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研究生:林螢美
研究生(外文):Ying-Mei Lin
論文名稱:Bcl-xL的去胺基化參與E1A提升卵巢癌細胞對cisplatin之敏感性
論文名稱(外文):Bcl-xL deamidation is involved in E1A-mediated sensitization to cisplatin in ovarian cancer cells
指導教授:陳一村
指導教授(外文):I-Tsuen Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:Bcl-xL去胺基化卵巢癌
外文關鍵詞:Bcl-xL deamiddationE1AOvarian cancer
相關次數:
  • 被引用被引用:1
  • 點閱點閱:124
  • 評分評分:
  • 下載下載:17
  • 收藏至我的研究室書目清單書目收藏:0
蛋白質的去胺基化是屬於後轉譯的修飾作用,其中還包括磷酸化、甲基化及乙醯化。天冬醯胺(Asn)和穀氨醯胺(Gln)這兩個氨基酸的去胺基化被認為是調控細胞反應的分子時鐘,這些反應包括蛋白質的轉換率,細胞發育和細胞老化等。目前有研究顯示在DNA損傷下,抗死蛋白Bcl-xL的去胺基化可調控細胞反應,而在抑癌基因p53和Rb 突變的癌細胞中也發現抗癌藥物cisplatin的處理會導致Bcl-xL 的去胺基化。過去已知腺病毒E1A會藉由與Rb結合而抑制它的正常功能;因此我們推論若E1A抑制Rb的正常功能之下,cisplatin的處理應該可以導致Bcl-xL 的去胺基化並且使沒有p53的卵巢癌細胞SKOV3-ip1對cisplatin較敏感。
由實驗結果發現,E1A可使卵巢癌細胞SKOV3-ip1對cisplatin所誘導的細胞死亡較為敏感;另外也發現Bcl-xL的修飾作用與cisplatin所誘導的細胞死亡有正相關的關係。由體外鹼性pH處理實驗及在Bcl-xL的兩個可能去胺基的位置進行點突變的實驗結果,我們推論Bcl-xL的修飾作用就是去胺基化。此外,外送去胺基化Bcl-xL的表現,也會使SKOV3-ip1細胞對cisplatin較為敏感;而其效果是與E1A增加cisplatin之敏感性相等。因此,由實驗結果可以推論誘導Bcl-xL去胺基化對於E1A所調控增加卵巢癌細胞SKOV3-ip1對cisplatin的敏感性扮演著一個重要的角色。然而E1A也被證實可以抑制Akt的活性,而Akt可磷化促死蛋白Bad。所以我們猜想Bad可能會與去胺基化的Bcl-xL結合而促使細胞凋亡。由免疫沉澱實驗結果,我們觀察到Bad可以和兩種形式的Bcl-xL結合,但較容易與去胺基化的Bcl-xL結合。由此實驗結果,我們認為Bad與去胺基化的Bcl-xL結合導致caspase活化並促使細胞死亡。
Protein deamidation is one of the post-translational modifications which also include phosphorylation, methylation and acetylation. Deamidation of asparaginyl and glutaminyl residues have been proposed to serve as a molecular clock regulating biological events such as protein turnover, development, and cellular aging. Recent studies showed that deamidation of Bcl-xL, an anti-apoptotic protein, regulates cellular response to DNA damage. This Bcl-xL deamidation induced by cisplatin appears to be mediated by p53 and Rb. It is known that adenovirus E1A inactivates Rb normal function by directly binding to Rb. Thus, we reason if E1A blocks the normal function of Rb, it may lead to cisplatin-induced deamidation of Bcl-xL and sensitize the p53-null ovarian cancer cell, SKOV3-ip1 cells, to cisplatin.
Our results showed that E1A transfectants were more sensitive to cisplatin-induced apoptosis as compared to parental cells (SKOV3-ip1). We also found that the level of Bcl-xL modification correlated with cisplatin-induced cell death. From in vitro alkaline pH assay and site-directed mutagenesis of Bcl-xL at two potential deamidation sites, where Asn 52 and 66 were replaced by Ala or Asp residues, we concluded that this Bcl-xL modification was due to deamidation. In addition, ectopic expression of deamidation forms of Bcl-xL sensitized the SKOV3-ip1 cells to cisplatin to a level equivalent to that caused by overexpression of E1A. Thus, our results suggested that induction of Bcl-xL deamidation play a key role in E1A-mediated sensitization to cisplatin in ovarian caner cells. Since, E1A has been reported to inhibit Akt activity which phosphorylates pro-apoptotic protein Bad, we examined whether Bad could interact with deamidated Bcl-xL to induce cell death. From immunoprecipitation assay, we observed that Bad bound to both forms of Bcl-xL, preferably deamidated Bcl-xL. These results suggest that Bad has higher ability to bind to deamidated Bcl-xL, thereby activating caspases and leading to cell death.
Deverman, B. E., Cook, B. L., Manson, S. R., Niederhoff, R. A., Langer, E. M., Rosova, I., Kulans, L. A., Fu, X., Weinberg, J. S., Heinecke, J. W., Roth, K. A., and Weintraub, S. J. Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage. Cell 111: 51–62, 2002.
Robinson, N.E, Robinson, Z. W., Robinson, B. R., Robinson, A. L., Robinson, J. A. Robinson, M. L., Robinson, A. B. Structure-dependent nonezymatic deamidation of glutaminyl and asparaginyl pentapeptides. J. Peptide Res. 63: 426-436, 2004.
Zhao, R., Yang, F.T., and Alexander, D.R. An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced Bcl-xL deamidation. Cancer Cell 5: 37-49, 2004.
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