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研究生(外文):Teng-Fa Hsu
論文名稱(外文):Stably expressed PIN1 in an ovarian cancer cell line and its effect on cell death
指導教授(外文):I-Tsuen Chen
外文關鍵詞:PIN1ovarian cancercell death
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Pin1是一種peptidyl-prolyl isomerase (PPIase),會專一性的辨認pSer/pThr-Pro這樣的胺基酸序列。當磷酸化的目標蛋白被Pin1辨認並結合後,此目標蛋白的構形在proline的位置會被改變,而影響了該蛋白的功能或穩定性,例如:cyclin D1、p53、c-myc,Bcl-2等。之前實驗室利用SKOV3.ip1/E1A的stable clone證實,在E1A的存在下,可以透過抑制Rb,使得在cisplatin處理下,提高Bcl-xL的去醯胺基化,而造成細胞死亡。本篇論文想探討在SKOV3.ip1細胞中,是否可以透過PIN1的大量表現影響cyclin D1,當cyclin D1的表現跟著增加,進而磷酸化Rb,當Rb失去功能,在處理cisplatin下,是否可提高Bcl-xL的去醯胺基化。首先我們建立了可以穩定表現Pin1之SKOV3.ip1/Pin1細胞株。由實驗結果發現,Pin1的確可以在SKOV3.ip1內,提高cyclin D1的表現,而E2F的活化也隨著cyclin D1表現的增加而提高,但是在Pin1表現量最多的細胞中,其細胞的生長速率卻是緩慢的,而在處理cisplatin的情況之下,過多Pin1表現並不會提高Bcl-xL的去醯胺基化,即使在有外送E1A的情形下,Bcl-xL的去醯胺基化也無明顯增加。但實驗中發現,在SKOV3.ip1/Pin1的stable clone中,處理cisplatin 48小時後,細胞的存活率都有明顯的下降,而Pin1表現最多的細胞,即使在沒有處理cisplatin的情況下,細胞的存活率明顯比其他兩株細胞低。因此推測,藉由Pin1間接磷酸化Rb似乎不足以提高Bcl-xL的去醯胺基化,Pin1的大量表現,可能不是透過Bcl-xL的去醯胺基化,而是引發細胞內的其它與細胞死亡相關的路徑,而造成細胞的死亡,目前我們正在進一步討論中。同時,我們也在建立表達NHE1之質體,NHE1似乎可以影響Bcl-xL的去醯胺基化,但是所建立的數個NHE1質體,均無法在細胞中表現,原因則正在探討中。
Pin1, a peptidyl-prolyl isomerase (PPIase), can recognize pSer/pThr-Pro sequence specifically, where Pin1 binds and interacts with the phosphoprotein. This will make conformational change of the target protein at proline residue which affects the function and stability of the target protein. Several phosphoproteins are recognized and affected by Pin1 including cyclin D1, p53, c-myc, Bcl-2 etc. Previously, we have shown that ectopic expression of E1A can sensitize SKOV3.ip1 cell to cisplatin by inducing Bcl-xL deamidation mediated by inactivated Rb. In this study, we are examining whether overexpression of Pin1 in SKOV3.ip1 cell could increase cyclin D1 level, and inactivate Rb by phosphorylation, where cells might be sensitized to cisplatin by inducing Bcl-xL deamidation. Firstly, a Pin1 plasmid was transfected into SKOV3.ip1 cell and several G418-resistant clones were isolated. One of the Pin1 stable clones, Pin1-D, shown 3-folds increase in Pin1 level was used for further study. Our data show that both cyclin D1 level and free E2F increased in Pin1-D cells, but Bcl-xL deamidation was not induced in the presence of cisplatin or E1A. Surprisingly, growth rate of pin-D reduced as compared to the vector-control cells. Moreover, the cell viability of Pin1-D decreased in the absence of cisplatin. Currently, we are investigating whether Pin1- induced apoptosis is mediated by Bcl-2 family or other pathways. At the same time, we have also constructed several NHE1-expressing clones. NHE1, a Na+/H+ exchanger 1 gene, shown recently, affects Bcl-xL deamidation. Nevertheless, none of the NHE1 clones expressed in transfected cells.
中文摘要 2
英文摘要 3
第一章 緒論 4
第二章 材料與方法 11
第三章 結果 26
第四章 討論 32
第五章 參考文獻 35
第六章 結果圖表 44
第七章 Supplemental data 52
第八章 附錄 53
1. Kun Ping Lu, Hanes SD, Hunter T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 1996;380(6574):544-7.

2. Yeh ES, Means AR. PIN1, the cell cycle and cancer. Nat Rev Cancer 2007;7(5):381-8.

3. Ranganathan R, Lu KP, Hunter T, Noel JP. Structural and Functional Analysis of the Mitotic Rotamase Pin1 Suggests Substrate Recognition Is Phosphorylation Dependent. Cell 1997;89(6):875-86.

4. Yaffe MB, Schutkowski M, Shen M, et al. Sequence-Specific and Phosphorylation-Dependent Proline Isomerization: A Potential Mitotic Regulatory Mechanism. Science 1997;278(5345):1957-60.

5. Lu KP. Prolyl isomerase Pin1 as a molecular target for cancer diagnostics and therapeutics. Cancer Cell 2003;4:175-80.

6. Lu P-J, Zhou XZ, Liou Y-C, Noel JP, Lu KP. Critical Role of WW Domain Phosphorylation in Regulating Phosphoserine Binding Activity and Pin1 Function. J Biol Chem 2002;277(4):2381-4.

7. Eckerdt F, Yuan J, Saxena K, et al. Polo-like Kinase 1-mediated Phosphorylation Stabilizes Pin1 by Inhibiting Its Ubiquitination in Human Cells. J Biol Chem 2005;280(44):36575-83.

8. Ryo A, Liou Y-C, Wulf G, Nakamura M, Lee SW, Lu KP. PIN1 Is an E2F Target Gene Essential for Neu/Ras-Induced Transformation of Mammary Epithelial Cells. Mol Cell Biol 2002;22(15):5281-95.

9. Yeh ES, Lew BO, Means AR. The Loss of PIN1 Deregulates Cyclin E and Sensitizes Mouse Embryo Fibroblasts to Genomic Instability. J Biol Chem 2006;281(1):241-51.

10. Junichi Kuramochi TASIJKHUKS. High Pin1 expression is associated with tumor progression in colorectal cancer. Journal of Surgical Oncology 2006;94(2):155-60.
11. Bao L, Kimzey A, Sauter G, Sowadski JM, Lu KP, Wang D-G. Prevalent Overexpression of Prolyl Isomerase Pin1 in Human Cancers. Am J Pathol 2004;164(5):1727-37.

12. Kun Ping Lu FS, Xiao Zhen Zhou, Greg Finn, Prudence Lam, Gerburg Wulf. Targeting carcinogenesis: A role for the prolyl isomerase Pin1? Molecular Carcinogenesis 2006;45(6):397-402.

13. Chang-Jae Kim Y-GC, Yong-Gyu Park, Suk-Woo Nam, Su-Young Kim, Sug-Hyung Lee, Nam-Jin Yoo, Jung-Young Lee, Won-Sang Park Pin1 overexpression in colorectal cancer and its correlation with aberrant b-catenin expression. World J Gastroenterol 2005;11:5006-9.

14. Masahiro Nakashima SM, Yuki Naruke, Hisayoshi Kondo, Vladimir Saenko, Tatiana Rogounovitch, Yuki Shimizu-Yoshida, Noboru Takamura, Hiroyuki Namba, Masahiro Ito, Aleksander Abrosimov, Eugeny Lushnikov, Pavel Roumiantsev, Anatoly Tsyb, Shunichi Yamashita, Ichiro Sekine. Cyclin D1 overexpression in thyroid tumours from a radio-contaminated area and its correlation with Pin1 and aberrant beta-catenin expression. The Journal of Pathology 2004;202(4):446-55.

15. van Drogen F, Sangfelt O, Malyukova A, et al. Ubiquitylation of Cyclin E Requires the Sequential Function of SCF Complexes Containing Distinct hCdc4 Isoforms. Molecular Cell 2006;23(1):37-48.

16. Yeh E, Cunningham M, Arnold H. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 2004;6(4):308-18.

17. Lu KP. Pinning down cell signaling, cancer and Alzheimer's disease. Trends in Biochemical Sciences 2004;29(4):200-9.

18. Kun Ping Lu Y-CLIV. Proline-directed phosphorylation and isomerization in mitotic regulation and in Alzheimer's Disease. BioEssays 2003;25(2):174-81.

19. Trimarchi JM, Lees JA. Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol 2002;3(1):11-20.

20. Wellcome/CRC Institute and Department of Pathology UoC, Tennis Court Road, Cambridge CB2 1QR, UK . Regulation of E2F1 activity by acetylation. EMBO J 2000;19:662-71.

21. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev 1998;12(15):2245-62.

22. Claudio PP, De Luca A, Howard CM, et al. Functional Analysis of pRb2/p130 Interaction with Cyclins. Cancer Res 1996;56(9):2003-8.

23. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 1995;81(3):323-30.

24. Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA, Weinberg RA. Effects of an Rb mutation in the mouse. Nature 1992;359(6393):295-300.

25. DeGregori J, Leone G, Miron A, Jakoi L, Nevins JR. Distinct roles for E2F proteins in cell growth control and apoptosis. Proceedings of the National Academy of Sciences 1997;94(14):7245-50.

26. Irwin M, Marin MC, Phillips AC, et al. Role for the p53 homologue p73 in E2F-1-induced apoptosis. Nature 2000;407(6804):645-8.

27. Phillips AC, Ernst MK, Bates S, Rice NR, Vousden KH. E2F-1 Potentiates Cell Death by Blocking Antiapoptotic Signaling Pathways. Molecular Cell 1999;4(5):771-81.

28. Stacey DW. Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Current Opinion in Cell Biology 2003;15(2):158-63.

29. Giacinti C, Giordano A. RB and cell cycle progression. oncogene 2006;25(38):5220-7.

30. Yoshitaka Hosokawa AA. Mechanism of cyclin D1(CCND1, PRAD1) overexpression in human cancer cells: Analysis of allele-specific expression. Genes, Chromosomes and Cancer 1998;22(1):66-71.

31. Wong E, Giandomenico CM. Current Status of Platinum-Based Antitumor Drugs. Chem Rev 1999;99(9):2451-66.

32. Weiss RBC, Michaele C. . New cisplatin analogs in development: a review. Drugs 1993;46:360-77.

33. Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. oncogene 2003;22(47):7265-79.

34. Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer 2007;7(8):573-84.

35. Eastman A. The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacology & Therapeutics 1987;34(2):155-66.

36. Pinto AL LS. Binding of the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin) to DNA. Biochim Biophys Acta 1985;780(3):167-80.

37. Kelland LR. Preclinical Perspectives on Platinum Resistance. Drugs 2000;59(6):1-8.

38. El-Khateeb M, Appleton TG, Gahan LR, Charles BG, Berners-Price SJ, Bolton A-M. Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques. Journal of Inorganic Biochemistry 1999;77(1-2):13-21.

39. Davies MS, Berners-Price SJ, Hambley TW. Rates of platination of -AG- and -GA- containing double-stranded oligonucleotides: effect of chloride concentration. Journal of Inorganic Biochemistry 2000;79(1-4):167-72.

40. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2(9):647-56.

41. Rathmell JC, Thompson CB. Pathways of Apoptosis in Lymphocyte Development, Homeostasis, and Disease. Cell 2002;109(2, Supplement 1):S97-S107.

42. Emily H.-Y.A. Cheng MCW, Solly Weiler,. BCL-2, BCL-XL Sequester BH3 Domain-Only Molecules Preventing BAX- and BAK-Mediated Mitochondrial Apoptosis. Molecular Cell 2001;8:705-11.

43. Kharbanda S, Pandey P, Schofield L, et al. Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced�apoptosis. Proceedings of the National Academy of Sciences 1997;94(13):6939-42.

44. Hsu Y-T, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of Bax and Bcl-XL during apoptosis. Proceedings of the National Academy of Sciences 1997;94(8):3668-72.

45. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 2008;9(1):47-59.

46. Muchmore SW, Sattler M, Liang H, et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 1996;381(6580):335-41.

47. Minn AJ, Velez P, Schendel SL, et al. Bcl-xL forms an ion channel in synthetic lipid membranes. Nature 1997;385(6614):353-7.

48. Dai Y, Grant S. Targeting Multiple Arms of the Apoptotic Regulatory Machinery. Cancer Res 2007;67(7):2908-11.

49. Shaomeng Wanga DY, Marc E Lippmana. Targeting Bcl-2 and Bcl-XL with nonpeptidic small-molecule antagonists. seminars in oncology 2003;30(16):133-42.

50. Robinson NE. Protein deamidation. Proceedings of the National Academy of Sciences 2002;99(8):5283-8.

51. Robinson NE, Robinson AB. Deamidation of human proteins. Proceedings of the National Academy of Sciences 2001;98(22):12409-13.

52. Robinson NE, Robinson AB. Molecular clocks. Proceedings of the National Academy of Sciences 2001;98(3):944-9.

53. Robinson AB, Robinson LR. Distribution of Glutamine and Asparagine Residues and Their Near Neighbors in Peptides and Proteins. Proceedings of the National Academy of Sciences 1991;88(20):8880-4.

54. Deverman BE, Cook BL, Manson SR, et al. Bcl-xL Deamidation Is a Critical Switch in the Regulation of the Response to DNA Damage. Cell 2002;111(1):51-62.

55. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell 2002;2(2):103-12.

56. Chang CY, Lin YM, Lee WP, Hsu HH, Chen EIT. Involvement of Bcl-XL deamidation in E1A-mediated cisplatin sensitization of ovarian cancer cells. oncogene 2006;25(18):2656-65.

57. Ryo A, Nakamura M, Wulf G, Liou Y-C, Lu KP. Pin1 regulates turnover and subcellular localization of β-catenin by inhibiting its interaction with APC. Nat Cell Biol 2001;3(9):793-801.

58. Henderson BR. Nuclear-cytoplasmic shuttling of APC regulates [beta]-catenin subcellular localization and turnover. Nat Cell Biol 2000;2(9):653-60.

59. Gerburg M. Wulf AR, Gerald G. Wulf,1 Sam W. Lee,2 Tianhua Niu,3 Victoria Petkova, and Kun Ping Lu4. Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1. EMBO J 2001;20:3459-72.

60. Liou YC RA, Huang HK, Lu PJ, Bronson R, Fujimori F, Uchida T, Hunter T, Lu KP. Loss of Pin1 function in the mouse causes phenotypes resembling cyclin D1-null phenotypes. Proc Natl Acad Sci U S A 2002;99:1335-40.

61. Malo ME FL. Physiological role and regulation of the Na+/H+ exchanger. Can J Physiol Pharmacol 2006;84(11):1081-95.

62. Aharonovitz O, Zaun HC, Balla T, York JD, Orlowski J, Grinstein S. Intracellular pH Regulation by Na+/H+ Exchange Requires Phosphatidylinositol 4,5-Bisphosphate. J Cell Biol 2000;150(1):213-24.

63. Pang T, Su X, Wakabayashi S, Shigekawa M. Calcineurin Homologous Protein as an Essential Cofactor for Na+/H+ Exchangers. J Biol Chem 2001;276(20):17367-72.

64. Bertrand B, Wakabayashi S, Ikeda T, Pouyssegur J, Shigekawa M. The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites. J Biol Chem 1994;269(18):13703-9.

65. Li X, Liu Y, Alvarez BV, Casey JR, Fliegel L. A Novel Carbonic Anhydrase II Binding Site Regulates NHE1 Activity. Biochemistry 2006;45(7):2414-24.

66. Counillon L, Pouyssegur J, Reithmeier RAF. The Na+/H+ Exchanger NHE-1 Possesses N- and O-Linked Glycosylation Restricted to the First N-Terminal Extracellular Domain. Biochemistry 1994;33(34):10463-9.

67. R S Haworth OF, and L Fliegel. Multiple carbohydrate moieties on the Na+/H+ exchanger. Biochem J 1993;1(289):637-40.

68. Yan W, Nehrke K, Choi J, Barber DL. The Nck-interacting Kinase (NIK) Phosphorylates the Na+-H+ Exchanger NHE1 and Regulates NHE1 Activation by Platelet-derived Growth Factor. J Biol Chem 2001;276(33):31349-56.

69. Takahashi E, Abe J-i, Gallis B, et al. p90RSK Is a Serum-stimulated Na+/H+ Exchanger Isoform-1 Kinase. . J Biol Chem 1999;274(29):20206-14.

70. Hooley R, Yu C-Y, Symons M, Barber DL. G alpha13 Stimulates Na+-H+ Exchange through Distinct Cdc42-dependent and RhoA-dependent Pathways. J Biol Chem 1996;271(11):6152-8.

71. Harguindey S, Orive G, Luis Pedraz J, Paradiso A, Reshkin SJ. The role of pH dynamics and the Na+/H+ antiporter in the etiopathogenesis and treatment of cancer. Two faces of the same coin--one single nature. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 2005;1756(1):1-24.

72. Rich IN, Worthington-White D, Garden OA, Musk P. Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+ exchanger. Blood 2000;95(4):1427-34.

73. Reshkin SJ, Bellizzi A, Caldeira S, et al. Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes. FASEB J 2000;14(14):2185-97.
74. Wu KL, Khan S, Lakhe-Reddy S. The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival. J Biol Chem 2004;279(25):26280-6.

75. Zhao R, Oxley D, Smith TS, Follows GA, Green AR, Alexander DR. DNA Damage-Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH. PLoS Biology 2007;5(1):39-53.

76. Reshkin SJ, Bellizzi A, Cardone RA, Tommasino M, Casavola V, Paradiso A. Paclitaxel Induces Apoptosis via Protein Kinase A- and p38 Mitogen-activated Protein-dependent Inhibition of the Na+/H+ Exchanger (NHE) NHE Isoform 1 in Human Breast Cancer Cells. Clin Cancer Res 2003;9(6):2366-73.

77. Wakabayashi S, Fafournoux P, Sardet C, Pouyssegur J. The Na+/H+ Antiporter Cytoplasmic Domain Mediates Growth Factor Signals and Controls "H+-Sensing". Proceedings of the National Academy of Sciences 1992;89(6):2424-8.

78. Kun Ping Lu, Hanes SD, Hunter T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 1996;380(6574):544-7.

79. Shen M, Stukenberg PT, Kirschner MW, Lu KP. The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosis-specific�phosphoproteins. Genes Dev 1998;12(5):706-20.

80. de Alboran IM, Malynn B, Davidson L, et al. Analysis of C-MYC Function in Normal Cells via Conditional Gene-Targeted Mutation. Immunity 2001;14(1):45-55.

81. Altieri DC. Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer 2008;8(1):61-70.

82. Dourlen P, Ando K, Hamdane M, Begard S, Buee L, Galas MC. The peptidyl prolyl cis/trans isomerase Pin1 downregulates the Inhibitor of Apoptosis Protein Survivin. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2007;1773(9):1428-37.

83. Mantovani F, Piazza S, Gostissa M, et al. Pin1 Links the Activities of c-Abl and p300 in Regulating p73 Function. Molecular Cell 2004;14(5):625-36.

84. De Nicola F, Bruno T, Iezzi S, et al. The Prolyl Isomerase Pin1 Affects Che-1 Stability in Response to Apoptotic DNA Damage. J Biol Chem 2007;282(27):19685-91.

85. Basu A DM, Qanungo S, Fan XJ, DuBois G, Haldar S. Proteasomal degradation of human peptidyl prolyl isomerase pin1-pointing phospho Bcl2 toward dephosphorylation. Neoplasia 2002;4(3):218-27.

86. Sherr CJ. D-type cyclins. Trends in Biochemical Sciences 1995;20(5):187-90.
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