( 您好!臺灣時間:2022/01/22 07:06
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::


研究生(外文):Shu-Lin Lee
論文名稱(外文):Molecular Analysis of the Alterations of Active-site Residues in Thymidylate Synthase and their Relations to Enzyme Activity
指導教授(外文):Eric I-chian Li
外文關鍵詞:Thymidylate SynthaseEnzyme Activity
  • 被引用被引用:0
  • 點閱點閱:255
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
胸苷酸合成酶(thymidylate synthase, TS)經由還原性甲基化轉移一個亞甲基 (methylene group),將dUMP轉變成dTMP,而dTMP為DNA的合成(synthesis)和修補 (repair)所必須。TS在細胞週期的合成期 (S phase)大量上升,此現象在快速增殖細胞中尤為明顯。因此,TS被認為是一個癌症化療的重要標的。癌症化療藥物pyrimidine analogues如:5-FU和5-FdUR,其活性代謝物FdUMP,會與TS及其輔因子CH2-THF形成一安定的化合物,抑制體內dTMP的產生,從而抑制DNA的合成。因為pyrimidine analogues被代謝後會影響TS的活性,所以被廣泛用來癌症的治療。本實驗室先前對TS基因研究中,利用V79易於被誘導產生突變的特性,以FAT培養液篩選出多種不具TS活性之突變株,其中以T163為易突變點(由threonine轉變成alanine)。TS2在受質結合位 (substrate binding site, dUMP site)發生改變,TS15和TS21在輔因子結合位 (cofactor binding site, folate site)發生改變,其TS活性明顯降低,顯示受質結合位及輔因子結合位與其TS活性具重要相關性。我利用實驗室既有的方法對突變株進行TS回復體實驗,自突變株TS2 R208K和TS12 G239D分別篩選出回復體之群聚(colony),基因序列比對結果,發現可將原本產生突變之位置進行回復,得知氨基酸208為易突變點,且所在之受質結合位為重要之TS活性決定部位,氨基酸239為易突變點,推測可能為重要的TS活性決定位置。針對folinic acid對TS活性之探討,改變FAT培養液中folinic acid濃度,可篩選出具部分TS活性之突變株,其TS的兩個主要結合位發生突變時,確實對TS活性造成影響。以V79細胞經化學致突變劑EMS處理後,篩選對5-FdUR產生抗性之突變株,其TS活性有過度表現 (over expression)的情況,得知氨基酸116,24,89和163的突變,所造成之5-FdUR抗性,而增加其TS活性。利用這些研究幫助我們瞭解哺乳類TS與其受質和輔因子間之相互作用,並增加TS對抗癌藥物5-FU產生耐藥性有更進一步的瞭解。
Thymidylate synthase (TS, EC catalyzes the do novo biosynthesis of thymidylate, which is necessary for DNA synthesis and repair. Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. Drug-resistant variants of thymidylate synthase can potentially be used in gene therapy to decrease the myelosuppressive side effects of TS-directed anticancer agents. Chinese hamster V79 cells can be easily induced to become TS deficient mutants due probably to its heterozygosity. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Our lab has also previously partially cloned and sequenced the Chinese hamster TS gene, in the present study I want to use random mutagenesis to gain structure-function information about the TS molecule. I treated Chinese hamster V79 cells with EMS and isolated TS mutants with a special selection FAT medium (folinic acid, aminopterin, thymidine).The TS mutants─TS2, TS15 and TS21─have low TS activities (TS-) and contain mutations within substrate binding site and cofactor binding site. Then I treated these TS deficient mutants with EMS and selected with a thymidine deficient medium, some TS revertant colonies were selected, the TS activities of these TS revertants are interestingly reversed. I also tried to select the TS mutants with elevated folinic acid in the FAT medium and found that these FAT resistant clones have partial TS activities. Finally I mutagenized V79 cells and treated them with 5-FdUR to obtain drug-resistant mutants. The TS activities of these drug-resistant mutants are the same or a bit higher than the wild-type V79 cells. These results illustrate the intricate relations between TS enzyme activity and mutations in the substrate and cofactor binding sites as well as mutations in these sites and the 5-FdUR resistance.
第一章 序論……………………………………………………1

第二章 研究目的與策略………………………………………7

第三章 實驗材料………………………………………………8

第四章 實驗方法………………………………………………11

第五章 實驗結果………………………………………………32

第六章 討論……………………………………………………39

第七章 參考文獻………………………………………………44
Berger SH. Barbour KW. Berger FG. A naturally occurring variation in thymidylate synthase structure is associated with a reduced response to 5-fluoro-2'-deoxyuridine in a human colon tumor cell line. Molecular Pharmacology. 34(4):480-4, 1988.

Berger SH. Hakala MT. Relationship of dUMP and free FdUMP pools to inhibition of thymidylate synthase by 5-fluorouracil. Molecular Pharmacology. 25(2):303-9, 1984.

Bertino JR. Biomodulation of 5-fluorouracil with antifolates. Seminars in Oncology. 24(5 Suppl 18):S18-52-S18-56, 1997.

Carreras CW. Santi DV. The catalytic mechanism and structure of thymidylate synthase. Annual Review of Biochemistry. 64:721-62, 1995.

Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 162(1):156-9, 1987.

Chu E. Koeller DM. Casey JL. Drake JC. Chabner BA. Elwood PC. Zinn S. Allegra CJ. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proceedings of the National Academy of Sciences of the United States of America. 88(20):8977-81, 1991.

Chu E. Koeller DM. Johnston PG. Zinn S. Allegra CJ. Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma. Molecular Pharmacology. 43(4):527-33, 1993.

CieSla J. Weiner KX. Weiner RS. Reston JT. Maley GF. Maley F. Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases. Biochimica et Biophysica Acta. 1261(2):233-42, 1995.

Clark JL. Berger SH. Mittelman A. Berger FG. Thymidylate synthase gene amplification in a colon tumor resistant to fluoropyrimidine chemotherapy. Cancer Treatment Reports. 71(3):261-5, 1987.

Cory JG. Breland JC. Carter GL. Effect of 5-fluorouracil on RNA metabolism in Novikoff hepatoma cells. Cancer Research. 39(12):4905-13, 1979.

Deng TL. Li DW. Jenh CH. Johnson LF. Structure of the gene for mouse thymidylate synthase. Locations of introns and multiple transcriptional start sites. Journal of Biological Chemistry. 261(34):16000-5, 1986.

Drake JC. Allegra CJ. Moran RG. Johnston PG. Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines. Biochemical Pharmacology. 51(10):1349-55, 1996.

Duschinsky R, Pleven E and Heidelberger C. The synthesis of 5-fluoropyrimidines. J Am Chem Soc. 79: 4559. 1957.

Finer-Moore JS. Montfort WR. Stroud RM. Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase. Biochemistry. 29(30):6977-86, 1990.

Ghoshal K. Jacob ST. An alternative molecular mechanism of action of 5-fluorouracil, a potent anticancer drug. Biochemical Pharmacology. 53(11):1569-75, 1997.

Glazer RI. Hartman KD. The effect of 5-fluorouracil on the synthesis and methylation of low molecular weight nuclear RNA in L1210 cells. Molecular Pharmacology. 17(2):245-9, 1980.

Glazer RI. Lloyd LS. Association of cell lethality with incorporation of 5-fluorouracil and 5-fluorouridine into nuclear RNA in human colon carcinoma cells in culture. Molecular Pharmacology. 21(2):468-73, 1982.

Grem JL. Hoth DF. Hamilton JM. King SA. Leyland-Jones B. Overview of current status and future direction of clinical trials with 5-fluorouracil in combination with folinic acid. Cancer Treatment Reports. 71(12):1249-64, 1987.

Herrick D. Kufe DW. Lethality associated with incorporation of 5-fluorouracil into preribosomal RNA. Molecular Pharmacology. 26(1):135-40, 1984.

Houghton JA. Houghton PJ. Wooten RS. Mechanism of induction of gastrointestinal toxicity in the mouse by 5-fluorouracil, 5-fluorouridine, and 5-fluoro-2'-deoxyuridine. Cancer Research. 39(7 Pt 1):2406-13, 1979.

Houghton JA. Torrance PM. Radparvar S. Williams LG. Houghton PJ. Binding of 5-fluorodeoxyuridylate to thymidylate synthase in human colon adenocarcinoma xenografts. European Journal of Cancer & Clinical Oncology. 22(4):505-10, 1986.

Jenh CH. Geyer PK. Baskin F. Johnson LF. Thymidylate synthase gene amplification in fluorodeoxyuridine-resistant mouse cell lines. Molecular Pharmacology. 28(1):80-5, 1985.

Jenh CH. Rao LG. Johnson LF. Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state. Journal of Cellular Physiology. 122(1):149-54, 1985.

Kaneda S. Nalbantoglu J. Takeishi K. Shimizu K. Gotoh O. Seno T. Ayusawa D. Structural and functional analysis of the human thymidylate synthase gene. Journal of Biological Chemistry. 265(33):20277-84, 1990.

Kufe DW. Major PP. 5-Fluorouracil incorporation into human breast carcinoma RNA correlates with cytotoxicity. Journal of Biological Chemistry. 256(19):9802-5, 1981.

Landis DM. Heindel CC. Loeb LA. Creation and characterization of 5-fluorodeoxyuridine-resistant Arg50 loop mutants of human thymidylate synthase. Cancer Research. 61(2):666-72, 2001.

Li IC. Chang CC. Trosko JE. Thymidylate synthetase gene as a quantitative mutation marker in Chinese hamster cells. Mutation Research. 243(3):233-9, 1990.

Li IC. Chu EH. Mutants of Chinese hamster cells deficient in thymidylate synthetase. Journal of Cellular Physiology. 120(2):109-16, 1984.

Liu Y. Barrett JE. Schultz PG. Santi DV. Tyrosine 146 of thymidylate synthase assists proton abstraction from the 5-position of 2'-deoxyuridine 5'-monophosphate. Biochemistry. 38(2):848-52, 1999.

Lonn U. Lonn S. Nylen U. Winblad G. 5-Fluoropyrimidine-induced DNA damage in human colon adenocarcinoma and its augmentation by the nucleoside transport inhibitor dipyridamole. Cancer Research. 49(5):1085-9, 1989.

Mader RM. Muller M. Steger GG. Resistance to 5-fluorouracil. General Pharmacology. 31(5):661-6, 1998.

Mandel HG. The target cell determinants of the antitumor actions of 5-FU: does FU incorporation into RNA play a role?. Cancer Treatment Reports. 65 Suppl 3:63-71, 1981.

Matthews DA. Appelt K. Oatley SJ. Xuong NH. Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate. Journal of Molecular Biology. 214(4):923-36, 1990.

Maybaum J. Ullman B. Mandel HG. Day JL. Sadee W. Regulation of RNA- and DNA-directed actions of 5-fluoropyrimidines in mouse T-lymphoma (S-49) cells. Cancer Research. 40(11):4209-15, 1980.

Mayer RJ. Systemic therapy for colorectal cancer: an overview. Seminars in Oncology. 18(5 Suppl 7):62-6, 1991.

Montfort WR. Perry KM. Fauman EB. Finer-Moore JS. Maley GF. Hardy L. Maley F. Stroud RM. Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate. Biochemistry. 29(30):6964-77, 1990.

Montfort WR. Weichsel A. Thymidylate synthase: structure, inhibition, and strained conformations during catalysis. Pharmacology & Therapeutics. 76(1-3):29-43, 1997.

Navalgund LG. Rossana C. Muench AJ. Johnson LF. Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. Journal of Biological Chemistry. 255(15):7386-90, 1980.

Peters GJ. Lankelma J. Kok RM. Noordhuis P. van Groeningen CJ. van der Wilt CL. Meyer S. Pinedo HM. Prolonged retention of high concentrations of 5-fluorouracil in human and murine tumors as compared with plasma. Cancer Chemotherapy & Pharmacology. 31(4):269-76, 1993.

Peters GJ. van der Wilt CL. van Triest B. Codacci-Pisanelli G. Johnston PG. van Groeningen CJ. Pinedo HM. Thymidylate synthase and drug resistance. European Journal of Cancer. 31A(7-8):1299-305, 1995.

Peters GJ. van Groeningen CJ. Clinical relevance of biochemical modulation of 5-fluorouracil. Annals of Oncology. 2(7):469-80, 1991.

Phan J. Koli S. Minor W. Dunlap RB. Berger SH. Lebioda L. Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug. Biochemistry. 40(7):1897-902, 2001.

Pinedo HM. Peters GF. Fluorouracil: biochemistry and pharmacology. Journal of Clinical Oncology. 6(10):1653-64, 1988.

Priest DG. Ledford BE. Doig MT. Increased thymidylate synthetase in 5-fluorodeoxyuridine resistant cultured hepatoma cells. Biochemical Pharmacology. 29(11):1549-53, 1980.

Roberts D. An isotopic assay for thymidylate synthetase. Biochemistry. 5(11):3546-8, 1966.

Rutman RJ, Cantarow A and Paschkis K. Studies in 2-acetylaminofluorene carcinogenesis. III. The utilization of uracil-2-C14 by preneoplastic rat liver and rat hepatoma. Cancer Res. 14: 119. 1954.

Santi DV. McHenry CS. Sommer H. Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate. Biochemistry. 13(3):471-81, 1974.

Schiffer CA. Clifton IJ. Davisson VJ. Santi DV. Stroud RM. Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site. Biochemistry. 34(50):16279-87, 1995.

Schmoll HJ. Buchele T. Grothey A. Dempke W. Where do we stand with 5-fluorouracil?. Seminars in Oncology. 26(6):589-605, 1999.

Spears CP. Gustavsson BG. Berne M. Frosing R. Bernstein L. Hayes AA. Mechanisms of innate resistance to thymidylate synthase inhibition after 5-fluorouracil. Cancer Research. 48(20):5894-900, 1988.

Spiegelman S. Sawyer R. Nayak R. Ritzi E. Stolfi R. Martin D. Improving the anti-tumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation. Proceedings of the National Academy of Sciences of the United States of America. 77(8):4966-70, 1980.

Steadman DJ. Spencer HT. Dunlap RB. Berger SH. Substitution at residue 214 of human thymidylate synthase alters nucleotide binding and isomerization of ligand-protein complexes. Biochemistry. 38(17):5582-7, 1999.

Takeishi K. Kaneda S. Ayusawa D. Shimizu K. Gotoh O. Seno T. Nucleotide sequence of a functional cDNA for human thymidylate synthase. Nucleic Acids Research. 13(6):2035-43, 1985.

Tobias JS. Current role of chemotherapy in head and neck cancer. Drugs. 43(3):333-45, 1992.

Van der Wilt CL. Braakhuis BJ. Pinedo HM. De Jong M. Smid K. Peters GJ. Addition of leucovorin in modulation of 5-fluorouracil with methotrexate: potentiating or reversing effect?. International Journal of Cancer. 61(5):672-8, 1995.

Van der Wilt CL. Pinedo HM. de Jong M. Peters GJ. Effect of folate diastereoisomers on the binding of 5-fluoro-2'-deoxyuridine-5'- monophosphate to thymidylate synthase. Biochemical Pharmacology. 45(5):1177-9, 1993.

Van der Wilt CL. Pinedo HM. Smid K. Peters GJ. Elevation of thymidylate synthase following 5-fluorouracil treatment is prevented by the addition of leucovorin in murine colon tumors. Cancer Research. 52(18):4922-8, 1992.

Wang FS. Aschele C. Sobrero A. Chang YM. Bertino JR. Decreased folylpolyglutamate synthetase expression: a novel mechanism of fluorouracil resistance. Cancer Research. 53(16):3677-80, 1993.

Weckbecker G. Biochemical pharmacology and analysis of fluoropyrimidines alone and in combination with modulators. Pharmacology & Therapeutics. 50(3):367-424, 1991.

Wilkinson DS. Crumley J. Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells. Journal of Biological Chemistry. 252(3):1051-6, 1977.

Yin MB. Zakrzewski SF. Hakala MT. Relationship of cellular folate cofactor pools to the activity of 5-fluorouracil. Molecular Pharmacology. 23(1):190-7, 1983.

Zhang ZG. Harstrick A. Rustum YM. Mechanisms of resistance to fluoropyrimidines. Seminars in Oncology. 19(2 Suppl 3):4-9, 1992.

林雅玲著,TS結構與功能之關係極其對5-FU抗性之探討,國立成功大學藥理學研究所,碩士論文 (2000)。

黃柏燈著,中國田鼠胸�P酸合成�@基因的選殖,定序與表現,國立成功大學藥理學研究所,碩士論文 (1997)。
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
第一頁 上一頁 下一頁 最後一頁 top