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研究生:黃婷筠
研究生(外文):Tyng-Yun Huang
論文名稱:人類去氧尿嘧啶三磷酸核苷酸水解酶對細胞生長及Methotrexate抗藥性影響之探討
論文名稱(外文):Functional analysis of human deoxyuridine triphosphatase in cell growth and methotrexate sensitivity
指導教授:張智芬
指導教授(外文):Zee-Fen Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:52
中文關鍵詞:酸水解酶酸水解酶酸水解酶酸水解酶
外文關鍵詞:deoxyuridine triphosphatase (dUTPase)methotrexatereplication stress
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去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)負責催化去氧尿苷三磷酸(dUTP)水解為去氧尿苷單磷酸(dUMP)和焦磷酸鹽(PPi)。有許多證據顯示去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)的表現量和腫瘤形成以及化療抗藥性有關連。本篇研究的目的在於了解去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)在細胞週期進行時的調控及其所扮演的必要角色。
在本篇研究中發現去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)的表現量隨著細胞週期改變,在S phase時大量表現。在293T細胞中抑制去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)的表現,會稍微減緩細胞生長速度。利用aphidicolin進行同步化(synchronization)後,進一步發現抑制去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)表現不但會影響細胞週期的進行,在細胞週期經過S phase時還會有去氧核糖核酸(DNA)損傷的訊號產生,並引發細胞凋亡(apoptosis),顯示去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)對複製壓力(replication stress)反應的重要性。
胸苷酸(dTMP)代謝一直是癌症化療應用的重要作用點。像是5-氟尿嘧啶(5-FU)或葉酸拮抗劑(antifolate)。抑制胸苷三磷酸新生成(de novo dTTP synthesis)的途徑會降低細胞內胸苷三磷酸(dTTP)含量,並導致去氧尿苷單磷酸(dUMP)累積,進一步磷酸化為去氧尿苷三磷酸(dUTP)。dUTP/dTTP比例的增加會導致去氧尿苷三磷酸(dUTP)誤嵌入DNA,引發DNA損傷反應。因此提出去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)的表現量是否會影響細胞對滅殺除癌錠(methotrexate)的感受性。結果顯示在293T細胞中降低去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)的表現量會增加細胞對滅殺除癌錠(methotrexate)的敏感度,並且增強DNA損傷反應。另一方面,表現去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)則可減少因滅殺除癌錠(methotrexate)所引起的損傷訊號。
總結本論文,說明了去氧尿嘧啶三磷酸核苷酸水解酶(dUTPase)在細胞從複製壓力(replication stress)復原時的重要性,以及在利用抑制胸苷三磷酸新生成(de novo dTTP synthesis)途徑化療藥物治療產生抗藥性所扮演的角色。



Deoxyuridine triphosphatase (dUTPase) catalyses the hydrolysis reaction of dUTP to form dUMP and pyrophosphate. Accumulating evidence has shown the association of dUTPase expression with tumor and chemotherapy resistance. The aim of my study is to understand the regulation and the essential role of dUTPase in the cell cycle progression.
I found that the expression of dUTPase is regulated in a cell cycle-dependent manner, being maximal in the S phase. Depletion of dUTPase in 293T cells reduced the cell growth rate slightly. By aphidicolin synchronization, I further found that depletion of dUTPase not only affected cell cycle progression, but also induced DNA damage and apoptosis when progression through S phase, indicating the importance of dUTPase in response to replication stress.
Thymidylate metabolism is an important target for chemotherapeutic agents such as fluoropyrimidines, and antifolates. Inhibition of de novo dTTP synthesis results in dTTP pool depletion and a subsequent accumulation of dUMP, which may then be phosphorylated to form dUTP. The increase in the dUTP/dTTP ratio induces dUTP misincorporation, resulting in DNA damage responses. Therefore, I addressed the question whether the expression level of dUTPase affects methotrexate sensitivity of cells. The results showed that knockdown of dUTPase sensitized 293T cells to methotrexate, accompanied by an enhancement of DNA damage response. In addition, enforced expression of dUTPase diminished methotrexate-induced damage signals.
Accordingly, these results elucidated the crucial role of dUTPase in cells resumed from replication stress and in resistance to anti-cancer treatment by blocking de novo dTTP synthesis.


口試委員會審定書…………………………i
謝誌…………………………………………ii
中文摘要……………………………………iii
Abstract……………………………………iv
Table of Contents…………………………v
Introduction………………………………1
Materials and Methods………………… 9
Results……………………………………17
Discussion……………………………… 24
Figures and Legends……………………27
Appendix………………………………… 47
References……………………………… 48


Arner, E.S., and Eriksson, S. (1995). Mammalian deoxyribonucleoside kinases. Pharmacol Ther 67, 155-186.

Bertani, L.E., Haeggmark, A., and Reichard, P. (1963). Enzymatic Synthesis of Deoxyribonucleotides. Ii. Formation and Interconversion of Deoxyuridine Phosphates. J Biol Chem 238, 3407-3413.

Bertani, L.E., Haggmark, A., and Reichard, P. (1961). Synthesis of pyrimidine deoxyribonucleoside diphosphates with enzymes from Escherichia coli. J Biol Chem 236, PC67-PC68.

Bessman, M.J., Lehman, I.R., Adler, J., Zimmerman, S.B., Simms, E.S., and Kornberg, A. (1958). Enzymatic Synthesis of Deoxyribonucleic Acid. Iii. The Incorporation of Pyrimidine and Purine Analogues into Deoxyribonucleic Acid. Proc Natl Acad Sci U S A 44, 633-640.

Blocker, R., and Roth, J.S. (1977). Factors affecting deoxycytidylate deaminase activity in some transplantable rat hepatomas. Cancer Res 37, 1918-1922.

Brynolf, K., Eliasson, R., and Reichard, P. (1978). Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil. Cell 13, 573-580.

Caradonna, S., and Muller-Weeks, S. (2001). The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity. Curr Protein Pept Sci 2, 335-347.

Chang, Z.F., Huang, D.Y., and Hsue, N.C. (1994). Differential phosphorylation of human thymidine kinase in proliferating and M phase-arrested human cells. J Biol Chem 269, 21249-21254.

Chimploy, K., and Mathews, C.K. (2001). Mouse ribonucleotide reductase control: influence of substrate binding upon interactions with allosteric effectors. J Biol Chem 276, 7093-7100.
Cohen, S.S. (1971). On the nature of thymineless death. Ann N Y Acad Sci 186, 292-301.

Conley, B.A., Kaplan, R.S., and Arbuck, S.G. (1998). National Cancer Institute Clinical Trials Program in Colorectal Cancer. Cancer Chemother Pharmacol 42 Suppl, S75-79.

Dube, D.K., Kunkel, T.A., Seal, G., and Loeb, L.A. (1979). Distinctive properties of mammalian DNA polymerases. Biochim Biophys Acta 561, 369-382.

el-Hajj, H.H., Zhang, H., and Weiss, B. (1988). Lethality of a dut (deoxyuridine triphosphatase) mutation in Escherichia coli. J Bacteriol 170, 1069-1075.

Gadsden, M.H., McIntosh, E.M., Game, J.C., Wilson, P.J., and Haynes, R.H. (1993). dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae. EMBO J 12, 4425-4431.

Goulian, M., Bleile, B., and Tseng, B.Y. (1980a). The effect of methotrexate on levels of dUTP in animal cells. J Biol Chem 255, 10630-10637.

Goulian, M., Bleile, B., and Tseng, B.Y. (1980b). Methotrexate-induced misincorporation of uracil into DNA. Proc Natl Acad Sci U S A 77, 1956-1960.

Goulian, M., Bleile, B.M., Dickey, L.M., Grafstrom, R.H., Ingraham, H.A., Neynaber, S.A., Peterson, M.S., and Tseng, B.Y. (1986). Mechanism of thymineless death. Adv Exp Med Biol 195 Pt B, 89-95.

Greenberg, G.R., and Somerville, R.L. (1962). Deoxyuridylate kinase activity and deoxyuridinetriphosphatase in Escherichia coli. Proc Natl Acad Sci U S A 48, 247-257.

Harris, J.M., McIntosh, E.M., and Muscat, G.E. (1999). Structure/function analysis of a dUTPase: catalytic mechanism of a potential chemotherapeutic target. J Mol Biol 288, 275-287.

Heidelberger, C., Chaudhuri, N.K., Danneberg, P., Mooren, D., Griesbach, L., Duschinsky, R., Schnitzer, R.J., Pleven, E., and Scheiner, J. (1957). Fluorinated pyrimidines, a new class of tumour-inhibitory compounds. Nature 179, 663-666.

Jacobs, S.A., Adamson, R.H., Chabner, B.A., Derr, C.J., and Johns, D.C. (1975). Stoichiometric inhibition of mammalian dihydrofolate reductase by the gamma-glutamyl metabolite of methotrexiate, 4-amino-4-deoxy-N-10-methylpteroylglutamyl-gamma-glutamate. Biochem Biophys Res Commun 63, 692-698.

Kawahara, A., Akagi, Y., Hattori, S., Mizobe, T., Shirouzu, K., Ono, M., Yanagawa, T., Kuwano, M., and Kage, M. (2009). Higher expression of deoxyuridine triphosphatase (dUTPase) may predict the metastasis potential of colorectal cancer. J Clin Pathol 62, 364-369.

Ladner, R.D. (2001). The role of dUTPase and uracil-DNA repair in cancer chemotherapy. Curr Protein Pept Sci 2, 361-370.

Ladner, R.D., and Caradonna, S.J. (1997). The human dUTPase gene encodes both nuclear and mitochondrial isoforms. Differential expression of the isoforms and characterization of a cDNA encoding the mitochondrial species. J Biol Chem 272, 19072-19080.

Ladner, R.D., Lynch, F.J., Groshen, S., Xiong, Y.P., Sherrod, A., Caradonna, S.J., Stoehlmacher, J., and Lenz, H.J. (2000). dUTP nucleotidohydrolase isoform expression in normal and neoplastic tissues: association with survival and response to 5-fluorouracil in colorectal cancer. Cancer Res 60, 3493-3503.

Ladner, R.D., McNulty, D.E., Carr, S.A., Roberts, G.D., and Caradonna, S.J. (1996). Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase. J Biol Chem 271, 7745-7751.

Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature 362, 709-715.

Longley, D.B., Harkin, D.P., and Johnston, P.G. (2003). 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 3, 330-338.

Lowndes, N.F., and Toh, G.W. (2005). DNA repair: the importance of phosphorylating histone H2AX. Curr Biol 15, R99-R102.

Mader, R.M., Muller, M., and Steger, G.G. (1998). Resistance to 5-fluorouracil. Gen Pharmacol 31, 661-666.


McIntosh, E.M., and Haynes, R.H. (1997). dUTP pyrophosphatase as a potential target for chemotherapeutic drug development. Acta Biochim Pol 44, 159-171.

Miwa, M., Ura, M., Nishida, M., Sawada, N., Ishikawa, T., Mori, K., Shimma, N., Umeda, I., and Ishitsuka, H. (1998). Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue. Eur J Cancer 34, 1274-1281.

Moertel, C.G. (1994). Chemotherapy for colorectal cancer. N Engl J Med 330, 1136-1142.

Neuberger, M.S., Di Noia, J.M., Beale, R.C., Williams, G.T., Yang, Z., and Rada, C. (2005). Somatic hypermutation at A.T pairs: polymerase error versus dUTP incorporation. Nat Rev Immunol 5, 171-178.

Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M., and Bonner, W.M. (2000). A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10, 886-895.

Pearl, L.H., and Savva, R. (1996). The problem with pyrimidines. Nat Struct Biol 3, 485-487.

Pyeon, D., Newton, M.A., Lambert, P.F., den Boon, J.A., Sengupta, S., Marsit, C.J., Woodworth, C.D., Connor, J.P., Haugen, T.H., Smith, E.M., et al. (2007). Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers. Cancer Res 67, 4605-4619.

Rahman, L., Voeller, D., Rahman, M., Lipkowitz, S., Allegra, C., Barrett, J.C., Kaye, F.J., and Zajac-Kaye, M. (2004). Thymidylate synthase as an oncogene: a novel role for an essential DNA synthesis enzyme. Cancer Cell 5, 341-351.

Reichard, P. (1988). Interactions between deoxyribonucleotide and DNA synthesis. Annu Rev Biochem 57, 349-374.

Schnell, J.R., Dyson, H.J., and Wright, P.E. (2004). Structure, dynamics, and catalytic function of dihydrofolate reductase. Annu Rev Biophys Biomol Struct 33, 119-140.

Shlomai, J., and Kornberg, A. (1978). Deoxyuridine triphosphatase of Escherichia coli. Purification, properties, and use as a reagent to reduce uracil incorporation into DNA. J Biol Chem 253, 3305-3312.

Sneider, T.W., Potter, V.R., and Morris, H.P. (1969). Enzymes of thymidine triphosphate synthesis in selected Morris hepatomas. Cancer Res 29, 40-54.

Strahler, J.R., Zhu, X.X., Hora, N., Wang, Y.K., Andrews, P.C., Roseman, N.A., Neel, J.V., Turka, L., and Hanash, S.M. (1993). Maturation stage and proliferation-dependent expression of dUTPase in human T cells. Proc Natl Acad Sci U S A 90, 4991-4995.

Takatori, H., Yamashita, T., Honda, M., Nishino, R., Arai, K., Takamura, H., Ohta, T., Zen, Y., and Kaneko, S. (2010). dUTP pyrophosphatase expression correlates with a poor prognosis in hepatocellular carcinoma. Liver Int 30, 438-446.

Vertessy, B.G., and Toth, J. (2009). Keeping uracil out of DNA: physiological role, structure and catalytic mechanism of dUTPases. Acc Chem Res 42, 97-106.

Whitehead, V.M. (1977). Synthesis of methotrexate polyglutamates in L1210 murine leukemia cells. Cancer Res 37, 408-412.
Williams, M.V., and Cheng, Y. (1979). Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia. J Biol Chem 254, 2897-2901.

Wilson, P.M., Fazzone, W., LaBonte, M.J., Deng, J., Neamati, N., and Ladner, R.D. (2008). Novel opportunities for thymidylate metabolism as a therapeutic target. Mol Cancer Ther 7, 3029-3037.

Yoshida, S., and Masaki, S. (1979). Utilization in vitro of deoxyuridine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus. Biochim Biophys Acta 561, 396-402.

Zhang, Y., Maley, F., Maley, G.F., Duncan, G., Dunigan, D.D., and Van Etten, J.L. (2007). Chloroviruses encode a bifunctional dCMP-dCTP deaminase that produces two key intermediates in dTTP formation. J Virol 81, 7662-7671.


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