JPWO2005007846A1 - Method for determining the sensitivity of tumor cells to anticancer agents - Google Patents

Abstract

According to the present invention, all or part of a cancer cell line selected from a breast cancer cell line, a liver cancer cell line, and a gastric cancer cell line is used, and the expression level of at least one gene in the cell line and the sensitivity to an anticancer agent are used. By measuring the strength of the tumor cells and statistically examining the presence or absence of their correlation, a method for identifying a gene group involved in the sensitivity of tumor cells to the anticancer drug used is provided. According to the present invention, genes that predict susceptibility to anticancer agents in tumor cells and cancer cell lines extracted from cancer patients can be selected, and the sensitivity of cancer patients to anticancer agents using these genes can be determined. .

Description

  The present invention relates to a method for identifying a gene group related to the sensitivity of tumor cells to an anticancer agent, a method for determining the sensitivity of cancer patients to an anticancer agent, and a method for screening a substance that enhances the sensitivity of tumor cells to an anticancer agent.

Cancer chemotherapy with various anticancer agents is one of the most important methods in the treatment of cancer. In order to perform effective treatment with an anticancer drug, it is important to predict the sensitivity of the patient to the anticancer drug and select an appropriate drug. Finding genes that determine susceptibility to anticancer drugs is one way to predict susceptibility. To date, many genes have been reported as determinants of sensitivity to multiple anticancer agents. For example, drug transporters MDR1 (Chen, CJ, et. Al., J. Biol. Chem., 265, 506-514, 1990), MRP2 (Kool, M, et. Al., Cancer Res) , 57, 3537-3547, 1997; and Taniguchi, K., Cancer Res., 56, 4124-4129, 1996), the cytochrome P450 (CYP) family of drug metabolizing enzymes (Patterson, L. H. et al.). and Murray, GI, Curr. Pharm. Des., 8, 1335-1347, 2002), glutathione-S-transferase (GST) (Bastist, G., et.al., J. Biol. Chem.,). 261 15544-15549,1986), N-acetyltransferase (Meisel, P., Pharmacogenomics, 3,349-366,2002), and the like. Further, for example, the following genes have been reported as the genes defining the sensitivity to each anticancer agent. γ-glutamyl hydrolase (Nair, MG, et. al., Biochemistry, 12, 3923-3927, 1973) and dihydrofolate ruductase (Schimke, RT, Cancer, 57, 1912-19x, t It is a resistance factor against (MTX). Moreover, thymylylate synthase (Pinedo, HM and Peters, GF, J. Clin. Oncol., 6,1653-1664, 1988), metallotionein (Eastman, A., Cancer, Treat Res., 57, 233-249, 1991), and cytidine deaminase (Cohen, SS, Cancer, 40, 509-518, 1977) are enhanced by 5-fluorouracile (5-FU), cisplatin (CDDP), and ara-C, respectively. Is a resistance factor against. Enhancement of DT-diaphorase activity is a susceptibility factor for mitomycin C (hereinafter also abbreviated as MMC) (Gustafson, DL and Pritosos, CA, Cancer Res., 52, 6936-6939, 1992). Thus, many genes are known as genes that determine sensitivity to anticancer agents. However, these known genes alone are insufficient to explain susceptibility to anticancer drugs and further research is needed.
On the other hand, attempts to predict sensitivity to anticancer agents or genes involved in sensitivity to anticancer agents using genome-wide analysis such as cDNA microarrays and SNPs are also progressing. Analysis of the correlation between gene expression in human cancer cell line panels and human cancer xenografts and susceptibility to anticancer agents has been reported, including the inventors' reports (Scherf, U., et. Al., Nat. Genet). Zembutsu, H., et al, Cancer Res., 62, 518-527, 2002; Dan, S., et al, Cancer Res., 62, 1139-1147 Sutton, JE et al., Proc. Natl. Acad. Sci. USA, 98, 10787-10792, 2001; and JP-A-2003-61678). The genes found in these studies can be used as markers for predicting susceptibility to anticancer drugs. In addition, some of these genes may actually determine susceptibility to anticancer drugs.

The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to select a gene that correlates with sensitivity to an anticancer agent with higher accuracy by providing a set of cell lines suitable for selection of a sensitivity-defining gene for an anticancer agent and performing analysis for each organ. One of them is to select genes that are actually involved in determining sensitivity to anticancer drugs. Still another object of the present invention is to provide a method for determining the sensitivity of a cancer patient to an anticancer agent using a gene involved in the determination of the sensitivity to the anticancer agent selected by the above method. Still another object of the present invention is to provide a sensitivity enhancer for an anticancer drug using a gene that exhibits an activity of enhancing the sensitivity to an anticancer drug by forcibly expressing or suppressing the expression in cancer cells among the genes selected by the above method. It is to be. Still another object of the present invention is to provide a method of screening for a substance that enhances the sensitivity of tumor cells to an anticancer agent using the gene selected by the above method.
The present inventors have intensively studied to solve the above problems, and firstly constructed a new human cancer cell panel composed of 45 cell lines derived from three kinds of tissues (ie, breast, liver and stomach) ( Tables 1-5). Furthermore, in these cancer cell panels composed of 45 cells, the sensitivity to anticancer agents and gene expression profiles were obtained and evaluated, and genes whose expression correlated with the sensitivity to anticancer agents were selected (Tables 6 to 11). Further, among these genes, genes that change the sensitivity to anticancer agents at the cellular level were screened. As a result, the present inventors succeeded in identifying a gene that can be a marker for predicting sensitivity to an anticancer drug. Moreover, it became possible to provide the method of determining the sensitivity with respect to the anticancer agent of a cancer patient by investigating the expression of those genes. Furthermore, it has become possible to provide a method for enhancing the sensitivity to anticancer agents by increasing or decreasing the expression of those genes, and a method for screening for substances that enhance the sensitivity to these anticancer agents. The present invention has been completed based on these findings.
That is, the present invention relates to the following (1) to (64).
(1) Breast cancer 12 cell lines (HBC-4, BSY-1, HBC-5, MCF-7, MDA-MB-231, KPL-3C, KPL-4, KPL-1, T-47D, HBC-9, ZR-75-1, HBC-8), liver cancer 12 cell line (HepG2, Hep3B, Li-7, PLC / PRF / 5, HuH7, HLE, HLF, HuH6, RBE, SSP-25, HuL-1, JHH- 1), and gastric cancer 21 cell lines (St-4, MKN1, MKN7, MKN28, MKN45, MKN74, GCIY, GT3TKB, HGC27, AZ521, 4-1st, NUGC-3, NUGC-3 / 5FU, HSC-42, AGS , KWS-1, TGS-11, GMK-2 (also known as OKIBA), Ist-1, GMK-3, GMK-5 (also known as AOTO)) By using all or part of a cell line, measuring the expression level of at least one gene in the cell line and the strength of sensitivity to an anticancer agent, and statistically testing the presence or absence of the correlation between them, tumor cells Of identifying genes related to susceptibility to anti-cancer drugs used by the drug.
(2) Gastric cancer 21 cell line (St-4, MKN1, MKN7, MKN28, MKN45, MKN74, GCIY, GT3TKB, HGC27, AZ521, 4-1st, NUGC-3, NUGC-3 / 5FU, HSC-42, AGS, Gastric cancer using all or part of a cancer cell line selected from KWS-1, TGS-11, GMK-2 (also known as OKIBA), Ist-1, GMK-3, GMK-5 (also known as AOTO)) The method according to (1), wherein a gene group related to the sensitivity of the cell to the anticancer agent used is identified.
(3) Breast cancer 12 cell lines (HBC-4, BSY-1, HBC-5, MCF-7, MDA-MB-231, KPL-3C, KPL-4, KPL-1, T-47D, HBC-9, (1) The method according to (1), wherein a gene group related to sensitivity to an anticancer drug used by breast cancer cells is identified using all or part of a cancer cell line selected from ZR-75-1 and HBC-8).
(4) Cancer cells selected from liver cancer 12 cell lines (HepG2, Hep3B, Li-7, PLC / PRF / 5, HuH7, HLE, HLF, HuH6, RBE, SSP-25, HuL-1, JHH-1) The method according to (1), wherein a gene group related to the sensitivity of the hepatoma cells to the anticancer agent used is identified using all or part of the strain.
(5) In a method for determining the sensitivity of a cancer patient to an anticancer agent,
(1) One or more genes are selected from the gene group identified by the method according to any one of (1) to (4) above, and a score corresponding to the expression level is selected for each of the selected genes. The process of determining,
(2) measuring the expression level of the gene for the test patient;
(3) assigning the score obtained in (1) based on the expression level measured in (2),
(4) a step of totaling the assigned points for all selected genes to obtain a total value; and
(5) a step of comparing the total value with a predetermined threshold value;
Including the above method.
(6) In the method for determining the sensitivity of a cancer patient to mitomycin C, SF1 (D26121), CBR3 (AB004854), EMS1 (M98343), JUN (J04111), SFRS9 (U30825) in tumor cells removed from the cancer patient , NMBR (M73482), RBMX (Z23064), SOD1 (M13267), NOL1 (X55504), PELP1 (U88153), ARHA (L25080), AARS (D32050), NME1 (X17620), HNRPA2B1 (M29065), NME2 (L2916), 78 , VAT1 (U18009), SERPINB10 (U35459), KIAA0436 (AB007896), DRPLA (D31840), MC3R (L06155), S PTBN1 (M96803), PET112L (AF026851), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770), RPLP2 (M17887), and ARF4 A method of determining the sensitivity to mitomycin C using the method described in (5) by measuring the expression level of one or more genes selected from the group consisting of (indicated by GenBank registration number in parentheses).
(7) In the method for determining the sensitivity of a cancer patient to vinorelbine, ARHA (L25080), NME2 (L16785), VIL2 (X51521), YWHAQ (X56468), HK1 (M75126) in tumor cells removed from the cancer patient SATB1 (M97287), CAMLG (U18242), CARS (L06845), CCNB1 (M25753), U2AF1 (M96982), PTMA (M26708), MLC1SA (M31212), NME1 (X17620), SARS (X91257), CDC20 (05) PPP4C (X70218), TNFAIP3 (M59465), EEF1D (Z21507), PFKP (D25328), ENTPD2 (U91510), CCL5 (M2 1121), ACAT1 (D90228), IQGAP1 (L33075), PAX5 (M96944), NRGN (Y09689), K-ALPHA-1 (K00558), NDUFB7 (M33374), HOXB1 (X16666), F10 (K03194), GPX2X ), NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (M36263), MAN2B1 (U60266), LSS (D63807), PIK3CG2 ), NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X6065) ), F2 (V00595), RARA (X06614), ITGB4 (X53587), IMPA1 (X66922), PACE (X17094), AGA (M64073), MVD (U49260), EHHADH (L07077), TFPI2 (D29992), MARCKS (68) ), FGB (J00129), and GPD1 (L34041), one or a plurality of genes selected from the group (in parentheses indicate GenBank registration numbers), and the method described in (5) A method for determining sensitivity to vinorelbine.
(8) In the method for determining the sensitivity of a cancer patient to paclitaxel, ADH6 (M68895), RAB28 (X94703), U2AF1 (M96982), GPC1 (X54232), HK1 (M75126) in tumor cells removed from the cancer patient, CARS (L06845), TNFAIP3 (M59465), K-ALPHA-1 (K00558), PFKP (D25328), GDI2 (D13988), VIL2 (X51521), RUNX2 (AF001450), NME2 (L16785), CDC20 (U05340), G052 (X04828), ARHA (L25080), CNR2 (X74328), PPP2R2B (M64930), SLC6A8 (L31409), DDX9 (L13848), A CAT1 (D90228), PI3 (Z18538), NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383), RPIN, RP4 AGA (M64073), BCL2L1 (Z23115), LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RA, 66, RA, 66, PTPRN (L18983), APOE (M12529), F10 (K03194), NR1I2 (AF061056), UBE2 3 (X92962) and one or more genes selected from the group consisting of FGB (J00129) (the parentheses indicate GenBank registration numbers) and the method described in (5) A method for determining sensitivity to paclitaxel.
(9) In a method for determining the sensitivity of a cancer patient to 5-fluorouracil, SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932) in tumor cells removed from the cancer patient ), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661), PLOD (M98252), CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X016673), PAFAH1B3 ), GBE1 (L07956), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and UBE2E1 (X92963) A method of measuring the expression level of one or a plurality of genes selected from the group consisting of (shown in GenBank registration number in parentheses) and determining the sensitivity to 5-fluorouracil using the method described in (5).
(10) In the method for determining the sensitivity of a cancer patient to doxorubicin, RPS7 (M77233), RPL23 (X52839), CLT (X72946), EEFID (Z21507), RPS15A (X84407), in tumor cells removed from the cancer patient, Expression level of one or more genes selected from the group consisting of ADCY1 (L05500), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232) (indicated by GenBank registration number in parentheses) And measuring the sensitivity to doxorubicin using the method described in (5).
(11) In a method for determining the sensitivity of a cancer patient to cisplatin, CALT (X72946), ZNF165 (X84801), GPC1 (X54232), TUBA1 (K00558), GCP2 (X78686), in tumor cells removed from the cancer patient, The expression level of one or a plurality of genes selected from the group consisting of COX5A (M22760), GLA (X00351) and GNS (X0695) (indicated by the GenBank registration number in parentheses) is measured, A method for determining sensitivity to cisplatin using the method.
(12) The method for determining sensitivity according to any one of (5) to (11), wherein the patient is a breast cancer patient.
(13) The method according to any one of (5) to (11), wherein the patient is a stomach cancer patient.
(14) The method according to any one of (5) to (11), wherein the patient is a liver cancer patient.
(15) In a method for determining the sensitivity of breast cancer patients to mitomycin C, INHBB (M31682), NK4 (M59807), HSPA1A (M11717), LOC54557 (AF075050), CD47 (Y00815) in tumor cells removed from breast cancer patients One selected from the group consisting of: RPN2 (Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950) and NRGN (Y09689) A method of measuring the expression level of a plurality of genes (indicated by GenBank registration numbers in parentheses) and determining the sensitivity to mitomycin C using the method described in (5).
(16) In the method for determining the sensitivity of a liver cancer patient to mitomycin C, EB1 (U24166), JUN (J04111), EIF3S8 (U46025), CCL5 (M21121), PHB (S85655) in tumor cells extracted from the liver cancer patient , HSPA1A (M11717), SPP1 (X13694), RAB7 (X93499), CTSD (M11233), ACTN1 (X15804), RXRB (M84820), PSME2 (D45248), HLA-C (M11886), RPL19 (X63527), MAPK X80692), GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M5) 7627), RFC4 (M87339), CRLF1 (AF059293), RPS6 (M20020), EMX1 (X68879), and TK2 (U77088), one or a plurality of genes (the parentheses indicate GenBank registration numbers) A method for determining the sensitivity to mitomycin C using the method according to (5).
(17) In a method for determining the sensitivity of a stomach cancer patient to mitomycin C, TEAD4 (U63824), NR2C2 (U10990), CSF1 (M37435), RAB28 (X94703), CBR3 (AB004854) in tumor cells removed from the stomach cancer patient , NFYC (Z74792), PGF (X54936), ERG (M21535), MLLT1 (L04285), FOS (K00650), TNFAIP3 (M59465), CNR2 (X74328), DRPLA (D31840), PSMB5 (D29011), SLC6A , SERPINB10 (U35459), VAT1 (U18009), TJP1 (L14837), PELP1 (U88153), C1QBP (L04636) , CDK10 (L33264), SERPINA6 (J02943), ACTB (X00351), SFRP4 (AF026992), EMX1 (X68879), RPS9 (U14971), AMD1 (M21154), RPL26 (X69392), HNRPF (L28010), 98MS STK12 (AF008552), NR2F6 (X12794), GBE1 (L07956), PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD2335 SDHA (D30648), PET112L (AF026851), DAD1 (D15057), HSPB1 X54079), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M9683), PACE (X17094), RPL24 (M94314), SPINT2 (X954) U07158), SIAT8B (U33551), CTSK (U13665), DCI (U24774), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D380) The expression level of one or a plurality of genes selected from (the parentheses indicate GenBank registration numbers) is measured, and the method according to (5) Method of determining sensitivity to mitomycin C with.
(18) In a method for determining the sensitivity of a liver cancer patient to paclitaxel, ALDH3B1 (U10868), SRPK1 (U09564), ALPP (M13077), EB1 (U24166), MAPKAPK (U12779) in tumor cells extracted from the liver cancer patient, GPI (K03515), CTBP1 (U37408), HMGCL (L07033), CAPZ (U03271), ACAT1 (D90228), COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J04568), SCD (Y13647) From YWHAZ (M86400), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 (M87338) A method of measuring the expression level of one or a plurality of genes selected from the group (indicated by GenBank registration number in parentheses) and determining the sensitivity to paclitaxel using the method described in (5).
(19) In a method for determining the sensitivity of a liver cancer patient to 5-fluorouracil, SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932) in tumor cells extracted from the liver cancer patient. ), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661), PLOD (M98252), CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X016673), PAFAH1B3 ), GBE1 (L07956), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and UBE2E1 (X9296) 3) measure the expression level of one or more genes selected from the group consisting of (the GenBank registration number is shown in parentheses), and determine the sensitivity to 5-fluorouracil using the method described in (5) Method.
(20) In the method for determining the sensitivity of a liver cancer patient to doxorubicin, RPS7 (M77233), RPL23 (X52839), CLT (X72946), EEF1D (Z21507), RPS15A (X84407) in tumor cells taken from the liver cancer patient Expression level of one or more genes selected from the group consisting of ADCY1 (L05500), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232) (indicated by GenBank registration number in parentheses) And measuring the sensitivity to doxorubicin using the method described in (5).
(21) In the method for determining the sensitivity of a liver cancer patient to cisplatin, CALT (X72946), ZNF165 (X84801), GPC1 (X54232), TUBA1 (K00558), GCP2 (X78686), in tumor cells removed from the liver cancer patient, The expression level of one or a plurality of genes selected from the group consisting of COX5A (M22760), GLA (X00351) and GNS (X0695) (indicated by the GenBank registration number in parentheses) is measured, A method for determining sensitivity to cisplatin using the method.
(22) The method according to any one of (1) to (21), wherein the gene expression level is measured by quantifying RNA, which is a transcription product of the gene, using a DNA microarray.
(23) The method according to any one of (1) to (21), wherein the expression level of the gene is measured by quantifying RNA, which is a transcription product of the gene, by quantitative PCR.
(24) The method according to any one of (1) to (21), wherein the expression level of the gene is measured by quantifying RNA, which is a transcription product of the gene, by Northern blotting.
(25) An RNA quantitative reagent containing an oligonucleotide complementary to the RNA for use in the method according to (23).
(26) The method according to any one of (1) to (21), wherein the expression level of the gene is measured by quantifying a protein that is a gene product of the gene by an immunochemical method.
(27) The method according to (26), wherein the expression level of the gene is measured by quantifying a protein that is a gene product of the gene by ELISA.
(28) The method according to (26), wherein the expression level of the gene is measured by quantifying a protein that is a gene product of the gene by Western blot.
(29) An immunoassay reagent comprising an antibody against the protein for use in the method according to (26).
(30) SF1 (D26121), CBR3 (AB004854), EMS1 (M98343), JUN (J04111), SFRS9 (U30825), NMBR (M73482), RBMX (Z23064), SOD1 (M13267), NOL1 (X55504), PELP1 ( U88153), ARHA (L25080), AARS (D32050), NME1 (X17620), HNRPA2B1 (M29065), NME2 (L16785), VAT1 (U18009), SERPINB10 (U35459), KIAA0436 (AB00783MC), DR40D L06155) is a single gene or a plurality of genes selected from the group consisting of (GenBank accession numbers in parentheses) and strong against cancer cells A sensitivity-enhancing drug for mitomycin C, which contains a gene exhibiting an activity of enhancing the sensitivity to mitomycin C by expressing it or a protein encoded by the gene.
(31) ARHA (L25080), NME2 (L16785), VIL2 (X51521), YWHAQ (X56468), HK1 (M75126), SATB1 (M97287), CAMLG (U18242), CARS (L06845), CCNB1 (M25753), U2 M96982), PTMA (M26708), MLC1SA (M3 21111), NME1 (X17620), SARS (X91257), CDC20 (U05340), PPP4C (X70218), TNFAIP3 (M59465), EEF1D (Z21507), PFKP (D253), 2853 U91510), CCL5 (M21121), ACAT1 (D90228), IQGAP1 (L33075), PAX5 (M96944), A single gene or a plurality of genes selected from the group consisting of NRGN (Y09688), K-ALPHA-1 (K00558), NDUFB7 (M33374) (indicated by GenBank registration number in parentheses), and forced expression in cancer cells A sensitizer for enhancing sensitivity to vinorelbine, which contains a gene exhibiting an activity to enhance sensitivity to vinorelbine or a protein encoded by the gene.
(32) ADH6 (M68895), RAB28 (X94703), U2AF1 (M96982), GPC1 (X54232), HK1 (M75126), CARS (L06845), TNFAIP3 (M59465), K-ALPHA-1 (K00558), PFKP (D25328) ), GDI2 (D13988), VIL2 (X51521), RUNX2 (AF001450), NME2 (L16785), CDC20 (U05340), GNAI2 (X04828), ARHA (L25080), CNR2 (X74328), PPP2R2B (M64930), LC6489 ), DDX9 (L13848), ACAT1 (D90228), and PI3 (Z18538). A sensitivity enhancer for paclitaxel containing a gene or protein encoded by a gene that exhibits an activity to enhance sensitivity to paclitaxel by forced expression in cancer cells.
(33) SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661) and PL M98252) is a gene or a plurality of genes selected from the group consisting of (GenBank accession number in parentheses), and a gene exhibiting an activity to enhance sensitivity to 5-fluorouracil by forced expression in cancer cells or the gene A drug for enhancing sensitivity to 5-fluorouracil containing a protein encoded by a gene.
(34) A single gene or a plurality of genes selected from the group consisting of RPS7 (M77233), RPL23 (X52839), CALT (X72946), EEF1D (Z21507), and RPS15A (X84407) (indicated by GenBank registration numbers in parentheses) A drug for enhancing sensitivity to doxorubicin, which comprises a gene exhibiting an activity of enhancing sensitivity to doxorubicin by forced expression in cancer cells or a protein encoded by the gene.
(35) A single gene or a plurality of genes selected from the group consisting of CALT (X72946), ZNF165 (X84801), and GPC1 (X54232) (indicated by the GenBank registration number in parentheses) are forced to be expressed in cancer cells A drug for enhancing sensitivity to cisplatin, which comprises a gene exhibiting an activity for enhancing sensitivity to cisplatin or a protein encoded by the gene.
(36) A single gene or a plurality of genes selected from the group consisting of INHBB (M31682), NK4 (M59807), HSPA1A (M11717), LOC54557 (AF0775050) and CD47 (Y00815) (indicated by GenBank registration numbers in parentheses) A breast cancer mitomycin C sensitivity-enhancing drug comprising a gene that exhibits an activity of enhancing sensitivity to mitomycin C by forced expression in cancer cells or a protein encoded by the gene.
(37) EB1 (U24166), JUN (J04111), EIF3S8 (U46025), CCL5 (M21121), PHB (S85655), HSPA1A (M11717), SPP1 (X13694), RAB7 (X93499), CTSD (M11233), ACTN1 X15804), RXRB (M84820), PSME2 (D45248), HLA-C (M11886) and RPL19 (X63527), or a single gene or a plurality of genes (indicated by GenBank accession numbers in parentheses), A drug for enhancing susceptibility to mitomycin C of liver cancer, comprising a gene showing an activity of enhancing sensitivity to mitomycin C by forced expression in cancer cells or a protein encoded by the gene.
(38) TEAD4 (U63824), NR2C2 (U10990), CSF1 (M37435), RAB28 (X94703), CBR3 (AB004854), NFYC (Z74792), PGF (X54936), ERG (M21535), MLLT1 (L04285), F K00650), TNFAIP3 (M59465), CNR2 (X74328), DRPLA (D31840), PSMB5 (D29011), SLC6A8 (L31409), SERPINB10 (U35459), VAT1 (U18009), TJP1 (L14837U, QB3U), PELP1U L04636), CDK10 (L33264), SERPINA6 (J02943), ACTB (X00351), SFRP4 (AF 026992), EMX1 (X68879), RPS9 (U14971), AMD1 (M21154), RPL26 (X69392), HNRPF (L28010), PTMS (M24398), STK12 (AF008552), NR2F6 (X12794) and GBE1 (L07956) A gene or a protein encoded by a gene or a protein encoded by the gene, which exhibits an activity to enhance sensitivity to mitomycin C by forced expression in a cancer cell. A drug for enhancing sensitivity to mitomycin C of gastric cancer.
(39) ALDH3B1 (U10868), SRPK1 (U09564), ALPP (M13077), EB1 (U24166), MAPKAPK (U12779), GPI (K03515), CTBP1 (U37408), HMGCL (L07033), CAPZ (U03271) D90228) is a single gene or a plurality of genes selected from the group consisting of (GenBank accession numbers in parentheses), and a gene exhibiting an activity to enhance sensitivity to paclitaxel by forced expression in cancer cells or the gene A drug for enhancing sensitivity to paclitaxel of liver cancer, comprising a protein to be encoded.
(40) SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661) and PL M98252) is a gene or a plurality of genes selected from the group consisting of (GenBank accession number in parentheses), and a gene exhibiting an activity to enhance sensitivity to 5-fluorouracil by forced expression in cancer cells or the gene A drug for enhancing sensitivity of liver cancer to 5-fluorouracil, comprising a protein encoded by a gene.
(41) A single gene or a plurality of genes selected from the group consisting of RPS7 (M77233), RPL23 (X52839), CALT (X72946), EEF1D (Z21507), and RPS15A (X84407) (indicated by GenBank registration numbers in parentheses) A drug for enhancing sensitivity to doxorubicin in liver cancer, which comprises a gene exhibiting an activity of enhancing sensitivity to doxorubicin by forced expression in cancer cells or a protein encoded by the gene.
(42) A single gene or a plurality of genes selected from the group consisting of CALT (X72946), ZNF165 (X84801) and GPC1 (X54232) (indicated by the GenBank registration number in parentheses), and forcedly expressed in cancer cells A drug for enhancing the sensitivity of liver cancer to cisplatin, which comprises a gene exhibiting an activity of enhancing the sensitivity to cisplatin or a protein encoded by the gene.
(43) A sensitivity enhancer for mitomycin C, which comprises the gene HSPA1A (GenBank accession number: M11717) or a protein encoded by the gene.
(44) A drug for enhancing sensitivity to mitomycin C, which comprises a gene JUN (GenBank accession number: J04111) or a protein encoded by the gene.
(45) A sensitivity enhancer for mitomycin C, which comprises the gene CTSD (GenBank accession number: M11233) or a protein encoded by the gene.
(46) adding a test substance to the cell,
SF1 (D26121), CBR3 (AB004854), EMS1 (M98343), JUN (J04111), SFRS9 (U30825), NMBR (M73482), RBMX (Z23064), SOD1 (M13267), NOL1 (X55504), PELP1 (U88153) ARHA (L25080), AARS (D32050), NME1 (X17620), HNRPA2B1 (M29065), NME2 (L16785), VAT1 (U18009), SERPINB10 (U35459), KIAA0436 (AB007896), DRPLA (D31840L and MC3D3840L)
ARHA (L25080), NME2 (L16785), VIL2 (X51521), YWHAQ (X56468), HK1 (M75126), SATB1 (M97287), CAMLG (U18242), CARS (L06845), CCNB1 (M25753), U2AF1 PTMA (M26708), MLC1SA (M31212), NME1 (X17620), SARS (X91257), CDC20 (U05340), PPP4C (X70218), TNFAIP3 (M59465), EEF1D (Z21507), PFKP (D25328), PD25328U, PD CCL5 (M21121), ACAT1 (D90228), IQGAP1 (L33075), PAX5 (M96944), NRGN Y09689), K-ALPHA-1 (K00558) and NDUFB7 (M33374);
ADH6 (M68895), RAB28 (X94703), U2AF1 (M96982), GPC1 (X54232), HK1 (M75126), CARS (L06845), TNFAIP3 (M59465), K-ALPHA-1 (K00558), PFKP (D25328), G2328 (D13988), VIL2 (X51521), RUNX2 (AF001450), NME2 (L16785), CDC20 (U05340), GNI2 (X04828), ARHA (L25080), CNR2 (X74328), PPP2R2B (M6499), X1409D (L13848), ACAT1 (D90228) and PI3 (Z18538);
INHBB (M31682), NK4 (M59807), HSPA1A (M11717), LOC54557 (AF0775050) and CD47 (Y00815);
EB1 (U24166), JUN (J04111), EIF3S8 (U46025), CCL5 (M21121), PHB (S85655), HSPA1A (M11717), SPP1 (X13694), RAB7 (X93499), CTSD (M11233), ACTN1 (X15) RXRB (M84820), PSME2 (D45248), HLA-C (M11886) and RPL19 (X63527);
TEAD4 (U63824), NR2C2 (U10990), CSF1 (M37435), RAB28 (X94703), CBR3 (AB004854), NFYC (Z74792), PGF (X54936), ERG (M21535), MLLT1 (L04285), FOS (00654) TNFAIP3 (M59465), CNR2 (X74328), DRPLA (D31840), PSMB5 (D29011), SLC6A8 (L31409), SERPINB10 (U35459), VAT1 (U18409), TJP1 (L14837), PELP1 (U881436), PLP1 (U881436) CDK10 (L33264), SERPINA6 (J02943), ACTB (X00351), SFRP4 (AF0266) 2), EMX1 (X68879), RPS9 (U14971), AMD1 (M21154), RPL26 (X69392), HNRPF (L28010), PTMS (M24398), STK12 (AF008552), NR2F6 (X12794) and GBE1 (L07956);
ALDH3B1 (U10868), SRPK1 (U09564), ALPP (M13077), EB1 (U24166), MAPKAPK (U12779), GPI (K03515), CTBP1 (U37408), HMGCL (L07033), CAPZ (U03271) and ACAT28D90
SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661) and PLODM;
RPS7 (M77233), RPL23 (X52839), CALT (X72946), EEF1D (Z21507) and RPS15A (X84407); and
CALT (X72946), ZNF165 (X84801) and GPC1 (X54232)
The expression level of at least one gene selected from the group consisting of (indicated by GenBank registration number in parentheses) is measured, and the expression level of the gene is compared with the expression level when the test substance is not added A method for screening a substance that enhances sensitivity to an anticancer agent, wherein the substance to be increased is selected as a candidate substance that enhances sensitivity to the anticancer agent.
(47) A substance that enhances sensitivity to an anticancer agent obtained by the method according to (46).
(48) A sensitivity enhancer for an anticancer agent, comprising the substance according to (47).
(49) SPTBN1 (M96803), PET112L (AF0266871), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770), RPLP2 (M17887L) (L38490) is a single gene or a plurality of genes selected from the group consisting of (GenBank accession numbers in parentheses), and the activity of enhancing the sensitivity to mitomycin C by suppressing the expression of the genes in cancer cells SiRNA or antisense polynucleotide which suppresses the expression of the gene which shows.
(50) HOXB1 (X16666), F10 (K03194), GPX2 (X53463), NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (3643), HSD17B1 (3643) U60266), LSS (D63807), PIK3CG (X83368), DBN1 (U00802), NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X60656), F2 (V00595), RARA (X0664), RARA (X06614) X53587), IMPA1 (X66922), PACE (X17094), AGA (M64073), MVD (U49260) ), EHHADH (L07077), TFPI2 (D29992), MARCKS (M68856), FGB (J00129), and GPD1 (L34041), or a single gene or a plurality of genes (indicated by the GenBank registration number in parentheses) An siRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing sensitivity to vinorelbine by suppressing the expression of the gene in cancer cells.
(51) NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383), SERPINF2 (D00174), AGA (M6403), AGA (M6403) Z23115), LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RARA (X06614), PTPRN (L18983) M12529), F10 (K03194), NR1I2 (AF061056), UBE2L3 (X92962), and FGB (J001) 29) a single gene or a plurality of genes selected from the group consisting of (GenBank registration number is shown in parentheses), and shows the activity of enhancing the sensitivity to paclitaxel by suppressing the expression of the gene in cancer cells SiRNA or antisense polynucleotide that suppresses gene expression.
(52) A single member selected from the group consisting of RPN2 (Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950) and NRGN (Y09687) Or a plurality of genes (indicated by GenBank accession numbers in parentheses), which suppresses the expression of the genes in cancer cells, thereby suppressing the expression of genes exhibiting an activity to enhance sensitivity to mitomycin C Sense polynucleotide.
(53) MAPK6 (X80692), GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M57627), RFC4 (M87339), CRLF1 (AF059293), SRP659293 M20020), EMX1 (X68879) and TK2 (U77088) are selected from the group consisting of a single gene or a plurality of genes (indicated by the GenBank registration number in parentheses) by suppressing the expression of the gene in cancer cells , SiRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity to enhance sensitivity to mitomycin C.
(54) PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD23A (D21235), SDHA (D30648), PET112L (AF026851), DAD1 D15057), HSPB1 (X54079), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M96803), PACE (X170942), RPL24 (M94), 14 U78095), STX4A (U07158), SIAT8B (U33551), CTSK (U13665), DCI Single or multiple genes selected from the group consisting of (U24774), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D38048) (GenBank in parentheses) An siRNA or an antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing sensitivity to mitomycin C by suppressing the expression of the gene in cancer cells.
(55) COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J0469), SCD (Y13647), YWHAZ (M86400), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 M87338) is a single gene or a plurality of genes selected from the group consisting of (GenBank accession numbers are shown in parentheses), and shows the activity of enhancing the sensitivity to paclitaxel by suppressing the expression of the genes in cancer cells SiRNA or antisense polynucleotide that suppresses gene expression.
(56) CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01677), PAFAH1B3 (D63391), GBE1 (L07756), DEK (X64229), PEA15 (X86809) PSMD7 (D3E921 and UBE1) ) One or a plurality of genes selected from the group consisting of (the GenBank registration number is shown in parentheses), and the activity of enhancing the sensitivity to 5-fluorouracil by suppressing the expression of the genes in cancer cells SiRNA or antisense polynucleotide which suppresses the expression of the gene which shows.
(57) One or more genes selected from the group consisting of ADCY1 (L05500), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982), and RPYR1 (U35232) (indicated by the GenBank registration number in parentheses) An siRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing the sensitivity to doxorubicin by suppressing the expression of the gene in cancer cells.
(58) One or more genes selected from the group consisting of TUBA1 (K00558), GCP2 (X78686), COX5A (M22760), GLA (X00351), and GNS (X0956) (indicated by the GenBank registration number in parentheses) An siRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing sensitivity to cisplatin by suppressing the expression of the gene in cancer cells.
(59) A vector for expressing the siRNA or antisense polynucleotide according to any one of (49) to (58).
(60) A sensitivity enhancer for an anticancer agent, comprising the siRNA or antisense polynucleotide according to any one of (49) to (58) or the vector according to (59).
(61) SPTBN1 (M96803), PET112L (AF0266871), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770), RPLP2 (M17887L) (L38490);
HOXB1 (X16666), F10 (K03194), GPX2 (X53463), NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (M36263), HSD17B1 (M36263) LSS (D63807), PIK3CG (X83368), DBN1 (U00802), NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X60656), F2 (V00595), RARA (X06614), ITGB4, 5GB35 IMPA1 (X66922), PACE (X17094), AGA (M64073), MVD (U49260), EH ADH (L07077), TFPI2 (D29992), MARCKS (M68956), FGB (J00129), and GPD1 (L34041);
NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383), SERPINF2 (D00174), AGA (M64073), BAZ23 LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RARA (X06614), PTPRN (L18983), APOE (L18983), APOE F10 (K03194), NR1I2 (AF061056), UBE2L3 (X92962), and FGB (J00129);
RPN2 (Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950) and NRGN (Y09689);
MAPK6 (X80692), GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M57627), RFC4 (M87339), CRLF1 (AF059293), RPS6 (MPS2020), RPS6 EMX1 (X68879) and TK2 (U77088);
PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD23A (D21235), SDHA (D30648), PET112L (AF0265681), DAD1 (D15057) HSPB1 (X54079), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M96803), PACE (X17094), RPL24 (M94314), SPIN80T STX4A (U07158), SIAT8B (U33551), CTSK (U13665), DCI (U24 74), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D38048);
COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J0469), SCD (Y13647), YWHAZ (M86400), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 (M8733)
CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01677), PAFAH1B3 (D63391), GBE1 (L07756), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and XBE92E1;
ADCY1 (L05500), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232); and
CALT (X72946), ZNF165 (X84801), GPC1 (X54232), TUBA1 (K00558), GCP2 (X78686), COX5A (M22760), GLA (X00351) and GNS (X06656)
A drug for enhancing the sensitivity to an anticancer agent, comprising an antibody against a protein encoded by a gene selected from the group consisting of (a GenBank registration number in parentheses).
(62) adding a test substance to the cell,
SPTBN1 (M96803), PET112L (AF0266871), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770), RPLP2 (M17887), and A4487L ;
HOXB1 (X16666), F10 (K03194), GPX2 (X53463), NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (M36263), HSD17B1 (M36263) LSS (D63807), PIK3CG (X83368), DBN1 (U00802), NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X60656), F2 (V00595), RARA (X06614), ITGB4, 5GB35 IMPA1 (X66922), PACE (X17094), AGA (M64073), MVD (U49260), EH ADH (L07077), TFPI2 (D29992), MARCKS (M68956), FGB (J00129), and GPD1 (L34041);
NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383), SERPINF2 (D00174), AGA (M64073), BAZ23 LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RARA (X06614), PTPRN (L18983), APOE (L18983), APOE F10 (K03194), NR1I2 (AF061056), UBE2L3 (X92962), and FGB (J00129);
RPN2 (Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950) and NRGN (Y09689);
MAPK6 (X80692), GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M57627), RFC4 (M87339), CRLF1 (AF059293), RPS6 (MPS2020), RPS6 EMX1 (X68879) and TK2 (U77088);
PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD23A (D21235), SDHA (D30648), PET112L (AF0265681), DAD1 (D15057) HSPB1 (X54079), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M96803), PACE (X17094), RPL24 (M94314), SPIN80T STX4A (U07158), SIAT8B (U33551), CTSK (U13665), DCI (U24 74), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D38048);
COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J0469), SCD (Y13647), YWHAZ (M86400), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 (M8733)
CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01667), PAFAH1B3 (D63391), GBE1 (L07756), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and XBE92E1;
ADCY1 (L05500), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232); and
CALT (X72946), ZNF165 (X84801), GPC1 (X54232), TUBA1 (K00558), GCP2 (X78686), COX5A (M22760), GLA (X00351) and GNS (X06656)
The expression level of at least one or more genes selected from the group consisting of (the GenBank registration number in parentheses) is measured, and the expression level of the gene is compared with the expression level of the gene when no test substance is added. A method for screening a substance that enhances sensitivity to an anticancer agent, wherein the substance to be reduced is selected as a candidate substance that enhances sensitivity to the anticancer agent.
(63) A substance that enhances sensitivity to an anticancer agent obtained by the method of (62).
(64) A sensitivity enhancer for an anticancer agent, comprising the substance according to (63).

FIG. 1 shows the results of hierarchical clustering of 51 anticancer agents based on the sensitivity of 45 human cancer cell lines.
FIG. 2 shows the result of hierarchical clustering of 42 human cancer cells according to the results of gene expression.
FIG. 3 shows the correlation between sensitivity to MMC and CSPD protein expression in hepatocyte cell lines.
FIG. 4A shows the correlation between sensitivity to MMC and HSPA1A gene expression in breast cancer cell lines.
FIG. 4B shows the correlation between sensitivity to MMC and JUN gene expression in all 42 cell lines. One dot represents one cell line. The X axis shows sensitivity to MMC, and the Y axis shows gene expression of HSPA1A or JUN. Pearson's correlation coefficient values between sensitivity to MMC and HSPA1A and JUN expression are 0.75 (p = 0.05) and 0.473 (p = 0.015), respectively.
C and D in FIG. 4 show growth inhibition curves when HT1080 cells transfected with each gene were treated with MMC. Each curve shows only the vector (black square: ■), LacZ (black rhombus: ◆), NQO1 (×), HSPA1A (black triangle: ▲), JUN (white triangle: △). When HT1080 cells are transfected with any of NQO1, HSPA1A, and JUN, the growth inhibitory activity of MMC is enhanced. An asterisk is a t-test with respect to the control and shows a significant difference at p <0.01.
FIG. 5 shows CTSD expression suppression by CTSD-specific siRNA. Lane 1 is a non-transfected cell, 2 is a cell transfected with lipofectamine 2000 alone, 3 is a CTSD # 271, 4 is a CTSD # 387, 5 is a Western blot of cells transfected with CTSD # 400, respectively. Indicates. The left arrow indicates the position and molecular weight of the detected CTSD band.
6A and 6B show growth inhibition curves when SSP-25 cells transfected with CTSD # 400 were treated with mitomycin C (FIG. 6A), paclitaxel (FIG. 6B left) or doxorubicin (FIG. 6B right). The X-axis represents the concentration (mol / L) of each anticancer agent, and the Y-axis represents the percentage (%) of cell proliferation relative to the case where no anticancer agent is added. Each curve shows a cell transfected with CTSD # 400 siRNA (black circle: ●) and a control not transfected with siRNA (white square: □).
FIG. 7 shows the correlation between sensitivity to MMC, paclitaxel and doxorubicin and CTSD gene expression in 12 liver cancer cell lines. One dot represents one cell line. The X axis shows the sensitivity to each anticancer agent, and the Y axis shows the expression of the CTSD gene.

  Hereinafter, embodiments of the present invention will be described in detail.
(A) A method for identifying a gene group related to the sensitivity of tumor cells to an anticancer agent
  As a method for identifying a gene involved in sensitivity to an anticancer drug according to the present invention,
(1) a step of measuring sensitivity to various anticancer agents in a cancer cell line panel;
(2) a step of measuring expression of a plurality of genes in the same cancer cell line panel as in (1) above;
(3) calculating a correlation coefficient between the sensitivity to the anticancer agent of (1) and the gene expression result of (2) above, and statistically selecting genes having a high correlation with the sensitivity to the anticancer agent;
It can consist of
  As the human cancer cell line panel used here, all or a part of the panel comprising 45 cell lines described in Tables 1 to 5 of the present specification can be used. In addition, by selecting and using cell lines belonging to gastric cancer, breast cancer, and liver cancer from the panels shown in Tables 1 to 5, a group of genes involved in sensitivity to anticancer drugs specialized in gastric cancer, breast cancer, and liver cancer are selected. be able to.
  As a method for measuring sensitivity to an anticancer agent in the present invention, anti-cell activity measurement, DNA synthesis rate measurement, total protein mass measurement, and the like can be used. As a method for measuring the anti-cell activity, a method such as MTT method or XTT method is used, in which cell growth is observed by a color reaction after contacting an anti-cancer agent with a cell for a certain period of time. In DNA synthesis rate measurement,³It can be measured, for example, by measuring the incorporation of H-labeled thymidine into cells. In the total protein mass measurement, sulfodhoamine B assay or the like can be used.
  In the present invention, the expression level of a gene can be measured by quantifying RNA that is a transcription product of a gene or protein that is a gene product.
(1) RNA or protein extraction
  RNA or protein can be extracted from cancer cell lines or cancer cells derived from cancer patients by the following general method. In the case of RNA, for example, when TRIZOL reagent (Invitrogen) is used, it may be performed according to the attached operation method. In the case of a protein, for example, it may be carried out in accordance with a book such as The Protein Handbook, 1996, Humana Press, New Jersey, USA.
(2) Quantification of RNA
  RNA can be quantified by techniques such as DNA microarray, Northern blot analysis, and RT-PCR. In addition, an RNA quantitative reagent containing a polynucleotide complementary to the RNA for use in RT-PCR is also included in the scope of the present invention.
(3) Protein quantification
  Protein quantification can be performed by immunochemical quantification using specific antibodies. As an antibody against the protein, a monoclonal antibody or a polyclonal antibody can be prepared according to the method described in the book Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, New York). Moreover, when a commercially available antibody is available, it can also be used after confirming the specificity. Using the obtained antibody, the protein can be quantified by performing ELISA or RIA according to a method described in a book (for example, enzyme immunoassay (Eiji Ishikawa et al., School of Medicine, 1982)). Moreover, an immunoassay reagent containing an antibody against the protein for use in the above method is also included in the scope of the present invention.
  In the present invention, a correlation coefficient between the sensitivity to the anticancer agent and the gene expression result is calculated, and genes having a high correlation with the sensitivity to the anticancer agent are statistically selected. The correlation analysis performed here can be performed, for example, by calculating Pearson's correlation coefficient (Peason correlation coefficients). Whether the Pearson correlation coefficient r is r = 0, for example, by assuming that the sensitivity to the anticancer drug correlates with the gene expression is a normal distribution when both are logarithmically expressed. This is done by testing. For example, in the correlation significance test, genes satisfying P <0.05 can be selected and identified as “a gene correlated with sensitivity to anticancer agents”.
  The kind of anticancer agent used in the present invention is not particularly limited, and any anticancer agent can be used, and a candidate substance for an anticancer agent can also be used. Specific examples of the anticancer agent include those listed in Table 1, and more specifically, mitomycin C, vinorelbine, paclitaxel 5-fluorouracil, doxorubicin, cisplatin (Cisplatin).
  Examples of genes that correlate with sensitivity to various anticancer agents identified by the above method include the following. Examples of genes that correlate with sensitivity to mitomycin C include the genes described in Table 6, Table 7, and Tables 12-14. Examples of genes that correlate with sensitivity to vinorelbine include the genes listed in Tables 8 and 9. Examples of genes that correlate with sensitivity to paclitaxel include the genes listed in Table 10, Table 11, and Table 15. Examples of genes that correlate with sensitivity to 5-fluorouracil include the genes listed in Table 16. Examples of genes that correlate with sensitivity to doxorubicin include the genes listed in Table 17. Examples of genes that correlate with sensitivity to cisplatin include the genes listed in Table 18. Further, the genes listed in Table 12 as genes that correlate with the sensitivity of breast cancer to mitomycin C, the genes listed in Table 13 as the genes that correlate with the sensitivity of liver cancer to mitomycin C, and the sensitivity of gastric cancer to mitomycin C. The genes listed in Table 14 can be listed as genes that correlate with. Furthermore, the genes listed in Table 15 as genes that correlate with the sensitivity of liver cancer to paclitaxel, the genes listed in Table 16 as the genes that correlate with the sensitivity of liver cancer to 5-fluorouracil, and those against liver cancer doxorubicin. Examples of the gene correlated with sensitivity include the genes described in Table 17, and examples of the gene correlated with sensitivity to cisplatin of liver cancer include the genes described in Table 18.
(B) Method for determining sensitivity of cancer patient to anticancer agent
  The present invention further provides a method for determining the sensitivity of a cancer patient to an anticancer agent. The gene expression in tumor cells taken out from cancer patients can be quantified by the method described in (A) above. As a determination method, one or more genes are selected from the gene group identified for each drug by the method (A), and for each of the selected genes, (1) a score corresponding to the expression level is determined. (2) Measure the expression level of the gene in tumor cells derived from the test patient, (3) assign the score obtained in (1) based on the expression level measured in (2), and (4) all selected The score assigned to the gene is summed to obtain a total value, and (5) the sensitivity to the given anticancer agent is judged by comparing the total value with a predetermined threshold value. can do. Multi-variate analysis methods such as multiple regression analysis, discriminant analysis, SVM (support vector machine) method, principal component analysis, self-organizing map method, etc. (by SAS) Analysis of experimental data, supervised by Kei Takeuchi, described in the University of Tokyo Press, 1990, etc.).
  The following method can be used to examine whether the gene expression change determined to have a correlation with the sensitivity to the anticancer agent by the above method actually changes the sensitivity to the anticancer agent.
(1) Forced gene expression
  By inserting a full length or partial length cDNA of the selected gene into an expression vector of an appropriate animal cell and introducing it into a cancer cell by transfection, the expression level of the gene can be increased. By adding an anticancer agent that is considered to be correlated therewith, it can be determined whether or not the sensitivity to the anticancer agent changes compared to the case where the gene is not expressed.
  Examples of expression vectors include pEGFP-C2 (Clontech), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotechnol. ,3133 (1990)], pAS3-3 (Japanese Patent Laid-Open No. 227075), pCDM8 [Nature,329, 840, (1987)], pCMV-Tag1 (Stratagene) pcDNA3.1 (+) (Invitrogen), pREP4 (Invitrogen), pMSG (Amersham Bioscience), pAMo [J. Biol. Chem. ,268, 22782 (1993)] and the like.
  As a method for introducing the recombinant vector, any method for introducing DNA into animal cells can be used. For example, electroporation [Miyaji H. et al. Et al., Cytotechnol. , 3, 133 (1990)], the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [Felner P. et al. L. Et al., Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
(2) Suppression of gene expression
  By introducing an antisense polynucleotide or siRNA corresponding to the selected gene into a cancer cell, the expression level of the gene can be lowered. Similarly, the expression level of the gene can be lowered by introducing an antisense polynucleotide or siRNA expression vector into a cancer cell.
  By adding an anticancer agent that is considered to be correlated therewith, it is possible to determine whether the gene expression is not suppressed and whether the sensitivity to the anticancer agent changes.
(C) Sensitivity enhancer for anticancer agent (i)
  When the sensitivity to an anticancer agent is enhanced by forced expression of a gene by the method described in (1) Forced expression of gene in (B) above, the gene therapy drug containing the gene is enhanced sensitivity to the anticancer agent. It can be used as a medicine. In addition, a protein preparation containing a protein encoded by the gene can be used as a sensitivity enhancer for the anticancer agent.
  Specifically, it is a single gene or a plurality of genes selected from the genes described in “Sensitivity” in Table 6 and Tables 12 to 14, and the activity to enhance the sensitivity to mitomycin C by forced expression in cancer cells. The gene shown or the protein encoded by the gene can be used as a sensitizer for mitomycin C.
  Similarly, a gene or a protein encoded by a gene or a protein encoded by the gene, which is an individual gene or a plurality of genes selected from the genes listed in Table 8 and that enhances the sensitivity to vinorelbine by forced expression in cancer cells, is used for vinorelbine. It can be used as a sensitivity enhancer.
  Similarly, a gene or a gene selected from the genes described in “Sensitivity” in Tables 10 and 15 and having an activity to enhance the sensitivity to paclitaxel by forced expression in cancer cells The protein encoded by can be used as a sensitivity enhancer for paclitaxel.
  Similarly, a gene or a gene selected from the genes described in “Sensitivity” in Table 16 that exhibits an activity of enhancing sensitivity to 5-fluorouracil by forced expression in cancer cells, or The encoded protein can be used as a sensitivity enhancer for 5-fluorouracil.
  Similarly, a gene or genes that are selected from the genes listed in “Sensitivity” in Table 17 and that exhibit the activity of enhancing the sensitivity to doxorubicin by forced expression in cancer cells or the gene encodes. The protein can be used as a sensitivity enhancer for doxorubicin.
  Similarly, a gene or a gene selected from the genes listed in “Sensitivity” in Table 18 that exhibits an activity of enhancing the sensitivity to cisplatin by forced expression in cancer cells or the gene encodes it. The protein can be used as a sensitizer for cisplatin.
  The above-mentioned gene showing the activity to enhance the sensitivity to various anticancer agents or the protein encoded by the gene can be used alone as a sensitivity enhancer for the anticancer agent, but usually one pharmacologically acceptable or It is desirable to mix it with a further carrier and use it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics. Details of such pharmaceutical preparations will be described later in (F) of the present specification.
(D) Sensitivity enhancer for anticancer drug (ii)
  When the sensitivity to a certain anticancer drug is enhanced by suppressing the expression of a gene by the method described in (2) Gene expression suppression of (B) above, an siRNA or antisense polynucleotide that suppresses the expression of the gene or A vector that expresses the siRNA or antisense polynucleotide can be used as a sensitivity enhancer for the anticancer agent. In addition, a drug containing a neutralizing antibody or the like that suppresses the function of the protein encoded by the gene can be used as a sensitivity enhancer for the anticancer drug.
  Specifically, it is a single gene or a plurality of genes selected from the genes described in “Resistance” in Table 7 and Tables 12 to 14, and suppresses the expression of the genes in cancer cells, thereby suppressing mitomycin C. An siRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity that enhances sensitivity, or a vector that expresses the siRNA or antisense polynucleotide can be used as a sensitizer for mitomycin C.
  Similarly, the expression of a gene selected from the genes listed in Table 9 or a plurality of genes, which suppresses the expression of the gene in cancer cells, thereby enhancing the sensitivity to vinorelbine is suppressed. SiRNA or antisense polynucleotide or a vector expressing the siRNA or antisense polynucleotide can be used as a sensitizer for vinorelbine.
  Similarly, it is a single gene or a plurality of genes selected from the genes described in “resistance” in Table 11 and Table 15, and the activity of enhancing the sensitivity to paclitaxel by suppressing the expression of the gene in cancer cells SiRNA or antisense polynucleotide that suppresses the expression of a gene or a vector that expresses the siRNA or antisense polynucleotide can be used as a sensitivity enhancer for paclitaxel.
  Similarly, it is a single gene or a plurality of genes selected from the genes described in “resistance” in Table 16, and has the activity of enhancing the sensitivity to 5-fluorouracil by suppressing the expression of the genes in cancer cells. The siRNA or antisense polynucleotide that suppresses the expression of the indicated gene or a vector that expresses the siRNA or antisense polynucleotide can be used as a sensitivity enhancer for 5-fluorouracil.
  Similarly, a single gene or a plurality of genes selected from the genes described in “Resistance” in Table 17, which exhibit an activity of enhancing the sensitivity to doxorubicin by suppressing the expression of the genes in cancer cells SiRNA or antisense polynucleotide that suppresses the expression of or a vector that expresses the siRNA or antisense polynucleotide can be used as an agent for enhancing sensitivity to doxorubicin.
  Similarly, a single gene or a plurality of genes selected from the genes described in “resistance” in Table 18, which exhibit an activity of enhancing the sensitivity to cisplatin by suppressing the expression of the genes in cancer cells. A siRNA or antisense polynucleotide that suppresses the expression of or a vector that expresses the siRNA or antisense polynucleotide can be used as a sensitizer for cisplatin.
  Next, siRNA used in the present invention will be described. The siRNA is a short double-stranded RNA containing a partial sequence of mRNA of a target gene, and can suppress the expression of the gene by RNAi (RNA interference). The sequence of siRNA can be determined from literature [Genes Dev. ,13, 3191, (1999)]. Preferably, a sequence of 19 bases following AA and a GC content of 30 to 70%, more preferably around 50% is selected in a region 50 to 100 bases downstream from the initiation codon in the translation region of mRNA. A siRNA can be prepared by synthesizing and annealing two RNAs having a sequence of 19 bases selected and a sequence having TT added to the 3 'end of each complementary sequence and annealing. In addition, the expression of the gene is suppressed by inserting DNA corresponding to the selected 19-base sequence into a siRNA expression vector such as pSilencer 1.0-U6 (Ambion) or pSUPER (OligoEngine). A vector that expresses an siRNA that can be produced can be produced.
  The antisense polynucleotide used in the present invention is a polynucleotide having a base sequence complementary to a sequence of 15 or more consecutive bases in the base sequence of the target gene. Polynucleotides include DNA, RNA, and polynucleotide derivatives. The polynucleotide derivative includes a polynucleotide derivative in which a phosphodiester bond in a polynucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in a polynucleotide is converted to an N3′-P5 ′ phosphoramidate bond. Polynucleotide derivatives, polynucleotide derivatives in which ribose and phosphodiester bonds in the polynucleotide are converted to peptide nucleic acid bonds, polynucleotide derivatives in which the uracil in the polynucleotide is substituted with C-5 propynyl uracil, in the polynucleotide Polynucleotide derivatives in which uracil is substituted with C-5 thiazole uracil, polynucleotide derivatives in which cytosine in the polynucleotide is substituted with C-5 propynylcytosine, cytosine in the polynucleotide is phenoxazine Polynucleotide derivative substituted with decorated cytosine, polynucleotide derivative substituted with 2'-o-propylribose in the polynucleotide, or polynucleotide substituted with 2'-methoxyethoxyribose in the polynucleotide Derivatives and the like can be given.
  Antisense RNA / DNA technology [Bioscience and Industry,50322 (1992), chemistry,46, 681 (1991), Biotechnology,9, 358 (1992), Trends Biotechnol. ,10, 87 (1992), Trends Biotechnol. ,10, 152 (1992), Cell engineering,16, 1463 (1997)], triple helix technology [Trends Biotechnol. ,10, 132 (1992)], the transcription or translation of the target gene in the cancer cell is suppressed by administering the antisense polynucleotide described above to the cancer cell, and as a result, the expression of the target gene is suppressed. Can do. In particular, a base sequence complementary to 15 to 100 bases including the start codon of the region encoding the target gene polypeptide is preferable. Moreover, the polynucleotide derivative which does not receive degradation by deoxyribonuclease and ribonuclease is preferable.
  The antisense polynucleotide can be produced using a commercially available DNA synthesizer, a method described in WO93 / 20095, WO02 / 10185, or the like. In addition, a recombinant vector in which the target gene is connected in the reverse direction is prepared downstream of the promoter of the expression vector used for forced expression of the gene of (B) (1), and this recombinant vector is introduced into cancer cells. Thus, antisense RNA can be expressed in cells.
  Furthermore, in the present invention, an antibody against a protein encoded by any of the genes described in “Resistance” in Tables 7, 9, 11 and 12 to 18 can also be used as a sensitivity enhancer for an anticancer agent. Preferably, the antibody is a neutralizing antibody that suppresses the function of the protein. Antibodies against such target proteins can be obtained by methods known to those skilled in the art.
  The siRNA or antisense polynucleotide showing the activity to enhance the sensitivity to various anticancer agents described above, the vector expressing the siRNA or antisense polynucleotide and the antibody can be used alone as a sensitivity enhancer for the anticancer agent, Usually, it is desirable to mix with one or more pharmacologically acceptable carriers and use it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics. Details of such pharmaceutical preparations will be described later in (F) of the present specification.
(E) Screening and sensitivity enhancer for anticancer drug obtained by the screening
  When a certain substance is administered to achieve expression enhancement or suppression of any of the genes described in Tables 6 to 18 of the present specification, thereby enhancing the sensitivity to a certain anticancer agent, the expression of the gene Substances having an activity to enhance or suppress the activity can also be used as a sensitivity enhancer for the anticancer agent.
  Substances having such activity can be screened by the following method. For example, the target substance can be obtained by binding the expression control region of the gene to an appropriate reporter gene and introducing it into a cell, and selecting a substance that increases or decreases the expression of the reporter gene by adding a test sample. . As the reporter gene, firefly luciferase gene, green fluorescent protein (GFP) gene, β-galactosidase gene, and the like can be used. Alternatively, mRNA of the gene can be directly quantified by RT-PCR method, Northern blotting method, quantitative PCR method, or the protein encoded by the gene can be quantified by an immunological method such as ELISA method or EIA method. The target substance can be screened.
  Test samples include synthetic compounds, naturally occurring proteins, artificially synthesized proteins, peptides, carbohydrates, lipids, modified products and derivatives thereof, and mammals (eg, mice, rats, guinea pigs, hamsters, Pigs, sheep, cows, horses, dogs, cats, monkeys, humans, etc.), urine, body fluids, tissue extracts, cell culture supernatants, cell extracts, non-peptide compounds, fermentation products, plants and other Examples include biological extracts.
  The substance obtained by the above-described screening method of the present invention is useful as a sensitivity enhancer for an anticancer agent, and the substance and a sensitivity enhancer for an anticancer agent containing the same also fall within the scope of the present invention.
(F) Pharmaceutical preparation of sensitivity enhancer for anticancer agent of the present invention
  Various genes, proteins, antibodies and substances as described in the above (C), (D) and (E) in the present specification can be used alone as sensitizers for anticancer agents. Is preferably mixed with one or more pharmaceutically acceptable carriers and used as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutical sciences.
  The route of administration is preferably the most effective for prevention or treatment, and can include oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous. . Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include saccharides such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoate Preservatives such as acid esters and flavors such as strawberry flavor and peppermint can be used as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin and the like can be used as additives.
  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, an injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the active ingredient itself or the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the active ingredient as fine particles. Specific examples of the carrier include lactose and glycerin. Depending on the properties of the protein and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
  The dose or frequency of administration varies depending on the type of active ingredient used, the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 0.01 μg / kg to 10 mg / kg per day for an adult.
  In the case of a gene therapy agent using a nucleic acid such as DNA or RNA as an active ingredient for enhancing sensitivity to an anticancer agent, the DNA or RNA is used alone or as a retrovirus vector, adenovirus vector, adeno-associated virus vector, etc. After inserting into an appropriate vector, the method of formulation, formulation and administration according to the conventional methods described above, or the method of administration by the non-viral gene transfer method can be used.
  A recombinant virus vector can be prepared according to the method described below.
  A fragment of DNA used as an active ingredient (hereinafter also referred to as target DNA) is prepared. A recombinant viral vector is constructed by inserting the DNA fragment downstream of the promoter in the viral vector.
  In the case of an RNA viral vector, a recombinant virus is constructed by preparing RNA fragments homologous to the target DNA and inserting them into the downstream of the promoter in the viral vector. In addition to the double strand, the RNA fragment selects either the sense strand or the antisense strand depending on the type of the viral vector. For example, in the case of a retroviral vector, RNA homologous to the sense strand is selected, and in the case of a sense viral vector, RNA homologous to the antisense strand is selected.
  The recombinant viral vector is introduced into packaging cells compatible with the vector. The packaging cell may be any cell as long as it can replenish the deficient protein of a recombinant viral vector lacking at least one gene encoding a protein necessary for virus packaging. For example, human kidney-derived HEK293 cells, mouse fibroblasts NIH3T3, and the like can be used. Proteins to be replenished by packaging cells include gag, pol, env, etc. derived from mouse retrovirus in the case of retrovirus vectors, gag, pol, env, vpr, vpu, vif, derived from HIV virus in the case of lentiviral vectors. Examples of tat, rev, nef, and the like include adenovirus-derived E1A and E1B, and adeno-associated viruses include Rep (p5, p19, p40), and Vp (Cap).
  As the viral vector, those containing a promoter at a position where the recombinant virus can be produced in the packaging cell and the target DNA can be transcribed in the target cell are used. As a plasmid vector, MFG [Proc. Natl. Acad. Sci. USA,92, 6733-6737 (1995)], pBabePuro [Nucleic Acids Res. ,183587-3596 (1990)], LL-CG, CL-CG, CS-CG, CLG [Journal of Virology,72, 8150-8157 (1998)], pAdex1 [Nucleic Acids Res. ,23, 3816-3821 (1995)]. Any promoter can be used as long as it functions in human tissues. For example, cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, Examples include metallothionein promoter, heat shock protein promoter, SRα promoter and the like. In addition, an enhancer of human CMV IE gene may be used together with a promoter.
  Examples of a method for introducing a recombinant virus vector into a packaging cell include a calcium phosphate method [Japanese Patent Laid-Open No. 2-227075] and a lipofection method [Proc. Natl. Acad. Sci. USA,847413 (1987)] and the like.
  In addition to the above method, the recombinant virus vector can be administered directly by in vivo using liposome delivery (in  vivo) Viral vectors can also be directed to pancreatic lesions in combination with gene transfer. That is, a virus vector is prepared by combining a target DNA of an appropriate size with a polylysine-conjugated antibody specific for adenovirus hexon protein and binding the resulting complex to an adenovirus vector. can do. The viral vector stably reaches the target cell, is taken up into the cell by the endosome, is degraded in the cell, and can efficiently express the gene.
  In the present invention, the vector expressing the target DNA or siRNA or antisense polynucleotide can be transported to the lesion also by the non-viral gene transfer method.
  Non-viral gene transfer methods known in the art include calcium phosphate coprecipitation [Virology,52456-467 (1973); Science,209, 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA,77, 5399-5403 (1980); Proc. Natl. Acad. Sci. USA,777380-7384 (1980); Cell,27, 223-231 (1981); Nature,294, 92-94 (1981)], membrane fusion-mediated transfer via liposomes [Proc. Natl. Acad. Sci. USA,84, 7413-7417 (1987); Biochemistry,289508-9514 (1989); Biol. Chem. ,H.264, 12126-12129 (1989); Hum. Gene Ther. ,3, 267-275, (1992); Science,249, 1285-1288 (1990); Circulation,83, 2007-2011 (1992)] or direct DNA uptake and receptor-mediated DNA transfer [Science,247, 1465-1468 (1990); Biol. Chem. ,266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA,873655-3659 (1991); Biol. Chem. ,H.264, 16985-16987 (1989); BioTechniques,11474-485 (1991); Proc. Natl. Acad. Sci. USA,873410-3414 (1990); Proc. Natl. Acad. Sci. USA,88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA,87, 4033-4037 (1990); Proc. Natl. Acad. Sci. USA,88, 8850-8854 (1991); Hum. Gene Ther. ,3, 147-154 (1991)].
  It has been reported in tumor studies that liposome-mediated membrane fusion-mediated transfer allows local administration and uptake of the gene by direct administration of the liposome preparation to the target tissue. [Hum. Gene Ther.3399-410 (1992)]. Therefore, the same effect is expected in the pancreatic lesion. Direct DNA uptake techniques are preferred for direct targeting of DNA to pancreatic lesions. Receptor-mediated DNA transfer is performed, for example, by conjugating DNA (typically in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. The ligand is selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell or tissue. The ligand-DNA conjugate can be injected directly into the blood vessel, if desired, and can be directed to a target tissue where receptor binding and internalization of the DNA-protein complex occurs. In order to prevent intracellular destruction of DNA, adenovirus can be co-infected to disrupt endosomal function.
  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

４５種のヒト癌細胞株の感受性を元に５１種の抗癌剤の階層的クラスタリングを行った結果を図１に示す。階層的クラスタリングはＧｅｎｅ Ｓｐｒｉｎｇソフトウェア（Ｓｉｌｉｃｏｎ Ｇｅｎｅｔｉｃｓ社）により、群平均法（ａｖｅｒａｇｅ ｌｉｎｋａｇｅ ｍｅｔｈｏｄ）を用いて行った。データ間の距離としては、２抗癌剤間の各細胞のｌｏｇ_１０ＧＩ_５０の絶対値の分布から求めたピアソンの相関係数を用いた。
２つの抗癌剤ｌｏｇ_１０ＧＩ_５０の絶対値の分布から求められるその結果、５１の薬剤を数個のクラスターに分けることが可能であったが、それぞれのクラスターは類似の作用機構を有する複数の薬剤を含んでいた。例えば、あるクラスターにはカンプトテシン、トポテカンなどのトポイソメラーゼＩの阻害剤が、第二のクラスターにはタキサン系抗癌剤（パクリタキセル、ドセタキセル）とビンカアルカロイド系抗癌剤（ビンブラスチン、ビンクリスチン、ビノレルビン）などの微小管作用薬（ｔｕｂｕｌｉｎ ｂｉｎｄｅｒ）が集まり、５−フルオロウラシルとその誘導体も一つのクラスターに集まった。この結果は、発明者らのこの４５細胞を用いるシステムにより、多数の薬剤を作用機構に基づいて分類分けすることが可能であることを示している。
［実施例２］：遺伝子発現プロファイルによる４２ヒト癌細胞のクラスタリング
上記４５細胞からなるヒト癌細胞パネルのうち、ＨＢＣ−９、ＨＢＣ−８、ＧＭＫ−３の３細胞を除く４２細胞株を用いて、３５３７の遺伝子の発現をｃＤＮＡアレイにより測定した。
＜ｃＤＮＡアレイによる遺伝子発現の測定＞
ｃＤＮＡアレイとしては、Ａｔｌａｓ^ＴＭｈｕｍａｎ３．６ａｒｒａｙ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ Ｃｌｏｎｔｅｃｈ社）を用い、２度繰り返した。実験はＣｌｏｎｔｅｃｈ社の製品説明書に従って行ったが、以下に簡単に述べる。細胞株は対数増殖期に処理し、全ＲＮＡ（Ｔｏｔａｌ ＲＮＡ）をＴｒｉｚｏｌ試薬（Ｉｎｖｉｔｒｏｇｅｎ社）を用いて抽出し、ＲＮｅａｓｙ Ｋｉｔ（Ｑｉａｇｅｎ社）を用いて精製した。精製したＴｏｔａｌ ＲＮＡをＡｔｌａｓ^ＴＭＰｕｒｅ Ｔｏｔａｌ ＲＮＡ Ｌａｂｅｌｉｎｇ Ｓｙｓｔｅｍ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ Ｃｌｏｎｔｅｃｈ社）を用いた逆転写により^３２Ｐで標識したｃＤＮＡプローブに変換した。このｃＤＮＡプローブは、Ａｔｌａｓ Ａｒｒａｙに６８℃、一晩、ハイブリダイズし、その後洗浄した。ハイブリダイズ済みのアレイは、ＰｈｏｓｐｈｏｒＩｍａｇｅｒ（Ｍｏｌｅｃｕｌａｒ Ｄｙｎａｍｉｃｓ社）によってスキャンしシグナルを検出した。検出したデータはＡｔｌａｓ^ＴＭＩｍａｇｅ ２．０ｓｏｆｔｗａｒｅ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ Ｃｌｏｎｔｅｃｈ社）によって数値化され、全ての遺伝子の９０パーセンタイルの値で割算することにより、標準化された。２回の繰り返し実験で２倍以上の異なる値を示した遺伝子は解析から除いた。９０パーセンタイルの値の３０％の値を閾値と設定し、閾値より低い値に対しては、閾値の値を与えた。このデータ処理を行った後で、２を底として対数値に変換した。
この発現プロファイルの結果を元に、細胞を階層的クラスタリングにより分類した。階層的クラスタリングはＧｅｎｅ Ｓｐｒｉｎｇソフトウェア（Ｓｉｌｉｃｏｎ Ｇｅｎｅｔｉｃｓ社）により、群平均法を用いて行った。データ間の距離としては、２細胞間の各抗癌剤のｌｏｇ_１０ＧＩ_５０の絶対値の分布から求めたピアソンの相関係数を用いた。
クラスタリングの結果を図２に示す。同じ組織由来の細胞株がクラスターを形成する傾向が見られた。即ち、ＫＰＬ−４を除く乳癌細胞株が一つのクラメターにまとまった。また、肝癌細胞株についても、数個の胃癌細胞株が間に挿入されたものの、ＨＬＥを除いて一つにまとまった。胃癌細胞株については、大枠で２つのグループにまとまった。以上の結果は、遺伝子発現プロファイルの結果から見る限り、樹立された細胞株も由来した組織の性質を維持していることを示すものである。
［実施例３］：遺伝子発現プロファイルと抗癌剤に対する感受性の相関解析
抗癌剤に対する感受性に関係する遺伝子群を探索するため、実施例１と実施例２でそれぞれ得られた遺伝子発現と抗癌剤に対する感受性の２つのデータベースを統合し、その相関解析を行った。４２の細胞株について、３５３７遺伝子の発現と５１の抗癌剤に対する感受性を用いて、ピアソンの相関係数の包括的な計算を行った。用いた計算式は以下の通りである。

ここで、ｘ_ｉは細胞ｉにおける遺伝子ｘの発現量対数表示を示し、ｙ_ｉは細胞ｉの抗癌剤ｙに対する感受性の対数表示の絶対値（｜ｌｏｇ_１０ＧＩ_５０｜）を示す。ｘ_ｍは遺伝子ｘの発現量の対数表示の平均値（ｍｅａｎ）を示し、ｙ_ｍは抗癌剤ｙに対する感受性の対数表示の絶対値（｜ｌｏｇ_１０ＧＩ_５０｜）の平均値を示す。Ｐ値が０．０５未満（ｐ〈０．０５）のものを有意な相関と見なした。
「Ｐ〈０．０５かつ全細胞の５０％以上で発現が見られる」という制限で、「抗癌剤に対する感受性と有意に相関のある発現を示す遺伝子」を選抜した。５１の抗癌剤のそれぞれに対して異なる遺伝子のセットが抽出された。表６から表１１には、全４２細胞を用いた際の、ＭＭＣ、ビノレルビン、パクリタキセルに対する感受性と相関する遺伝子のセットを示す。即ち、表６から表１１は、抗癌剤に対する感受性と有意に相関する遺伝子のリストを示す。ＭＭＣ（表６及び表７）、ビノレルビン（表８及び表９）並びにパクリタキセル（表１０及び表１１）について、４２細胞株での相関解析結果を示す。“遺伝子”の欄はＨＵＧＯ ｄａｔａｂａｓｅでの遺伝子名を示す。“相関係数”は抗癌剤に対する感受性とその遺伝子の発現の間のピアソンの相関係数の値を示す。表６、８及び１０（“感受性”と付記）は、発現が強いと感受性である遺伝子を示し、表７、９及び１１（“耐性”と付記）は、発現が強いと耐性である遺伝子を示す。

表６及び表７に示す通り、ＭＭＣの場合、２０個の遺伝子が感受性遺伝子（感受性株で高発現）、１０個の遺伝子が耐性遺伝子（耐性株で高発現）、として選抜された。これらの遺伝子のうち、ＪＵＮ，ＥＭＳ１，ＮＭＢＲは細胞増殖に関係する遺伝子である。他にはＳＯＤ１，ＰＥＬＰ１，ＳＦＲＳ９など様々な遺伝子が含まれていた。また、表１０に示す通り、微小管作用薬であるパクリタキセルにおいては、細胞骨格系に関連するｅｚｒｉｎをコードするＶＩＬ２遺伝子が選抜された。同様にして、多くの遺伝子がそれぞれの抗癌剤について、感受性あるいは耐性遺伝子として選抜された。
さらに、ピアソンの相関係数による解析を、同じ組織由来の細胞株毎にも行った。ＭＭＣに関して、１０種の乳癌細胞株、１２種の肝癌細胞株、２０種の胃癌細胞株において、それぞれ抗癌剤に対する感受性と相関のある遺伝子として選抜された遺伝子群を表１２から表１４に示す。即ち、表１２は乳癌、表１３は肝癌、表１４−１、２は胃癌において、それぞれマイトマイシンＣに対する感受性と発現が相関する遺伝子のリストを示す。カラムの説明は表６から１１と同一である。これらの遺伝子群は抗癌剤に対する感受性予測のマーカーとして利用できるものを含む他、あるものは実際に抗癌剤に対する感受性を規定していると考えられる。

また、１２種の肝癌細胞株において、マイトマイシンＣ以外の各種抗癌剤に対する感受性と相関のある遺伝子として選抜された遺伝子群を表１５から表１８に示す。即ち、肝癌において、表１５はパクリタキセル、表１６は５−フルオロウラシル、表１７はドキソルビシン、表１８はシスプラチンそれぞれに対する感受性と発現が相関する遺伝子のリストを示す。カラムの説明は表６から１１と同一である。これらの遺伝子群は抗癌剤に対する感受性予測のマーカーとして利用できるものを含む他、あるものは実際に抗癌剤に対する感受性を規定していると考えられる。

［実施例４］：選抜された遺伝子発現のＲＴ−ＰＣＲ法による検証
実験例３で示したＤＮＡアレイの実験結果から肝癌および胃癌においてＭＭＣに対する感受性と相関があるとして選抜された遺伝子について、ＲＴ−ＰＣＲ法によって検証を行った。
胃癌については表１９に示す感受性遺伝子６種、耐性遺伝子７種について特異的ＰＣＲプライマーを作製し、ＲＴ−ＰＣＲにより、それぞれの遺伝子の各胃癌細胞株における発現量を測定し、実施例１で得られたＭＭＣに対する感受性との相関を調べた。ＲＴ−ＰＣＲは、実施例２で調製したｔｏｔａｌ ＲＮＡ ２μｇ、ＲＮａｓｅインヒビター、オリゴｄ（Ｔ）１６、ＭｕＬＶリバーストランスクリプターゼを含む反応液を４２℃で６０分間インキュベートして、ｃＤＮＡの合成を行い、この反応液の１／１０量を鋳型として、各遺伝子特異的ＰＣＲプライマーを用いたＰＣＲを行った。ＰＣＲは、９５℃で５分間加熱後、９５℃で１分間（変性）、５５〜６０℃で１分間（アニーリング）、７２℃で１〜２分間（伸長）の反応サイクルを２５〜３０サイクル繰り返し、７２℃で５分間加熱する条件（アニーリング温度、伸長時間、サイクル数は遺伝子により異なる）で行った。
その結果、感受性遺伝子としてＴＥＡ ｄｏｍａｉｎ ｆａｍｉｌｙ ｍｅｍｂｅｒ ４（ＴＥＡＤ４，ＧｅｎＢａｎｋ登録番号：Ｕ６３８２４）、Ｓｏｌｕｔｅ Ｃａｒｒｉｅｒ Ｆａｍｉｌｙ ６（ＳＬＣ６Ａ８，ＧｅｎＢａｎｋ登録番号：Ｌ３１４０９）の２遺伝子が、耐性遺伝子としてＰｒｏｔｅａｓｏｍｅ ２６Ｓ ｓｕｂｕｎｉｔ，ｎｏｎ−ＡＴＰａｓｅ ８（ＰＳＭＤ８，ＧｅｎＢａｎｋ登録番号：Ｄ３８０４７），ｃａｔｈｅｐｓｉｎ Ｄ（ＣＴＳＤ，ＧｅｎＢａｎｋ登録番号：Ｍ１１２３３），ａｎｎｅｘｉｎ Ａ４（ＡＮＸＡ４，ＧｅｎＢａｎｋ登録番号：Ｍ１９３８３），ＫＤＥＬ ｅｎｄｏｐｌａｓｍｉｃ ｒｅｔｉｃｕｌｕｍ ｐｒｏｔｅｉｎ ｒｅｔｅｎｔｉｏｎ ｒｅｃｅｐｔｏｒ１（ＫＤＥＬＲ１，ＧｅｎＢａｎｋ登録番号：Ｘ５５８８５）の４遺伝子に、ＲＴ−ＰＣＲによって測定された発現量とＭＭＣに対する感受性との相関が認められ、確かに発現と感受性に相関がある遺伝子として確認された（表１９）。

また、肝癌については、感受性遺伝子として表２０に示す３種について同様に検証したところ、ｃ−ｊｕｎ（ＪＵＮ，ＧｅｎＢａｎｋ登録番号：Ｊ０４１１１）、ｃａｔｈｅｐｓｉｎ Ｄ（ＣＴＳＤ，ＧｅｎＢａｎｋ登録番号：Ｍ１１２３３）の遺伝子に、ＲＴ−ＰＣＲによって測定された発現量とＭＭＣに対する感受性との相関が認められ、発現量と感受性との相関が確認された（表２０）。

さらに、同じく肝癌においてパクリタキセルの感受性遺伝子候補についても同様にＲＴ−ＰＣＲによる検証を行ったところ、感受性遺伝子としてａｃｅｔｙｌＣｏＡ−ａｃｅｔｙｌｔｒａｎｓｆｅｒａｓｅ（ＡＣＡＴ１，ＧｅｎＢａｎｋ登録番号：Ｄ９０２２８、表１５中の順位１０）、耐性遺伝子としてｂｃｌ−ＸＬ（ＢＣＬ２Ｌ１，ＧｅｎＢａｎｋ登録番号：Ｚ２３１１５、表１５中のランク９）について、ＲＴ−ＰＣＲによって測定された発現量とパクリタキセルに対する感受性との相関が認められ、ｃＤＮＡアレイによって見出された相関が確認された。
［実施例５］：選抜された遺伝子発現のウェスタンブロットによる検証
実験例３および４で、肝癌において、ＤＮＡアレイまたはＲＴ−ＰＣＲにより測定した遺伝子の発現量がＭＭＣに対する感受性と相関があることが確認されたカテプシンＤ（ＣＴＳＤ）について、遺伝子の発現量を、遺伝子産物である蛋白質量をウェスタンブロットにより測定した場合に、遺伝子の発現量とＭＭＣに対する感受性とに相関があるかどうかについて、検証を行った。
細胞をプレートに培養後、ＰＢＳ（−）で２回洗浄した。プレートから細胞をはがし、溶解バッファー〔６２．５ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ ｐＨ６．８、２％（Ｗ／Ｖ）ドデシル硫酸ナトリウム（ＳＤＳ）、５％（ｖ／ｖ）２−メルカプトエタノール、１０％（ｖ／ｖ）グリセロール、１ｍｍｏｌ／Ｌフェニルメチルスルフォニルフルオライド〕中で溶解させた。細胞溶解液の蛋白質濃度をＤＣプロテインアッセイキット（バイオラッド社）により定量し、１０μｇの蛋白質を含む細胞溶解液を、１０％ポリアクリルアミド−ＳＤＳゲル上で電気泳動により分離した。ゲルから蛋白質を膜に移し、以下のようにウェスタンブロットによる解析を行った。蛋白質を移した膜を、１：２５０に希釈したＣＴＳＤ特異的マウスモノクローナル抗体Ａｂ−１（オンコジーン・リサーチ・プロダクツ社）と反応させた。膜を洗浄し、さらに１：１０００に希釈したホースラディッシュ結合抗マウスＩｇＧ抗体（アマシャム・バイオサイエンス社）を反応させた。ＥＣＬ（エンハンスト・ケミルミネッセンス）検出システム（アマシャム・バイオサイエンス社）を用いて、ＣＴＳＤ蛋白質を検出し、そのシグナル強度を測定した。
ＣＴＳＤ蛋白質のシグナル強度と実施例１で得られたＭＭＣに対する感受性との相関を調べたところ、ピアソンの相関係数は０．８６２となった。したがって、ウェスタンブロットによって測定されたＣＴＳＤ遺伝子の発現量とＭＭＣに対する感受性との相関が認められた（図３）。
［実施例６］：抗癌剤の抗癌剤に対する感受性を変化させる遺伝子の同定（１）
抗癌剤に対する感受性を決定する遺伝子を探索するため、細胞増殖の阻害によって抗癌剤に対する感受性の変化を検出できる遺伝子スクリーニングシステムを確立した。遺伝子を細胞に導入することによる抗癌剤に対する感受性の変化を検出するシステムである。細胞増殖の指標としては、［^３Ｈ］−チミジン（ｔｈｙｍｉｄｉｎｅ）のとりこみを用いた。わずかな感受性の変化を検出するために、高いトランスフェクション効率が要求される。そこで、９０％以上の効率で遺伝子導入が可能なヒト線維芽肉腫細胞株であるＨＴ１０８０を用いた。
この系を用いて、ＭＭＣの抗癌剤に対する感受性を決定する遺伝子のスクリーニングを行った。候補となる遺伝子のｃＤＮＡの翻訳領域全長をクローニングし、動物細胞発現ベクターであるｐｃＤＮＡ３．１／ｍｙｃ−ＨｉｓＡ（Ｉｎｖｉｔｒｏｇｅｎ社）にクローニングした。これら発現プラスミドをＬｉｐｏｆｅｃｔａｍｉｎｅ Ｐｌｕｓ試薬（Ｉｎｖｉｔｒｏｇｅｎ社）を用いて、ＨＴ１０８０細胞に導入した。トランスフェクション後２４時間後に、適切な濃度のＭＭＣを添加し、さらに２４時間培養した。０．０６７ＭＢｑの［^３Ｈ］−チミジンを添加し、３７℃で４５分間保温した後、予め暖めておいたＰＢＳ（−）で洗い、１０％ＴＣＡで氷上に２時間処理し、細胞を固定した。固定後、細胞を１０％ＴＣＡで洗い、０．１％ＳＤＳ／０．２％ＮａＯＨで溶解させた。溶解した細胞を３７℃で保温した後、０．２５Ｍ酢酸溶液を加えて中和した。［^３Ｈ］−チミジンの取り込みは、シンチレーションカウンターで測定した。
このシステムの有効性を確認するため、ＭＭＣに対する感受性を規定することが知られているＤＴ−ｄｉａｐｈｏｒａｓｅ（Ｇｕｓｔａｆｓｏｎ，Ｄ．Ｌ．，＆ Ｐｒｉｔｓｏｓ，Ｃ．Ａ．，Ｃａｎｃｅｒ Ｒｅｓ．，５２，６９３６−６９３９，１９９２．）をコードする遺伝子ＮＱＯ１について調べた。ＮＱＯ１を導入したＨＴ１０８０細胞はＭＭＣに対する感受性が上昇した（図４Ｃ）。すなわち、このシステムは抗癌剤に対する感受性の変化を検出するのに有効なシステムであると考えられる。
このシステムを用いて、表６及び表１２から１４に選抜された遺伝子のいずれが、実際に抗癌剤に対する感受性を変化させるかを検討した。その結果、ＨＳＰ７０−１蛋白質をコードしているＨＳＰＡ１Ａ遺伝子（ＧｅｎＢａｎｋ登録番号：Ｍ１１７１７）の導入が、ＭＭＣに対する感受性を有意に上昇させることが示された（図４Ｃ）。ＨＳＰＡ１Ａの発現は乳癌（図４Ａ、表１２）、肝癌（表１３）では有意にＭＭＣに対する感受性と相関しているが、胃癌では有意な相関は見られていない（表１４）。また、全細胞株の解析で発現とＭＭＣに対する感受性との相関が見られたＪＵＮ遺伝子（ＧｅｎＢａｎｋ登録番号：Ｊ０４１１１）（図４Ｂ、表６）の導入でも、同じくＭＭＣに対する感受性が有意に上昇した（図４Ｄ）。以上の結果はバイオインフォマティクス的アプローチを用いてＤＮＡアレイの実験結果から抽出した結果が、抗癌剤に対する感受性を決定する遺伝子の予測に有効であることを示している。
［実施例７］：抗癌剤の抗癌剤に対する感受性を変化させる遺伝子の同定（２）
抗癌剤に対する感受性を決定する遺伝子を探索するため、遺伝子発現を抑制した場合の抗癌剤に対する感受性の変化を検出するスクリーニングシステムを確立した。以下に、肝癌において、遺伝子発現とＭＭＣに対する感受性の相関が見られたＣＴＳＤ遺伝子が、感受性決定遺伝子であることを確認した例をあげる。
（１）ｓｉＲＮＡによるＣＴＳＤ発現の抑制の確認
ＣＴＳＤ遺伝子の発現を抑制するための、ＣＴＳＤ特異的なｓｉＲＮＡ（ＣＴＳＤ ＃２７１、ＣＴＳＤ ＃３８７およびＣＴＳＤ ＃４００）を以下のようにして調製した。配列番号１〜６の配列それぞれからなるオリゴヌクレオチド〔ダーマコン・リサーチ社（Ｄｈａｒｍａｃｏｎ Ｒｅｓｅａｒｃｈ Ｉｎｃ．）の委託合成品〕を、それぞれダーマコン・リサーチ社から供給されたバッファーに溶解させ、配列番号１と２、３と４、５と６の組み合わせで混合した。それぞれ、６０℃で４５分間保温した後、室温に３０分置いてアニールさせ、２本鎖オリゴヌクレオチドＣＴＳＤ ＃２７１（センス鎖：配列番号１、アンチセンス鎖：配列番号２）、ＣＴＳＤ ＃３８７（センス鎖：配列番号３、アンチセンス鎖：配列番号４）およびＣＴＳＤ ＃４００（センス鎖：配列番号５、アンチセンス鎖：配列番号６）を生成した。それぞれを沈殿させた後、最終的に２０μｍｏｌ／Ｌの濃度になるように、１×アニーリング・バッファー〔３０ｍｍｏｌ／Ｌ ＨＥＰＥＳ−ＫＯＨ（ｐＨ７．９）、１００ｍｍｏｌ／Ｌ酢酸カリウム、２ｍｍｏｌ／Ｌ酢酸マグネシウム〕に溶解させた。
肝癌細胞株ＳＳＰ−２５を、５％ウシ血清を含むダルベッコ改変イーグル培地（ＤＭＥＭ）に懸濁させ、６ウェルプレート上に２．５×１０^５個／ウェルずつ分注した。１６時間後、上記のｓｉＲＮＡ（ＣＴＳＤ ＃２７１、ＣＴＳＤ ＃３８７およびＣＴＳＤ ＃４００）をそれぞれリポフェクトアミン２０００（インビトロジェン社）を用いて、最終的にウェルあたり１００ｎｍｏｌ／Ｌの濃度で細胞にトランスフェクションした。
ｓｉＲＮＡをトランスフェクションしてから４８時間後に細胞を回収し、実施例５と同様にして、ウェスタン・ブロットにより細胞のＣＴＳＤ蛋白質を検出した。その結果、図５に示すように、ＣＴＳＤ ＃２７１、ＣＴＳＤ ＃３８７およびＣＴＳＤ ＃４００それぞれを導入した細胞では、ＣＴＳＤ蛋白質がほとんど検出されず、ｓｉＲＮＡによりＣＴＳＤ遺伝子の発現が抑制されていることが確認された。
（２）ＣＴＳＤ発現の抑制によるＭＭＣ感受性の変化
（１）と同様にしてＣＴＳＤ特異的なｓｉＲＮＡ ＣＴＳＤ ＃４００を肝癌細胞株ＳＳＰ−２５にトランスフェクションした。トランスフェクションしてから４８時間後に細胞を回収し、９６ウェルプレートに１×１０^４個／ウェルずつ分注し、５％ＦＢＳ、１００単位／ｍＬペニシリンおよび１００ｍｇ／ｍＬストレプトマイシンを含むＲＰＭＩ １６４０培地１００μＬで一晩培養した。ＭＭＣ、パクリタキセル、ドキソルビシンを、ＭＭＣおよびドキソルビシンは１０^−５〜１０^−９ｍｏｌ／Ｌの濃度、パクリタキセルは１０^−６〜１０^−１０ｍｏｌ／Ｌの濃度になるようにそれぞれ添加して培養した。薬剤を添加してから４８時間後に、ｓｕｌｆｏｒｈｏｄａｍｉｎｅ Ｂアッセイ法により細胞の増殖抑制を測定し、抗癌剤に対する感受性を評価した。
その結果、図６Ａに示すように、ＣＴＳＤ ＃４００をトランスフェクトしなかったコントロールの細胞と比べ、ＣＴＳＤ ＃４００をトランスフェクトし、ＣＴＳＤ遺伝子の発現を抑制した細胞では、ＭＭＣに対する感受性が低下することが確認された。このことから、ＣＴＳＤ遺伝子は、肝癌において、発現を抑制することによりＭＭＣに対する感受性が低下するような感受性決定遺伝子であることが確認された。肝癌においては、ＣＴＳＤ遺伝子の発現は有意にＭＭＣに対する感受性と相関しているが、パクリタキセルおよびドキソルビシンに対する感受性とは有意な相関は見られていない（図７、表１３）。ＣＴＳＤ遺伝子の発現を抑制した細胞において、パクリタキセルおよびドキソルビシンに対する感受性には変化はみられなかった（図６Ｂ）。
以上の結果はバイオインフォマティクス的アプローチを用いてＤＮＡアレイの実験結果から抽出した結果が、抗癌剤に対する感受性を決定する遺伝子の予測に有効であることを示している。[Example 1]: Sensitivity test for anticancer drugs comprising a panel of 45 human cancer cell lines
The following 45 types of cell lines were used. Those without individual citations are described below (Yamori, T., et.al., Cancer Res, 59: 4042-4049, 1999.).
Breast cancer 12 cell lines: HBC-4, BSY-1, HBC-5, MCF-7, MDA-MB-231, KPL-3C (Kurebayashi, J., et.al., Br J Cancer, 74: 200-207 , 1996.) KPL-4 (Kurebayashi, J., et.al., Br J Cancer, 79: 707-717, 1999.), KPL-1 (Kurebayashi, J., et.al., Br J Cancer, 71: 845-853, 1995.), T-47D (Shiu, RP, J Biol Chem, 255: 4278-4281, 1980.), HBC-9, ZR-75-1 (Engel, L.W.). , Et al, Cancer Res, 38: 3352-3364, 1978.), HBC-8;
Hepatoma 12 cell line: HepG2 (Schwartz, AL, et. Al., J Biol Chem, 256: 8878-8881, 1981; ATCC No. HB-8065), Hep3B (Liu, P., et.al. , Int J Oncol, 16: 599-610, 2000. Tohoku University Institute of Aging Medicine, Medical Cell Resource Center TKG 0291), Li-7 (Tohoku University Institute of Aging Medicine, Medical Cell Resource Center TKG 0368), PLC / PRF / 5 (MacNab, GM, et.al., Br J Cancer, 34: 509-515, 1976 .; Human Science Research Resource Bank JCRB0406), HuH7 (Kaneko, S., et.al., Cancer) Res, 55: 5283-5287, 1995. Huma Science Research Resource Bank JCRB0403), HLE (Dor, I., et. Al., Gann, 66: 385-392, 1975 .; Human Science Research Resource Bank JCRB0404), HLF (Dor, I., et.al., Gann, 66: 385-392, 1975 .; Human Science Research Resource Bank JCRB0405), HuH6 (Tanaka, M., et.al., Biochem Biophys Res Commun, 110: 837-841, 1983 .; RIKEN BioResource Center RCB1367), RBE (Fukutomi, M., et.al., Cell Biochem Funct, 19: 65-68, 2001 .; RIKEN BioResource Center RCB1292), SSP- 25 (Fukutomi, M., et.al., Cell Biochem Funct, 19: 65-68, 2001 .; RIKEN BioResource Center RCB1293), HuL-1 (Sorimachi, K., et.al., Cell Struct Funct) , 13: 1-11, 1988.), JHH-1 (Fujise, K., et.al., Hepatogastroenterology, 37: 457-460, 1990.);
Gastric cancer 21 cell lines: St-4, MKN1, MKN7, MKN28, MKN45, MKN74, GCIY (Nozue, M., et.al., Hum Cell, 4: 71-75, 1991), GT3TKB (Tamura, G., et.al., Jpn J Cancer Res, 87: 1153-1159, 1996), HGC27 (Hassan, S., et.al., Dig Dis Sci, 43: 8-14, 1998), AZ521 (Imanishi, K.). , Et.al., Br J Cancer, 59: 761-765, 1989.), 4-1st (Morisada, S., et.al., Jpn J Cancer Res, 80: 69-76, 1989.), NUGC. -3, NUGC-3 / 5FU (Inaba, M., et. Al. Jpn J Cancer Res, 87: 212-220, 1996), HSC-42 (Nishiyama, M., et.al., Int J Cancer, 72: 649-656, 1997), AGS (Barranco, S.C.,). et.al., Cancer Res, 43: 1703-1709, 1983), KWS-1, TGS-11, GMK-2 (also known as OKIBA), Ist-1 (Terashima, M., et.al., Jpn J Cancer). Res, 82: 883-885, 1991), GMK-3, GMK-5 (also known as AOTO).
All cell lines were grown in RPMI 1640 (Nishii Pharmaceutical) medium (containing 5% calf serum, penicillin (100 units / ml), streptomycin (100 μg / ml)) at 37 ° C., 5% CO 2. ₂ Cultured in the presence.
The sensitivity to anticancer agents was evaluated by inhibiting growth. For the measurement of growth inhibition, the change in the total amount of intracellular protein after drug contact for 48 hours was measured by the sulfohodamine B assay. GI ₅₀ Calculation of the value (concentration of anticancer agent showing growth inhibition of 50%, unit mol / L) was according to the following literature (Yamori, T., et.al., Cancer Res, 59: 4042-4049, 1999. Monks, A., et.al., J Natl Cancer Inst, 83: 757-766, 1991.). GI of median value of triplicate experiment ₅₀ Value.
Presumed mechanism of action of the 51 anticancer agents used and log for each of 45 cell lines ₁₀ GI ₅₀ The absolute values of are shown in Tables 1-5.

FIG. 1 shows the result of hierarchical clustering of 51 anticancer agents based on the sensitivity of 45 human cancer cell lines. Hierarchical clustering was performed using Gene Spring software (Silicon Genetics) using a group average method. The distance between the data is the log of each cell between the two anticancer drugs. ₁₀ GI ₅₀ Pearson's correlation coefficient obtained from the absolute value distribution was used.
Two anticancer drugs log ₁₀ GI ₅₀ As a result, it was possible to divide 51 drugs into several clusters, and each cluster contained a plurality of drugs having a similar mechanism of action. For example, some clusters have inhibitors of topoisomerase I such as camptothecin and topotecan, and the second cluster has microtubule agents such as taxane anticancer agents (paclitaxel, docetaxel) and vinca alkaloid anticancer agents (vinblastine, vincristine, vinorelbine). (Tubulin binder) gathered, and 5-fluorouracil and its derivatives also gathered in one cluster. This result shows that the system using the 45 cells of the inventors can classify a large number of drugs based on the mechanism of action.
[Example 2]: Clustering of 42 human cancer cells by gene expression profile
Among the above-mentioned human cancer cell panel comprising 45 cells, expression of 3537 genes was measured by cDNA array using 42 cell lines excluding 3 cells of HBC-9, HBC-8 and GMK-3.
<Measurement of gene expression by cDNA array>
As an cDNA array, Atlas ^TM This was repeated twice using human 3.6array (BD Biosciences Clontech). The experiment was performed according to the Clontech product instructions, but is briefly described below. The cell line was treated in the logarithmic growth phase, and total RNA (Total RNA) was extracted using Trizol reagent (Invitrogen) and purified using RNeasy Kit (Qiagen). Purified Total RNA ^TM By reverse transcription using Pure Total RNA Labeling System (BD Biosciences Clontech) ³² It was converted to a cDNA probe labeled with P. This cDNA probe was hybridized to Atlas Array at 68 ° C. overnight and then washed. The hybridized array was scanned by PhosphorImager (Molecular Dynamics) to detect the signal. The detected data is Atlas ^TM Quantified by Image 2.0 software (BD Biosciences Clontech) and normalized by dividing by the 90th percentile value of all genes. Genes that showed different values more than twice in two repeated experiments were excluded from the analysis. A value of 30% of the 90th percentile value was set as a threshold value, and a threshold value was given to values lower than the threshold value. After performing this data processing, it was converted to a logarithmic value with 2 as the base.
Based on the results of this expression profile, the cells were classified by hierarchical clustering. Hierarchical clustering was performed using the group average method with Gene Spring software (Silicon Genetics). The distance between the data is the log of each anticancer drug between the two cells. ₁₀ GI ₅₀ Pearson's correlation coefficient obtained from the absolute value distribution was used.
The result of clustering is shown in FIG. There was a tendency for cell lines derived from the same tissue to form clusters. That is, breast cancer cell lines excluding KPL-4 were combined into one clusterer. In addition, although several gastric cancer cell lines were inserted between the liver cancer cell lines, they were combined into one except for HLE. The stomach cancer cell lines were roughly divided into two groups. The above results indicate that, as far as the results of gene expression profiles are concerned, the established cell line also maintains the nature of the tissue from which it was derived.
[Example 3]: Correlation analysis of gene expression profile and sensitivity to anticancer agents
In order to search for gene groups related to susceptibility to anticancer agents, the two databases of gene expression and susceptibility to anticancer agents obtained in Example 1 and Example 2 were integrated and the correlation analysis was performed. A comprehensive calculation of Pearson's correlation coefficient was performed for 42 cell lines using expression of 3537 gene and sensitivity to 51 anticancer agents. The calculation formula used is as follows.

Where x _i Indicates a logarithmic expression of gene x in cell i, y _i Is the logarithmic absolute value of the sensitivity of cell i to anticancer agent y (| log ₁₀ GI ₅₀ |). x _m Indicates the mean value (mean) of the logarithm of the expression level of gene x, y _m Is the absolute value of the logarithm of sensitivity to the anticancer agent y (| log ₁₀ GI ₅₀ The average value of | A P value of less than 0.05 (p <0.05) was considered a significant correlation.
With the restriction that “P <0.05 and expression is observed in 50% or more of all cells”, “a gene showing expression significantly correlated with sensitivity to an anticancer agent” was selected. A different set of genes was extracted for each of the 51 anticancer agents. Tables 6 to 11 show a set of genes that correlate with sensitivity to MMC, vinorelbine, and paclitaxel when all 42 cells are used. That is, Tables 6 to 11 show a list of genes that significantly correlate with sensitivity to anticancer agents. The correlation analysis result in 42 cell lines is shown about MMC (Table 6 and Table 7), vinorelbine (Table 8 and Table 9), and paclitaxel (Table 10 and Table 11). The column “gene” indicates the gene name in HUGO database. “Correlation coefficient” indicates the value of Pearson's correlation coefficient between the sensitivity to an anticancer drug and the expression of the gene. Tables 6, 8 and 10 (supplied with “susceptibility”) show genes that are sensitive when strongly expressed, and Tables 7, 9 and 11 (supplied with “resistance”) show genes that are resistant with strong expression. Show.

As shown in Tables 6 and 7, in the case of MMC, 20 genes were selected as sensitive genes (highly expressed in sensitive strains) and 10 genes were selected as resistant genes (highly expressed in resistant strains). Among these genes, JUN, EMS1, and NMBR are genes related to cell proliferation. In addition, various genes such as SOD1, PELP1, and SFRS9 were included. Further, as shown in Table 10, in paclitaxel, which is a microtubule acting drug, the VIL2 gene encoding ezrin related to the cytoskeletal system was selected. Similarly, many genes were selected as susceptibility or resistance genes for each anticancer agent.
Furthermore, analysis using Pearson's correlation coefficient was performed for each cell line derived from the same tissue. Tables 12 to 14 show gene groups selected as genes correlated with sensitivity to anticancer agents in 10 breast cancer cell lines, 12 liver cancer cell lines, and 20 gastric cancer cell lines, respectively. That is, Table 12 shows breast cancer, Table 13 shows liver cancer, Tables 14-1 and 2 show gastric cancer, and shows a list of genes whose sensitivity and expression correlate with mitomycin C, respectively. The column descriptions are the same as in Tables 6-11. These gene groups include those that can be used as markers for predicting susceptibility to anticancer drugs, and some genes may actually regulate susceptibility to anticancer drugs.

Tables 15 to 18 show gene groups selected as genes correlated with susceptibility to various anticancer agents other than mitomycin C in 12 liver cancer cell lines. That is, in liver cancer, Table 15 shows paclitaxel, Table 16 shows 5-fluorouracil, Table 17 shows doxorubicin, and Table 18 shows a list of genes whose sensitivity and expression correlate with cisplatin. The column descriptions are the same as in Tables 6-11. These gene groups include those that can be used as markers for predicting susceptibility to anticancer drugs, and some genes may actually regulate susceptibility to anticancer drugs.

[Example 4]: Verification of selected gene expression by RT-PCR method
From the experimental results of the DNA array shown in Experimental Example 3, genes selected as having a correlation with sensitivity to MMC in liver cancer and gastric cancer were verified by the RT-PCR method.
For gastric cancer, specific PCR primers were prepared for 6 types of sensitive genes and 7 types of resistant genes shown in Table 19, and the expression level of each gene in each gastric cancer cell line was measured by RT-PCR. The correlation with the susceptibility to MMC was examined. RT-PCR was performed by incubating a reaction solution containing 2 μg of total RNA prepared in Example 2, RNase inhibitor, oligo d (T) 16, MuLV reverse transcriptase at 42 ° C. for 60 minutes to synthesize cDNA, PCR using each gene-specific PCR primer was performed using 1/10 of this reaction solution as a template. In PCR, after heating at 95 ° C for 5 minutes, the reaction cycle of 95 ° C for 1 minute (denaturation), 55-60 ° C for 1 minute (annealing), and 72 ° C for 1-2 minutes (extension) is repeated 25-30 cycles , Under the conditions of heating at 72 ° C. for 5 minutes (annealing temperature, extension time, cycle number varies depending on the gene).
As a result, two genes, TEA domain family member 4 (TEAD4, GenBank accession number: U63824) and Solute Carrier Family 6 (SLC6A8, GenBank accession number: L31409) as susceptibility genes, and Proteasome ATunSit26Sit26Sunt26Sun26 (PSMD8, GenBank registration number: D38047), catthepsin D (CTSD, GenBank registration number: M11233), annexin A4 (ANXA4, GenBank registration number: M19383), KDEL endopretic reticulumG1 4 gene 885), correlation with susceptibility was observed on the expression amount and MMC measured by RT-PCR, was confirmed as the gene does correlate to expression and sensitivity (Table 19).

Further, for liver cancer, three types shown in Table 20 as susceptibility genes were similarly verified, and the genes of c-jun (JUN, GenBank registration number: J04111), catthepsin D (CTSD, GenBank registration number: M11233), A correlation between the expression level measured by RT-PCR and sensitivity to MMC was observed, and a correlation between the expression level and sensitivity was confirmed (Table 20).

Furthermore, the same susceptibility gene candidate for paclitaxel in liver cancer was similarly verified by RT-PCR. As a susceptibility gene, acetylCoA-acetyltransferase (ACAT1, GenBank accession number: D90228, rank 10 in Table 15), and resistance gene For bcl-XL (BCL2L1, GenBank accession number: Z23115, rank 9 in Table 15), there was a correlation between the expression level measured by RT-PCR and sensitivity to paclitaxel, and the correlation found by the cDNA array confirmed.
[Example 5]: Verification of selected gene expression by Western blot
In Experiments 3 and 4, in the case of cathepsin D (CTSD) in which the expression level of the gene measured by DNA array or RT-PCR in liver cancer was confirmed to be correlated with the sensitivity to MMC, It was verified whether there was a correlation between the gene expression level and the sensitivity to MMC when the amount of protein as a product was measured by Western blot.
The cells were cultured on a plate and then washed twice with PBS (−). Cells are peeled from the plate, and lysis buffer [62.5 mmol / L Tris-HCl pH 6.8, 2% (W / V) sodium dodecyl sulfate (SDS), 5% (v / v) 2-mercaptoethanol, 10% ( v / v) glycerol, 1 mmol / L phenylmethylsulfonyl fluoride]. The protein concentration of the cell lysate was quantified using a DC protein assay kit (Bio-Rad), and the cell lysate containing 10 μg of protein was separated by electrophoresis on a 10% polyacrylamide-SDS gel. Proteins were transferred from the gel to the membrane and analyzed by Western blot as follows. The protein-transferred membrane was reacted with CTSD-specific mouse monoclonal antibody Ab-1 (Oncogene Research Products) diluted 1: 250. The membrane was washed and further reacted with horseradish-conjugated anti-mouse IgG antibody (Amersham Biosciences) diluted 1: 1000. CTSD protein was detected using an ECL (Enhanced Chemiluminescence) detection system (Amersham Biosciences), and the signal intensity was measured.
When the correlation between the signal intensity of the CTSD protein and the sensitivity to MMC obtained in Example 1 was examined, the Pearson correlation coefficient was 0.862. Therefore, a correlation was observed between the expression level of CTSD gene measured by Western blot and sensitivity to MMC (FIG. 3).
[Example 6]: Identification of gene that changes sensitivity of anticancer drug to anticancer drug (1)
In order to search for genes that determine susceptibility to anticancer drugs, we established a genetic screening system that can detect changes in susceptibility to anticancer drugs by inhibiting cell proliferation. This is a system for detecting a change in sensitivity to an anticancer agent by introducing a gene into a cell. As an index of cell proliferation, [ ³ Incorporation of H] -thymidine was used. High transfection efficiency is required to detect slight changes in sensitivity. Therefore, HT1080 which is a human fibroblastoma cell line capable of gene transfer with an efficiency of 90% or more was used.
This system was used to screen for genes that determine the sensitivity of MMC to anticancer agents. The entire translation region of the candidate gene cDNA was cloned and cloned into an animal cell expression vector pcDNA3.1 / myc-HisA (Invitrogen). These expression plasmids were introduced into HT1080 cells using Lipofectamine Plus reagent (Invitrogen). 24 hours after transfection, an appropriate concentration of MMC was added, and the cells were further cultured for 24 hours. 0.067MBq [ ³ H] -thymidine was added and incubated at 37 ° C. for 45 minutes, followed by washing with pre-warmed PBS (−) and treatment with 10% TCA on ice for 2 hours to fix the cells. After fixation, the cells were washed with 10% TCA and lysed with 0.1% SDS / 0.2% NaOH. The lysed cells were incubated at 37 ° C. and then neutralized by adding a 0.25 M acetic acid solution. [ ³ H] -thymidine incorporation was measured with a scintillation counter.
To confirm the effectiveness of this system, DT-diaphorase (Gustafson, DL, & Pritosos, CA, Cancer Res., 52, 6936-6939, known to define susceptibility to MMC, is confirmed. , 1992.) was examined for the gene NQO1. HT1080 cells into which NQO1 had been introduced had increased sensitivity to MMC (FIG. 4C). That is, this system is considered to be an effective system for detecting changes in sensitivity to anticancer agents.
Using this system, it was examined which of the genes selected in Table 6 and Tables 12 to 14 actually changes the sensitivity to anticancer agents. As a result, it was shown that introduction of the HSPA1A gene (GenBank accession number: M11717) encoding the HSP70-1 protein significantly increased sensitivity to MMC (FIG. 4C). The expression of HSPA1A is significantly correlated with sensitivity to MMC in breast cancer (FIG. 4A, Table 12) and liver cancer (Table 13), but no significant correlation is observed in gastric cancer (Table 14). Moreover, the introduction of the JUN gene (GenBank accession number: J04111) (FIG. 4B, Table 6), whose correlation between expression and sensitivity to MMC was found in the analysis of all cell lines, also significantly increased the sensitivity to MMC ( FIG. 4D). The above results indicate that the results extracted from the DNA array experimental results using a bioinformatics approach are effective in predicting genes that determine the sensitivity to anticancer agents.
[Example 7]: Identification of gene that changes sensitivity of anticancer drug to anticancer drug (2)
In order to search for genes that determine sensitivity to anticancer drugs, a screening system was established to detect changes in sensitivity to anticancer drugs when gene expression was suppressed. The following is an example of confirming that the CTSD gene in which correlation between gene expression and sensitivity to MMC is found in liver cancer is a sensitivity determining gene.
(1) Confirmation of suppression of CTSD expression by siRNA
CTSD-specific siRNA (CTSD # 271, CTSD # 387 and CTSD # 400) for suppressing the expression of the CTSD gene was prepared as follows. Oligonucleotides comprising each of the sequences of SEQ ID NOs: 1 to 6 (consigned synthetic products from Dharmacon Research Inc.) were dissolved in buffers supplied from Dermacon Research, respectively, and SEQ ID NOs: 1 and 2, Mixed with combinations of 3 and 4, 5 and 6. Each was incubated at 60 ° C. for 45 minutes, and then annealed at room temperature for 30 minutes to be annealed, double-stranded oligonucleotide CTSD # 271 (sense strand: SEQ ID NO: 1, antisense strand: SEQ ID NO: 2), CTSD # 387 (sense Strand: SEQ ID NO: 3, antisense strand: SEQ ID NO: 4) and CTSD # 400 (sense strand: SEQ ID NO: 5, antisense strand: SEQ ID NO: 6). After each precipitation, 1 × annealing buffer [30 mmol / L HEPES-KOH (pH 7.9), 100 mmol / L potassium acetate, 2 mmol / L magnesium acetate] to a final concentration of 20 μmol / L. Dissolved in.
The liver cancer cell line SSP-25 was suspended in Dulbecco's modified Eagle medium (DMEM) containing 5% bovine serum and 2.5 × 10 6 on a 6-well plate. ⁵ Individual / well was dispensed. After 16 hours, the above siRNAs (CTSD # 271, CTSD # 387 and CTSD # 400) were each transfected into cells using Lipofectamine 2000 (Invitrogen) at a concentration of 100 nmol / L per well. .
Cells were collected 48 hours after siRNA transfection, and the CTSD protein of the cells was detected by Western blot in the same manner as in Example 5. As a result, as shown in FIG. 5, in the cells into which CTSD # 271, CTSD # 387 and CTSD # 400 were introduced, almost no CTSD protein was detected, and it was confirmed that the expression of the CTSD gene was suppressed by siRNA. It was done.
(2) Changes in MMC sensitivity due to suppression of CTSD expression
In the same manner as in (1), CTSD-specific siRNA CTSD # 400 was transfected into hepatoma cell line SSP-25. Cells were harvested 48 hours after transfection and placed in a 96 well plate at 1 × 10 ⁴ Individual / well were dispensed and cultured overnight in 100 μL of RPMI 1640 medium containing 5% FBS, 100 units / mL penicillin and 100 mg / mL streptomycin. MMC, paclitaxel, doxorubicin, 10 MMC and doxorubicin ^-5 -10 ^-9 mol / L concentration, paclitaxel is 10 ^-6 -10 ^-10 Each was added and cultured to a concentration of mol / L. Forty-eight hours after the addition of the drug, inhibition of cell proliferation was measured by the sulfohodamine B assay, and the sensitivity to the anticancer drug was evaluated.
As a result, as shown in FIG. 6A, the sensitivity to MMC is decreased in cells transfected with CTSD # 400 and suppressed in expression of the CTSD gene, compared to control cells not transfected with CTSD # 400. Was confirmed. From this, it was confirmed that the CTSD gene is a sensitivity determining gene whose sensitivity to MMC is decreased by suppressing expression in liver cancer. In liver cancer, CTSD gene expression is significantly correlated with sensitivity to MMC, but no significant correlation is observed with sensitivity to paclitaxel and doxorubicin (FIG. 7, Table 13). In the cells in which the expression of the CTSD gene was suppressed, there was no change in sensitivity to paclitaxel and doxorubicin (FIG. 6B).
The above results indicate that the results extracted from the DNA array experimental results using a bioinformatics approach are effective in predicting genes that determine the sensitivity to anticancer agents.

  According to the present invention, genes that predict susceptibility to anticancer agents in tumor cells and cancer cell lines extracted from cancer patients can be selected, and the sensitivity of cancer patients to anticancer agents using these genes can be determined. . Moreover, it becomes possible to enhance the sensitivity to anticancer agents by using these genes or by controlling the expression of these genes.

[Sequence Listing]

Claims (64)

Breast cancer 12 cell lines (HBC-4, BSY-1, HBC-5, MCF-7, MDA-MB-231, KPL-3C, KPL-4, KPL-1, T-47D, HBC-9, ZR-75) -1, HBC-8), liver cancer 12 cell lines (HepG2, Hep3B, Li-7, PLC / PRF / 5, HuH7, HLE, HLF, HuH6, RBE, SSP-25, HuL-1, JHH-1), And gastric cancer 21 cell lines (St-4, MKN1, MKN7, MKN28, MKN45, MKN74, GCIY, GT3TKB, HGC27, AZ521, 4-1st, NUGC-3, NUGC-3 / 5FU, HSC-42, AGS, KWS- 1, TGS-11, GMK-2 (also known as OKIBA), Ist-1, GMK-3, GMK-5 (also known as AOTO)) Using all or part of the tumor cells, the expression level of at least one gene in the cell line and the sensitivity to anticancer agents were measured, and the presence or absence of a correlation between them was statistically tested. A method for identifying genes related to susceptibility to anticancer drugs. Gastric cancer 21 cell lines (St-4, MKN1, MKN7, MKN28, MKN45, MKN74, GCIY, GT3TKB, HGC27, AZ521, 4-1st, NUGC-3, NUGC-3 / 5FU, HSC-42, AGS, KWS-1 , TGS-11, GMK-2 (also known as OKIBA), Ist-1, GMK-3, and GMK-5 (also known as AOTO)), and all or part of the cancer cell line is used for gastric cancer cells. The method according to claim 1, wherein a gene group involved in susceptibility to an anticancer drug is identified. Breast cancer 12 cell lines (HBC-4, BSY-1, HBC-5, MCF-7, MDA-MB-231, KPL-3C, KPL-4, KPL-1, T-47D, HBC-9, ZR-75) The method according to claim 1, wherein a gene group relating to the sensitivity of a breast cancer cell to an anticancer agent is identified using all or part of a cancer cell line selected from -1, HBC-8). All cancer cell lines selected from 12 liver cancer cell lines (HepG2, Hep3B, Li-7, PLC / PRF / 5, HuH7, HLE, HLF, HuH6, RBE, SSP-25, HuL-1, JHH-1) Or the method of Claim 1 which identifies the gene group which concerns on the sensitivity with respect to the anticancer agent which hepatoma cell used using a part. In a method for determining the sensitivity of a cancer patient to an anticancer agent,
(1) A step of selecting one or more genes from the gene group identified by the method according to any one of claims 1 to 4 and determining a score corresponding to the expression level for each of the selected genes. ,
(2) measuring the expression level of the gene for the test patient;
(3) assigning the score obtained in (1) based on the expression level measured in (2),
(4) a step of summing up the assigned points for all selected genes to obtain a total value, and (5) a step of comparing the total value with a predetermined threshold value,
Including the above method. In a method for determining the sensitivity of a cancer patient to mitomycin C, SF1 (D26121), CBR3 (AB004854), EMS1 (M98343), JUN (J04111), SFRS9 (U30825), NMBR ( M73482), RBMX (Z23064), SOD1 (M13267), NOL1 (X55504), PELP1 (U88153), ARHA (L25080), AARS (D32050), NME1 (X17620), HNRPA2B1 (M29065), NME2 (L16785), VME U18009), SERPINB10 (U35459), KIAA0436 (AB007896), DRPLA (D31840), MC3R (L06155), SPTB 1 (M96803), PET112L (AF026851), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770), RPLP2 (M17887), and ARF4L (L4490) A method for determining the sensitivity to mitomycin C using the method according to claim 5 by measuring the expression level of one or a plurality of genes selected from the group consisting of: In a method for determining the sensitivity of a cancer patient to vinorelbine, ARHA (L25080), NME2 (L16785), VIL2 (X51521), YWHAQ (X56468), HK1 (M75126), SATB1 (M97287) in tumor cells removed from cancer patients. ), CAMLG (U18242), CARS (L06845), CCNB1 (M25753), U2AF1 (M96982), PTMA (M26708), MLC1SA (M31212), NME1 (X17620), SARS (X91257), CDC20 (U05340), PPP4C ), TNFAIP3 (M59465), EEF1D (Z21507), PFKP (D25328), ENTPD2 (U91510), CCL5 (M2112) ), ACAT1 (D90228), IQGAP1 (L33075), PAX5 (M96944), NRGN (Y09689), K-ALPHA-1 (K00558), NDUFB7 (M33374), HOXB1 (X16666), F10 (K03194), GPX2 (X46) , NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (M36263), MAN2B1 (U60266), LSS (D63807), P3368G, 833 , NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X60656), 2 (V00595), RARA (X06614), ITGB4 (X53587), IMPA1 (X66922), PACE (X17094), AGA (M64073), MVD (U49260), EHHADH (L07077), TFPI2 (D29992), MARCKS (M68856) The expression level of one or more genes selected from the group consisting of FGB (J00129) and GPD1 (L34041) (the parentheses indicate GenBank registration numbers) is measured, and the method according to claim 5 is used. A method for determining sensitivity to vinorelbine. In a method for determining the sensitivity of a cancer patient to paclitaxel, ADH6 (M68895), RAB28 (X94703), U2AF1 (M96982), GPC1 (X54232), HK1 (M75126), CARS (L06845) in tumor cells removed from the cancer patient. ), TNFAIP3 (M59465), K-ALPHA-1 (K00558), PFKP (D25328), GDI2 (D13988), VIL2 (X51521), RUNX2 (AF001450), NME2 (L16785), CDC20 (U05340), GNAI2 (X028) , ARHA (L25080), CNR2 (X74328), PPP2R2B (M64930), SLC6A8 (L31409), DDX9 (L13848), ACAT (D90228), PI3 (Z18538), NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383A, DRP4) (M64073), BCL2L1 (Z23115), LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RARA (PT066) (L18983), APOE (M12529), F10 (K03194), NR1I2 (AF061056), UBE2L3 ( 92962), and the expression level of one or more genes selected from the group consisting of FGB (J00129) (indicated by the GenBank registration number in parentheses) are used to measure paclitaxel using the method according to claim 5. A method of determining sensitivity. In a method for determining the sensitivity of a cancer patient to 5-fluorouracil, SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 in tumor cells removed from the cancer patient. (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661), PLOD (M98252), CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01667), PAFAH1B3 (D63391) (L07956), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and UBE2E1 (X92963) One or more genes (in parentheses indicate the GenBank accession number) measuring the expression level of, method of determining sensitivity to 5-fluorouracil by using a method according to claim 5 selected from. In a method for determining the sensitivity of a cancer patient to doxorubicin, RPS7 (M77233), RPL23 (X52839), CLT (X72946), EEF1D (Z21507), RPS15A (X84407), ADCY1 (L05500) in tumor cells removed from the cancer patient. ), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232), the expression level of one or a plurality of genes (indicated by the GenBank registration number) The method of determining the sensitivity with respect to doxorubicin using the method of Claim 5. In a method for determining the sensitivity of a cancer patient to cisplatin, CALT (X72946), ZNF165 (X84801), GPC1 (X54232), TUBA1 (K00558), GCP2 (X78686), COX5A (M22760) in tumor cells removed from the cancer patient. ), GLA (X00351) and GNS (X0695) are used to measure the expression level of one or more genes selected from the group consisting of GenBank registration numbers in parentheses, and the method according to claim 5 is used. To determine sensitivity to cisplatin. The method according to claim 5, wherein the patient is a breast cancer patient. The method for determining sensitivity according to claim 5, wherein the patient is a stomach cancer patient. The method according to claim 5, wherein the patient is a liver cancer patient. In a method for determining the sensitivity of a breast cancer patient to mitomycin C, INHBB (M31682), NK4 (M59807), HSPA1A (M11717), LOC54557 (AF0775050), CD47 (Y00815), RPN2 ( One or more genes selected from the group consisting of Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950) and NRGN (Y09689) A method for determining the sensitivity to mitomycin C using the method according to claim 5 by measuring the expression level (indicated by GenBank registration number in parentheses). In a method for determining the sensitivity of a liver cancer patient to mitomycin C, EB1 (U24166), JUN (J04111), EIF3S8 (U46025), CCL5 (M21121), PHB (S85655), HSPA1A ( M11717), SPP1 (X13694), RAB7 (X93499), CTSD (M11233), ACTN1 (X15804), RXRB (M84820), PSME2 (D45248), HLA-C (M11886), RPL19 (X63527), MAPK6 (X80692) GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M57627) Expression of one or more genes selected from the group consisting of RFC4 (M87339), CRLF1 (AF059293), RPS6 (M20020), EMX1 (X68879) and TK2 (U77088) (indicated by GenBank registration numbers in parentheses) A method for determining the sensitivity to mitomycin C using the method according to claim 5 by measuring the amount. In a method for determining the sensitivity of a gastric cancer patient to mitomycin C, TEAD4 (U63824), NR2C2 (U10990), CSF1 (M37435), RAB28 (X94703), CBR3 (AB004854), NFYC ( Z74792), PGF (X54936), ERG (M21535), MLLT1 (L04285), FOS (K00650), TNFAIP3 (M59465), CNR2 (X74328), DRPLA (D31840), PSMB5 (D29011), SLC6A8SE (L3140B) U35459), VAT1 (U18009), TJP1 (L14837), PELP1 (U88153), C1QBP (L04636), CDK 0 (L33264), SERPINA6 (J02943), ACTB (X00351), SFRP4 (AF026669), EMX1 (X68879), RPS9 (U14971), AMD1 (M21154), RPL26 (X69392), HNRPF (L28098), PTMS (M24398) STK12 (AF008552), NR2F6 (X12794), GBE1 (L07756), PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD23A SDHA (D30648), PET112L (AF026851), DAD1 (D15057), HSPB1 (X54 79), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M96803), PACE (X17094), RPL24 (M94314), SPINT2 (X78095) U07158), SIAT8B (U33551), CTSK (U13665), DCI (U24774), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D380) The expression level of one or more genes selected from (the GenBank registration number is shown in parentheses) is measured, and the method according to claim 5 is used. A method for determining sensitivity to mitomycin C. In a method of determining the sensitivity of a liver cancer patient to paclitaxel, ALDH3B1 (U10868), SRPK1 (U09564), ALPP (M13077), EB1 (U24166), MAPKAPK (U12779), GPI (K03515) in tumor cells removed from the liver cancer patient. ), CTBP1 (U37408), HMGCL (L07033), CAPZ (U03271), ACAT1 (D90228), COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J0469), SCD (Y13647), M86400 ), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 (M87338)? One or more genes (in parentheses indicate the GenBank accession number) measuring the expression level of, method of determining sensitivity to paclitaxel using a method according to claim 5 is selected. In a method for determining the sensitivity of liver cancer patients to 5-fluorouracil, SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 in tumor cells removed from liver cancer patients. (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661), PLOD (M98252), CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01667), PAFAH1B3 (D63391) (L07956), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and UBE2E1 (X92963) One or (in brackets shows the GenBank accession number) multiple genes measuring the expression level of, method of determining sensitivity to 5-fluorouracil by using a method according to claim 5 selected from that group. In a method for determining the sensitivity of a liver cancer patient to doxorubicin, RPS7 (M77233), RPL23 (X52839), CLT (X72946), EEF1D (Z21507), RPS15A (X84407), ADCY1 (L05500) in tumor cells removed from the liver cancer patient ), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232), the expression level of one or a plurality of genes (indicated by the GenBank registration number) The method of determining the sensitivity with respect to doxorubicin using the method of Claim 5. In a method for determining the sensitivity of a liver cancer patient to cisplatin, CALT (X72946), ZNF165 (X84801), GPC1 (X54232), TUBA1 (K00558), GCP2 (X78686), COX5A (M22760) in tumor cells removed from the liver cancer patient. ), GLA (X00351) and GNS (X0695) are used to measure the expression level of one or more genes selected from the group consisting of GenBank registration numbers in parentheses, and the method according to claim 5 is used. To determine sensitivity to cisplatin. The method according to any one of claims 1 to 21, wherein the gene expression level is measured by quantifying RNA, which is a transcription product of the gene, using a DNA microarray. The method according to any one of claims 1 to 21, wherein the gene expression level is measured by quantifying RNA, which is a transcription product of the gene, by quantitative PCR. The method according to any one of claims 1 to 21, wherein the expression level of the gene is measured by quantifying RNA, which is a transcription product of the gene, by Northern blotting. 24. An RNA quantification reagent comprising an oligonucleotide complementary to the RNA for use in the method of claim 23. The method according to any one of claims 1 to 21, wherein the expression level of the gene is measured by quantifying a protein that is a gene product of the gene by an immunochemical method. 27. The method according to claim 26, wherein the expression level of the gene is measured by quantifying the protein that is the gene product of the gene by ELISA. 27. The method according to claim 26, wherein the expression level of the gene is measured by quantifying a protein that is a gene product of the gene by Western blot. An immunoassay reagent comprising an antibody against the protein for use in the method of claim 26. SF1 (D26121), CBR3 (AB004854), EMS1 (M98343), JUN (J04111), SFRS9 (U30825), NMBR (M73482), RBMX (Z23064), SOD1 (M13267), NOL1 (X55504), PELP1 (U88153) ARHA (L25080), AARS (D32050), NME1 (X17620), HNRPA2B1 (M29065), NME2 (L16785), VAT1 (U18009), SERPINB10 (U35459), KIAA0436 (AB007896), DRPLA (D31840L and MC3R3840L) A single gene or a plurality of genes selected from the group consisting of (GenBank registration number in parentheses) and forced expression in cancer cells Gene or the gene shows activity enhancing sensitivity to mitomycin C contains a protein encoded by Rukoto, sensitizing agents against mitomycin C. ARHA (L25080), NME2 (L16785), VIL2 (X51521), YWHAQ (X56468), HK1 (M75126), SATB1 (M97287), CAMLG (U18242), CARS (L06845), CCNB1 (M25753), U2AF1 PTMA (M26708), MLC1SA (M31 211), NME1 (X17620), SARS (X91257), CDC20 (U05340), PPP4C (X70218), TNFAIP3 (M59465), EEF1D (Z21507), PFKP (D25328), PD25328U, PD CCL5 (M21121), ACAT1 (D90228), IQGAP1 (L33075), PAX5 (M96944), NRGN Y08989), K-ALPHA-1 (K00558), NDUFB7 (M33374), a single gene or a plurality of genes (indicated by GenBank accession numbers in parentheses), and forced expression in cancer cells by vinorelbine A drug for enhancing sensitivity to vinorelbine, which contains a gene exhibiting an activity to enhance sensitivity to or a protein encoded by the gene. ADH6 (M68895), RAB28 (X94703), U2AF1 (M96982), GPC1 (X54232), HK1 (M75126), CARS (L06845), TNFAIP3 (M59465), K-ALPHA-1 (K00558), PFKP (D25328), G2328 (D13988), VIL2 (X51521), RUNX2 (AF001450), NME2 (L16785), CDC20 (U05340), GNAI2 (X04828), ARHA (L25080), CNR2 (X74328), PPP2R2B (M64930), SLC6A8D (L13848), ACAT1 (D90228) and PI3 (Z18538) selected from the group consisting of one or more genes (in parentheses are G Gene or the gene shows activity enhancing sensitivity to paclitaxel by a showing a nBank registration number) forced expression in cancer cells containing the protein which encodes, sensitizing agents to paclitaxel. SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661) and PLODM (25236) A single gene or a plurality of genes selected from the group consisting of GenBank registration numbers in parentheses, and a gene exhibiting an activity to enhance sensitivity to 5-fluorouracil by forced expression in cancer cells or the gene encodes The sensitivity enhancement agent with respect to 5-fluorouracil containing the protein to do. A single gene or a plurality of genes selected from the group consisting of RPS7 (M77233), RPL23 (X52839), CALT (X72946), EEF1D (Z21507) and RPS15A (X84407) (indicated by the GenBank registration number in parentheses) A drug for enhancing sensitivity to doxorubicin, which comprises a gene exhibiting an activity of enhancing sensitivity to doxorubicin by forced expression in cancer cells or a protein encoded by the gene. A single gene or a plurality of genes selected from the group consisting of CALT (X72946), ZNF165 (X84801) and GPC1 (X54232) (indicated by the GenBank accession number in parentheses), which are expressed against cancer cells by forced expression in cancer cells A drug for enhancing sensitivity to cisplatin, comprising a gene exhibiting an activity for enhancing sensitivity or a protein encoded by the gene. A single gene or a plurality of genes selected from the group consisting of INHBB (M31682), NK4 (M59807), HSPA1A (M11717), LOC54557 (AF0775050) and CD47 (Y00815) (indicated by the GenBank registration number in parentheses), A drug for enhancing susceptibility to mitomycin C of breast cancer, comprising a gene exhibiting an activity of enhancing sensitivity to mitomycin C by forced expression in cancer cells or a protein encoded by the gene. EB1 (U24166), JUN (J04111), EIF3S8 (U46025), CCL5 (M21121), PHB (S85655), HSPA1A (M11717), SPP1 (X13694), RAB7 (X93499), CTSD (M11233), ACTN1 (X15) RXRB (M84820), PSME2 (D45248), HLA-C (M11886) and RPL19 (X63527) selected from the group or a single gene (in parentheses indicate GenBank registration number), A sensitivity enhancer for mitomycin C of liver cancer, comprising a gene exhibiting an activity of enhancing sensitivity to mitomycin C by forced expression or a protein encoded by the gene. TEAD4 (U63824), NR2C2 (U10990), CSF1 (M37435) RAB28 (X94703), CBR3 (AB004854), NFYC (Z74792), PGF (X54936), ERG (M21535), MLLT1 (L04285), FOS (K040), FOS (K0065) (M59465), CNR2 (X74328), DRPLA (D31840), PSMB5 (D29011), SLC6A8 (L31409), SERPINB10 (U35459), VAT1 (U18409), TJP1 (L14837), PELP1 (U88153), C1QPBP, C1QPBP (L33264), SERPINA6 (J02943), ACTB (X00351), SFRP4 (AF02669) ), EMX1 (X68879), RPS9 (U14971), AMD1 (M21154), RPL26 (X69392), HNRPF (L28010), PTMS (M24398), STK12 (AF008552), NR2F6 (X12794) and GBE1 (L07756). A single gene or a plurality of genes to be selected (indicated by GenBank accession numbers in parentheses), containing a gene that exhibits an activity of enhancing sensitivity to mitomycin C by forced expression in cancer cells or a protein encoded by the gene A sensitizer for gastric cancer to mitomycin C. ALDH3B1 (U10868), SRPK1 (U09564), ALPP (M13077), EB1 (U24166), MAPKAPK (U12779), GPI (K03515), CTBP1 (U37408), HMGCL (L07033), CAPZ (U03271) and ACAT2890D2 A gene selected from the group consisting of a single gene or a plurality of genes (indicated by the GenBank registration number in parentheses), which is a gene encoded by a cancer cell or a protein encoded by the gene that exhibits an activity to enhance sensitivity to paclitaxel by forced expression in cancer cells A sensitizer for liver cancer paclitaxel, comprising: SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661) and PLODM (2523625) A single gene or a plurality of genes selected from the group consisting of GenBank registration numbers in parentheses, and a gene exhibiting an activity to enhance sensitivity to 5-fluorouracil by forced expression in cancer cells or the gene encodes The sensitivity enhancement agent with respect to 5-fluorouracil of liver cancer containing the protein to do. A single gene or a plurality of genes selected from the group consisting of RPS7 (M77233), RPL23 (X52839), CALT (X72946), EEF1D (Z21507) and RPS15A (X84407) (indicated by the GenBank registration number in parentheses) A drug for enhancing sensitivity to doxorubicin in liver cancer, which comprises a gene showing an activity of enhancing sensitivity to doxorubicin by forced expression in cancer cells or a protein encoded by the gene. A single gene or a plurality of genes selected from the group consisting of CALT (X72946), ZNF165 (X84801) and GPC1 (X54232) (indicated by the GenBank accession number in parentheses), which are expressed against cancer cells by forced expression in cancer cells A drug for enhancing sensitivity to cisplatin of liver cancer, comprising a gene exhibiting an activity that enhances sensitivity or a protein encoded by the gene. A sensitivity enhancer for mitomycin C, which comprises the gene HSPA1A (GenBank accession number: M11717) or a protein encoded by the gene. A susceptibility enhancer for mitomycin C, which contains the gene JUN (GenBank accession number: J04111) or the protein encoded by the gene. A drug for enhancing sensitivity to mitomycin C, which comprises the gene CTSD (GenBank accession number: M11233) or a protein encoded by the gene. A test substance is added to the cells, and SF1 (D26121), CBR3 (AB004854), EMS1 (M98343), JUN (J04111), SFRS9 (U30825), NMBR (M73482), RBMX (Z23064), SOD1 (M13267) , NOL1 (X55504), PELP1 (U88153), ARHA (L25080), AARS (D32050), NME1 (X17620), HNRPA2B1 (M29065), NME2 (L16785), VAT1 (U18009), SERPINB10 (U35459), IA00785936 DRPLA (D31840) and MC3R (L06155);
ARHA (L25080), NME2 (L16785), VIL2 (X51521), YWHAQ (X56468), HK1 (M75126), SATB1 (M97287), CAMLG (U18242), CARS (L06845), CCNB1 (M25753), U2AF1 PTMA (M26708), MLC1SA (M31212), NME1 (X17620), SARS (X91257), CDC20 (U05340), PPP4C (X70218), TNFAIP3 (M59465), EEF1D (Z21507), PFKP (D25328), PD25328U, PD CCL5 (M21121), ACAT1 (D90228), IQGAP1 (L33075), PAX5 (M96944), NRGN Y09689), K-ALPHA-1 (K00558) and NDUFB7 (M33374);
ADH6 (M68895), RAB28 (X94703), U2AF1 (M96982), GPC1 (X54232), HK1 (M75126), CARS (L06845), TNFAIP3 (M59465), K-ALPHA-1 (K00558), PFKP (D25328), G2328 (D13988), VIL2 (X51521), RUNX2 (AF001450), NME2 (L16785), CDC20 (U05340), GNI2 (X04828), ARHA (L25080), CNR2 (X74328), PPP2R2B (M6499), X1409D (L13848), ACAT1 (D90228) and PI3 (Z18538);
INHBB (M31682), NK4 (M59807), HSPA1A (M11717), LOC54557 (AF0775050) and CD47 (Y00815);
EB1 (U24166), JUN (J04111), EIF3S8 (U46025), CCL5 (M21121), PHB (S85655), HSPA1A (M11717), SPP1 (X13694), RAB7 (X93499), CTSD (M11233), ACTN1 (X15) RXRB (M84820), PSME2 (D45248), HLA-C (M11886) and RPL19 (X63527);
TEAD4 (U63824), NR2C2 (U10990), CSF1 (M37435), RAB28 (X94703), CBR3 (AB004854), NFYC (Z74792), PGF (X54936), ERG (M21535), MLLT1 (L04285), FOS (00654) TNFAIP3 (M59465), CNR2 (X74328), DRPLA (D31840), PSMB5 (D29011), SLC6A8 (L31409), SERPINB10 (U35459), VAT1 (U18409), TJP1 (L14837), PELP1 (U881436), PLP1 (U881436) CDK10 (L33264), SERPINA6 (J02943), ACTB (X00351), SFRP4 (AF0266) 2), EMX1 (X68879), RPS9 (U14971), AMD1 (M21154), RPL26 (X69392), HNRPF (L28010), PTMS (M24398), STK12 (AF008552), NR2F6 (X12794) and GBE1 (L07956);
ALDH3B1 (U10868), SRPK1 (U09564), ALPP (M13077), EB1 (U24166), MAPKAPK (U12779), GPI (K03515), CTBP1 (U37408), HMGCL (L07033), CAPZ (U03271) and ACAT28D90
SRPK1 (U09564), OAZ1 (D78361), RPL34 (L38941), ARHB (X06820), RPS17 (M13932), RPL28 (U14969), TRIP11 (L40380), ZNF75 (S67970), RPL37 (D23661) and PLODM;
RPS7 (M77233), RPL23 (X52839), CALT (X72946), EEF1D (Z21507) and RPS15A (X84407); and CALT (X72946), ZNF165 (X84801) and GPC1 (X54232)
The expression level of at least one gene selected from the group consisting of (indicated by GenBank registration number in parentheses) is measured, and the expression level of the gene is compared with the expression level when the test substance is not added A method for screening a substance that enhances sensitivity to an anticancer agent, wherein the substance to be increased is selected as a candidate substance that enhances sensitivity to the anticancer agent. The substance which enhances the sensitivity with respect to the anticancer agent acquired by the method of Claim 46. 48. A drug for enhancing sensitivity to an anticancer agent, comprising the substance according to claim 47. SPTBN1 (M96803), PET112L (AF0266871), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770), RPLP2 (M17887), and A4487L A gene or a plurality of genes selected from the group consisting of (indicated by GenBank registration number in parentheses), which suppresses the expression of the gene in cancer cells and exhibits an activity to enhance sensitivity to mitomycin C SiRNA or antisense polynucleotide that suppresses the expression of. HOXB1 (X16666), F10 (K03194), GPX2 (X53463), NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (M36263), HSD17B1 (M36263) LSS (D63807), PIK3CG (X83368), DBN1 (U00802), NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X60656), F2 (V00595), RARA (X06614), ITGB4, 5GB35 IMPA1 (X66922), PACE (X17094), AGA (M64073), MVD (U49260), EH A single gene or a plurality of genes selected from the group consisting of ADH (L07077), TFPI2 (D29992), MARCKS (M68956), FGB (J00129), and GPD1 (L34041) (indicated by the GenBank registration number in parentheses) An siRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing the sensitivity to vinorelbine by suppressing the expression of the gene in cancer cells. NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383), SERPINF2 (D00174), AGA (M64073), BAZ23 LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RARA (X06614), PTPRN (L18983), APOE (L18983), APOE F10 (K03194), NR1I2 (AF061056), UBE2L3 (X92962), and FGB (J00129) Expression of a gene selected from the group consisting of a single gene or a plurality of genes (indicated by GenBank registration number in parentheses), which suppresses the expression of the gene in cancer cells, thereby enhancing the sensitivity to paclitaxel SiRNA or antisense polynucleotide that suppresses One or more selected from the group consisting of RPN2 (Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950), and NRGN (Y09689) SiRNA or antisense polynucleotide which is a gene (indicated by GenBank registration number in parentheses) and suppresses the expression of the gene exhibiting an activity to enhance sensitivity to mitomycin C by suppressing the expression of the gene in cancer cells . MAPK6 (X80692), GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M57627), RFC4 (M87339), CRLF1 (AF059293), RPS6 (MPS2020), RPS6 A single gene or a plurality of genes selected from the group consisting of EMX1 (X68879) and TK2 (U77088) (indicated by the GenBank accession number in parentheses), and by suppressing the expression of the gene in cancer cells, mitomycin C SiRNA or antisense polynucleotide which suppresses the expression of the gene which shows the activity which enhances the sensitivity with respect to. PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD23A (D21235), SDHA (D30648), PET112L (AF0265681), DAD1 (D15057) HSPB1 (X54079), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M96803), PACE (X17094), RPL24 (M94314), SPIN80T STX4A (U07158), SIAT8B (U33551), CTSK (U13665), DCI (U24 74), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D38048) or a single gene or multiple genes (inside parentheses are GenBank registrations) SiRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing sensitivity to mitomycin C by suppressing the expression of the gene in cancer cells. COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J0469), SCD (Y13647), YWHAZ (M86400), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 (M8733) Expression of a gene selected from the group consisting of a single gene or a plurality of genes (indicated by the GenBank registration number in parentheses), which suppresses the expression of the gene in cancer cells, thereby enhancing the sensitivity to paclitaxel SiRNA or antisense polynucleotide that suppresses CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01677), PAFAH1B3 (D63391), GBE1 (L07956), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and UBE2E1 One or a plurality of genes selected from the group (indicated by the GenBank registration number in parentheses) are genes that exhibit an activity of enhancing sensitivity to 5-fluorouracil by suppressing the expression of the gene in cancer cells SiRNA or antisense polynucleotide that suppresses the expression of. One or more genes selected from the group consisting of ADCY1 (L05500) HNRPU (X65488), COX5A (M22760), U2AF1 (M96982), and RPYR1 (U35232) (indicated by the GenBank registration number in parentheses) An siRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing sensitivity to doxorubicin by suppressing the expression of the gene in cancer cells. One or a plurality of genes selected from the group consisting of TUBA1 (K00558), GCP2 (X78686), COX5A (M22760), GLA (X00351) and GNS (X0695) (indicated by the GenBank registration number in parentheses) An siRNA or antisense polynucleotide that suppresses the expression of a gene exhibiting an activity of enhancing the sensitivity to cisplatin by suppressing the expression of the gene in cancer cells. A vector that expresses the siRNA or antisense polynucleotide according to any one of claims 49 to 58. 59. A sensitivity enhancer for an anticancer agent, comprising the siRNA or antisense polynucleotide according to any one of claims 49 to 58 or the vector according to claim 59. SPTBN1 (M96803), PET112L (AF0266871), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770), RPLP2 (M17887), and A4487L ;
HOXB1 (X16666), F10 (K03194), GPX2 (X53463), NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (M3622L) (D63807), PIK3CG (X83368), DBN1 (U00802), NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X60656), F2 (V00595), RARA (X06614), ITGB4 (X35) (X66922), PACE (X17094), AGA (M64073), MVD (U49260), EHH DH (L07077), TFPI2 (D29992), MARCKS (M68956), FGB (J00129), and GPD1 (L34041);
NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383), SERPINF2 (D00174), AGA (M64073), BAZ23 LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RARA (X06614), PTPRN (L18983), APOE (L18983), APOE F10 (K03194), NR1I2 (AF061056), UBE2L3 (X92962), and FGB (J00129);
RPN2 (Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950) and NRGN (Y09689);
MAPK6 (X80692), GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M57627), RFC4 (M87339), CRLF1 (AF059293), RPS6 (MPS2020), RPS6 EMX1 (X68879) and TK2 (U77088);
PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD23A (D21235), SDHA (D30648), PET112L (AF0265681), DAD1 (D15057) HSPB1 (X54079), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M96803), PACE (X17094), RPL24 (M94314), SPIN80T STX4A (U07158), SIAT8B (U33551), CTSK (U13665), DCI (U24 74), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D38048);
COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J0469), SCD (Y13647), YWHAZ (M86400), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 (M8733)
CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01667), PAFAH1B3 (D63391), GBE1 (L07756), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and XBE92E1;
ADCY1 (L05500), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232); , COX5A (M22760), GLA (X00351) and GNS (X0695)
A drug for enhancing the sensitivity to an anticancer agent, comprising an antibody against a protein encoded by a gene selected from the group consisting of (a GenBank registration number in parentheses). A test substance is added to the cells, and SPTBN1 (M96803), PET112L (AF026851), CAPN1 (X04366), MEL (X56741), PACE (X17094), DVL2 (AF006012), LOC54543 (AJ011007), PAPOLA (X76770) , RPLP2 (M17887), and ARF4L (L38490);
HOXB1 (X16666), F10 (K03194), GPX2 (X53463), NR1I2 (AF061056), ANXA4 (M19383), PDLIM1 (U90878), LIPC (X07228), SERPINF2 (D00174), HSD17B1 (M36263), HSD17B1 (M36263) LSS (D63807), PIK3CG (X83368), DBN1 (U00802), NDUFA4 (U94586), BDH (M93107), BCL2L1 (Z23115), EEF1B2 (X60656), F2 (V00595), RARA (X06614), ITGB4, 5GB35 IMPA1 (X66922), PACE (X17094), AGA (M64073), MVD (U49260), EH ADH (L07077), TFPI2 (D29992), MARCKS (M68956), FGB (J00129), and GPD1 (L34041);
NAP1L1 (M86667), HOXB1 (X16666), PACE (X17094), MAN2B1 (U60266), GPX2 (X53463), DBN1 (U00802), ANXA4 (M19383), SERPINF2 (D00174), AGA (M64073), BAZ23 LIPC (X07228), BDH (M93107), LSS (D63807), PDLIM1 (U90878), ZNF161 (D28118), UBE2E1 (X92963), TLE1 (M99435), RARA (X06614), PTPRN (L18983), APOE (L18983), APOE F10 (K03194), NR1I2 (AF061056), UBE2L3 (X92962), and FGB (J00129);
RPN2 (Y00282), ATP50 (X83218), CAST (D50827), HPCA (D16593), ZNF9 (M28372), A2LP (U70671), IL18 (D49950) and NRGN (Y09689);
MAPK6 (X80692), GCSH (M69175), G22P1 (M32865), USP11 (U44839), ACTB (X00351), YWHAZ (M86400), IL10 (M57627), RFC4 (M87339), CRLF1 (AF059293), RPS6 (MPS2020), RPS6 EMX1 (X68879) and TK2 (U77088);
PSMD8 (D38047), LAMP2 (J04183), CTSD (M11233), ADORA2B (M97759), ANXA4 (M19383), PTPRK (Z70660), RAD23A (D21235), SDHA (D30648), PET112L (AF0265681), DAD1 (D15057) HSPB1 (X54079), PSMA6 (X61972), KDELR1 (X55885), B2M (AB021288), M6PR (M16985), GCLC (M90656), SPTBN1 (M96803), PACE (X17094), RPL24 (M94314), SPIN80T STX4A (U07158), SIAT8B (U33551), CTSK (U13665), DCI (U24 74), MEL (X56741), PITPNB (D30037), YY1 (M76541), RAB1 (M28209), UBE2L6 (AF031141) and PSMB7 (D38048);
COX4AL (AF005888), RPS27A (X63237), F2 (V00595), GFAP (J0469), SCD (Y13647), YWHAZ (M86400), GCSH (M69175), SQLLE (D78130), BCL2L1 (Z23115) and RFC40 (M8733)
CD81 (M33680), HNRPC (M16342), AP2S1 (M97074), GAPD (X01667), PAFAH1B3 (D63391), GBE1 (L07756), DEK (X64229), PEA15 (X86809) PSMD7 (D50063) and XBE92E1;
ADCY1 (L05500), HNRPU (X65488), COX5A (M22760), U2AF1 (M96982) and RPYR1 (U35232); , COX5A (M22760), GLA (X00351) and GNS (X0695)
The expression level of at least one or more genes selected from the group consisting of (the GenBank registration number in parentheses) is measured, and the expression level of the gene is compared with the expression level of the gene when no test substance is added. A method for screening a substance that enhances sensitivity to an anticancer agent, wherein the substance to be reduced is selected as a candidate substance that enhances sensitivity to the anticancer agent. 63. A substance that enhances sensitivity to an anticancer agent obtained by the method of claim 62. 64. A drug for enhancing sensitivity to an anticancer agent, comprising the substance according to claim 63.

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