0.99 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 13 
                 0.00 
                 1.00 
               
               
                 
                   Membrane 
                 
                 0016020 
                 1976 
                 48 
                 0.07 
                 1.00 
                 32 
                 0.93 
                 1.00 
                 6 
                 0.92 
                 1.00 
                 24 
                 1.00 
                 1.00 
               
               
                 endomembrane system 
                 0012505 
                 112 
                 2 
                 0.66 
                 1.00 
                 4 
                 0.18 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.93 
                 1.00 
               
               
                 integral to membrane 
                 0016021 
                 1549 
                 26 
                 0.88 
                 1.00 
                 21 
                 0.99 
                 1.00 
                 5 
                 0.86 
                 1.00 
                 18 
                 1.00 
                 1.00 
               
               
                 mitochondrial membrane 
                 0005740 
                 75 
                 0 
                 1.00 
                 1.00 
                 4 
                 0.06 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
               
               
                 plasma membrane 
                 0005886 
                 1041 
                 27 
                 0.09 
                 1.00 
                 10 
                 1.00 
                 1.00 
                 3 
                 0.87 
                 1.00 
                 13 
                 1.00 
                 1.00 
               
               
                 
                   Cytoplasm 
                 
                 0005737 
                 1705 
                 51 
                 0.00 
                 1.00 
                 39 
                 0.16 
                 1.00 
                 9 
                 0.35 
                 1.00 
                 30 
                 0.98 
                 1.00 
               
               
                 
                   Cytoplasmic vesicle 
                 
                 0016023 
                 59 
                 2 
                 0.33 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 2 
                 0.40 
                 1.00 
               
               
                 
                   Cytoskeleton 
                 
                 0005856 
                 334 
                 19 
                 0.00 
                 0.00 
                 11 
                 0.07 
                 1.00 
                 1 
                 0.79 
                 1.00 
                 4 
                 0.96 
                 1.00 
               
               
                 actin cytoskeleton 
                 0015629 
                 120 
                 8 
                 0.00 
                 1.00 
                 2 
                 0.69 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
               
               
                 microtubule cytoskeleton 
                 0015630 
                 78 
                 3 
                 0.21 
                 1.00 
                 6 
                 0.00 
                 1.00 
                 1 
                 0.30 
                 1.00 
                 0 
                 1.00 
                 1.00 
               
               
                 
                   Cytosol 
                 
                 0005829 
                 176 
                 6 
                 0.14 
                 1.00 
                 2 
                 0.87 
                 1.00 
                 1 
                 0.55 
                 1.00 
                 2 
                 0.92 
                 1.00 
               
               
                 
                   Endoplasmic reticulum 
                 
                 0005783 
                 244 
                 8 
                 0.12 
                 1.00 
                 1 
                 0.99 
                 1.00 
                 1 
                 0.67 
                 1.00 
                 4 
                 0.83 
                 1.00 
               
               
                 
                   Golgi apparatus 
                 
                 0005794 
                 183 
                 3 
                 0.72 
                 1.00 
                 1 
                 0.98 
                 1.00 
                 2 
                 0.20 
                 1.00 
                 3 
                 0.80 
                 1.00 
               
               
                 
                   Mitochondrion 
                 
                 0005739 
                 315 
                 4 
                 0.89 
                 1.00 
                 14 
                 0.00 
                 1.00 
                 2 
                 0.42 
                 1.00 
                 3 
                 0.98 
                 1.00 
               
               
                 
                   Ribosome 
                 
                 0005840 
                 61 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.71 
                 1.00 
                 1 
                 0.24 
                 1.00 
                 1 
                 0.76 
                 1.00 
               
               
                 
                   Nucleus 
                 
                 0005634 
                 1483 
                 15 
                 1.00 
                 1.00 
                 51 
                 0.00 
                 0.00 
                 7 
                 0.51 
                 1.00 
                 44 
                 0.04 
                 1.00 
               
               
                 nuclear membrane 
                 0005635 
                 50 
                 2 
                 0.27 
                 1.00 
                 3 
                 0.08 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
               
               
                 nucleolus 
                 0005730 
                 39 
                 0 
                 1.00 
                 1.00 
                 5 
                 0.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.60 
                 1.00 
               
               
                 nucleoplasm 
                 0005654 
                 91 
                 1 
                 0.84 
                 1.00 
                 3 
                 0.27 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 5 
                 0.06 
                 1.00 
               
               
                 
                   Chromosome 
                 
                 0005694 
                 108 
                 0 
                 1.00 
                 1.00 
                 8 
                 0.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 10 
                 0.00 
                 0.00 
               
               
                 
                   Cellular 
                   — 
                   component unknown 
                 
                 0008372 
                 266 
                 3 
                 0.91 
                 1.00 
                 2 
                 0.97 
                 1.00 
                 2 
                 0.34 
                 1.00 
                 8 
                 0.28 
                 1.00 
               
             
          
           
               
                 C. Molecular function 
               
             
          
           
               
                 
                   Apoptosis regulator activity 
                 
                 0016329 
                 44 
                 2 
                 0.22 
                 1.00 
                 1 
                 0.59 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 4 
                 0.02 
                 1.00 
               
               
                 
                   Binding 
                 
                 0005488 
                 3368 
                 58 
                 0.97 
                 1.00 
                 72 
                 0.18 
                 1.00 
                 9 
                 0.99 
                 1.00 
                 83 
                 0.24 
                 1.00 
               
               
                 lipid binding 
                 0008289 
                 94 
                 2 
                 0.57 
                 1.00 
                 5 
                 0.04 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.89 
                 1.00 
               
               
                 metal ion binding 
                 0046872 
                 631 
                 22 
                 0.01 
                 1.00 
                 6 
                 0.99 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 12 
                 0.81 
                 1.00 
               
               
                 nucleic acid binding 
                 0003676 
                 1232 
                 6 
                 1.00 
                 1.00 
                 34 
                 0.02 
                 1.00 
                 6 
                 0.49 
                 1.00 
                 36 
                 0.08 
                 1.00 
               
               
                 nucleotide binding 
                 0000166 
                 763 
                 11 
                 0.91 
                 1.00 
                 26 
                 0.00 
                 1.00 
                 3 
                 0.68 
                 1.00 
                 10 
                 0.99 
                 1.00 
               
               
                 protein binding 
                 0005515 
                 814 
                 20 
                 0.20 
                 1.00 
                 18 
                 0.34 
                 1.00 
                 1 
                 0.98 
                 1.00 
                 26 
                 0.05 
                 1.00 
               
               
                 receptor binding 
                 0005102 
                 301 
                 7 
                 0.40 
                 1.00 
                 2 
                 0.98 
                 1.00 
                 1 
                 0.75 
                 1.00 
                 14 
                 0.01 
                 1.00 
               
               
                 
                   Catalytic activity 
                 
                 0003824 
                 2129 
                 37 
                 0.89 
                 1.00 
                 55 
                 0.01 
                 1.00 
                 12 
                 0.21 
                 1.00 
                 43 
                 0.90 
                 1.00 
               
               
                 hydrolase activity 
                 0016787 
                 917 
                 14 
                 0.90 
                 1.00 
                 20 
                 0.35 
                 1.00 
                 4 
                 0.61 
                 1.00 
                 25 
                 0.22 
                 1.00 
               
               
                 kinase activity 
                 0016301 
                 409 
                 5 
                 0.92 
                 1.00 
                 16 
                 0.01 
                 1.00 
                 3 
                 0.28 
                 1.00 
                 2 
                 1.00 
                 1.00 
               
               
                 oxidoreductase activity 
                 0016491 
                 320 
                 10 
                 0.11 
                 1.00 
                 10 
                 0.10 
                 1.00 
                 2 
                 0.42 
                 1.00 
                 6 
                 0.76 
                 1.00 
               
               
                 transferase activity 
                 0016740 
                 706 
                 8 
                 0.98 
                 1.00 
                 22 
                 0.02 
                 1.00 
                 5 
                 0.20 
                 1.00 
                 8 
                 1.00 
                 1.00 
               
               
                 
                   Cell adhesion molecule activity 
                 
                 0005194 
                 209 
                 9 
                 0.02 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 3 
                 0.87 
                 1.00 
               
               
                 
                   Defense/immunity protein 
                 
                 0003793 
                 36 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.57 
                 1.00 
               
               
                 
                   activity 
                 
               
               
                 
                   Enzyme regulator activity 
                 
                 0030234 
                 306 
                 8 
                 0.27 
                 1.00 
                 5 
                 0.73 
                 1.00 
                 5 
                 0.01 
                 1.00 
                 6 
                 0.72 
                 1.00 
               
               
                 enzyme inhibitor activity 
                 0004857 
                 132 
                 7 
                 0.02 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 2 
                 0.12 
                 1.00 
                 4 
                 0.37 
                 1.00 
               
               
                 
                   Motor activity 
                 
                 0003774 
                 61 
                 1 
                 0.71 
                 1.00 
                 1 
                 0.71 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
               
               
                 
                   Obsolete molecular function 
                 
                 0008369 
                 419 
                 12 
                 0.13 
                 1.00 
                 4 
                 0.97 
                 1.00 
                 1 
                 0.86 
                 1.00 
                 12 
                 0.27 
                 1.00 
               
               
                 
                   Signal transducer activity 
                 
                 0004871 
                 1157 
                 16 
                 0.97 
                 1.00 
                 17 
                 0.94 
                 1.00 
                 5 
                 0.61 
                 1.00 
                 23 
                 0.83 
                 1.00 
               
               
                 receptor activity 
                 0004872 
                 678 
                 7 
                 0.99 
                 1.00 
                 9 
                 0.93 
                 1.00 
                 4 
                 0.36 
                 1.00 
                 11 
                 0.93 
                 1.00 
               
               
                 receptor binding 
                 0005102 
                 301 
                 7 
                 0.40 
                 1.00 
                 2 
                 0.98 
                 1.00 
                 1 
                 0.75 
                 1.00 
                 14 
                 0.01 
                 1.00 
               
               
                 receptor signaling protein 
                 0005057 
                 127 
                 2 
                 0.73 
                 1.00 
                 4 
                 0.24 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
               
               
                 activity 
               
               
                 
                   Structural molecule activity 
                 
                 0005198 
                 333 
                 17 
                 0.00 
                 1.00 
                 5 
                 0.80 
                 1.00 
                 3 
                 0.19 
                 1.00 
                 4 
                 0:96 
                 1.00 
               
               
                 extracellular matrix structural 
                 0005201 
                 60 
                 5 
                 0.01 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.76 
                 1.00 
               
               
                 constituent 
               
               
                 structural constituent of 
                 0005200 
                 62 
                 6 
                 0.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.77 
                 1.00 
               
               
                 cytoskeleton 
               
               
                 
                   Transcription regulator activity 
                 
                 0030528 
                 630 
                 4 
                 1.00 
                 1.00 
                 16 
                 0.18 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 18 
                 0.21 
                 1.00 
               
               
                 transcription cofactor activity 
                 0003712 
                 153 
                 0 
                 1.00 
                 1.00 
                 5 
                 0.19 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 7 
                 0.07 
                 1.00 
               
               
                 transcription factor activity 
                 0003700 
                 472 
                 3 
                 1.00 
                 1.00 
                 11 
                 0.33 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 12 
                 0.42 
                 1.00 
               
               
                 
                   Translation regulator activity 
                 
                 0045182 
                 43 
                 0 
                 1.00 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 1 
                 0.18 
                 1.00 
                 1 
                 0.64 
                 1.00 
               
               
                 
                   Transporter activity 
                 
                 0005215 
                 822 
                 20 
                 0.21 
                 1.00 
                 16 
                 0.57 
                 1.00 
                 1 
                 0.98 
                 1.00 
                 14 
                 0.92 
                 1.00 
               
               
                 carrier activity 
                 0005386 
                 239 
                 7 
                 0.20 
                 1.00 
                 4 
                 0.70 
                 1.00 
                 1 
                 0.67 
                 1.00 
                 3 
                 0.92 
                 1.00 
               
               
                 electron transporter activity 
                 0005489 
                 138 
                 8 
                 0.01 
                 1.00 
                 6 
                 0.06 
                 1.00 
                 1 
                 0.47 
                 1.00 
                 5 
                 0.22 
                 1.00 
               
               
                 ion transporter activity 
                 0015075 
                 170 
                 6 
                 0.13 
                 1.00 
                 5 
                 0.25 
                 1.00 
                 1 
                 0.54 
                 1.00 
                 2 
                 0.91 
                 1.00 
               
               
                 protein transporter activity 
                 0008565 
                 143 
                 3 
                 0.55 
                 1.00 
                 2 
                 0.78 
                 1.00 
                 0 
                 1.00 
                 1.00 
                 4 
                 0.43 
                 1.00 
               
               
                   Molecular _ function unknown   
                 0005554 
                 257 
                 2 
                 0.97 
                 1.00 
                 4 
                 0.76 
                 1.00 
                 1 
                 0.69 
                 1.00 
                 6 
                 0.56 
                 1.00 
               
               
                   
               
               
                   Table 14    
               
               
                   GoMiner analysis of dysregulated genes in four immortal LFS cell lines. The genes, which were dysregulated (up- or down-regulated) during immortalization and 5aza-CdR treatment in MDAH041, MDAH087-N, MDAH087-1, MDAH087-10 cells were analyzed by GoMiner according to biological process (A), cellular component (B) and molecular function (C).    
               
               
                   The GO categories plotted in  FIG. 2  are denoted by bold font.    
               
               
                   Total: total gene number associated with the GO term on Affymetrix HGU95av2 GeneChip ®;    
               
               
                   Immortal: genes dysregulated during immortalization;    
               
               
                   5aza: genes dysregulated during 5aza-CdR treatment of immortal cells.    
               
               
                   P*: corrected p-value (p &lt; 0.005 were rounded to 0.00; p* &gt; 1 were reduced to 1.00)    
               
             
          
         
       
     
         [0189]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                   
               
               
                 35 Genes upregulated during immortalization in gene 
               
               
                 ontology cell proliferation category 0008283 
               
             
          
           
               
                   
                 Affymetrix HGU95Av2 
                   
                 Average 
                 Fold 
               
               
                 Symbol 
                 Probe ID 
                 LocusLink 
                 Signal Log 2   
                 Change 
               
               
                   
               
             
          
           
               
                 BAX 
                 2065_s_at 
                 581 
                 0.79 
                 1.73 
               
               
                 BCAT1 
                 38201_at 
                 586 
                 2.06 
                 4.18 
               
               
                 BCR 
                 1635_at 
                 613 
                 0.92 
                 1.89 
               
               
                 BIN1 
                 32238_at 
                 274 
                 1.04 
                 2.05 
               
               
                 CDC20 
                 38414_at 
                 991 
                 1.06 
                 2.09 
               
               
                 CDC25B 
                 1347_at 
                 994 
                 0.64 
                 1.56 
               
               
                 CDKN3 
                 1599_at 
                 1033 
                 1.05 
                 2.07 
               
               
                 CENPB 
                 37931_at 
                 1059 
                 0.98 
                 1.97 
               
               
                 CHC1 
                 37927_at 
                 1104 
                 1.04 
                 2.05 
               
               
                 CKS2 
                 40690_at 
                 1164 
                 1.02 
                 2.02 
               
               
                 E2F4 
                 1703_g_at 
                 1874 
                 1.24 
                 2.36 
               
               
                 EGFR 
                 1537_at 
                 1956 
                 2.97 
                 7.86 
               
               
                 EMP1 
                 1321_s_at 
                 2012 
                 2.58 
                 5.96 
               
               
                 ERF 
                 38996_at 
                 2077 
                 1.12 
                 2.17 
               
               
                 IGF1R 
                 34718_at 
                 3480 
                 0.86 
                 1.81 
               
               
                 ILF3 
                 40845_at 
                 3609 
                 1.33 
                 2.51 
               
               
                 ILF3 
                 40846_g_at 
                 3609 
                 0.91 
                 1.88 
               
               
                 MAPK1 
                 976_s_at 
                 5594 
                 0.82 
                 1.77 
               
               
                 MET 
                 35684_at 
                 4233 
                 1.15 
                 2.23 
               
               
                 MYC 
                 1827_s_at 
                 4609 
                 1.14 
                 2.20 
               
               
                 MYC 
                 1973_s_at 
                 4609 
                 1.63 
                 3.10 
               
               
                 MYC 
                 37724_at 
                 4609 
                 1.31 
                 2.48 
               
               
                 NOL1 
                 1979_s_at 
                 4839 
                 1.31 
                 2.49 
               
               
                 NRAS 
                 1539_at 
                 4893 
                 1.38 
                 2.60 
               
               
                 NRP1 
                 36836_at 
                 8829 
                 1.54 
                 2.91 
               
               
                 PES1 
                 41869_at 
                 23481 
                 0.87 
                 1.82 
               
               
                 PLK 
                 37228_at 
                 5347 
                 1.08 
                 2.12 
               
               
                 PPP5C 
                 392_g_at 
                 5536 
                 1.72 
                 3.30 
               
               
                 PPP5C 
                 391_at 
                 5536 
                 1.18 
                 2.26 
               
               
                 PRIM1 
                 798_at 
                 5557 
                 1.75 
                 3.35 
               
               
                 PRIM2A 
                 122_at 
                 5558 
                 0.90 
                 1.86 
               
               
                 RAD21 
                 38114_at 
                 5885 
                 0.77 
                 1.70 
               
               
                 RAF1 
                 1917_at 
                 5894 
                 0.85 
                 1.80 
               
               
                 RFC5 
                 653_at 
                 5985 
                 1.42 
                 2.68 
               
               
                 SMC2L1 
                 37502_at 
                 10592 
                 1.18 
                 2.27 
               
               
                 TOP1 
                 1710_s_at 
                 7150 
                 1.80 
                 3.48 
               
               
                 TOP2B 
                 1581_s_at 
                 7155 
                 1.42 
                 2.68 
               
               
                 UBE2C 
                 1651_at 
                 11065 
                 1.08 
                 2.11 
               
               
                 VEGF 
                 1953_at 
                 7422 
                 1.08 
                 2.11 
               
               
                 VEGF 
                 36100_at 
                 7422 
                 1.04 
                 2.06 
               
               
                 VEGF 
                 36101_s_at 
                 7422 
                 3.52 
                 11.49 
               
               
                   
               
             
          
         
       
     
         [0190]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                   
               
               
                 Sixteen of the 19 genes identified in the wounding category 
               
               
                 (GO: 009611) are interferon and/or cytokine regulated genes. 
               
             
          
           
               
                   
                 Affymetrix 
                   
                 Average 
                   
                   
                   
                   
               
               
                   
                 HGU95Av2 
                   
                 Signal 
                 Fold 
                 Table I 
               
               
                 Symbol 
                 Probe ID 
                 LocusLink 
                 Log 2   
                 Change 
                 Category 
                 IFN 
                 Cytokine 
               
               
                   
               
             
          
           
               
                 CD97 
                 35625_at 
                 976 
                 −0.70 
                 −1.63 
                 B 
                 • 
                   
               
               
                 CXCL12 
                 32666_at 
                 6387 
                 −2.08 
                 −4.22 
                 B 
                   
                 • 
               
               
                 FGF7 
                 1466_s_at 
                 2252 
                 −2.03 
                 −4.09 
                 B 
                   
                 • 
               
               
                 MAP2K3 
                 1622_at 
                 5606 
                 −0.41 
                 −1.33 
                 B 
               
               
                 F2R 
                 41700_at 
                 2149 
                 −0.79 
                 −1.73 
                 C 
               
               
                 CCL20 
                 40385_at 
                 6364 
                 6.10 
                 68.57 
                 D 
                 • 
                 • 
               
               
                 CXCL2 
                 37187_at 
                 2920 
                 3.22 
                 9.33 
                 D 
                   
                 • 
               
               
                 CXCL3 
                 34022_at 
                 2921 
                 3.37 
                 10.36 
                 D 
                   
                 • 
               
               
                 CXCL6 
                 35410_at 
                 6372 
                 2.36 
                 5.12 
                 D 
                   
                 • 
               
               
                 GAGE1 
                 31497_at 
                 2543 
                 1.92 
                 3.79 
                 D 
               
               
                 IL1B 
                 39402_at 
                 3553 
                 2.95 
                 7.75 
                 D 
                   
                 • 
               
               
                 IL8 
                 35372_r_at 
                 3576 
                 3.20 
                 9.20 
                 D 
                 • 
                 • 
               
               
                 MAP2K3 
                 2075_s_at 
                 5606 
                 0.72 
                 1.64 
                 D 
               
               
                 MICB 
                 35937_at 
                 4277 
                 0.91 
                 1.88 
                 D 
                 • 
               
               
                 MYD88 
                 38369_at 
                 4615 
                 0.96 
                 1.95 
                 D 
                 • 
               
               
                 NFKB1 
                 1377_at 
                 4790 
                 0.92 
                 1.89 
                 D 
               
               
                 NFKB1 
                 38438_at 
                 4790 
                 0.87 
                 1.83 
                 D 
               
               
                 NFKB1 
                 1378_g_at 
                 4790 
                 0.61 
                 1.53 
                 D 
               
               
                 NMI 
                 36472_at 
                 9111 
                 0.92 
                 1.90 
                 D 
                 • 
               
               
                 SAA1 
                 33272_at 
                 6288 
                 2.74 
                 6.66 
                 D 
               
               
                 TAP1 
                 40153_at 
                 6890 
                 1.48 
                 2.79 
                 D 
                 • 
               
               
                   
               
             
          
         
       
     
         [0191]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                   
               
               
                 Genes with decreased expression during immortalization that are in GO categories structural 
               
               
                 molecular activity genes (GO: 0005198), cell adhesion molecular activity (GO: 0005194) 
               
               
                 and cytoskeletal category (GO: 0005856). Sixteen of the 24 genes from structural 
               
               
                 molecular activity genes (GO: 0005198), and 1 of the 9 genes in the cell adhesion molecular 
               
               
                 activity (GO: 0005194), overlap with the genes in the cytoskeletal category (GO: 0005856) 
               
             
          
           
               
                   
                 Affymetrix 
                   
                 Average 
                   
                   
                   
                   
               
               
                   
                 HGU95Av2 
                   
                 Signal 
                 Fold 
                 Adhesion 
                 Structural 
                 Cyto           
               
               
                 Symbol 
                 Probe ID 
                 LocusLink 
                 Log 2   
                 Change 
                 (GO: 0005194) 
                 (GO: 0005198) 
                 (GO           
               
               
                   
               
             
          
           
               
                 ACTA2 
                 32755_at 
                 59 
                 −2.48 
                 −5.57 
                   
                 • 
                   
               
               
                 ACTC 
                 39063_at 
                 70 
                 −6.09 
                 −67.90 
                   
                 • 
               
               
                 ACTR1A 
                 40052_at 
                 10121 
                 −0.58 
                 −1.50 
                   
                 • 
               
               
                 ADD1 
                 32145_at 
                 118 
                 −0.48 
                 −1.40 
                   
                 • 
               
               
                 ARPC1B 
                 39043_at 
                 10095 
                 −1.10 
                 −2.14 
                   
                 • 
               
               
                 BPAG1 
                 32780_at 
                 667 
                 −0.54 
                 −1.45 
                 • 
                 • 
               
               
                 CAP2 
                 33405_at 
                 10486 
                 −1.34 
                 −2.53 
                   
                 • 
               
               
                 CAPG 
                 38391_at 
                 822 
                 −2.50 
                 −5.65 
               
               
                 CD97 
                 35625_at 
                 976 
                 −0.70 
                 −1.63 
                 • 
               
               
                 CD99 
                 41138_at 
                 4267 
                 −1.35 
                 −2.55 
                 • 
               
               
                 CNN1 
                 34203_at 
                 1264 
                 −3.39 
                 −10.50 
               
               
                 COL4A1 
                 39333_at 
                 1282 
                 −2.82 
                 −7.04 
                   
                 • 
               
               
                 COL4A2 
                 36659_at 
                 1284 
                 −1.93 
                 −3.82 
                   
                 • 
               
               
                 CRYAB 
                 32242_at 
                 1410 
                 −3.95 
                 −15.41 
                   
                 • 
               
               
                 CRYAB 
                 32243_g_at 
                 1410 
                 −3.84 
                 −14.32 
                   
                 • 
               
               
                 DSP 
                 36133_at 
                 1832 
                 −3.91 
                 −15.04 
                   
                 • 
               
               
                 ECM1 
                 37600_at 
                 1893 
                 −1.77 
                 −3.40 
                   
                 • 
               
               
                 ELN 
                 39098_at 
                 2006 
                 −3.36 
                 −10.23 
                   
                 • 
               
               
                 EMS1 
                 39861_at 
                 2017 
                 −1.35 
                 −2.56 
               
               
                 ENG 
                 32562_at 
                 2022 
                 −0.93 
                 −1.90 
                 • 
               
               
                 EPB41L3 
                 41385_at 
                 23136 
                 −4.68 
                 −25.66 
                   
                 • 
               
               
                 FARP1 
                 32148_at 
                 10160 
                 −2.27 
                 −4.82 
               
               
                 FBLN5 
                 39038_at 
                 10516 
                 −1.70 
                 −3.24 
                 • 
               
               
                 FEZ1 
                 37743_at 
                 9638 
                 −3.53 
                 −11.58 
               
               
                 FEZ2 
                 38651_at 
                 9637 
                 −0.84 
                 −1.79 
               
               
                 GSN 
                 32612_at 
                 2934 
                 −1.27 
                 −2.42 
                   
                 • 
               
               
                 ITGA1 
                 37484_at 
                 3672 
                 −1.90 
                 −3.72 
                 • 
               
               
                 ITGA7 
                 36892_at 
                 3679 
                 −2.00 
                 −3.99 
                 • 
               
               
                 KNS2 
                 39057_at 
                 3831 
                 −0.69 
                 −1.62 
               
               
                 MAP1A 
                 35917_at 
                 4130 
                 −1.30 
                 −2.46 
                   
                 • 
               
               
                 ME1 
                 31824_at 
                 4199 
                 −1.38 
                 −2.61 
                 • 
               
               
                 MYL9 
                 39145_at 
                 10398 
                 −1.49 
                 −2.81 
                   
                 • 
               
               
                 NID 
                 35366_at 
                 4811 
                 −1.25 
                 −2.38 
                 • 
                 • 
               
               
                 NOTCH3 
                 38750_at 
                 4854 
                 −3.66 
                 −12.62 
                   
                 • 
               
               
                 PEA15 
                 32260_at 
                 8682 
                 −1.34 
                 −2.53 
               
               
                 SGCD 
                 41378_at 
                 6444 
                 −2.59 
                 −6.00 
               
               
                 SGCD 
                 34993_at 
                 6444 
                 −2.07 
                 −4.21 
               
               
                 SGCD 
                 34991_at 
                 6444 
                 −2.00 
                 −4.00 
               
               
                 SPTAN1 
                 33833_at 
                 6709 
                 −0.72 
                 −1.64 
                   
                 • 
               
               
                 STOM 
                 40419_at 
                 2040 
                 −1.88 
                 −3.69 
               
               
                 STX6 
                 41663_at 
                 10228 
                 −0.77 
                 −1.71 
               
               
                 STX7 
                 38774_at 
                 8417 
                 −0.84 
                 −1.79 
               
               
                 TEK 
                 1596_g_at 
                 7010 
                 −4.15 
                 −17.71 
                   
                 • 
               
               
                 TPM2 
                 32313_at 
                 7169 
                 −1.14 
                 −2.21 
                   
                 • 
               
               
                 TPM2 
                 32314_g_at 
                 7169 
                 −0.83 
                 −1.78 
                   
                 • 
               
               
                 TUBB 
                 39331_at 
                 7280 
                 −1.05 
                 −2.07 
                   
                 • 
               
               
                 VAMP3 
                 35783_at 
                 9341 
                 −0.47 
                 −1.39 
               
               
                 VAMP5 
                 32533_s_at 
                 10791 
                 −1.71 
                 −3.27 
               
               
                 VIL2 
                 40103_at 
                 7430 
                 −1.19 
                 −2.29 
                   
                 • 
               
               
                   
               
             
          
         
       
     
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