Patent Publication Number: US-2004053288-A1

Title: Method for estimating therapeutic efficacy of tumor necrosis factor

Description:
BACKGROUND OF THE INVENTION  
       [0001] 1. Field of the Invention  
       [0002] The present invention relates to a method for estimating the potential therapeutic efficacy of physiologically active substances or anti-tumor agents, particularly, tumor necrosis factor (hereinafter abbreviated as “TNF-α” throughout the specification) in the treatment of cancers.  
       [0003] 2. Description of the Prior Art  
       [0004] Treatments with antitumor agents have been used in medical fields as effective therapeutic methods for treating cancers. Conventional antitumor agents, however, are only effective on restricted types of cancers, and this narrows their applicability. In addition, there are not so many antitumor agents with satisfactory therapeutic efficacy. Most of conventional antitumor agents have relatively strong side effects, and this results in a heavy burden to cancer patients as a serious demerit.  
       [0005] Recently, as novel antitumor agents, the use of physiologically active substances have been highlighted. Comparing with conventional antitumor agents mainly produced by chemical synthesis, such physiologically active substances have the merit that they have a relatively wide applicability because of their satisfactory antitumor effects on more various types of cancers. Among the physiologically active substances, particularly, TNF-α, which was discovered by L. J. Old et al. in 1975 as a cytotoxic factor secreted in sera of animals such as rabbits and mice which had been sequentially administered with BCG and intracellular toxin, has been focused on as a physiologically active substance with a strong antitumor activity on a variety of antitumor cells since the discovery. However, even the above TNF-α has serious side effects similarly as in conventional antitumor agents and subsidiary acts on some types of cells to cause fever, and therefore, TNF-α has not yet been actually used in medical fields.  
       [0006] Due to the genetic progress such as the total human gene analysis as a result of the worldwide project, gene-related screening tools such as DNA microarray technology have been rapidly progressed.  Jikken - Igaku,  extra number, edited by H. Aburatani, Vol. 19, No. 19, pp. 2,518-2,524 (2001) discloses a trial of applying the analysis of gene expression profiles using DNA microarray technology to accurately evaluate the properties of cancers in diagnosing cancers; and  Jikken - Igaku,  extra number, edited by K. Chiba et al, Vol. 19, No. 19, pp. 2,507-2,512 (2001) proposes a trial of estimating the individual difference in effects of pharmaceuticals such as antitumor agents, i.e., pharmacogenomics. Since the above trials would directly enable an appropriate selection of a potent therapy without actually conducting therapies with trials and errors in diagnosing and treating diseases such as cancers, it is surely expected that such trials will lower patients&#39; physical burdens and the doses of expensive medicines and effectively reduce patients&#39; economical burdens such as medical costs. Although there are not so many actually applicable cases to which the above methods for estimating efficacy of medicines are applicable, future researches would increase such cases. Although there has not yet been established such methods applicable to antitumor agents including TNF-α, such a method, when established, will possibly be a breakthrough in applying to cancer treatment antitumor agents including TNF-α, which could not have been used in clinical treatments because of their undesirable side effects in spite of their outstanding physiological activities.  
       SUMMARY OF THE INVENTION  
       [0007] The present invention aims to provide a method for estimating the therapeutic efficacy of physiologically active substances or anti-tumor agents, particularly, TNF-α in treating cancers.  
       [0008] To overcome the above object, the present inventors widely screened the expression profiles of genes such as apoptosis-related genes and TNF-α-related genes in established cell lines derived from cancers, found genes which are deeply related to sensitivity to TNF-α; and examined the expression levels of these genes to establish the method for estimating the therapeutic efficacy of TNF-α in cancer treatment. Thus, the present inventors accomplished this invention.  
       [0009] The present invention estimates the therapeutic efficacy of TNF-α in cancer treatment based on the gene expression of TNF-α-related gene, particularly, a protein kinase B (Akt-1) gene, death receptor (DR3) gene, multidrug resistance-associated protein (MRP5) gene, or multidrug resistance-associated protein (MRP6) gene.  
     
    
    
     BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING  
     [0010]FIG. 1 shows a relative expression level of Akt-1 gene with respect to the mRNA level of TNF-α sensitive cells in each rank.  
     [0011]FIG. 2 shows a relative expression level of DR3 gene with respect to the mRNA level of TNF-α sensitive cells in each rank.  
     [0012]FIG. 3 shows a relative expression level of MRP5 gene with respect to the mRNA level of TNF-α sensitive cells in each rank.  
     [0013]FIG. 4 shows a relative expression level of MRP6 gene with respect to the mRNA level of TNF-α sensitive cells in each rank.  
     [0014]FIG. 5 is a comparison of the expression level of Akt-1 gene with respect to mRNA level in each type of cells from different origins.  
     [0015]FIG. 6 is a result of cluster analysis of TNF-α-related gene.  
     [0016]FIG. 7 shows a relationship between the sensitivity of cells to TNF-α and the expression level of Akt-1 gene or ICAM-1, where the symbols “▾” and “∘” mean TNF-α-sensitive cells and non-TNF-α-sensitive cells, respectively. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0017] The term “TNF-α” as referred to as in the present invention means TNF-α in general obtainable from humans or other warm-blooded animals; those produced by culturing cells of humans or other warm-blooded animals in an appropriate manner, and contacting the cells with appropriate TNF-α inducers to produce TNF-α; those produced by preparing appropriate expression vectors introduced with TNF-α genes of humans or other warm-blooded animals, introducing the expression vectors into microorganisms such as  Escherichia coli  or yeasts, animal- or plant-bodies, or cultured animal- or plant-cells, and optionally allowing the resulting transformants to express TNF-α using TNF-α inducers; and those which are totally or partially produced by chemical syntheses by the protein engineering. These TNF-α preparations include natural and recombinant TNF-αs independently of their preparation methods and origins, and further include those which are produced in vivo by administering TNF-α inducers to patients or by allowing to express external TNF-α genes introduced into patients&#39; bodies by means of gene therapy. In addition, to control the stability, action and effect of TNF-α, the TNF-αs usable in the present invention include those which are modified with N-glycosylated or O-glycosylated saccharide chains composed of monosaccharides such as glucose, galactose, N-acetyl glucosamine, N-acetyl galactosamine, fucose, mannose, xylose, and sialic acid; those which are modified with saccharide chains composed of saccharides sulfonated with hyaluronic acid or heparan sulfonate; those which are modified with water-soluble high molecules such as polyethylene glycol and poly vinyl alcohol; and those which are partially modified in their amino acid sequences without losing TNF-α activity.  
     [0018] In the present invention, TNF-α can be used in combination with one or more other substances, for example, physiologically active substances such as interferons, interleukins, and growth hormones; and pharmaceuticals such as antitumor agents, antibiotics, vaccines, crude drugs, and herbal medicines.  
     [0019] The term “TNF-α-related genes” as referred to as in the present invention means genes which are induced their expression by TNF-α or which are related to the expression of TNF-α induction, for example, TNF-α-receptor genes, apoptosis-related genes, TNF-α-signal-transduction-related genes, multidrug resistance-associated genes, etc., particularly, the genes in Table 3 as described later. Specifically, prokinase B (hereinafter may be abbreviated as “Akt-1” throughout the specification) gene, a TNF-α-signal-related-gene; death receptor 3 (hereinafter may be abbreviated as “DR3” throughout the specification), receptor-related gene; multidrug resistance-associated protein 5 (hereinafter may be abbreviated as “MRP5” throughout the specification); and multidrug resistance-associated protein 6 (hereinafter may be abbreviated as “MRP6” throughout the specification) which are all deeply related to the therapeutic efficacy of TNF-α because cells, in which these genes are expressed in quantity, have a relatively high sensitivity to TNF-α. Thus, the examination of the expression level of these genes will be advantageously, effectively used in estimating the therapeutic efficacy of TNF-α.  
     [0020] The term “cancer cells” as referred to as in the present invention means those which are derived from cancer tissues, i.e., cancer cells collected from cancer patients or established cell lines, particularly, cancer cells from tissues of cancer patients to be treated are preferable.  
     [0021] The methods used for quantifying the expression level of TNF-α-related genes in the present invention are those for determining the expression level of the genes with respect to the expression level of protein, i.e., those for quantifying the expression level of TNF-α-related proteins using cancer cells intact or after extracting the proteins from the cells with appropriate methods; enzyme immunoassay (EIA), radioimmunoassay (RIA), immunoprecipitation, immunocyte staining method, electrophoresis, western blotting, panning method, high-performance liquid chromatography (HPLC), peptide sequencing method, etc., which can be used in an appropriate combination.  
     [0022] The methods used for determining the expression level of mRNAs of TNF-α-related genes in the present invention are those for quantifying the mRNAs expressed in cancer cells by using the cells intact or by extracting the mRNAs from the cells, or quantifying the level of cDNAs synthesized by reverse transcriptase using the mRNAs as templates; conventional Northern blot technique, Southern blot technique, hybridization method, magnetic bead technology, electrophoresis, polymerase chain reaction (PCR) method, and DNA sequencing method, etc., which can be used in an appropriate combination. In the present invention, the later described real time PCR method as a modified method of PCR method is most preferably used because of its superior processing speed and quantifying accuracy. Without the need of extracting from cancer cells, the mRNAs or their corresponding cDNAs usable in the present invention can be obtainable by appropriately selecting them from RNAs or their corresponding cDNAs, which are derived from commercially available cells, organs, or tissues; as well as their processed products in the form of a pre-blotting membrane or DNA microarray technology.  
     [0023] Explaining the methods used for examining the sensitivity of cancer cells to TNF-α in the present invention, either cancer cells collected from patients suffering from cancers such as intestinal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, hepatoma, renal cancer, neural cancer, skin cancer, cancer of pharynx, sarcoma, and carcinoma uteri; or established cell lines derived from the above cancer cells are cultured in conventional nutrient culture media, supplemented with 0.1 to 10 μg/ml of TNF-α, and incubated for a prescribed period of time, followed by collecting and staining the resulting cells with a dye such as propidium iodide to count death cells or apoptosed cells by a spectrophotometer, flow cytometry, etc. The sensitivity of cells to TNF-α can be determined by comparing the number of death cells or apoptosed cells in the above test cultures with that for a control culture, cultured similarly as in the test cultures but with no addition of TNF-α. Also the sensitivity of cells to TNF-α can be determined by transplanting cancer cells, collected from a cancer patient, to experimental animals such as nude mice, breeding the animals while administering TNF-α and measuring and evaluating the size of the grown tumor masses in the animals.  
     [0024] The methods used for evaluating the expression level of TNF-α-related genes in the present invention include those which calculate the relative expression level of TNF-α-related genes in patients to be administered with TNF-α based on the expression level of genes, as an internal standard, whose expression levels between cells and tissues are not so different, such as glycolytic pathway enzymes such as glyceraldehyde-3-phosphate dehydrogenase or cytoskeleton proteins such as β-actin; those which calculate an increased or decreased level of TNF-α-related genes in patients to be administered with TNF-α based on the expression level of TNF-α-related genes in normal cells of the same patient&#39;s tissue as the cancer cells tested. The data on the expression levels thus obtained should preferably be examined whether there exist the desired significant differences under a prescribed level of significant difference by means of Mann-Whitney U test, Student&#39;s Welch&#39;s t-test, cluster analysis, etc.  
     [0025] The following experiments explain the present invention in detail:  
     Experiment 1  
     [0026] Sensitivity of Cell Lines, Derived from Different Organs to TNF-α 
     [0027] Twenty-one different types of cancer cells collected from patients suffering from cancers and 69 different types of established cell lines obtained from American Type Culture Collection (ATCC), Manassas, USA; Japanese Collection of Research Bioresources (JCRB), Tokyo, Japan; European Collection of Cell Cultures (ECACC), North Carolina, USA; German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany; National Institute of Health and Nutrition (NIHN), Tokyo, Japan; and Institute of Development, Aging, and Cancer (IDAC), Miyagi, Japan, were respectively inoculated into an RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum at a cell density of 1×10 4  cells/ml to 3×10 4  cells/ml, cultured for a prescribed period of time, admixed with 5 ng/ml of TNF-α, and incubated at 37° C. for 48 hours under 5% (v/v) CO 2  atmospheric conditions. As negative controls, cell culture systems free of TNF-α for each type of cells were provided and cultured similarly as above. After culturing, the cells were collected from the resulting cultures and allowed to stand in a cold 70% (v/v) aqueous ethanol solution for two hours to immobilize the cells. The immobilized cells were stained by keeping in a phosphate-buffered saline (pH 7.2) containing 40 μg/ml of propidium iodide for 20 min. The DNA level in each type of cells was measured on “EPICS XL”, a flow cytometry commercialized by Beckman Coulter Inc., CA, USA, and the percentage (%) of apoptosed cells was analyzed on “WINCYCLE”, a prescribed software commercialized by Beckman Coulter Inc., CA, USA. The percentages (%) of apoptosed cells for each type of cells treated with TNF-α were minused those which corresponded to negative controls with no TNF-α, and the obtained data were graded into four ranks: A rank which means that it had a percentage (%) of less than 5%; B rank, a percentage (%) of more than 5% but less than 10%; C rank, a percentage (%) of more than 10% but less than 20%; and D rank, a percentage (%) of more than 20%. The results are in Tables 1 and 2.  
                               TABLE 1                                      Cancer cell   Percentage (%)                                                         Code       of apoptosed           No.   Origin   Name   No.   Obtention   cells   Rank                                         1   Intestinal   From colon cancer patient A   3.7   A           cancer                                         2   Intestinal   LoVo   CCL229   ATCC   0.1   A           cancer       3   Intestinal   HCT-15   CCL225   ATCC   28.4   D           cancer       4   Intestinal   WiDr   CCL218   ATCC   5.4   B           cancer       5   Intestinal   LS174T   CCL188   ATCC   7.5   B           cancer       6   Intestinal   CoLo 205   CCL222   ATCC   29.3   D           cancer       7   Intestinal   HT-29   HTB38   ATCC   1.6   A           cancer       8   Intestinal   CoLo 678   ACC194   DSMZ   0.3   A           cancer       9   Intestinal   SW 1116   CCL233   ATCC   23.8   D           cancer       10   Intestinal   SW 480   CCL228   ATCC   5.8   B           cancer       11   Intestinal   CoLo 206   ACC21   DSMZ   10.7   C           cancer       12   Intestinal   DLD-1   CCL221   ATCC   1.4   A           cancer                                 13   Lung   From lung cancer patient A   5.0   B           cancer       14   Lung   From lung cancer patient B   21.2   D           cancer       15   Lung   From lung cancer patient C   5.4   B           cancer       16   Lung   From lung cancer patient D   0.2   A           cancer       17   Lung   From lung cancer patient E   0.3   A           cancer       18   Lung   From lung cancer patient F   6.3   B           cancer                                         19   Lung   EBC-1   JCRB0820   JCRB   18.6   C           cancer       20   Lung   MRC-5   CCL171   ATCC   1.2   A           cancer       21   Lung   CALU6   HTB56   ATCC   1.9   A           cancer       22   Lung   WI-38VA13   CCL75.1   ATCC   8.3   B           cancer       23   Lung   OBA-LK-1   TKG0572   IDAC   22.2   D           cancer       24   Lung   CALU3   HTB55   ATCC   1.7   A           cancer       25   Lung   A 549   CCL185   ATCC   0.1   A           cancer       26   Lung   CoLo 699   ACC196   DSMZ   0.8   A           cancer       27   Lung   LU 65   JCRB0079   JCRB   3.0   A           cancer       28   Pancreatic   PK-1   TKG0239   IDAC   6.2   C           cancer       29   Pancreatic   PK-9   TKG0240   IDAC   4.0   A           cancer       30   Pancreatic   KLM-1   TKG0490   IDAC   1.4   A           cancer       31   Pancreatic   MIA Paca2   CRL1420   ATCC   7.4   B           cancer       32   Pancreatic   PANC-1   CRL1469   ATCC   7.6   B           cancer       33   Pancreatic   PK-59   TKG0492   IDAC   3.0   A           cancer       34   Pancreatic   PK-8   TKG0383   IDAC   9.6   B           cancer       35   Pancreatic   PK-45H   TKG0493   IDAC   0.6   A           cancer       36   Breast   ZR-75-1   CRL1500   ATCC   23.4   D           cancer       37   Breast   YMB-1-E   JCRB0825   JCRB   39.4   D           cancer       38   Breast   MCF-7   HTB22   ATCC   9.5   B           cancer                                 39   Gastric   From gastric cancer patient A   2.8   A           cancer                                         40   Gastric   MKN-7   TKG0228   IDAC   0.6   A           cancer       41   Gastric   MKN-74   JCRB0255   JCRB   0.7   A           cancer       42   Gastric   MKN-45   JCRB0254   JCRB   7.4   B           cancer       43   Gastric   MKN-1   JCRB0252   JCRB   2.3   A           cancer       44   Gastric   MKN-28   JCRB0253   JCRB   1.5   A           cancer       45   Gastric   AZ 521   JCRB0061   JCRB   0.2   A           cancer       46   Gastric   SCH   JCRB0251   JCRB   30.5   D           cancer                                 47   Hepatoma   From hepatoma patient A   0.2   A       48   Hepatoma   From hepatoma patient B   2.0   A       49   Hepatoma   From hepatoma patient C   10.7   C       50   Hepatoma   From hepatoma patient D   0.1   A       51   Hepatoma   From hepatoma patient E   13.4   C       52   Hepatoma   From hepatoma patient F   0.1   A                                         53   Hepatoma   HuH28   JCRB0426   JCRB   0.9   A       54   Hepatoma   HuCCT1   JCRB0425   JCRB   0.1   A       55   Hepatoma   HuL-1       NIHN   5.8   B       56   Hepatoma   HepG2   HB8065   ATCC   2.9   A       57   Hepatoma   PLC/PRF/5   CRL8024   ATCC   0.1   A       58   Hepatoma   Li-7   TKG0368   IDAC   3.6   A       59   Hepatoma   HuH7   JCRB0403   JCRB   1.1   A       60   Hepatoma   Hep 3B   HB8064   ATCC   3.6   A                                 61   Renal   From renal cancer patient A   3.5   A           cancer       62   Renal   From renal cancer patient B   0.2   A           cancer       63   Renal   From renal cancer patient C   5.3   B           cancer       64   Renal   From renal cancer patient D   0.1   A           cancer                                         65   Renal cancer   ACHN   CRL1611   ATCC   0.6   A       66   Renal cancer   VMRC-RCW   TKG0447   IDAC   0.1   A       67   Renal cancer   Caki-1   HTB46   ATCC   0.2   A                                 68   Neurologic   From neurologic cancer   0.2   A           cancer   cancer patient A                                         69   Neurologic   U251   JRCB0461   JCRB   1.0   A           cancer       70   Neurologic   U373 MG   89081403   ECACC   1.2   A           cancer       71   Neurologic   SCCH-26   JCRB0106   JCRB   2.7   A           cancer       72   Neurologic   TN-2   TKG0278   IDAC   1.8   A           cancer       73   Neurologic   HEPM   CRL1486   ATCC   0.1   A           cancer       74   Neurologic   KINGS-1   IF050435   JCRB   6.3   B           cancer                                 75   Skin cancer   From skin cancer patient A   12.0   C       76   Skin cancer   From skin cancer patient B   4.2   A                                         77   Skin cancer   RPMI 7932   94072246   ECACC   0.1   A       78   Skin cancer   G-361   CRL1424   ATCC   0.1   A       79   Skin cancer   A375   CRL1619   ATCC   2.8   A       80   Skin cancer   SK-MEL-28   HTB72   ATCC   5.9   B       81   Throat   Detroit   CCL138   ATCC   8.8   B           cancer   562       82   Throat   HO-1-u-1   JCRB0828   JCRB   0.4   A           cancer       83   Throat   HO-1-N-1   JCRB0831   JCRB   3.2   A           cancer       84   Throat   FADu   HTB43   ATCC   0.1   A           cancer       85   Sarcoma   RD   CCL136   ATCC   22.5   D       86   Sarcoma   Hu-09N2   JCRB0428   JCRB   11.6   C       87   Sarcoma   Saos-2   HTB85   ATCC   1.3   A       88   Carcinoma   Ca Ski   CRL1550   ATCC   12.1   C           uteri       89   Carcinoma   ME-180   HTB33   ATCC   3.0   A           uteri       90   —   KB   CCL171   ATCC   4.7   A                  
 
     [0028] As shown in Tables 1 and 2, 56, 18, 7 and 9 out of 90 different types of cells tested were respectively graded into the ranks A to D.  
     Experiment 2  
     [0029] Expression of Factors in Cells  
     [0030] Using “RNEASY MIDI KIT”, an RNA kit commercialized by Qiagen GmbH, Hilden, Germany, RNAs were respectively prepared in usual manner from the 90 different types of cells in Tables 1 and 2. One microgram of each of the RNAs was reacted with 100 μl of a reaction mixture containing one microgram of a separately prepared random hexamer and 100 units of a murine breast viral reverse transcriptase sequentially at 25° C. for 10 min, at 42° C. for 30 min, and 99° C. for 5 min, and then the reaction was suspended to obtain a cDNA.  
     [0031] Primers for PCR in SEQ ID NOs: 1 to 100, which corresponded to 49 different types of TNF-α-related genes, registered at Unigene, a DNA database; and to a β-actin gene as an internal standard gene, were prepared and in usual manner subjected to real time PCR using “ABIPRISM 7700 SEQUENCE DETECTION SYSTEM”, an apparatus for PCR commercialized by Applied Biosystems Japan, Ltd., Tokyo, Japan: Twenty nanograms of a cDNA, 100 nM of a sense primer, and 100 nM of an antisense primer were dissolved in water to obtain a 12.5 μl aqueous solution which was then admixed with 12.5 μl of “SYBR GREEN PCR MASTERMIX” commercialized by Applied Biosystems Japan, Inc., Tokyo, Japan, to obtain a reaction mixture. Then, the reaction mixture was subjected to 45 cycles of PCR with sequential incubations at 90° C. for 15 sec and at 60° C. for one min, followed by comparing the expression levels for each gene with that of the internal β-actin gene to express the levels in a numerical manner as their relative expression levels. According to the grading of the ranks in Experiment 1, the ranks B, C and D against the rank A in each gene were examined according to Mann-Whitney U test, and the results were evaluated whether they had a significant difference at p&lt;0.01 or p&lt;0.05. The results are in Table 3.  
                       TABLE 3                                      Group                                 No.   Gene   B   C   D                                         1   TNF-R55   +   −   −       2   TNF-R75   −   −   −       3   TIMP2   −   −   −       4   SODD   −   −   −       5   TACE   −   −   −       6   TNF-α   −   −   −       7   TRAF1   +   +   −       8   TRAF2   −   −   −       9   FAN   −   −   −       10   Caspase-8   −   −   −       11   Caspase-3   −   −   −       12   Caspase-9   −   −   −       13   HIAP1   −   −   −       14   HIAP2   −   −   −       15   Akt-1   +   +   ++       16   Bcl-1   −   −   −       17   Bcl-xL   −   −   −       18   BAD   −   −   −       19   Bax   −   −   −       20   p53   −   −   +       21   Mn-SOD   −   −   −       22   IKK1   −   −   −       23   DR3   +   +   +       24   PKC-β1   −   −   −       25   NF-kBp50   −   −   −       26   NF-kBp65   −   −   −       27   N-SMase   −   −   −       28   ERK1   −   −   −       29   ERK2   −   −   −       30   p38   −   −   −       31   CyclinG1   −   −   −       32   apoptosis   −   −   −           inhibitor 4       33   cdc25b   −   −   −       34   CyclinD1   −   −   −       35   JAB   −   −   −       36   PKR   −   −   −       37   2,5-OA   −   −   −       38   MDR1   −   −   −       39   MDR3   −   −       40   MRP1   −   −   −       41   MRP2   −   −   −       42   MRP3   −   −   −       43   MRP4   −   −   −       44   MRP5   +   +   +       45   MRP6   −   +   ++       46   BCRP   −   −   −       47   COX-2   −   −   −       48   iNOS   −   −   −       49   TGF-β   −   −   −                                          
 
     [0032] As evident from Table 3, the expression levels of Akt-1, DR3, MRP5 and MRP6 genes with respect to the mRNA levels in the cells with a relatively high sensitivity to TNF-α showed a statistically significantly high expression level. FIGS.  1  to  4  show the results of statistical works of all the above expression levels. As shown in FIG. 5, the data on the expression level of Akt-1 gene with respect to the mRNA level in respective established cell lines from intestinal, breast, lung, and pancreatic organs revealed that the cell lines had a higher expression level of Akt-1 gene, resulting in an estimation that the cancers in such organs would have a high potential of being cured by TNF-α.  
     Experiment 3  
     [0033] Cluster Analysis  
     [0034] Based on the profiles of the 49 different types of genes as the results in Experiment 2, the cluster analysis between the genes was conducted by conventional analysis for factors and main ingredients using “EPCLUST”, a commercially available computer software of European Bioinfomatics Institute, Cambridge, UK. The results are in FIG. 6, revealing that Akt-1, MRP5 and MRP6 genes can be classified into the same cluster which exhibits substantially the same dynamics. The data indicates that there exist at least two representative groups of Akt-1 and DR3 genes as gene groups which enable the estimation of therapeutic efficacy of TNF-α.  
     Experiment 4  
     [0035] Expression of Cell Membrane Antigen  
     [0036] In addition to the action of inducing cells to death through the induction of apoptosis, TNF-α has also the action of promoting the expression of ICAM-1 as a cell membrane antigen. It was examined whether there exists any correlation between the expressions of Akt-1 gene and ICAM-1: With reference to the results in Experiment 1, seven types of TNF-α sensitive cells, i.e., those from the lung cancer patient B, HCT-15 cells, RD cells, OBA-LK-1 cells, CoLo 205 cells, CoLo 206 cells, a YMB-1-E cells; and seven types of non-TNF-α-sensitive cells, i.e., those from the colon cancer patient A, the skin cancer patient A, the lung cancer patient E, the neurologic cancer patient A, the hepatoma patient D, PK-45H cells, and MKN-7 cells were selected, and each of which was suspended in an RPMI 1640 medium supplemented with 10% (v/v) of fetal calf serum to give a cell density of 2×10 4  cells/ml. To three milliliters of each of the resulting cell suspensions, placed in a commercialized 6-well-plate, was added 5 ng/ml of a human TNF-α preparation, and the cell suspensions were incubated at 37° C. for 24 hours under 5% CO 2  atmospheric conditions. After culturing, the cells were collected from each culture, and then successively treated with “BBA-3”, a murine anti-human ICAM-1 antibody, commercialized by R &amp; D Systems Inc., Minneapolis, USA, at 4° C. for 40 min, successively washed with a phosphate buffered saline containing 1% (v/v) of calf serum albumin, a fluorescein-labelled anti-mouse immunoglobulin G antibody at 4° C. for 40 min, and 3% (v/v) formalin. The resulting cells were measured for fluorescent intensity on “EPICS XL”, a flow cytometry commercialized by Beckman Coulter Inc., CA, USA, to examine the expression level of ICAM-1 on the surface of cell membranes. The relationship between the data from the flow cytometry and the expression data of Akt-1 gene in Experiment 2 was studied. The results are in FIG. 7. In FIG. 7, the symbols “▾” and “∘” mean TNF-α-sensitive cells and non-TNF-α-sensitive cells, respectively.  
     [0037] As found in FIG. 7, two out of the seven different types of TNF-α-sensitive cells (“▾”) increased in the expression level of ICAM-1 but the resting five types of cells did not show any change in the expression level, while two out of the seven different types of non-TNF-α-sensitive cells (“∘”) did not exhibit any change in the expression level of ICAM-1 but the resting five types of cells increased in the expression level, and this gave no statistically significant relationship between the TNF-α sensitivity and the expression level of ICAM-1. The TNF-α-sensitive cells “▾” was significantly high in the expression level of Akt-1 gene with respect to its corresponding mRNA level, and there was found a relatively high correlation between the TNF-α-sensitivity and the expression level of Akt-1 gene when examined on Mann-Whitney U test. These results show that the estimation of the therapeutic efficacy of TNF-α by examining the expression level of Akt-1 gene is more preferable in estimating the antitumor effect of TNF-α through the induction of apoptosis than by estimating the expression level of Akt-1 gene as one of the various functions and effects of TNF-α.  
     [0038] The following example explain the present invention in detail, but should not limit the present invention:  
     EXAMPLE  
     [0039] Cancer cells, extracted from five patients suffering from lung cancer, were treated in usual manner to collect RNAs. Using the RNAs as templates, their corresponding cDNAs were prepared with a murine breast viral reverse transcriptase. A reaction mixture was prepared by adding to 20 μg of each of the cDNAs as templates 12.5 μg of “SYBR GREEN PCR MASTERMIX” containing a thermostable DNA polymerase etc., commercialized by Applied Biosystems Japan, Inc., Tokyo, Japan, and either 100 nM of primers for detection of Akt-1 mRNA having nucleotide sequences of SEQ ID NOs:29 and 30, or 100 nM of primers for detection of β-actin mRNA having nucleotide sequences of SEQ ID NOs:99 and 100. The resulting reaction mixtures were subjected to 45 cycles of PCR sequentially at 90° C. for 15 sec and at 60° C. for one minute using “ABIPRISM 7700 SEQUENCE DETECTION SYSTEM”, a real time PCR apparatus. With reference to the expression level of β-actin mRNA as an internal standard, the expression level of Akt-1 gene was calculated.  
     [0040] The cells from each of the above five patients suffering from lung cancer were intraperitoneally transplanted to 10 nude mice at a cell density of 1×10 4  cells/head. On two days after the transplantation, five nude mice out of the 10 mice were intravenously administered with 5 ng/head of TNF-α. On three days after the administration, the tumor masses in the mice were measured according to the method disclosed by K. Nakahara in “ International Journal of Medicine ”, Vol. 34, pp. 263-267 (1984) and compared with those of control nude mice with no administration of TNF-α to evaluate the antitumor effect of TNF-α. As a result, the more the expression level of Akt-1 gene with respect to mRNA level the higher the antitumor effect of TNF-α.  
     [0041] As described above, according to the present invention, the therapeutic efficacy of TNF-α on cancer cells will be estimated by measuring the expression level of genes such as of Akt-1, DR3, etc., because the sensitivity of TNF-α on cancer cells highly correlates with the expression level of such genes. Thus, the application of the present invention to patients prior to the administration of TNF-α, the desired therapeutic efficacy of TNF-α will be estimated in a safe and sure manner. The measurement of the expression level of Akt-1 gene is suitable for estimating the antitumor effect of TNF-α among the actions and functions of TNF-α. Thus, the present invention enables the screening of the possibility of pharmaceutical uses of TNF-α which the uses had been deemed actually impossible. In addition, the present invention can be applied to examination of the types of cancers treatable with antitumor agents such as TNF-α.  
     [0042] While there has been described what is at present considered to be the preferred embodiments of the invention, it will be understood the various modifications may be made therein, and it is intended to cover in the appended claims all such modifications as fall within the true spirits and scope of the invention.  
    
     
       
         1 
         
           
             100  
           
           
             1  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TNF-R55 mRNA  
             
           
            1 

cctgccagga gaaacagaac ac                                              22 

 
           
             2  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TNF-R55 mRNA  
             
           
            2 

gggactgaag ctttgggttt gg                                              22 

 
           
             3  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TNF-R75 mRNA  
             
           
            3 

gccccaccag atctgtaacg tg                                              22 

 
           
             4  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TNF-R75 mRNA  
             
           
            4 

tgaggcacct tggcttctct c                                               21 

 
           
             5  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TIMP2 mRNA  
             
           
            5 

gcggtcagtg agaaggaagt gg                                              22 

 
           
             6  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TIMP2 mRNA  
             
           
            6 

ggagatgtag cacgggatca tgg                                             23 

 
           
             7  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of SODD mRNA  
             
           
            7 

acagcccaac tccagtctct                                                 20 

 
           
             8  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of SODD mRNA  
             
           
            8 

aagttgtgcc ggttcatgct                                                 20 

 
           
             9  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TACE(ADAM17) 
      mRNA  
             
           
            9 

ctgcacaggt aatagcagtg ag                                              22 

 
           
             10  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TACE(ADAM17) mRNA  
             
           
            10 

ctcagctggt caatgaaatc cc                                              22 

 
           
             11  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TNF-alpha mRNA  
             
           
            11 

ttctcgaacc ccgagtgaca ag                                              22 

 
           
             12  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TNF-alpha mRNA  
             
           
            12 

cccttctcca gctggaagac c                                               21 

 
           
             13  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TRAF1 mRNA  
             
           
            13 

gcactttcct gtggaagatc acc                                             23 

 
           
             14  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TRAF1 mRNA  
             
           
            14 

ctggccacgt tggtttcact c                                               21 

 
           
             15  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TRAF2 mRNA  
             
           
            15 

aacattgtct gcgtcctgaa cc                                              22 

 
           
             16  
             24  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TRAF2 mRNA  
             
           
            16 

cgttcaggta gatacgcaga caca                                            24 

 
           
             17  
             19  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of FAN mRNA  
             
           
            17 

tgcctctggg cttggaagt                                                  19 

 
           
             18  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of FAN mRNA  
             
           
            18 

tcaggcattt cctggtagcg t                                               21 

 
           
             19  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Caspase-8 mRNA  
             
           
            19 

cacgggagaa agtgcccaaa c                                               21 

 
           
             20  
             24  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Caspase-8 mRNA  
             
           
            20 

ggttgctcct ctgaatcagt ctca                                            24 

 
           
             21  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Caspase-3 mRNA  
             
           
            21 

gctgtctacg gcacatggtg a                                               21 

 
           
             22  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Caspase-3 mRNA  
             
           
            22 

gttgccacct ttcggttaac cc                                              22 

 
           
             23  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Caspase-9 mRNA  
             
           
            23 

gctgtctacg gcacagatgg                                                 20 

 
           
             24  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Caspase-9 mRNA  
             
           
            24 

gatgtcgtcc agggtctcaa c                                               21 

 
           
             25  
             24  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of HIAP1 mRNA  
             
           
            25 

actacatagg acctggagac agag                                            24 

 
           
             26  
             24  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of HIAP1 mRNA  
             
           
            26 

aagtactcac accttggaaa ccac                                            24 

 
           
             27  
             24  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of HIAP2 mRNA  
             
           
            27 

tcagtaactg ggaaccaaag gatg                                            24 

 
           
             28  
             24  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of HIAP2 mRNA  
             
           
            28 

aagtactcac accttggaaa ccac                                            24 

 
           
             29  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Akt-1 mRNA  
             
           
            29 

tgtgtcagcc ctggactacc                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Akt-1 mRNA  
             
           
            30 

tgagcagccc tgaaagcaag                                                 20 

 
           
             31  
             19  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Bcl-2 mRNA  
             
           
            31 

tgcacctgac gcccttcac                                                  19 

 
           
             32  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Bcl-2 mRNA  
             
           
            32 

cacttgtggc ccagataggc a                                               21 

 
           
             33  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Bcl-XL mRNA  
             
           
            33 

aagggactga atcggagatg ga                                              22 

 
           
             34  
             19  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Bcl-XL mRNA  
             
           
            34 

catgcccgtc aggaaccag                                                  19 

 
           
             35  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of BAD mRNA  
             
           
            35 

cgagtgagca ggaagactcc a                                               21 

 
           
             36  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of BAD mRNA  
             
           
            36 

aggagtccac aaactcgtca ct                                              22 

 
           
             37  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Bax mRNA  
             
           
            37 

gagctgcaga ggatgattgc c                                               21 

 
           
             38  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Bax mRNA  
             
           
            38 

ccactgtgac ctgctccaga                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of p53 mRNA  
             
           
            39 

tgcagctgtg ggttgattcc                                                 20 

 
           
             40  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of p53 mRNA  
             
           
            40 

aacacgcacc tcaaagctgt tc                                              22 

 
           
             41  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Mn-SOD mRNA  
             
           
            41 

aacgtcaccg aggagaagta cc                                              22 

 
           
             42  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of Mn-SOD mRNA  
             
           
            42 

cagcagtgga ataaggcctg tt                                              22 

 
           
             43  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of IKK1 mRNA  
             
           
            43 

gcgagcagat gacgtatggg a                                               21 

 
           
             44  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of IKK1 mRNA  
             
           
            44 

gcttacagcc caacaacttg ct                                              22 

 
           
             45  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of DR3 mRNA  
             
           
            45 

actgccaacc atgcctagac tg                                              22 

 
           
             46  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of DR3 mRNA  
             
           
            46 

agagcctcca tcccagcttc                                                 20 

 
           
             47  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of PKC-beta1 mRNA  
             
           
            47 

tgagagggcc aagatcagtc ag                                              22 

 
           
             48  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of PKC-beta1 mRNA  
             
           
            48 

agtacaggcg gtccatggtc                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of NF-?Bp50 mRNA  
             
           
            49 

gcagcgagcc attgcctttc                                                 20 

 
           
             50  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of NF-?Bp50 mRNA  
             
           
            50 

ggtccagcat ggtgaagagt gt                                              22 

 
           
             51  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of NF-?Bp65 mRNA  
             
           
            51 

ctgatgtgca ccgacaagtg g                                               21 

 
           
             52  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of NF-?Bp65 mRNA  
             
           
            52 

gttgatggtg ctcagggatg ac                                              22 

 
           
             53  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of N-Smase mRNA  
             
           
            53 

ggcctctgtg tcttctccaa ac                                              22 

 
           
             54  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of N-Smase mRNA  
             
           
            54 

aagccctgtc cactccttca                                                 20 

 
           
             55  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of ERK1 mRNA  
             
           
            55 

gcaggacctg atggagactg ac                                              22 

 
           
             56  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of ERK1 mRNA  
             
           
            56 

ccagaatgca gcccacagac                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of ERK2 mRNA  
             
           
            57 

gcgctacacc aacctctcgt                                                 20 

 
           
             58  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of ERK2 mRNA  
             
           
            58 

cacggtgcag aacgttagct g                                               21 

 
           
             59  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of p38 mRNA  
             
           
            59 

gccgagctgt tgactggaag                                                 20 

 
           
             60  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of p38 mRNA  
             
           
            60 

ggaggtccct gctttcaaag g                                               21 

 
           
             61  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of cyclin G1 mRNA  
             
           
            61 

ttggcaactg acttgatccg aa                                              22 

 
           
             62  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of cyclin G1 mRNA  
             
           
            62 

caagctcttg ccagaaggtc a                                               21 

 
           
             63  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of apoptosis inhibitor 4 mRNA  
             
           
            63 

cgaggctggc ttcatccact                                                 20 

 
           
             64  
             19  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of apoptosis inhibitor 4 mRNA  
             
           
            64 

acggcgcact ttcttcgca                                                  19 

 
           
             65  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of cdc25B mRNA  
             
           
            65 

gctctgggga agacaaggag aa                                              22 

 
           
             66  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of cdc25B mRNA  
             
           
            66 

tggcacttgc tgtacatgac ga                                              22 

 
           
             67  
             19  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of cyclin D1 mRNA  
             
           
            67 

acgaaggtct gcgcgtgtt                                                  19 

 
           
             68  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of cyclin D1 mRNA  
             
           
            68 

ccgctggcca tgaactgcct                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of JAB mRNA  
             
           
            69 

gtggcagccg acaatgcagt                                                 20 

 
           
             70  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of JAB mRNA  
             
           
            70 

cgaggccatc ttcacgctaa gg                                              22 

 
           
             71  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of PKR mRNA  
             
           
            71 

cgcagccaaa ttagctgttg ag                                              22 

 
           
             72  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of PKR mRNA  
             
           
            72 

ttgctttggg actcacacgt ag                                              22 

 
           
             73  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of 2′,5′-OAS mRNA  
             
           
            73 

cgtgcgctca gcttcgtact                                                 20 

 
           
             74  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of 2′,5′-OAS mRNA  
             
           
            74 

tactgaggtg gcagcttccc a                                               21 

 
           
             75  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MDR1 mRNA  
             
           
            75 

tagcggctct tccaagctca a                                               21 

 
           
             76  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MDR1 mRNA  
             
           
            76 

caacatggtc cagtgccact ac                                              22 

 
           
             77  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MDR3 mRNA  
             
           
            77 

tggccctggt tggaagtagt g                                               21 

 
           
             78  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MDR3 mRNA  
             
           
            78 

agaaggatct tggggttgcg aa                                              22 

 
           
             79  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP1 mRNA  
             
           
            79 

cggaaaccat ccacgaccct aa                                              22 

 
           
             80  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP1 mRNA  
             
           
            80 

tcatgaggaa gtagggccca aa                                              22 

 
           
             81  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP2 mRNA  
             
           
            81 

gtcctggctg gagtcgcttt                                                 20 

 
           
             82  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP2 mRNA  
             
           
            82 

ggcgtccagc acattgtttg g                                               21 

 
           
             83  
             19  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP3 mRNA  
             
           
            83 

tcctggctgg agtcgcttt                                                  19 

 
           
             84  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP3 mRNA  
             
           
            84 

cgtccagcac attgtttggg t                                               21 

 
           
             85  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP4 mRNA  
             
           
            85 

gatcgcagtg actgccctac t                                               21 

 
           
             86  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP4 mRNA  
             
           
            86 

gtggtgaagg tcacaaacac ga                                              22 

 
           
             87  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP5 mRNA  
             
           
            87 

acggaaagag gcacccatga g                                               21 

 
           
             88  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP5 mRNA  
             
           
            88 

tgttcccgct tccttgcttg a                                               21 

 
           
             89  
             20  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP6 mRNA  
             
           
            89 

tggatcgtgg tctgcttcgt                                                 20 

 
           
             90  
             19  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of MRP6 mRNA  
             
           
            90 

ttctcggcca ccagagtgt                                                  19 

 
           
             91  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of BCRP mRNA  
             
           
            91 

gccgtggaac tctttgtggt ag                                              22 

 
           
             92  
             22  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of BCRP mRNA  
             
           
            92 

acagccaaga tgcaatggtt gt                                              22 

 
           
             93  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of COX-2 mRNA  
             
           
            93 

agtccctgag catctacggt ttg                                             23 

 
           
             94  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of COX-2 mRNA  
             
           
            94 

gaaggggatg ccagtgatag agg                                             23 

 
           
             95  
             21  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of iNOS mRNA  
             
           
            95 

ccccacctca ggaaaacagt c                                               21 

 
           
             96  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of iNOS mRNA  
             
           
            96 

ctctgtgtcc ttgagctggt aag                                             23 

 
           
             97  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TGF-beta mRNA  
             
           
            97 

gcaacaattc ctggcgatac ctc                                             23 

 
           
             98  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of TGF-beta mRNA  
             
           
            98 

agttcttctc cgtggagctg aag                                             23 

 
           
             99  
             23  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of beta-actin mRNA  
             
           
            99 

gtaccactgg catcgtgatg gac                                             23 

 
           
             100  
             24  
             DNA  
             Artificial  
             
               Oligonucleotide used as primer for PCR 
      detection of beta-actin mRNA  
             
           
            100 

gctcattgcc aatggtgatg acct                                            24