bluuebunny/crossref_metadata_2025 huggingface dataset:
Telomerase- and Alternative Telomere Lengthening–Independent Telomere Stabilization in a Metastasis-Derived Human Non–Small Cell Lung Cancer Cell Line: Effect of Ectopic hTERT
Authors: Brachner Andreas , Sasgary Soleman , Pirker Christine , Rodgarkia Chantal , Mikula Mario , Mikulits Wolfgang , Bergmeister Helga , Setinek Ulrike , Wieser Matthias , Chin Suet-Feung , Caldas Carlos , Micksche Michael , Cerni Christa , Berger Walter
Published: April 2006

In the majority of human malignancies, maintenance of telomeres is achieved by reactivation of telomerase, whereas a smaller fraction uses an alternative telomere lengthening (ALT) mechanism. Here, we used 16 non–small cell lung cancer (NSCLC) cell lines to investigate telomere stabilization mechanisms and their effect on tumor aggressiveness. Three of 16 NSCLC cell lines (VL-9, SK-LU-1, and VL-7) lacked telomerase activity, correlating with significantly reduced tumorigenicity in vitro and in vivo. Of the three telomerase-negative cell lines, only SK-LU-1 displayed characteristics of an ALT mechanism (i.e., highly heterogeneous telomeres and ALT-associated promyelocytic leukemia bodies). VL-9 cells gained telomerase during in vitro propagation, indicating incomplete immortalization in vivo. In contrast, NSCLC metastasis-derived VL-7 cells remained telomerase and ALT negative up to high passage numbers and following transplantation in severe combined immunodeficient mice. Telomeres of VL-7 cells were homogenously short, and chromosomal instability (CIN) was comparable with most telomerase-positive cell lines. This indicates the presence of an efficient telomere stabilization mechanism different from telomerase and ALT in VL-7 cells. To test the effect of ectopic telomerase reverse transcriptase (hTERT) in these unique ALT- and telomerase-negative tumor backgrounds, hTERT was transfected into VL-7 cells. The activation of telomerase led to an excessively rapid gain of telomeric sequences resulting in very long (∼14 kb), uniform telomeres. Additionally, hTERT expression induced a more aggressive growth behavior in vitro and in vivo without altering the level of CIN. These data provide further evidence for a direct oncogenic activity of hTERT not based on the inhibition of CIN. (Cancer Res 2006; 66(7): 3584-92)
Abnormal Gene Expression Profiles in Unaffected Parents of Patients with Hereditary-Type Retinoblastoma
Authors: Chuang Eric Y. , Chen Xi , Tsai Mong-Hsun , Yan Hailing , Li Chuan-Yuan , Mitchell James B. , Nagasawa Hatsumi , Wilson Paul F. , Peng Yuanlin , Fitzek Markus M. , Bedford Joel S. , Little John B.
Published: April 2006

The hereditary form of retinoblastoma (Rb) is associated with a germ line mutation in one RB allele and is characterized by the occurrence of multiple, bilateral Rb tumors and a predisposition to the development of second cancers. In an earlier study, we observed an unexpected hypersensitivity to ionizing radiation in skin fibroblasts derived from unaffected parents of children with hereditary Rb. In at least four of these five families, there was no family history of Rb, indicating a new germ line mutation. We hypothesize that the increased parental cell sensitivity to radiation may reflect the presence of an as yet unrecognized genetic abnormality occurring in one or both parents of children with Rb. In the present study, we use DNA microarray technology to determine whether differences in gene expression profiles occurred in the unaffected parents of patients with hereditary Rb relative to normal individuals. Microarray analyses were validated by quantitative reverse transcription-PCR measurements. A distinct difference was observed in the patterns of gene expression between unaffected Rb parents and normal controls. By use of the prediction analysis for microarrays and principal component analysis methodologies, significant differences between the two groups were identified when as few as nine genes were analyzed. Further study of this phenomenon may offer a new insight into the genetic mechanisms of Rb and perhaps more broadly in cancer biology. (Cancer Res 2006; 66(7): 3428-33)
The Transcription Factor ZEB1 Is Aberrantly Expressed in Aggressive Uterine Cancers
Authors: Spoelstra Nicole S. , Manning Nicole G. , Higashi Yujiro , Darling Douglas , Singh Meenakshi , Shroyer Kenneth R. , Broaddus Russell R. , Horwitz Kathryn B. , Richer Jennifer K.
Published: April 2006

The transcription factor ZEB1 (δEF1 in mice) has been implicated in cellular processes during development and tumor progression including epithelial to mesenchymal transition. δEF1 null mice die at birth, but heterozygotes expressing a LacZ reporter inserted into the δEF1 gene live and reproduce. Using these mice, we observed ZEB1 promoter activity in the virgin myometrium, and stroma and myometrium of the pregnant uterus. ZEB1 protein is up-regulated in the myometrium and endometrial stroma after progesterone or estrogen treatment of ovariectomized mice. In the normal human uterus, ZEB1 protein is increased in the myometrium and stroma during the secretory stage of the menstrual cycle. ZEB1 is not expressed in the normal endometrial epithelium. In malignancies of the uterus, we find that ZEB1 (a) is overexpressed in malignant tumors derived from the myometrium (leiomyosarcomas), (b) is overexpressed in tumor-associated stroma of low-grade endometrioid adenocarcinomas, and (c) is aberrantly expressed in the tumor epithelial cells of aggressive endometrial cancers. Specifically, in grade 3 endometrioid adenocarcinomas and uterine papillary serous carcinomas, ZEB1 could be expressed in the epithelial-derived carcinoma cells as well as in the stroma. In malignant mixed Müllerian tumors, the sarcomatous component always expresses ZEB1, and the carcinomatous component can also be positive. In summary, ZEB1 is normally regulated by both estrogen and progesterone receptors, but in uterine cancers, it is likely no longer under control of steroid hormone receptors and becomes aberrantly expressed in epithelial-derived tumor cells, supporting a role for ZEB1 in epithelial to mesenchymal transitions associated with aggressive tumors. (Cancer Res 2006; 66(7): 3893-902)
FoxM1B Is Overexpressed in Human Glioblastomas and Critically Regulates the Tumorigenicity of Glioma Cells
Authors: Liu Mingguang , Dai Bingbing , Kang Shin-Hyuk , Ban Kechen , Huang Feng-Ju , Lang Frederick F. , Aldape Kenneth D. , Xie Tong-xin , Pelloski Christopher E. , Xie Keping , Sawaya Raymond , Huang Suyun
Published: April 2006

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in malignant glioma. However, the functional importance of this factor in human glioma is not known. In the present study, we found that FoxM1B was the predominant FoxM1 isoform expressed in human glioma but not in normal brain tissue. The level of FoxM1 protein expression in human glioma tissues was directly correlated with the glioma grade. The level of FoxM1 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced FoxM1B expression caused SW1783 and Hs683 glioma cells, which do not form tumor xenografts, to regain tumorigenicity in nude mouse model systems. Moreover, gliomas that arose from FoxM1B-transfected anaplastic astrocytoma SW1783 cells displayed glioblastoma multiforme phenotypes. Inhibition of FoxM1 expression in glioblastoma U-87MG cells suppressed their anchorage-independent growth in vitro and tumorigenicity in vivo. Furthermore, we found that FoxM1 regulates the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27Kip1. These results showed that FoxM1 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity. Therefore, FoxM1 might be a new potential target of therapy for human malignant gliomas. (Cancer Res 2006; 66(7): 3593-602)
A Novel Endocytic Mechanism of Epidermal Growth Factor Receptor Sequestration and Internalization
Authors: Orth James D. , Krueger Eugene W. , Weller Shaun G. , McNiven Mark A.
Published: April 2006

Cells form transient, circular dorsal ruffles or “waves” in response to stimulation of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor. These dynamic structures progress inward on the dorsal surface and disappear, occurring concomitantly with a marked reorganization of F-actin. The cellular function of these structures is largely unknown. Here we show that EGF-induced waves selectively sequester and internalize ∼50% of ligand-bound EGFR from the cell surface. This process requires receptor phosphorylation, active phosphatidylinositol 3-kinase, and dynamin 2, although clathrin-coated pits or caveolae are not required. Epithelial and fibroblast cells stimulated with EGF sequestered EGFR rapidly into waves that subsequently generated numerous receptor-positive tubular-vesicular structures. Electron microscopy confirmed that waves formed along the dorsal membrane surface and extended numerous tubules into the cytoplasm. These findings characterize a structure that selectively sequesters large numbers of activated EGFR for their subsequent internalization, independent of traditional endocytic mechanisms such as clathrin pits or caveolae. (Cancer Res 2006; 66(7): 3603-10)
Aggresome Disruption: A Novel Strategy to Enhance Bortezomib-Induced Apoptosis in Pancreatic Cancer Cells
Authors: Nawrocki Steffan T. , Carew Jennifer S. , Pino Maria S. , Highshaw Ralph A. , Andtbacka Robert H.I. , Dunner Kenneth , Pal Ashutosh , Bornmann William G. , Chiao Paul J. , Huang Peng , Xiong Henry , Abbruzzese James L. , McConkey David J.
Published: April 2006

The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration approval for the treatment of multiple myeloma, and its activity is currently being evaluated in solid tumors. Bortezomib triggers apoptosis in pancreatic cancer cells, but the mechanisms involved have not been fully elucidated. Here, we show that pancreatic cancer cells exposed to bortezomib formed aggregates of ubiquitin-conjugated proteins (“aggresomes”) in vitro and in vivo. Bortezomib-induced aggresome formation was determined to be cytoprotective and could be disrupted using histone deacetylase (HDAC) 6 small interfering RNA or chemical HDAC inhibitors, which resulted in endoplasmic reticulum stress and synergistic levels of apoptosis in vitro and in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation in immortalized normal human pancreatic epithelial cells in vitro or in murine pancreatic epithelial cells in vivo. In addition, these cells did not undergo apoptosis following treatment with bortezomib, suberoylanilide hydroxamic acid, or the combination, showing tumor selectivity. Taken together, our study shows that inhibition of aggresome formation can strongly potentiate the efficacy of bortezomib and provides the foundation for clinical trials of bortezomib in combination with HDAC inhibitors for the treatment of pancreatic cancer. (Cancer Res 2006; 66(7): 3773-81)
Antibodies Neutralizing Hepsin Protease Activity Do Not Impact Cell Growth but Inhibit Invasion of Prostate and Ovarian Tumor Cells in Culture
Authors: Xuan Jian-Ai , Schneider Doug , Toy Pam , Lin Rick , Newton Alicia , Zhu Ying , Finster Silke , Vogel David , Mintzer Bob , Dinter Harald , Light David , Parry Renate , Polokoff Mark , Whitlow Marc , Wu Qingyu , Parry Gordon
Published: April 2006

Hepsin is a type II transmembrane serine protease that is expressed in normal liver, and at lower levels in kidney, pancreas, and testis. Several studies have shown that hepsin mRNA is significantly elevated in most prostate tumors, as well as a significant fraction of ovarian and renal cell carcinomas and hepatomas. Although the overexpression of mRNA in these tumors has been extensively documented, there has been conflicting literature on whether hepsin plays a role in tumor cell growth and progression. Early literature implied a role for hepsin in human tumor cell proliferation, whereas recent studies with a transgenic mouse model for prostate cancer support a role for hepsin in tumor progression and metastases. To evaluate this issue further, we have expressed an activatable form of hepsin, and have generated a set of monoclonal antibodies that neutralize enzyme activity. The neutralizing antibodies inhibit hepsin enzymatic activity in biochemical and cell-based assays. Selected neutralizing and nonneutralizing antibodies were used in cell-based assays with tumor cells to evaluate the effect of antibodies on tumor cell growth and invasion. Neutralizing antibodies failed to inhibit the growth of prostate, ovarian, and hepatoma cell lines in culture. However, potent inhibitory effects of the antibodies were seen on invasion of ovarian and prostate cells in transwell-based invasion assays. These results support a role for hepsin in tumor cell progression but not in primary tumor growth. Consistent with this, immunohistochemical experiments with a mouse monoclonal antibody reveal progressively increased staining of prostate tumors with advanced disease, and in particular, extensive staining of bone metastatic lesions. (Cancer Res 2006; 66(7): 3611-9)
Regeneration and Tolerance Factor: A Novel Mediator of Glioblastoma-Associated Immunosuppression
Authors: Roth Patrick , Aulwurm Steffen , Gekel Isabella , Beier Dagmar , Sperry Roxanne G. , Mittelbronn Michel , Meyermann Richard , Beaman Kenneth D. , Weller Michael , Wischhusen Jörg
Published: April 2006

Regeneration and tolerance factor (RTF) was originally identified in placenta where it is thought to be essential for fetal allograft survival. Here we report that RTF mRNA and protein are also expressed in human glioma cells in vitro and in vivo. Suppression of RTF expression by RNA interference promotes the lysis of glioma cells by natural killer (NK) and T cells in vitro. Moreover, RTF-depleted glioma cells are less tumorigenic than control cells in nude mice in vivo. Depletion of NK cells in these animals abolished this effect. RTF is thus a novel aberrantly expressed molecule which confers immune privilege to human malignant gliomas. (Cancer Res 2006; 66(7): 3852-8)
Osteoprotegerin Overexpression by Breast Cancer Cells Enhances Orthotopic and Osseous Tumor Growth and Contrasts with That Delivered Therapeutically
Authors: Fisher Jane L. , Thomas-Mudge Rachel J. , Elliott Jan , Hards Daphne K. , Sims Natalie A. , Slavin John , Martin T. John , Gillespie Matthew T.
Published: April 2006

Osteoprotegerin (OPG) acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL), which is a pivotal molecule required for osteoclast formation. In vitro OPG inhibits osteoclast formation and in vivo (administered as Fc-OPG) it reduces hypercalcemia and the establishment of osteolytic lesions in mouse models of tumor cell growth in bone. Osteolysis can be induced by parathyroid hormone–related protein (PTHrP) produced by breast cancer cells that results in an increased osteoblastic RANKL/OPG ratio. We examined the effect of local tumor production of OPG on the ability of breast cancer cells to establish and grow in bone and mammary fat pad. MCF-7 cells or MCF-7 cells overexpressing PTHrP were transfected with full-length OPG and inoculated into the proximal tibiae of athymic nude mice. Mice injected with cells overexpressing PTHrP and OPG showed enhanced tumor growth, increased osteolysis (2-fold compared with MCF-7 cells overexpressing PTHrP), and altered histology that was reflective of a less differentiated (more aggressive) phenotype compared with MCF-7 cells. In contrast, administration of recombinant Fc-OPG reduced tumor growth and limited osteolysis even in mice inoculated with OPG overexpressing cells. Similarly, OPG overexpression by breast cancer cells enhanced tumor growth following orthotopic inoculation. These results indicate that OPG overexpression by breast cancer cells increases tumor growth in vivo and that there are strikingly different responses between therapeutically administered Fc-OPG and full-length OPG produced by tumor cells. (Cancer Res 2006; 66(7): 3620-8)
Cannibalism of Live Lymphocytes by Human Metastatic but Not Primary Melanoma Cells
Authors: Lugini Luana , Matarrese Paola , Tinari Antonella , Lozupone Francesco , Federici Cristina , Iessi Elisabetta , Gentile Massimo , Luciani Francesca , Parmiani Giorgio , Rivoltini Licia , Malorni Walter , Fais Stefano
Published: April 2006

The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8+ T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to “feed” in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of “feeding” activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. (Cancer Res 2006; 66(7): 3629-38)
Combined Array-Comparative Genomic Hybridization and Single-Nucleotide Polymorphism-Loss of Heterozygosity Analysis Reveals Complex Changes and Multiple Forms of Chromosomal Instability in Colorectal Cancers
Authors: Gaasenbeek Michelle , Howarth Kimberley , Rowan Andrew J. , Gorman Patricia A. , Jones Angela , Chaplin Tracy , Liu Ying , Bicknell David , Davison Eleanor J. , Fiegler Heike , Carter Nigel P. , Roylance Rebecca R. , Tomlinson Ian P.M.
Published: April 2006

Cancers with chromosomal instability (CIN) are held to be aneuploid/polyploid with multiple large-scale gains/deletions, but the processes underlying CIN are unclear and different types of CIN might exist. We investigated colorectal cancer cell lines using array-comparative genomic hybridization (CGH) for copy number changes and single-copy number polymorphism (SNP) microarrays for allelic loss (LOH). Many array-based CGH changes were not found by LOH because they did not cause true reduction-to-homozygosity. Conversely, many regions of SNP-LOH occurred in the absence of copy number change, comprising an average per cell line of 2 chromosomes with complete LOH; 1-2 terminal regions of LOH (mitotic recombination); and 1 interstitial region of LOH. SNP-LOH detected many novel changes, representing possible locations of uncharacterized tumor suppressor loci. Microsatellite unstable (MSI+) lines infrequently showed gains/deletions or whole-chromosome LOH, but their near-diploid karyotypes concealed mitotic recombination frequencies similar to those of MSI− lines. We analyzed p53 and chromosome 18q (SMAD4) in detail, including mutation screening. Almost all MSI− lines showed LOH and/or deletion of p53 and 18q; some near-triploid lines had acquired three independent changes at these loci. We found consistent results in primary colorectal cancers. Overall, the distributions of mitotic recombination and whole-chromosome LOH in the MSI− cell lines differed significantly from random, with some lines having much higher than expected levels of these changes. Moreover, lines with more LOH changes had significantly fewer copy number changes. These data suggest that CIN is not synonymous with copy number change and some cancers have a specific tendency to whole-chromosome deletion and regain or to mitotic recombination. (Cancer Res 2006; 66(7): 3471-9)
Targeted Anti–Vascular Endothelial Growth Factor Receptor-2 Therapy Leads to Short-term and Long-term Impairment of Vascular Function and Increase in Tumor Hypoxia
Authors: Franco Marcela , Man Shan , Chen Limor , Emmenegger Urban , Shaked Yuval , Cheung Alison M. , Brown Allison S. , Hicklin Daniel J. , Foster F. Stuart , Kerbel Robert S.
Published: April 2006

Because antiangiogenic therapies inhibit the growth of new tumor-associated blood vessels, as well as prune newly formed vasculature, they would be expected to reduce the supply of oxygen and thus increase tumor hypoxia. However, it is not clear if antiangiogenic treatments lead only to consistent and sustained increases in hypoxia, or transient decreases in tumor hypoxia along with periods of increased hypoxia. We undertook a detailed analysis of an orthotopically transplanted human breast carcinoma (MDA-MB-231) over a 3-week treatment period using DC101, an anti–vascular endothelial growth factor receptor 2 antibody. We observed consistent reductions in microvascular density, blood flow (measured by high-frequency micro-ultrasound), and perfusion. These effects resulted in an increase in the hypoxic tumor fraction, measured with an exogenous marker, pimonidazole, concurrent with an elevation in hypoxia-inducible factor-1α expression, an endogenous marker. The increase in tumor hypoxia was evident within 5 days and remained so throughout the entire course of treatment. Vascular perfusion and flow were impaired at days 2, 5, 7, 8, 14, and 21 after the first injection, but not at 4 hours. A modest increase in the vessel maturation index was detected after the 3-week treatment period, but this was not accompanied by an improvement in vascular function. These results suggest that sustained hypoxia and impairment of vascular function can be two consistent consequences of antiangiogenic drug treatment. The implications of the results are discussed, particularly with respect to how they relate to different theories for the counterintuitive chemosensitizing effects of antiangiogenic drugs, even when hypoxia is increased. (Cancer Res 2006; 66(7): 3639-48)
CD1d-Restricted Natural Killer T Cells Can Down-regulate Tumor Immunosurveillance Independent of Interleukin-4 Receptor-Signal Transducer and Activator of Transcription 6 or Transforming Growth Factor-β
Authors: Terabe Masaki , Khanna Chand , Bose Seuli , Melchionda Fraia , Mendoza Arnulfo , Mackall Crystal L. , Helman Lee J. , Berzofsky Jay A.
Published: April 2006

It has been shown previously that the suppression of tumor immunosurveillance may be a mechanism by which tumors resist immune detection and elimination. In this study, we evaluated the role of the immunoregulatory natural killer T (NKT) cells in the biology of immunosurveillance of osteosarcoma. The K7M2 mouse osteosarcoma cell line was implanted orthotopically into wild-type and NKT cell–deficient CD1d knockout (KO) BALB/c mice, and mice were monitored for growth of primary tumors. Further, we examined the role of CD4+ and/or CD8+ cells by depleting the cells in vivo and measuring CTL activity in vitro. We also asked the role of interleukin (IL)-4 receptor α (IL-4Rα)-signal transducer and activator of transcription 6 (STAT6) signaling, including IL-13, and transforming growth factor β (TGF-β) by using gene-disrupted mice or treating mice with cytokine antagonists. We were surprised to find a high rate of rejection of osteosarcoma primary tumors in 88% (14 of 16) of CD1d KO mice compared with syngeneic wild-type BALB/c mice that showed rejection of tumor in <24% of mice. Further studies suggested that the rejection of tumor in CD1d KO mice was dependent on CD8+ lymphocytes. Distinct from other murine tumor models, the negative regulation induced by CD1d-restricted NKT cells was not dependent on IL-4Rα-STAT6 signaling, including IL-13, or on TGF-β. These data suggest that a novel CD1d-restricted NKT cell–mediated mechanism for tumor immunosuppression is active in the K7M2 osteosarcoma model and that NKT cells can regulate immunosurveillance through more than one pathway. (Cancer Res 2006; 66(7): 3869-75)
Complement Activated by Chimeric Anti–Folate Receptor Antibodies Is an Efficient Effector System to Control Ovarian Carcinoma
Authors: Macor Paolo , Mezzanzanica Delia , Cossetti Chiara , Alberti Paola , Figini Mariangela , Canevari Silvana , Tedesco Francesco
Published: April 2006

Two chimeric monoclonal antibodies (mAb), cMOV18 and cMOV19, recognizing distinct epitopes of folate receptor highly expressed on epithelial ovarian cancer (EOC) cells were analyzed for their ability to activate complement (C) as a means to enhance their antitumor activity. The individual cMOVs failed to activate C on six EOC cell lines as documented by the marginal deposition of C components and the negligible C-dependent cytotoxicity (CDC). Conversely, the mixture of cMOVs was more effective, although the percentage of cell killing did not exceed 25%. Fluorescence-activated cell sorting analysis of EOC cells for surface expression of the membrane C regulatory proteins (mCRP) revealed high levels of CD46, variable expression of CD59, and absence of CD55. This finding was confirmed in tumor tissue specimens obtained from advanced-stage EOC patients and analyzed for the expression of mCRPs mRNA using a cDNA microarray and for the presence of proteins by immunohistochemistry. Incubation of EOC cells with neutralizing mAbs to CD46 and CD59 led to a significant increase in the CDC from 10% - 20% to 45% - 50%. The relative contribution of antibody-dependent cell cytoxicity (ADCC) and C-dependent killing of two EOC cell lines induced by the mixture of cMOV18 and cMOV19 was about 15% and 25% - 35%, respectively, bringing the total killing to about 40% - 50%. This value increased to 60% - 70% after neutralization of CD46 and CD59 without an appreciable change of ADCC. These results suggest that C is the major contributor to the killing of EOC cells induced by the mixture of cMOV18 and cMOV19. (Cancer Res 2006; 66(7): 3876-83)
SOX4 Expression in Bladder Carcinoma: Clinical Aspects and In vitro Functional Characterization
Authors: Aaboe Mads , Birkenkamp-Demtroder Karin , Wiuf Carsten , Sørensen Flemming Brandt , Thykjaer Thomas , Sauter Guido , Jensen Klaus Møller-Ernst , Dyrskjøt Lars , Ørntoft Torben
Published: April 2006

The human transcription factor SOX4 was 5-fold up-regulated in bladder tumors compared with normal tissue based on whole-genome expression profiling of 166 clinical bladder tumor samples and 27 normal urothelium samples. Using a SOX4-specific antibody, we found that the cancer cells expressed the SOX4 protein and, thus, did an evaluation of SOX4 protein expression in 2,360 bladder tumors using a tissue microarray with clinical annotation. We found a correlation (P < 0.05) between strong SOX4 expression and increased patient survival. When overexpressed in the bladder cell line HU609, SOX4 strongly impaired cell viability and promoted apoptosis. To characterize downstream target genes and SOX4-induced pathways, we used a time-course global expression study of the overexpressed SOX4. Analysis of the microarray data showed 130 novel SOX4-related genes, some involved in signal transduction (MAP2K5), angiogenesis (NRP2), and cell cycle arrest (PIK3R3) and others with unknown functions (CGI-62). Among the genes regulated by SOX4, 25 contained at least one SOX4-binding motif in the promoter sequence, suggesting a direct binding of SOX4. The gene set identified in vitro was analyzed in the clinical bladder material and a small subset of the genes showed a high correlation to SOX4 expression. The present data suggest a role of SOX4 in the bladder cancer disease. (Cancer Res 2006; 66(7): 3434-42)
Fruit and Vegetable Consumption and Colorectal Adenomas in the Nurses' Health Study
Authors: Michels Karin B. , Giovannucci Edward , Chan Andrew T. , Singhania Rohit , Fuchs Charles S. , Willett Walter C.
Published: April 2006

Consumption of fruits and vegetables may confer protection from colorectal adenomas, but the limited observational and interventional evidence is inconclusive. We examined the association between fruit and vegetable consumption and the prevalence and incidence of adenomas of the distal colon and rectum in the Nurses' Health Study (NHS). We used data from 34,467 women in the NHS who had undergone colonoscopy or sigmoidoscopy during follow-up between 1980 and 1998. Consumption of fruits and vegetables was assessed in 1980, 1984, 1986, 1990, and 1994 using a semiquantitative food frequency questionnaire; 1,720 prevalent cases of adenoma of the distal colon and rectum were diagnosed between 1980 and 1998. Frequent consumption of fruit was inversely related to the risk of being diagnosed with polyps, whereas little association was found for vegetable consumption. Women who reported consuming five or more servings of fruit a day had an odds ratio (OR) of 0.60 [95% confidence interval (95% CI), 0.44-0.81] for developing colorectal adenomas compared with women who consumed only one or fewer servings of fruit per day, after adjustment for relevant covariates (Ptrend = 0.001). The respective OR for vegetable consumption was 0.82 (95% CI, 0.65-1.05; Ptrend = 0.1). Women who consumed four or more servings of legumes per week had a lower incidence of colorectal adenomas than women who reported consuming one serving per week or less (OR, 0.67; 95% CI, 0.51-0.90; Ptrend = 0.005). Frequent consumption of fruit may reduce the risk of colorectal adenomas. (Cancer Res 2006; 66(7): 3942-53)
Hydroxyurea Induces Bystander Cytotoxicity in Cocultures of Herpes Simplex Virus Thymidine Kinase–Expressing and Nonexpressing HeLa Cells Incubated with Ganciclovir
Authors: Gentry Brian G. , Boucher Paul D. , Shewach Donna S.
Published: April 2006

Suicide gene therapy with the herpes simplex virus thymidine kinase (HSV-TK) cDNA and ganciclovir can elicit cytotoxicity to transgene-expressing and nonexpressing bystander cells via transfer of ganciclovir phosphates through gap junctions. HeLa cells do not exhibit bystander cytotoxicity, although we showed recently that they transfer low levels of ganciclovir phosphates to bystander cells. Here, we attempted to induce bystander cytotoxicity using hydroxyurea, an inhibitor of ribonucleotide reductase, to decrease the endogenous dGTP pool, which should lessen competition with ganciclovir triphosphate for DNA incorporation. Addition of hydroxyurea to cocultures of HSV-TK-expressing and bystander cells synergistically increased ganciclovir-mediated cytotoxicity to both cell populations while producing primarily an additive effect in cultures of 100% HSV-TK-expressing cells. Whereas HSV-TK-expressing cells in coculture were ∼50-fold less sensitive to ganciclovir compared with cultures of 100% HSV-TK-expressing cells, addition of hydroxyurea restored ganciclovir sensitivity. Quantification of deoxynucleoside triphosphate pools showed that hydroxyurea decreased dGTP pools without significantly affecting ganciclovir triphosphate levels. Although hydroxyurea significantly increased the ganciclovir triphosphate:dGTP value for 12 to 24 hours in HSV-TK-expressing and bystander cells from coculture (1.4- to 4.9-fold), this value was increased for <12 hours (2.5-fold) in 100% HSV-TK-expressing cells. These data suggest that the prolonged increase in the ganciclovir triphosphate:dGTP value in cells in coculture resulted in synergistic cytotoxicity. Compared with enhancement of bystander cytotoxicity through modulation of gap junction intercellular communication, this strategy is superior because it increased cytotoxicity to both HSV-TK-expressing and bystander cells in coculture. This approach may improve clinical efficacy. (Cancer Res 2006; 66(7): 3845-51)
Early Loss of Fhit in the Respiratory Tract of Rodents Exposed to Environmental Cigarette Smoke
Authors: D'Agostini Francesco , Izzotti Alberto , Balansky Roumen , Zanesi Nicola , Croce Carlo M. , De Flora Silvio
Published: April 2006

The Fhit gene, encompassing the most active common human chromosomal fragile region, FRA3B, has been shown to act as a tumor suppressor. Several studies have shown significant Fhit alterations or Fhit protein loss in lung cancers from smokers compared with lung cancers from nonsmokers. To evaluate the role of Fhit under controlled experimental conditions, we exposed rodents to environmental cigarette smoke (ECS) and evaluated Fhit expression or Fhit protein in the respiratory tract. After 14 days of exposure to ECS, loss of Fhit protein in the bronchial/bronchiolar epithelium affected half of the tested B6-129(F1) mice, either wild type or Fhit+/−. After 28 days, it affected the vast majority of the tested SKH-1 hairless mice and of A/J mice and all (UL53-3 x A/J)F1 mice, either wild type or P53+/−. In Sprague-Dawley rats, exposure to ECS for up to 30 days caused a time-dependent loss of Fhit in pulmonary alveolar macrophages. Moreover, ECS down-regulated Fhit expression and significantly decreased Fhit protein in the rat bronchial epithelium. The oral administration of N-acetylcysteine attenuated the ECS-related loss of Fhit, whereas oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate, and indole 3-carbinol, and their combinations had no significant effect. Parallel studies evaluated a variety of molecular, biochemical, and cytogenetic alterations in the respiratory tract of the same animals. In conclusion, there is unequivocal evidence that Fhit is an early, critical target in smoke-related lung carcinogenesis in rodents, and that certain chemopreventive agents can attenuate the occurrence of this gene alteration. (Cancer Res 2006; 66(7): 3936-41)
Immortalization of Primary Endothelial Cells by the K1 Protein of Kaposi's Sarcoma–Associated Herpesvirus
Authors: Wang Ling , Dittmer Dirk P. , Tomlinson Christine C. , Fakhari Farnaz D. , Damania Blossom
Published: April 2006

Kaposi's sarcoma–associated herpesvirus (KSHV) is linked to three different human cancers: Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The Kaposi's sarcoma lesion expresses high levels of angiogenic factors and is comprised of a mixed cell population, including endothelial cells that are infected with KSHV. We find that the KSHV K1 protein is expressed in Kaposi's sarcoma lesions and can immortalize and extend the life span of primary human umbilical vein endothelial cells in culture. Vascular endothelial growth factor (VEGF) is critical for the survival of endothelial cells, and we show that expression of K1 in endothelial cells resulted in increased levels of secreted VEGF and the activation of key signaling pathways, including the VEGF/VEGF receptor and the phosphatidylinositol-3′-OH-kinase (PI3K) pathway. The SH2 binding motifs present in the cytoplasmic tail of K1 were critical for K1's ability to activate these pathways. Activation of PI3K by K1 results in activation of Akt kinase and mammalian target of rapamycin and inactivation of the proapoptotic proteins FKHR, glycogen synthase kinase-3, and Bad, which are events indicative of cell survival. Because activation of the PI3K pathway is critical for transformation of many human cells, we suggest that PI3K activation by K1 is involved in endothelial cell immortalization and contributes to KSHV-associated tumorigenesis. We also report that K1 enhances angiogenesis in vivo and increases tumor vasculature and tumor size. (Cancer Res 2006; 66(7): 3658-66)
Transforming Growth Factor α Expression Drives Constitutive Epidermal Growth Factor Receptor Pathway Activation and Sensitivity to Gefitinib (Iressa) in Human Pancreatic Cancer Cell Lines
Authors: Pino Maria S. , Shrader Marissa , Baker Cheryl H. , Cognetti Francesco , Xiong Henry Q. , Abbruzzese James L. , McConkey David J.
Published: April 2006

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-α (TGF-α)] expression (r2 = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-α reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-α expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-α expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-α expression may therefore represent an effective means of identifying EGFR-responsive primary tumors. (Cancer Res 2006; 66(7): 3802–12)
Testicular Xenografts: A Novel Approach to Study Cytotoxic Damage in Juvenile Primate Testis
Authors: Jahnukainen Kirsi , Ehmcke Jens , Schlatt Stefan
Published: April 2006

The underlying primary damage to the testis caused by chemotherapeutic regimens during childhood is largely unknown. Xenografting of monkey testes was successfully applied in maturation of juvenile testis to the point of complete spermatogenesis. This allows us to manipulate developing primate testis without direct treatment of patients. This new model is validated establishing the effects of cytotoxic treatment in the immature primate testis. Male castrated nude mice received eight s.c. grafts of juvenile monkey testicular tissue and, 28 weeks later, were injected with busulfan (38 mg/kg, i.p.) or vehicle. Graft numbers, size, and histology were examined. Grafts showed pubertal induction of spermatogenesis to the level of pachytene spermatocytes at point of busulfan treatment and further progressed to the level of round spermatids in control samples at 4 weeks. Busulfan treatment caused a statistically significant decrease in the number of seminiferous tubules containing germ cells. Type B spermatogonia and more advanced stages of spermatogenesis were depleted. A statistically significant decrease to pretreatment level was observed in the number of type A pale and centrally located spermatogonia. Busulfan did not affect type A dark spermatogonia. Occasionally, elongating spermatids were detected in busulfan-treated grafts. Observations show that busulfan selectively destroys differentiating spermatogonia whereas some of the spermatocytes present at the moment of cytotoxic insult are able to continue differentiation. Data indicate that xenografting of testicular monkey tissue is a valid approach to detect the busulfan-induced germ cell damage and serves as a powerful experimental tool to study cytotoxic effects in developing primate testis. (Cancer Res 2006; 66(7): 3813-8)
Genetically Engineered Models Have Advantages over Xenografts for Preclinical Studies
Authors: Becher Oren J. , Holland Eric C.
Published: April 2006

Mouse models of human cancer are valuable tools for cancer research. Although xenografts and genetically engineered models (GEMs) possess limitations as well as advantages, each system plays a significant role in preclinical testing. Tumor xenografts are easy to use, relatively inexpensive, and reproducible. The main drawback of xenografts is that the genetics and histology of the tumors are frequently not representative of the respective human tumor and, thus far, these models have not been as predictive of therapeutic success as one would like. By contrast, GEMs are histologically and genetically accurate models of human cancer but have disadvantages of heterogeneity with regard to frequency, latency, and growth. These disadvantages are reminiscent of the variable behavior of actual human tumors. Recently, these shortcomings have been partly overcome with the development of anatomic and molecular in vivo imaging techniques such as magnetic resonance imaging and bioluminescence imaging. These new technologies will hopefully support the use of GEMs in preclinical trials and help determine if trials in GEMs are more predicative than xenografts of human responses. (Cancer Res 2006; 66(7): 3355-9)
Prediction of Sensitivity of Rectal Cancer Cells in Response to Preoperative Radiotherapy by DNA Microarray Analysis of Gene Expression Profiles
Authors: Watanabe Toshiaki , Komuro Yasuhiro , Kiyomatsu Tomomichi , Kanazawa Takamitsu , Kazama Yoshihiro , Tanaka Junichiro , Tanaka Toshiaki , Yamamoto Yoko , Shirane Masatoshi , Muto Tetsuichiro , Nagawa Hirokazu
Published: April 2006

Preoperative radiotherapy has been widely used to improve local control of disease and to improve survival in the treatment of rectal cancer. However, the response to radiotherapy differs among individual tumors. Our objective here was to identify a set of discriminating genes that can be used for characterization and prediction of response to radiotherapy in rectal cancer. Fifty-two rectal cancer patients who underwent preoperative radiotherapy were studied. Biopsy specimens were obtained from rectal cancer before preoperative radiotherapy. Response to radiotherapy was determined by histopathologic examination of surgically resected specimens and classified as responders or nonresponders. By determining gene expression profiles using human U95Av2 Gene Chip, we identified 33 novel discriminating genes of which the expression differed significantly between responders and nonresponders. Using this gene set, we were able to establish a new model to predict response to radiotherapy in rectal cancer with an accuracy of 82.4%. The list of discriminating genes included growth factor, apoptosis, cell proliferation, signal transduction, or cell adhesion–related genes. Among 33 discriminating genes, apoptosis inducers (lumican, thrombospondin 2, and galectin-1) showed higher expression in responders whereas apoptosis inhibitors (cyclophilin 40 and glutathione peroxidase) showed higher expression in nonresponders. The present study suggested the possibility that gene expression profiling may be useful in predicting response to radiotherapy to establish an individualized tailored therapy for rectal cancer. Global expression profiles of responders and nonresponders may provide insights into the development of novel therapeutic targets. (Cancer Res 2006; 66(7): 3370-4)
γ-Secretase-Dependent Proteolysis of CD44 Promotes Neoplastic Transformation of Rat Fibroblastic Cells
Authors: Pelletier Ludivine , Guillaumot Patricia , Frêche Barbara , Luquain Céline , Christiansen Dale , Brugière Sabine , Garin Jérome , Manié Serge N.
Published: April 2006

The metalloprotease-dependent extracellular domain cleavage of the adhesion molecule CD44 is frequently observed in human tumors and is thought to promote metastasis. This cleavage is followed by γ-secretase-dependent release of CD44 intracellular domain (CD44-ICD), which exhibits nuclear signaling activity. Using a reversible Ret-dependent oncogenic conversion model and a restricted proteomic approach, we identified a positive correlation between the neoplastic transformation of Rat-1 cells and the expression of standard CD44. In these transformed cells, CD44 was found to undergo a sequential metalloprotease and γ-secretase cleavage, resulting in an increase in expression of CD44-ICD. We showed that this proteolytic fragment possesses a transforming activity. In support of this role, a significant and specific reduction in Ret-induced transformation of Rat-1 cells was observed following drug-mediated inhibition of γ-secretase. Taken together, these findings suggest that the shedding of CD44 may not only modulate metastasis but also affects earlier events in tumorigenesis through the release of CD44-ICD. (Cancer Res 2006; 66(7): 3681-7)
Carcinogenic Cr(VI) and the Nutritional Supplement Cr(III) Induce DNA Deletions in Yeast and Mice
Authors: Kirpnick-Sobol Zhanna , Reliene Ramune , Schiestl Robert H.
Published: April 2006

Industrial Cr(VI) emissions contaminate drinking water sources across the U.S., and many people take Cr(III) nutritional supplements. Cr(VI) is a human pulmonary carcinogen, but whether it is carcinogenic in the drinking water is not known. Due to widespread human exposure, it is imperative to determine the carcinogenic potential of Cr(VI) and Cr(III). DNA deletions and other genome rearrangements are involved in carcinogenesis. We determined the effects of Cr(VI) as potassium dichromate and Cr(III) as chromium(III) chloride on the frequencies of DNA deletions measured with the deletion assay in Saccharomyces cerevisiae and the in vivo pun reversion assay in C57BL/6J pun/pun mice. Exposing yeast and mice via drinking water to Cr(VI) and Cr(III) significantly increased the frequency of DNA deletions. We quantified intracellular chromium concentrations in yeast and tissue chromium concentrations in mice after exposure. Surprisingly, this revealed that Cr(III) is a more potent inducer of DNA deletions than Cr(VI) once Cr(III) is absorbed. This study concludes that both the environmental contaminant Cr(VI) and the nutritional supplement Cr(III) increase DNA deletions in vitro and in vivo, when ingested via drinking water. (Cancer Res 2006; 66(7): 3480-4)
TLR-4 Signaling Promotes Tumor Growth and Paclitaxel Chemoresistance in Ovarian Cancer
Authors: Kelly Michael G. , Alvero Ayesha B. , Chen Rui , Silasi Dan-Arin , Abrahams Vikki M. , Chan Serena , Visintin Irene , Rutherford Thomas , Mor Gil
Published: April 2006

Evidence suggests that an inflammatory profile of cytokines and chemokines persisting at a particular site would lead to the development of a chronic disease. Recent studies implicate bacterial infection as one possible link between inflammation and carcinogenesis; however, the crucial molecular pathways involved remain unknown. We hypothesized that one possible upstream signaling pathway leading to inflammation in carcinogenesis may be mediated by Toll-like receptors (TLR). We describe for the first time an adaptive mechanism acquired by ovarian cancer cells that allows them to promote a proinflammatory environment and develop chemoresistance. We propose that the TLR-4-MyD88 signaling pathway may be a risk factor for developing cancer and may represent a novel target for the development of biomodulators. Our work explains how bacterial products, such as lipopolysaccharide, can promote, directly from the tumor, the production of proinflammatory cytokines and the enhancement of tumor survival. In addition, we provide new evidence that links TLR-4 signaling, inflammation, and chemoresistance in ovarian cancer cells. (Cancer Res 2006; 66(7): 3859-68)