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系統識別號 U0026-0812200910462587
論文名稱(中文) 前胸腺激素與腫瘤轉移之研究
論文名稱(英文) Study of the Correlation between Prothymosin α and Tumor Metastasis
校院名稱 成功大學
系所名稱(中) 生物化學研究所
系所名稱(英) Department of Biochemistry
學年度 91
學期 2
出版年 92
研究生(中文) 劉長青
研究生(英文) Chang-Ching Liu
電子信箱 evergreen0215@yahoo.com.tw
學號 s1690408
學位類別 碩士
語文別 英文
論文頁數 40頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-賴明德
口試委員-謝達斌
口試委員-陳麗珠
中文關鍵字 前胸腺激素  腫瘤轉移 
英文關鍵字 prothymosin alpha  tumor metastasis 
學科別分類
中文摘要 前胸腺激素(prothymosin α)首先由大鼠胸腺所分離出來,當時曾一度被認定為胸腺荷爾蒙。其為一廣泛分部於各哺乳類細胞之高度酸化蛋白質。前胸腺激素表現出許多關於免疫系統激發與哺乳類細胞增生之生物性功能。這些過去的發現顯示前胸腺激素於生理與免疫方面扮演多重重要角色,同時它也牽涉到腫瘤發生的一些關係。而為了要理解究竟當前胸腺激素於細胞中過量表現會有哪些因子發生關聯,微矩陣分析技術結果顯示NIH3T3纖維母細胞與pp23(前胸腺激素過量表現之NIH3T3細胞),本實驗室發現osteopontin基因表現在pp23細胞中明顯下降。Osteopontin為一酸化附著性醣蛋白,且其為一具有RGD domain可與細胞表面integrin作用之非膠質性細胞間質,而在一些轉型的細胞中通常可發現osteopontin有著過量的表現,例如乳癌、前列腺癌、骨癌、神經膠細胞瘤、鱗狀細胞癌。許多研究指出osteopontin可作為癌症治療的適切目標,起因於其牽涉到腫瘤擴散以及於骨癌中血管新生與蝕骨現象等等。此外另有研究指出osteopontin缺失的小鼠模式明顯降低了實驗中腫瘤轉移至骨與軟組織等情形。
根據以上的發現,我們利用螢火蟲螢光酵素報導基因方式測試,發現PC3(前列腺癌骨組織轉移細胞)細胞中前胸腺激素可顯著降低osteopontin表現。反轉錄聚合脢連鎖反應與螢光酵素分析直接證實了先前微矩陣結果,並進一步提示了前胸腺激素可用來作為抑制腫瘤轉移的可能應用性。另一方面,本實驗進一步利用osteopontin promoter驅動前胸腺激素,並將此構築送入小鼠B16F10黑色素瘤細胞中。此轉型後之B16F10細胞表現出較原始株高的增生能力,顯示出前胸腺激素影響了原始B16F10的生長速率。此外並利用此轉型株細胞作進一步的小鼠肺臟腫瘤轉移實驗,結果戲劇性的顯示轉型株細胞轉移至肺臟所產生的黑色顆粒明顯較原始B16F10細胞來得少,顯示前胸腺激素可能牽涉到腫瘤轉移中的事件。因此本研究提供了一些訊息,不單是有關於前胸腺激素與腫瘤轉移之間的關聯性,並且可用來進一步作為癌症治療的目標物。
英文摘要 Prothymosin alpha (ProTα) was first isolated from rat thymus as a precursor of thymosin α1 and once considered as a thymic hormone. It is a highly acidic protein widely distributed in mammalian cells. ProTα exerts many biological functions involved in immune system stimulation and proliferation of mammalian cells. These findings suggest that proTα plays multiple roles in physiological and immunological functions; it also may be employed as factors of tumorgenesis. In order to elucidate what factors may be involved after proTα overexpression in cells, our previous result of microarray analysis was applied to this approach. Comparing NIH3T3 and pp23 (a proTα-overexpressing stable clone) cells, we found that osteopontin (OPN) was significantly repressed in pp23 cells. OPN is an acidic adhesion glycoprotein and noncollagenous bone extracellular matrix with a functional RGD cell binding domain that interacts with the αvβ3 cell surface integrin heterodimer. Some transformed cells are often characterized by abundant secretion of OPN, such as breast cancer, prostate cancer, osteosarcoma, glioblastoma and squamous cell carcinoma. Many studies suggest that OPN may be a candidate target for gene therapy because it contributes not only to tumor cell dissemination but also to angiogenesis and osteolysis in bone metastasis. Mice with OPN deficiency have been shown to reduce experimental tumor metastasis to bone and soft tissues.
Base on these facts, we examined PC3 cells (originally isolated from prostate cancer cells metastasized to bone) using luciferase reporter gene assay and found that proTα could significantly suppress the promoter activity of OPN. The results of RT-PCR and luciferase assay directly confirm the microarray result and further suggest a possible application of proTα for inhibiting tumor metastasis. On the other hand, we also transduced proTα driven by the OPN promoter into B16F10 melanoma cells and investigated its effect on cell migration and tumor invasion. The OPN-proTα stable clones exhibited higher proliferation ability than wild-type B16F10 cells, suggesting that proTα influenced the growth rate of B16F10 cells. In addition, we tested these OPN-proTα clones for pulmonary metastasis in C57BL/6 mice. Dramatically, our result showed that the number of pulmonary metastatic nodules was decreased markedly in mice injected with OPN-proTα clones compared with those injected with wild-type B16F10 cells, indicating that proTα may be involved in metastasis of tumor cells. This study may provide information about not only the correlation between proTα and tumor metastasis, but also the target for cancer gene therapy.
論文目次 Chinese abstract……………………………………………………….... I Abstract………………………………………………………………….III Acknowledgement.................................................................................... IV Contents……………………………………………………………….... V
Figure contents……………………………………………………..........VII Abbreviation…………………………………………………………..... VIII
Introduction
Prothymosin alpha...………………………………………………….1
Tumor metastasis and osteopontin……..…………………………….3
The aims of this study…………….………………………………….5
Materials and methods
Plasmid construction and DNA manipulation………………………..6
Cell culture……………………………………………………………9
Transfection…………………………………………………………..10
Immunohistochemical stain…………………………………………..10
Proliferation assay…………………………………………………….11
Luciferase assay………………………………………………………11
RNA isolation and RT-PCR…………………………………………..12
Wound healing assay………………………………………………….14
Filter membrane assay………………………………………………..14
Experimental Animals………………………………………………...15
Animal model of B16F10 melanoma pulmonary metastasis…………15
Statistical analysis…………………………………………………….15
Results
Osteopontin expression in prothymosin α-overexpressing NIH3T3

cells…………………………………………………………………..16
Promoter activity of the osteopontin gene was regulated by prothymosin α………………………………………………………..16
Establishment of stable B16F10 clones overexpressing prothymosin α driven by the osteopontin promoter………………………………..17
Prothymosin α suppressed the migration and pulmonary metastasis of B16F10 cells………………………………………………………17 Discussion……………………………………………………………… 19 References……………………………………………………………….22 Appendix………………………………………………………………...39
The
author……………………………………………………………….40
參考文獻 1. Haritos, A. A., Goodall, G. J., and Horecker, B. L. Prothymosin α: isolation and properties of the major immunoreactive form of thymosin α 1 in rat thymus. Proc.Natl.Acad.Sci.U.S.A, 81: 1008-1011, 1984.
2. Eschenfeldt, W. H. and Berger, S. L. The human prothymosin α gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proc.Natl.Acad.Sci.U.S.A, 83: 9403-9407, 1986.
3. Gomez-Marquez, J., Segade, F., Dosil, M., Pichel, J. G., Bustelo, X. R., and Freire, M. The expression of prothymosin α gene in T lymphocytes and leukemic lymphoid cells is tied to lymphocyte proliferation. J.Biol.Chem., 264: 8451-8454, 1989.
4. Gomez-Marquez, J. and Segade, F. Prothymosin α is a nuclear protein. FEBS Lett., 226: 217-219, 1988.
5. Manrow, R. E., Sburlati, A. R., Hanover, J. A., and Berger, S. L. Nuclear targeting of prothymosin α. J.Biol.Chem., 266: 3916-3924, 1991.
6. Watts, J. D., Cary, P. D., and Crane-Robinson, C. Prothymosin α is a nuclear protein. FEBS Lett., 245: 17-20, 1989.
7. Smith, M. R. Prothymosin α: in search of a function. Leuk.Lymphoma, 18: 209-214, 1995.
8. Bustelo, X. R., Otero, A., Gomez-Marquez, J., and Freire, M. Expression of the rat prothymosin α gene during T-lymphocyte proliferation and liver regeneration. J.Biol.Chem., 266: 1443-1447, 1991.
9. Eilers, M., Schirm, S., and Bishop, J. M. The MYC protein activates transcription of the α-prothymosin gene. EMBO J., 10: 133-141, 1991.
10. Sburlati, A. R., Manrow, R. E., and Berger, S. L. Prothymosin α antisense oligomers inhibit myeloma cell division. Proc.Natl.Acad.Sci.U.S.A, 88: 253-257, 1991.
11. Rodriguez, P., Vinuela, J. E., Alvarez-Fernandez, L., and Gomez-Marquez, J. Prothymosin α antisense oligonucleotides induce apoptosis in HL-60 cells. Cell Death.Differ., 6: 3-5, 1999.
12. Rodriguez, P., Vinuela, J. E., Alvarez-Fernandez, L., Buceta, M., Vidal, A., Dominguez, F., and Gomez-Marquez, J. Overexpression of prothymosin α accelerates proliferation and retards differentiation in HL-60 cells. Biochem.J., 331 ( Pt 3): 753-761, 1998.
13. Wu, C. L., Shiau, A. L., and Lin, C. S. Prothymosin α promotes cell proliferation in NIH3T3 cells. Life Sci., 61: 2091-2101, 1997.
14. Shiau, A. L., Lin, P. R., Chang, M. Y., and Wu, C. L. Retrovirus-mediated transfer of prothymosin gene inhibits tumor growth and prolongs survival in murine bladder cancer. Gene Ther., 8: 1609-1617, 2001.
15. Shiau, A. L., Chen, Y. L., Liao, C. Y., Huang, Y. S., and Wu, C. L. Prothymosin α enhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis. Vaccine, 19: 3947-3956, 2001.
16. Shiau, A. L., Chu, C. Y., Su, W. C., and Wu, C. L. Vaccination with the glycoprotein D gene of pseudorabies virus delivered by nonpathogenic Escherichia coli elicits protective immune responses. Vaccine, 19: 3277-3284, 2001.
17. Weber, G. F. and Ashkar, S. Stress response genes: the genes that make cancer metastasize. J.Mol.Med., 78: 404-408, 2000.
18. O'Regan, A. and Berman, J. S. Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation. Int.J.Exp.Pathol., 81: 373-390, 2000.
19. Singh, R. P., Patarca, R., Schwartz, J., Singh, P., and Cantor, H. Definition of a specific interaction between the early T lymphocyte activation 1 (Eta-1) protein and murine macrophages in vitro and its effect upon macrophages in vivo. J.Exp.Med., 171: 1931-1942, 1990.
20. Denhardt, D. T. and Chambers, A. F. Overcoming obstacles to metastasis--defenses against host defenses: osteopontin (OPN) as a shield against attack by cytotoxic host cells. J.Cell Biochem., 56: 48-51, 1994.
21. Weber, G. F., Ashkar, S., Glimcher, M. J., and Cantor, H. Receptor-ligand interaction between CD44 and osteopontin (Eta-1). Science, 271: 509-512, 1996.
22. Senger, D. R. and Perruzzi, C. A. Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin. Biochim.Biophys.Acta, 1314: 13-24, 1996.
23. Ashkar, S., Weber, G. F., Panoutsakopoulou, V., Sanchirico, M. E., Jansson, M., Zawaideh, S., Rittling, S. R., Denhardt, D. T., Glimcher, M. J., and Cantor, H. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science, 287: 860-864, 2000.
24. Prols, F., Loser, B., and Marx, M. Differential expression of osteopontin, PC4, and CEC5, a novel mRNA species, during in vitro angiogenesis. Exp.Cell Res., 239: 1-10, 1998.
25. Wiener, J., Lombardi, D. M., Su, J. E., and Schwartz, S. M. Immunohistochemical and molecular characterization of the differential response of the rat mesenteric microvasculature to angiotensin-II infusion. J.Vasc.Res., 33: 195-208, 1996.
26. deBlois, D., Lombardi, D. M., Su, E. J., Clowes, A. W., Schwartz, S. M., and Giachelli, C. M. Angiotensin II induction of osteopontin expression and DNA replication in rat arteries. Hypertension, 28: 1055-1063, 1996.
27. Henke, C. A., Roongta, U., Mickelson, D. J., Knutson, J. R., and McCarthy, J. B. CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix. J.Clin.Invest, 97: 2541-2552, 1996.
28. Trochon, V., Mabilat, C., Bertrand, P., Legrand, Y., Smadja-Joffe, F., Soria, C., Delpech, B., and Lu, H. Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro. Int.J.Cancer, 66: 664-668, 1996.
29. Liaw, L., Skinner, M. P., Raines, E. W., Ross, R., Cheresh, D. A., Schwartz, S. M., and Giachelli, C. M. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of αvβ3 in smooth muscle cell migration to osteopontin in vitro. J.Clin.Invest, 95: 713-724, 1995.
30. Senger, D. R., Ledbetter, S. R., Claffey, K. P., Papadopoulos-Sergiou, A., Peruzzi, C. A., and Detmar, M. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the αvβ3 integrin, osteopontin, and thrombin. Am.J.Pathol., 149: 293-305, 1996.
31. Senger, D. R., Asch, B. B., Smith, B. D., Perruzzi, C. A., and Dvorak, H. F. A secreted phosphoprotein marker for neoplastic transformation of both epithelial and fibroblastic cells. Nature, 302: 714-715, 1983.
32. Senger, D. R. and Perruzzi, C. A. Secreted phosphoprotein markers for neoplastic transformation of human epithelial and fibroblastic cells. Cancer Res., 45: 5818-5823, 1985.
33. Chambers, A. F., Behrend, E. I., Wilson, S. M., and Denhardt, D. T. Induction of expression of osteopontin (OPN; secreted phosphoprotein) in metastatic, ras-transformed NIH 3T3 cells. Anticancer Res., 12: 43-47, 1992.
34. Barraclough, R., Chen, H. J., Davies, B. R., Davies, M. P., Ke, Y., Lloyd, B. H., Oates, A., and Rudland, P. S. Use of DNA transfer in the induction of metastasis in experimental mammary systems. Biochem.Soc.Symp., 63: 273-294, 1998.
35. Ish-Horowicz, D. and Burke, J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res., 9: 2989-2998, 1981.
36. Saporito-Irwin, S. M., Geist, R. T., and Gutmann, D. H. Ammonium acetate protocol for the preparation of plasmid DNA suitable for mammalian cell transfections. Biotechniques, 23: 424-427, 1997.
37. Chung, C. T. and Miller, R. H. A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res., 16: 3580, 1988.
38. Iizuka, K., Murakami, T., and Kawaguchi, H. Pure atmospheric pressure promotes an expression of osteopontin in human aortic smooth muscle cells. Biochem.Biophys.Res.Commun., 283: 493-498, 2001.
39. Weber, G. F. The metastasis gene osteopontin: a candidate target for cancer therapy. Biochim.Biophys.Acta, 1552: 61-85, 2001.
40. Yumoto, K., Ishijima, M., Rittling, S. R., Tsuji, K., Tsuchiya, Y., Kon, S., Nifuji, A., Uede, T., Denhardt, D. T., and Noda, M. Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice. Proc.Natl.Acad.Sci.U.S.A, 99: 4556-4561, 2002. 41. Magdalena, C., Dominguez, F., Loidi, L., and Puente, J. L. Tumour prothymosin α content, a potential prognostic marker for primary breast cancer. Br.J.Cancer, 82: 584-590, 2000.
42. Orre, R. S., Cotter, M. A., Subramanian, C., and Robertson, E. S. Prothymosin α functions as a cellular oncoprotein by inducing transformation of rodent fibroblasts in vitro. J.Biol.Chem., 276: 1794-1799, 2001.
43. Subramanian, C., Hasan, S., Rowe, M., Hottiger, M., Orre, R., and Robertson, E. S. Epstein-Barr virus nuclear antigen 3C and prothymosin α interact with the p300 transcriptional coactivator at the CH1 and CH3/HAT domains and cooperate in regulation of transcription and histone acetylation. J.Virol., 76: 4699-4708, 2002.
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