研究生: |
張玳瑋 Tai-Wei Chang |
---|---|
論文名稱: |
秋水仙胺對細胞修復紫外線引起之基因損傷的效應 The effect of colcemid on the repair of UVC-induced DNA damages in Chinese hamster ovary cells |
指導教授: |
劉銀樟
Yin-Chang Liu |
口試委員: | |
學位類別: |
碩士 Master |
系所名稱: |
生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine |
論文出版年: | 2005 |
畢業學年度: | 93 |
語文別: | 英文 |
論文頁數: | 49 |
中文關鍵詞: | 秋水仙胺 、中國倉鼠卵巢細胞 、紫外線 、基因修復 |
外文關鍵詞: | Colcemid, CHO-K1 cell, UVC, DNA repair |
相關次數: | 點閱:2 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
廣泛使用的細胞分裂紡錘體抑制劑─秋水仙胺,於先前研究中顯示,在中國倉鼠卵巢細胞內具有加重紫外線所引起細胞死亡的效應。本研究主要探討秋水仙胺對於基因修復的影響。研究中係利用一敏感的基因損傷針測技術-彗星測量法 (comet assay) 為主軸,去探究秋水仙胺對於細胞DNA的影響性,其結果如下所示:首先,在照射過紫外線的細胞被處以秋水仙胺的實驗中顯示,秋水仙胺對於DNA修復過程中有累積DNA鏈斷的效應,此效應並非來自於秋水仙胺本身對DNA鏈鎖的影響;再者,這樣的效應亦非出自於細胞凋亡的引發。進一步的實驗顯示,即使細胞在不同的細胞週期中,秋水仙胺仍然可顯示此效應;然而,在施以相同的處理對核苷酸切除修復機制缺陷的細胞 (Nucleotide excision repair-deficient cells),秋水仙胺便無法顯示其影響;故表示秋水仙胺的效應跟核苷酸切除修復機制有關。於更進一步的機制探討中顯示,秋水仙胺抑制了DNA修復過程中的鏈鎖連黏步驟,造成細胞進行核苷酸切除之後無法執行正常的DNA接連功能,因此,這可能即為秋水仙胺在中國倉鼠卵巢細胞內具有加重紫外線所引起細胞死亡效應的原因。此一特殊抑制基因修復的分子機制尚待釐清。
Colcemid, a well-known inhibitor of mitotic spindle, enhances UVC-induced cell death in Chinese hamster ovary cells (CHO-K1) according to previous studies. In this study, the enhancement of UV-induced cell death by colcemid was related to its effect on DNA repair. The evidence for this correlation was obtained from the experimental results described below. First, an accumulation of DNA breaks was detected when colcemid was added to cells following UV-irradiation. The increase of DNA breaks was not formed in cells treated with colcemid alone, neither was the phenomenon a result of DNA fragmentation, a hallmark associated with the programmed cell death. Furthermore, the increase of DNA breaks is not cell-cycle dependent but depends on nucleotide excision repair. Second, colcemid inhibited the rejoining of DNA breaks accumulated by hydroxyurea and cytosine-β-D-arabinofuranoside following UV irradiation in a dose dependent manner. On the other hand, colcemid did not affect the excision of UV-induced DNA lesions. Thus, colcemid may inhibit the steps of gap-filling of nucleotide excision repair by a yet-unknown mechanism.
Allen, R.D., D.G. Weiss, J.H. Hayden, D.T. Brown, H. Fujiwake, and M. Simpson. 1985. Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol 100: 1736-52.
Blocker, A., F.F. Severin, J.K. Burkhardt, J.B. Bingham, H. Yu, J.C. Olivo, T.A. Schroer, A.A. Hyman, and G. Griffiths. 1997. Molecular requirements for bi-directional movement of phagosomes along microtubules. J Cell Biol 137: 113-29.
Choucroun, P., D. Gillet, G. Dorange, B. Sawicki, and J.D. Dewitte. 2001. Comet assay and early apoptosis. Mutat Res 478: 89-96.
Collis, S.J., M.J. Swartz, W.G. Nelson, and T.L. DeWeese. 2003. Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res 63: 1550-4.
Conlon, K.A., H. Miller, T.A. Rosenquist, D.O. Zharkov, and M. Berrios. 2005. The murine DNA glycosylase NEIL2 (mNEIL2) and human DNA polymerase beta bind microtubules in situ and in vitro. DNA Repair (Amst) 4: 419-31.
De Boer, J. and J.H. Hoeijmakers. 2000. Nucleotide excision repair and human syndromes. Carcinogenesis 21: 453-60.
De Laat, W.L., N.G. Jaspers, and J.H. Hoeijmakers. 1999. Molecular mechanism of nucleotide excision repair. Genes Dev 13: 768-85.
Derry, W.B., L. Wilson, and M.A. Jordan. 1995. Substoichiometric binding of taxol suppresses microtubule dynamics. Biochemistry 34: 2203-11.
Florent, M., T. Godard, J.J. Ballet, P. Gauduchon, and B. Sola. 1999. Detection by the comet assay of apoptosis induced in lymphoid cell lines after growth factor deprivation. Cell Biol Toxicol 15: 185-92.
Fortini, P., G. Raspaglio, M. Falchi, and E. Dogliotti. 1996. Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay. Mutagenesis 11: 169-75.
Friedberg, E.C. 2001. How nucleotide excision repair protects against cancer. Nat Rev Cancer 1: 22-33.
Giannakakou, P., D.L. Sackett, Y. Ward, K.R. Webster, M.V. Blagosklonny, and T. Fojo. 2000. p53 is associated with cellular microtubules and is transported to the nucleus by dynein. Nat Cell Biol 2: 709-17.
Higurashi, M., T. Ohtsuki, A. Inase, R. Kusumoto, C. Masutani, F. Hanaoka, and S. Iwai. 2003. Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA. J Biol Chem 278: 51968-73.
Hoeijmakers, J.H. 2001. Genome maintenance mechanisms for preventing cancer. Nature 411: 366-74.
Jha, M.N., J.R. Bamburg, and J.S. Bedford. 1994. Cell cycle arrest by Colcemid differs in human normal and tumor cells. Cancer Res 54: 5011-5.
Kondo, S., G.H. Barnett, H. Hara, T. Morimura, and J. Takeuchi. 1995. MDM2 protein confers the resistance of a human glioblastoma cell line to cisplatin-induced apoptosis. Oncogene 10: 2001-6.
Lindahl, T. and R.D. Wood. 1999. Quality control by DNA repair. Science 286: 1897-905.
Martin, F.L., K.J. Cole, M.H. Orme, P.L. Grover, D.H. Phillips, and S. Venitt. 1999. The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells. Mutat Res 445: 21-43.
Middleton, M.R. and G.P. Margison. 2003. Improvement of chemotherapy efficacy by inactivation of a DNA-repair pathway. Lancet Oncol 4: 37-44.
Nasmyth, K. 2001. A prize for proliferation. Cell 107: 689-701.
Pachter, J.S. 1992. Association of mRNA with the cytoskeletal framework: its role in the regulation of gene expression. Crit Rev Eukaryot Gene Expr 2: 1-18.
Rosell, R., R.V. Lord, M. Taron, and N. Reguart. 2002. DNA repair and cisplatin resistance in non-small-cell lung cancer. Lung Cancer 38: 217-27.
Sancar, A., L.A. Lindsey-Boltz, K. Unsal-Kacmaz, and S. Linn. 2004. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73: 39-85.
Singh, N.P., M.T. McCoy, R.R. Tice, and E.L. Schneider. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175: 184-91.
Tice, R.R., E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J.C. Ryu, and Y.F. Sasaki. 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35: 206-21.
Tzang, B.S., Y.J. Chiang, H.C. Lan, C.B. Liao, and Y.C. Liu. 2002. Tuning up or down the UV-induced apoptosis in Chinese hamster ovary cells with cell cycle inhibitors. Photochem Photobiol 75: 662-7.
Tzang, B.S., Y.C. Lai, and Y.C. Liu. 1999. UV-induced but P53 independent apoptotic death in CHO.K1 cells is promoted by M phase inhibitors. In Vitro Cell Dev Biol Anim 35: 17-8.
Zegura, B., B. Sedmak, and M. Filipic. 2003. Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 41: 41-8.