跳到主要內容

臺灣博碩士論文加值系統

(18.205.192.201) 您好!臺灣時間:2021/08/06 04:12
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳芊菱
研究生(外文):Chien-Ling Chen
論文名稱:研究Rad9蛋白在調節MDM2及p53上所扮演的生物功能
論文名稱(外文):Study of the biological functions for Rad9 in regulating MDM2 and p53
指導教授:張敏政張敏政引用關係
指導教授(外文):Ming C. Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
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:2008
畢業學年度:96
語文別:中文
論文頁數:93
中文關鍵詞:生物功能p53Rad9 蛋白MDM2
外文關鍵詞:p53MDM2Rad9biological functions
相關次數:
  • 被引用被引用:0
  • 點閱點閱:148
  • 評分評分:
  • 下載下載:15
  • 收藏至我的研究室書目清單書目收藏:0
人類的Rad9蛋白為Schizosaccharomyces pombe Rad9蛋白的同源蛋白,其為checkpoint rad genes產物中的一員。Rad9為一391個胺基酸蛋白,其在許多重要生物功能上扮演重要的角色,包括調控DNA受損反應、調節細胞週期的進行、執行DNA的修復、誘導細胞凋亡、調控部分基因的轉錄、具有3’端到5’端核酸外切酵素(exonuclease)的活性、促進核糖核甘酸(ribonucleotide)的生合成以及參與胚胎發育過程。先前便有文獻指出Rad9在在調節細胞週期的進行、誘導細胞凋亡及維持整個基因體的完整性等生物功能都和p53極為相似,因此Rad9及p53此兩蛋白在功能上可能具有相互協調。在我們先前的研究中,利用體內及體外實驗(in vivo and in vitro assay)證明Rad9可與MDM2進行交互作用(interaction)。所以接下來利用in vitro pull down assay探討MDM2的哪個區域(domain)具有與Rad9連接的能力(Rad9-binding capacity)。由我的實驗結果中證明MDM2是利用其RING-finger motif去與Rad9連接,此motif為MDM2執行E3泛素連接酶(E3 ubiquitin ligase)功能的區域,因此進ㄧ步推測Rad9可能藉由與MDM2連接而影響到MDM2 E3泛素連接酶的活性。為了確立此推測,以核醣核酸干擾技術(si-RNA)進行處理後抑制掉細胞內Rad9蛋白的表現,再以西方墨點法(Western blotting)去偵測MDM2 E3泛素連接酶的受質(substrates) p53及鈣黏著素(E-cadherin)的蛋白表現量。由結果中可觀察到在Rad9-siRNA處理的細胞中,p53及鈣黏著素的蛋白表現量有下降的現象產生。此外,在Rad9-siRNA處理的細胞中亦觀察到MDM2的蛋白表現量有明顯呈現上升的趨勢,意指Rad9可能藉由與MDM2連接而影響到MDM2的蛋白穩定度。Rad9在過去研究中已知為一DNA的修復過程中的DNA受損反應感受器,因此我們進ㄧ步去觀察在UV照射造成DNA受損情況下,經Rad9-siRNA處理的細胞中MDM2及p53蛋白表現量是否有所改變。由我的實驗結果中觀察到在對照組中p53蛋白表現量增加,然而MDM2蛋白表現量卻減少的現象。相對的,經過UV照射後,以核醣核酸干擾技術(si-RNA)抑制掉HEK-293T細胞內Rad9蛋白的表現,則可觀察到相對於對照組有較低的p53蛋白表現量及較高的MDM2蛋白表現量。此外,先以核醣核酸干擾技術抑制掉HEK-293T細胞內Rad9蛋白的表現後,再經camptothecin(CPT)藥物處理後,可觀察到明顯的p53累積現象的延遲。為了更進ㄧ步了解在Rad9-siRNA處理的細胞中是否會影響到p53的功能,我們分析p53下游的目標蛋白p21及PUMA的蛋白表現量,並偵測p53相關細胞凋亡途徑中caspase-3 酵素活性。由我的實驗結果中觀察到在Rad9-siRNA處理的A549細胞中皆有較高的p21及PUMA蛋白表現量,但有較低的caspase-3 酵素活性。此外,我們也觀察到在Rad9-siRNA處理的A549細胞具有較高的細胞遷移能力(cell migration activity);而在相對於對照組的Rad9-siRNA處理的MDA-MB-435S 細胞中則具有較高的細胞侵犯能力(cell invasion activity)。接下來對於研究 Rad9 蛋白在調節 MDM2及p53上所扮演的生物功能上,要再加以探討在一般情況下,在Rad9-siRNA處理或是Rad9過度表現的細胞中,Rad9對於p53的轉錄活性是否有所影響。此外,Rad9在經DNA損傷或抗癌藥物處理後對於細胞凋亡途徑及癌症形成過程的影響,亦是我們接下來所要再加以釐清的方向。
The human Rad9 (Rad9) is the homologue of the fission yeast Schizosaccharomyces pombe Rad9 protein, a member of the checkpoint rad genes products. The Rad9 is a 391-amino acid protein that plays multiple roles in fundamental biological processes, including the regulation of the DNA damage response, cell cycle checkpoint control, DNA repair, apoptosis, transcriptional regulation and embryogenesis. Previous studies indicated that the biological functions of hRad9 in controlling cell cycle checkpoints, apoptosis, and genetic stability are much like p53, suggesting that Rad 9 and p53 might function coordinately in these important cellular bioprocesses. In our previous studies, we found that Rad9 could interact with MDM2 by using in vivo and in vitro assays. In this study, in vitro pull down assay was used to determine which domain of MDM2 has a Rad9-binding capacity. The results indicated that MDM2 interacts with Rad9 through its RING-finger motif, which is known to administer E3 ligase activity of MDM2, suggesting that Rad9 may affect the E3 ligase activity of MDM2 when Rad9 interacts with MDM2. To investigate this, Rad9 expression was knocked down by si-RNA, and the protein levels of p53 and E-cadherin, both of which are substrates for the MDM2 E3 ubiquitin ligase, were detected by Western blotting. The results revealed that Rad9-siRNA cells exhibited a significant decrease in the protein levels of p53 and E-cadherin. In addition, we also found that the Rad9-siRNA cells exhibited a significant increase in MDM2 protein level, indicating the Rad9 could influence MDM2 stability by interacting with MDM2. Since Rad9 is also known to act as a DNA damage sensor for DNA repair, we determined the protein levels of MDM2 and p53 in Rad9-siRNA cells under UV irradiation damage condition. Our present data reveals that protein levels of p53 increased, whereas protein levels of MDM2 decreased after UV irradiation in control cells. In contrast, after UV irradiation, inhibiting Rad9 by siRNA in HEK-293T cells resulted in lower levels of p53 expression and higher levels of MDM2 expression compared to those of control cells. Furthermore, after camptothecin treatment, inhibiting Rad9 by siRNA in HEK-293T cells resulted in markedly delayed in the accumulation of p53. To further investigate whether depletion of Rad9 could also impact on the functions of p53 in Rad9-siRNA cells, we examined the protein levels of p21 and PUMA, both of which are p53 downstream target proteins, and detected the caspase-3 activity, which is involved in the p53-dependent apoptosis pathway. We found that in Rad9-siRNA A549 cells, both p21 and PUMA had higher level expressions, but caspase-3 enzyme had lower activity. In addition, we also found that the Rad9-siRNA A549 cells possessed a higher cell migration activity, and the Rad9-siRNA cells possessed a higher cell invasion activity than that of the parental cell, MDA-MB-435S cell. To further study of the biological functions for Rad9 in regulating MDM2 and p53, the effects of Rad9 on transcriptional activity of p53 in Rad9-siRNA cells or in Rad9-overexpressing cells under normal condition are undertaken. In addition, the roles of Rad9 in apoptotic pathway and in tumorigenesis in response to DNA damage or anti-cancer drugs treatment will also be investigated.
中文摘要..................................................1
英文摘要..................................................4
誌謝......................................................6
目錄......................................................7
圖表目錄.................................................10
附錄目錄.................................................12
縮寫檢索表...............................................13
緒論
一、Rad9蛋白.......................................................15
1、Rad9蛋白分子量 ( Mr )
2、Rad9蛋白的功能性區域( functional domain )
3、Rad9蛋白的主要生理功能 ( biological functions )
(1) Rad9與基因體完整性( genomic integrity )之影響
(2) Rad9與細胞凋亡( apoptosis )的調控關係
(3) Rad9與DNA修復( DNA repair )的調控機制
(4) Rad9與細胞週期( cell cycle )的調節機制
(5) Rad9與癌症的關係
二、MDM2 ( Mouse Double Minute 2 )蛋白...................22
1、MDM2蛋白分子量 ( Mr )
2、MDM2蛋白的功能性區域 ( functional domain )
3、MDM2蛋白的主要生理功能 ( biological functions )
(1) MDM2與抑癌蛋白p53的調控關係
(2) MDM2與其交互作用蛋白的調控關係
(3) MDM2與與癌症的關係
三、p53蛋白.......................................................27
1、p53與細胞生長週期的調控關係
2、p53與細胞凋亡( apoptosis )的調控關係
3、p53與與癌症的關係
研究動機.......................................................30
材料與方法.......................................................33
實驗結果.......................................................51
一、探討MDM2具有與Rad9連接的能力( Rad9-binding capacity )的區域
二、利用核醣核酸干擾技術觀察Rad9對MDM2及p53蛋白穩定性之影

三、觀察細胞在遭受紫外光照射(UV irradiation)後,Rad9是否會影響
MDM2及p53蛋白表現量
四、觀察細胞在處理DNA損傷藥物CPT(camptothecin)後,Rad9是否會
影響MDM2及p53蛋白表現量
五、觀察Rad9對p53下游的目標蛋白p21及PUMA之影響
六、觀察Rad9對p53蛋白相關細胞凋亡途徑( p53 - dependent apoptosis
pathway )中的半胱氨酸蛋白水解酶- 3活性之影響
七、利用核醣核酸干擾技術觀察Rad9對鈣黏著素(E-cadherin)蛋白穩定性
之影響
八、探討Rad9對細胞遷移能力( cell migration activity )之影響
九、探討Rad9對細胞侵犯能力( cell invasion activity )之影響
討論.....................................................61
參考文獻.................................................65
圖表.....................................................69
附錄.....................................................87
1.Murray, J.M., Carr, A.M., Lehmann, A.R. & Watts, F.Z. Cloning and characterisation of the rad9 DNA repair gene from Schizosaccharomyces pombe. Nucleic Acids Res 19, 3525-3531 (1991).
2.Lieberman, H.B. Rad9, an evolutionarily conserved gene with multiple functions for preserving genomic integrity. J Cell Biochem 97, 690-697 (2006).
3.Prakash, L. Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6 and rad9 of Saccharomyces cerevisiae. Mutat Res 45, 13-20 (1977).
4.White, J.H., Lusnak, K. & Fogel, S. Mismatch-specific post-meiotic segregation frequency in yeast suggests a heteroduplex recombination intermediate. Nature 315, 350-352 (1985).
5.Terleth, C., Schenk, P., Poot, R., Brouwer, J. & van de Putte, P. Differential repair of UV damage in rad mutants of Saccharomyces cerevisiae: a possible function of G2 arrest upon UV irradiation. Mol Cell Biol 10, 4678-4684 (1990).
6.Komatsu, K., et al. Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis. Nat Cell Biol 2, 1-6 (2000).
7.Ishikawa, K., Ishii, H., Saito, T. & Ichimura, K. Multiple functions of rad9 for preserving genomic integrity. Curr Genomics 7, 477-480 (2006).
8.Yin, Y., et al. Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21. Proc Natl Acad Sci U S A 101, 8864-8869 (2004).
9.Hirai, I. & Wang, H.G. A role of the C-terminal region of human Rad9 (hRad9) in nuclear transport of the hRad9 checkpoint complex. J Biol Chem 277, 25722-25727 (2002).
10.Yoshida, K., Komatsu, K., Wang, H.G. & Kufe, D. c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage. Mol Cell Biol 22, 3292-3300 (2002).
11.St Onge, R.P., Besley, B.D., Pelley, J.L. & Davey, S. A role for the phosphorylation of hRad9 in checkpoint signaling. J Biol Chem 278, 26620-26628 (2003).
12.St Onge, R.P., Besley, B.D., Park, M., Casselman, R. & Davey, S. DNA damage-dependent and -independent phosphorylation of the hRad9 checkpoint protein. J Biol Chem 276, 41898-41905 (2001).
13.Roos-Mattjus, P., et al. Phosphorylation of human Rad9 is required for genotoxin-activated checkpoint signaling. J Biol Chem 278, 24428-24437 (2003).
14.Hopkins, K.M., et al. Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality. Mol Cell Biol 24, 7235-7248 (2004).
15.Dang, T., Bao, S. & Wang, X.F. Human Rad9 is required for the activation of S-phase checkpoint and the maintenance of chromosomal stability. Genes Cells 10, 287-295 (2005).
16.Aravind, L., Dixit, V.M. & Koonin, E.V. Apoptotic molecular machinery: vastly increased complexity in vertebrates revealed by genome comparisons. Science 291, 1279-1284 (2001).
17.Komatsu, K., Hopkins, K.M., Lieberman, H.B. & Wang, H. Schizosaccharomyces pombe Rad9 contains a BH3-like region and interacts with the anti-apoptotic protein Bcl-2. FEBS Lett 481, 122-126 (2000).
18.Yoshida, K., Wang, H.G., Miki, Y. & Kufe, D. Protein kinase Cdelta is responsible for constitutive and DNA damage-induced phosphorylation of Rad9. EMBO J 22, 1431-1441 (2003).
19.Lee, M.W., Hirai, I. & Wang, H.G. Caspase-3-mediated cleavage of Rad9 during apoptosis. Oncogene 22, 6340-6346 (2003).
20.Bao, S., et al. Disruption of the Rad9/Rad1/Hus1 (9-1-1) complex leads to checkpoint signaling and replication defects. Oncogene 23, 5586-5593 (2004).
21.Griffith, J.D., Lindsey-Boltz, L.A. & Sancar, A. Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy. J Biol Chem 277, 15233-15236 (2002).
22.Parrilla-Castellar, E.R., Arlander, S.J. & Karnitz, L. Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex. DNA Repair (Amst) 3, 1009-1014 (2004).
23.Wang, W., et al. The human Rad9-Rad1-Hus1 checkpoint complex stimulates flap endonuclease 1. Proc Natl Acad Sci U S A 101, 16762-16767 (2004).
24.Toueille, M., et al. The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilisation efficiency: implications for DNA repair. Nucleic Acids Res 32, 3316-3324 (2004).
25.Blankley, R.T. & Lydall, D. A domain of Rad9 specifically required for activation of Chk1 in budding yeast. J Cell Sci 117, 601-608 (2004).
26.Sanchez, Y., et al. Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 286, 1166-1171 (1999).
27.Sorensen, C.S., Syljuasen, R.G., Lukas, J. & Bartek, J. ATR, Claspin and the Rad9-Rad1-Hus1 complex regulate Chk1 and Cdc25A in the absence of DNA damage. Cell Cycle 3, 941-945 (2004).
28.Lieberman, H.B. & Yin, Y. A novel function for human Rad9 protein as a transcriptional activator of gene expression. Cell Cycle 3, 1008-1010 (2004).
29.Ishikawa, K., et al. Rad9 modulates the P21WAF1 pathway by direct association with p53. BMC Mol Biol 8, 37 (2007).
30.Pandita, R.K., et al. Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair. Mol Cell Biol 26, 1850-1864 (2006).
31.Maniwa, Y., et al. Accumulation of hRad9 protein in the nuclei of nonsmall cell lung carcinoma cells. Cancer 103, 126-132 (2005).
32.Maniwa, Y., et al. His239Arg SNP of HRAD9 is associated with lung adenocarcinoma. Cancer 106, 1117-1122 (2006).
33.Cheng, C.K., Chow, L.W., Loo, W.T., Chan, T.K. & Chan, V. The cell cycle checkpoint gene Rad9 is a novel oncogene activated by 11q13 amplification and DNA methylation in breast cancer. Cancer Res 65, 8646-8654 (2005).
34.Zhu, A., Zhang, C.X. & Lieberman, H.B. Rad9 has a functional role in human prostate carcinogenesis. Cancer Res 68, 1267-1274 (2008).
35.Fakharzadeh, S.S., Trusko, S.P. & George, D.L. Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. EMBO J 10, 1565-1569 (1991).
36.Momand, J., Zambetti, G.P., Olson, D.C., George, D. & Levine, A.J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69, 1237-1245 (1992).
37.Oliner, J.D., Kinzler, K.W., Meltzer, P.S., George, D.L. & Vogelstein, B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 358, 80-83 (1992).
38.Roth, J., Dobbelstein, M., Freedman, D.A., Shenk, T. & Levine, A.J. Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J 17, 554-564 (1998).
39.Marechal, V., Elenbaas, B., Piette, J., Nicolas, J.C. & Levine, A.J. The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes. Mol Cell Biol 14, 7414-7420 (1994).
40.Dai, M.S., et al. Regulation of the MDM2-p53 pathway by ribosomal protein L11 involves a post-ubiquitination mechanism. J Biol Chem 281, 24304-24313 (2006).
41.Iwakuma, T. & Lozano, G. MDM2, an introduction. Mol Cancer Res 1, 993-1000 (2003).
42.Barak, Y., Juven, T., Haffner, R. & Oren, M. mdm2 expression is induced by wild type p53 activity. EMBO J 12, 461-468 (1993).
43.Wu, X., Bayle, J.H., Olson, D. & Levine, A.J. The p53-mdm-2 autoregulatory feedback loop. Genes Dev 7, 1126-1132 (1993).
44.Freedman, D.A., Wu, L. & Levine, A.J. Functions of the MDM2 oncoprotein. Cell Mol Life Sci 55, 96-107 (1999).
45.Ladanyi, M., Wang, S., Niesvizky, R., Feiner, H. & Michaeli, J. Proto-oncogene analysis in multiple myeloma. Am J Pathol 141, 949-953 (1992).
46.Cadwell, C. & Zambetti, G.P. The effects of wild-type p53 tumor suppressor activity and mutant p53 gain-of-function on cell growth. Gene 277, 15-30 (2001).
47.Aylon, Y. & Oren, M. Living with p53, dying of p53. Cell 130, 597-600 (2007).
48.Ou, Y.H., Chung, P.H., Sun, T.P. & Shieh, S.Y. p53 C-terminal phosphorylation by CHK1 and CHK2 participates in the regulation of DNA-damage-induced C-terminal acetylation. Mol Biol Cell 16, 1684-1695 (2005).
49.Vogelstein, B., Lane, D. & Levine, A.J. Surfing the p53 network. Nature 408, 307-310 (2000).
50.Vousden, K.H. Apoptosis. p53 and PUMA: a deadly duo. Science 309, 1685-1686 (2005).
51.Hengartner, M.O. Apoptosis. Death cycle and Swiss army knives. Nature 391, 441-442 (1998).
52.Yang, J.Y., et al. MDM2 promotes cell motility and invasiveness by regulating E-cadherin degradation. Mol Cell Biol 26, 7269-7282 (2006).
53.Thiery, J.P. & Sleeman, J.P. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7, 131-142 (2006).
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊