順鉑(cisplatin)是一種含鉑的化合物是目前最常用的化療藥物。用在治療肺癌、卵巢癌、頭頸癌和睾丸癌等。然而，癌細胞對藥物的抗藥性一直是治療癌症的一大障礙。近年來，有少部分的研究開始指出DNA修復路徑可能是癌症細胞增強抗藥性的機制。范可尼貧血(Fanconi anemia, FA)和同源重組(homologous recombination, HR)修復路徑為兩個主要解決順鉑藥物在細胞週期S時期所造成的DNA損傷。在本篇研究，我們發現FA和HR相關的基因在具有順鉑藥物抗性的鼻咽癌(nasopharyngeal carcinoma, NPC)細胞株中有大量表現的現象，說明了FA和HR可能對抗順鉑藥物的抗藥性扮演重要的角色。我們發現在在具有抗順鉑藥物的鼻咽癌細胞(NPC)中，有高度的姊妹染色體的互換率(sister chromatid exchange, SCE)發生。若缺法HR基因BRCA1以及FA基因FANCD2的細胞株中則會抑制SCE的交換，並且會對順鉑藥物產生敏感性，而且細胞週期會停滯在S時期的現象。此外，在抗順鉑藥物的神經膠質瘤細胞株A172也發現到HR與FA的基因也有大量表現的現象，顯示著若增強FA和HR的修復路徑與抗順鉑藥物的特性有著密切的關係。有趣的是，在肺癌細胞中我們發現FA的基因FANCD2與HR的基因BRCA1有過表現的現象。總結我們的結果顯示，FA與HR在抗順鉑藥物的特性與癌症演化扮演重要的角色。在治療對抗順鉑藥物的癌細胞時，我們可以藉由抑制FA與HR兩路徑的基因，以提供一個新的抗順鉑藥物的癌症療效。

Cisplatin is a platinum-containing compound that is commonly used to treat lung, ovarian, head and neck, and testicular cancer. However, the major obstacle for the treatment is drug resistance. Recently, a few studies start to show that DNA repair pathways may contribute to the drug resistance phenotype of cancer. The Fanconi anemia (FA) and homologous recombination (HR) pathways are two major pathways to resolve cisplatin-caused DNA damage during S-phase. In this study, we identified several genes in the FA and HR pathways are highly expressed in the cisplatin resistance nasopharyngeal carcinoma (NPC) cells, indicating the importance of FA and HR in the cisplatin-resistant phenotype. In support this notion, a high frequency of sister chromatid exchange (SCE) occurs in the cisplatin resistance nasopharyngeal carcinoma (NPC) cells. Depletion of the HR gene BRCA1 or the FA gene FANCD2 suppresses SCE, sensitizes cells to cisplatin, and accumulates cells in S-phase in the presence of low-dose cisplatin. In addition, elevated expression of HR and FA genes is also observed in the cisplatin-resistant glioblastoma cell line A172, suggesting enhanced FA and HR pathways may underlie the cisplatin-resistance of cancer. Interestingly, the FA gene FANCD2 and HR gene BRCA1 are highly expressed in lung cancer cells. Our results suggest that FA and HR play an important role in cisplatin-resistant phenotype, as well as cancer development. Targeting the HR and FA pathways could be a potential therapeutic strategy to treat the cisplatin-resistant cancer.

Bekker-Jensen, S., C. Lukas, R. Kitagawa, F. Melander, M.B. Kastan, J. Bartek, and J. Lukas. 2006. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. The Journal of cell biology. 173:195-206.
Bielas, J.H., and L.A. Loeb. 2005. Mutator phenotype in cancer: timing and perspectives. Environmental and molecular mutagenesis. 45:206-213.
Bienko, M., C.M. Green, N. Crosetto, F. Rudolf, G. Zapart, B. Coull, P. Kannouche, G. Wider, M. Peter, A.R. Lehmann, K. Hofmann, and I. Dikic. 2005. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science. 310:1821-1824.
Branzei, D., and M. Foiani. 2010. Maintaining genome stability at the replication fork. Nature reviews. Molecular cell biology. 11:208-219.
Chaksangchaichot, P., P. Punyarit, and S. Petmitr. 2007. Novel hMSH2, hMSH6 and hMLH1 gene mutations and microsatellite instability in sporadic colorectal cancer. Journal of cancer research and clinical oncology. 133:65-70.
Chang, D.J., and K.A. Cimprich. 2009. DNA damage tolerance: when it's OK to make mistakes. Nature chemical biology. 5:82-90.
Chapman, J.R., M.R. Taylor, and S.J. Boulton. 2012. Playing the end game: DNA double-strand break repair pathway choice. Molecular cell. 47:497-510.
Chen, L., C.J. Nievera, A.Y. Lee, and X. Wu. 2008. Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair. The Journal of biological chemistry. 283:7713-7720.
Ciccia, A., C. Ling, R. Coulthard, Z. Yan, Y. Xue, A.R. Meetei, H. Laghmani el, H. Joenje, N. McDonald, J.P. de Winter, W. Wang, and S.C. West. 2007. Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM. Molecular cell. 25:331-343.
Ciccia, A., N. McDonald, and S.C. West. 2008. Structural and functional relationships of the XPF/MUS81 family of proteins. Annual review of biochemistry. 77:259-287.
Cohn, M.A., and A.D. D'Andrea. 2008. Chromatin recruitment of DNA repair proteins: lessons from the fanconi anemia and double-strand break repair pathways. Molecular cell. 32:306-312.
Cole, A.R., L.P. Lewis, and H. Walden. 2010. The structure of the catalytic subunit FANCL of the Fanconi anemia core complex. Nature structural & molecular biology. 17:294-298.
D'Andrea, A.D. 2013. BRCA1: a missing link in the Fanconi anemia/BRCA pathway. Cancer discovery. 3:376-378.
Daigaku, Y., A.A. Davies, and H.D. Ulrich. 2010. Ubiquitin-dependent DNA damage bypass is separable from genome replication. Nature. 465:951-955.
Deng, C.X., and S.G. Brodie. 2000. Roles of BRCA1 and its interacting proteins. BioEssays : news and reviews in molecular, cellular and developmental biology. 22:728-737.
Eastman, A. 1987. The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacology & therapeutics. 34:155-166.
Esteller, M., J.M. Silva, G. Dominguez, F. Bonilla, X. Matias-Guiu, E. Lerma, E. Bussaglia, J. Prat, I.C. Harkes, E.A. Repasky, E. Gabrielson, M. Schutte, S.B. Baylin, and J.G. Herman. 2000. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. Journal of the National Cancer Institute. 92:564-569.
Fanconi, G. 1967. Familial constitutional panmyelocytopathy, Fanconi's anemia (F.A.). I. Clinical aspects. Seminars in hematology. 4:233-240.
Fekairi, S., S. Scaglione, C. Chahwan, E.R. Taylor, A. Tissier, S. Coulon, M.Q. Dong, C. Ruse, J.R. Yates, 3rd, P. Russell, R.P. Fuchs, C.H. McGowan, and P.H. Gaillard. 2009. Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases. Cell. 138:78-89.
Fishel, R., M.K. Lescoe, M.R. Rao, N.G. Copeland, N.A. Jenkins, J. Garber, M. Kane, and R. Kolodner. 1993. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell. 75:1027-1038.
Galanski, M. 2006. Recent developments in the field of anticancer platinum complexes. Recent patents on anti-cancer drug discovery. 1:285-295.
Gao, C., K. Furge, J. Koeman, K. Dykema, Y. Su, M.L. Cutler, A. Werts, P. Haak, and G.F. Vande Woude. 2007. Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations. Proceedings of the National Academy of Sciences of the United States of America. 104:8995-9000.
Garcia-Higuera, I., T. Taniguchi, S. Ganesan, M.S. Meyn, C. Timmers, J. Hejna, M. Grompe, and A.D. D'Andrea. 2001. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Molecular cell. 7:249-262.
Garner, E., and A. Smogorzewska. 2011. Ubiquitylation and the Fanconi anemia pathway. FEBS letters. 585:2853-2860.
Ghosal, G., and J. Chen. 2013. DNA damage tolerance: a double-edged sword guarding the genome. Translational cancer research. 2:107-129.
Gudmundsdottir, K., and A. Ashworth. 2006. The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability. Oncogene. 25:5864-5874.
Hansemann, D. 1890. Ueber asymmetrische Zelltheilung in Epithelkrebsen und deren biologische Bedeutung. Archiv f. pathol. Anat. 119:299-326.
Helleday, T. 2010. Homologous recombination in cancer development, treatment and development of drug resistance. Carcinogenesis. 31:955-960.
Hodskinson, M.R., J. Silhan, G.P. Crossan, J.I. Garaycoechea, S. Mukherjee, C.M. Johnson, O.D. Scharer, and K.J. Patel. 2014. Mouse SLX4 Is a Tumor Suppressor that Stimulates the Activity of the Nuclease XPF-ERCC1 in DNA Crosslink Repair. Molecular cell. 54:472-484.
Hoege, C., B. Pfander, G.L. Moldovan, G. Pyrowolakis, and S. Jentsch. 2002. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 419:135-141.
Houtgraaf, J.H., J. Versmissen, and W.J. van der Giessen. 2006. A concise review of DNA damage checkpoints and repair in mammalian cells. Cardiovascular revascularization medicine : including molecular interventions. 7:165-172.
Huang, M., and A.D. D'Andrea. 2010. A new nuclease member of the FAN club. Nature structural & molecular biology. 17:926-928.
Huertas, P., and S.P. Jackson. 2009. Human CtIP mediates cell cycle control of DNA end resection and double strand break repair. The Journal of biological chemistry. 284:9558-9565.
Johnson, R.E., S. Prakash, and L. Prakash. 1999. Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Poleta. Science. 283:1001-1004.
Johnson, R.E., M.T. Washington, L. Haracska, S. Prakash, and L. Prakash. 2000. Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. Nature. 406:1015-1019.
Kannouche, P., A.R. Fernandez de Henestrosa, B. Coull, A.E. Vidal, C. Gray, D. Zicha, R. Woodgate, and A.R. Lehmann. 2002. Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells. The EMBO journal. 21:6246-6256.
Kannouche, P.L., and A.R. Lehmann. 2004. Ubiquitination of PCNA and the polymerase switch in human cells. Cell cycle. 3:1011-1013.
Kee, Y., and A.D. D'Andrea. 2010. Expanded roles of the Fanconi anemia pathway in preserving genomic stability. Genes & development. 24:1680-1694.
Kim, H., and A.D. D'Andrea. 2012. Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway. Genes & development. 26:1393-1408.
Klarer, A.C., and W. McGregor. 2011. Replication of damaged genomes. Critical reviews in eukaryotic gene expression. 21:323-336.
Kolodner, R.D., C.D. Putnam, and K. Myung. 2002. Maintenance of genome stability in Saccharomyces cerevisiae. Science. 297:552-557.
Kuraoka, I., W.R. Kobertz, R.R. Ariza, M. Biggerstaff, J.M. Essigmann, and R.D. Wood. 2000. Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease. The Journal of biological chemistry. 275:26632-26636.
Kurzrock, R., H.M. Kantarjian, B.J. Druker, and M. Talpaz. 2003. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics. Annals of internal medicine. 138:819-830.
Leach, F.S., N.C. Nicolaides, N. Papadopoulos, B. Liu, J. Jen, R. Parsons, P. Peltomaki, P. Sistonen, L.A. Aaltonen, M. Nystrom-Lahti, and et al. 1993. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell. 75:1215-1225.
Lebwohl, D., and R. Canetta. 1998. Clinical development of platinum complexes in cancer therapy: an historical perspective and an update. European journal of cancer. 34:1522-1534.
Lee, K.Y., and K. Myung. 2008. PCNA modifications for regulation of post-replication repair pathways. Molecules and cells. 26:5-11.
Liu, Y., and S.C. West. 2004. Happy Hollidays: 40th anniversary of the Holliday junction. Nature reviews. Molecular cell biology. 5:937-944.
Loeb, L.A. 2001. A mutator phenotype in cancer. Cancer research. 61:3230-3239.
Loeb, L.A., K.R. Loeb, and J.P. Anderson. 2003. Multiple mutations and cancer. Proceedings of the National Academy of Sciences of the United States of America. 100:776-781.
Loeb, L.A., C.F. Springgate, and N. Battula. 1974. Errors in DNA replication as a basis of malignant changes. Cancer research. 34:2311-2321.
Minca, E.C., and D. Kowalski. 2010. Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks. Molecular cell. 38:649-661.
Mitchell, R.J., S.M. Farrington, M.G. Dunlop, and H. Campbell. 2002. Mismatch repair genes hMLH1 and hMSH2 and colorectal cancer: a HuGE review. American journal of epidemiology. 156:885-902.
Moynahan, M.E., and M. Jasin. 2010. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nature reviews. Molecular cell biology. 11:196-207.
Murakami, T., I. Shibuya, T. Ise, Z.S. Chen, S. Akiyama, M. Nakagawa, H. Izumi, T. Nakamura, K. Matsuo, Y. Yamada, and K. Kohno. 2001. Elevated expression of vacuolar proton pump genes and cellular PH in cisplatin resistance. International journal of cancer. Journal international du cancer. 93:869-874.
Myung, K., A. Datta, and R.D. Kolodner. 2001. Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae. Cell. 104:397-408.
Negrini, S., V.G. Gorgoulis, and T.D. Halazonetis. 2010. Genomic instability--an evolving hallmark of cancer. Nature reviews. Molecular cell biology. 11:220-228.
Nowell, P.C. 1976. The clonal evolution of tumor cell populations. Science. 194:23-28.
Nyberg, K.A., R.J. Michelson, C.W. Putnam, and T.A. Weinert. 2002. Toward maintaining the genome: DNA damage and replication checkpoints. Annual review of genetics. 36:617-656.
Ogi, T., Y. Shinkai, K. Tanaka, and H. Ohmori. 2002. Polkappa protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene. Proceedings of the National Academy of Sciences of the United States of America. 99:15548-15553.
Petermann, E., and T. Helleday. 2010. Pathways of mammalian replication fork restart. Nature reviews. Molecular cell biology. 11:683-687.
Pfeiffer, P., W. Goedecke, and G. Obe. 2000. Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis. 15:289-302.
Prakash, S., R.E. Johnson, and L. Prakash. 2005. Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annual review of biochemistry. 74:317-353.
Prestayko, A.W., J.C. D'Aoust, B.F. Issell, and S.T. Crooke. 1979. Cisplatin (cis-diamminedichloroplatinum II). Cancer treatment reviews. 6:17-39.
Rogakou, E.P., D.R. Pilch, A.H. Orr, V.S. Ivanova, and W.M. Bonner. 1998. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. The Journal of biological chemistry. 273:5858-5868.
Russell, P.A., P.D. Pharoah, K. De Foy, S.J. Ramus, I. Symmonds, A. Wilson, I. Scott, B.A. Ponder, and S.A. Gayther. 2000. Frequent loss of BRCA1 mRNA and protein expression in sporadic ovarian cancers. International journal of cancer. Journal international du cancer. 87:317-321.
Sale, J.E. 2013. Translesion DNA synthesis and mutagenesis in eukaryotes. Cold Spring Harbor perspectives in biology. 5:a012708.
San Filippo, J., P. Sung, and H. Klein. 2008. Mechanism of eukaryotic homologous recombination. Annual review of biochemistry. 77:229-257.
Sancar, A., L.A. Lindsey-Boltz, K. Unsal-Kacmaz, and S. Linn. 2004. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual review of biochemistry. 73:39-85.
Schwab, R.A., A.N. Blackford, and W. Niedzwiedz. 2010. ATR activation and replication fork restart are defective in FANCM-deficient cells. The EMBO journal. 29:806-818.
Smeaton, M.B., E.M. Hlavin, T. McGregor Mason, A.M. Noronha, C.J. Wilds, and P.S. Miller. 2008. Distortion-dependent unhooking of interstrand cross-links in mammalian cell extracts. Biochemistry. 47:9920-9930.
Stelter, P., and H.D. Ulrich. 2003. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature. 425:188-191.
Su, W.P., S.H. Hsu, C.K. Wu, S.B. Chang, Y.J. Lin, W.B. Yang, J.J. Hung, W.T. Chiu, S.F. Tzeng, Y.L. Tseng, J.Y. Chang, W.C. Su, and H. Liaw. 2014. Chronic treatment with cisplatin induces replication-dependent sister chromatid recombination to confer cisplatin-resistant phenotype in nasopharyngeal carcinoma. Oncotarget.
Sung, P., L. Krejci, S. Van Komen, and M.G. Sehorn. 2003. Rad51 recombinase and recombination mediators. The Journal of biological chemistry. 278:42729-42732.
Svendsen, J.M., A. Smogorzewska, M.E. Sowa, B.C. O'Connell, S.P. Gygi, S.J. Elledge, and J.W. Harper. 2009. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair. Cell. 138:63-77.
Symington, L.S., and J. Gautier. 2011. Double-strand break end resection and repair pathway choice. Annual review of genetics. 45:247-271.
Thompson, M.E., R.A. Jensen, P.S. Obermiller, D.L. Page, and J.T. Holt. 1995. Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression. Nature genetics. 9:444-450.
Ulrich, H.D. 2009. Regulating post-translational modifications of the eukaryotic replication clamp PCNA. DNA repair. 8:461-469.
Wang, W. 2007. Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins. Nature reviews. Genetics. 8:735-748.
Waters, L.S., B.K. Minesinger, M.E. Wiltrout, S. D'Souza, R.V. Woodruff, and G.C. Walker. 2009. Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance. Microbiology and molecular biology reviews : MMBR. 73:134-154.
Wilson, C.A., L. Ramos, M.R. Villasenor, K.H. Anders, M.F. Press, K. Clarke, B. Karlan, J.J. Chen, R. Scully, D. Livingston, R.H. Zuch, M.H. Kanter, S. Cohen, F.J. Calzone, and D.J. Slamon. 1999. Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas. Nature genetics. 21:236-240.
Winge, Ö. 1930. Zytologische Untersuchungen über die Natur maligner Tumoren. Z.Zellforsch. 10:683-735.
Wray, J., J. Liu, J.A. Nickoloff, and Z. Shen. 2008. Distinct RAD51 associations with RAD52 and BCCIP in response to DNA damage and replication stress. Cancer research. 68:2699-2707.
Yu, X., and J. Chen. 2004. DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains. Molecular and cellular biology. 24:9478-9486.
Zhou, B.B., and S.J. Elledge. 2000. The DNA damage response: putting checkpoints in perspective. Nature. 408:433-439.
許森惠. 2013. 後複製修復在DNA損害修復和鼻咽癌抗藥性的功能研究.