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研究生:潘菡君
研究生(外文):Han-chun Pan
論文名稱:探討人類乳癌細胞中P0及GCIP蛋白和tumorprogression之關係
論文名稱(外文):Characterize the Relationships between P0 and GCIPwith Tumor Progression in Human Breast Cancer Cells
指導教授:張敏政張敏政引用關係
指導教授(外文):Ming-Chung Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
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:100
中文關鍵詞:P0GCIPEMT (Epithelial-mesenchymal transition)cyclin D1
外文關鍵詞:P0GCIPEMTcyclin D1
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抑制癌細胞的轉移一直是最主要的癌症治療重點,癌症轉移的相關機制在近年來也就逐漸成為癌症研究的目標。而對於癌症轉移的研究經常是在探討tumor progression (癌化)這個特殊的過程,意指腫瘤開始呈現侵犯性成長並有機會經由血液或淋巴系統擴散或轉移(metastasis)到身體其他區域,而在新的位置繼續形成新的腫瘤細胞。近年來,有許多科學家發現EMT 這個現象可以作為tumor progression(癌化)過程的正向指標。EMT (Epithelial-mesenchymal transition)是指從上皮細胞型態(epithelium)轉變成間葉細胞mesenchyme)型態的一種特殊過程,最初被定義為胚胎時期與器官發育現象相關,而近年來有許多文獻指出,EMT 參與癌細胞自良性腫瘤變成轉移性癌細胞的過程,被視為主要的癌化指標之一。真核核糖體蛋白P0 是真核生物中核糖體stalk 結構中非常重要的成分之ㄧ,除了在真核生物蛋白質合成的轉譯過程中,扮演絕對重要的角色之外,近年來被發現P0 蛋白在大腸癌、肝癌以及乳癌中有過度表現的情形。在實驗室先前的研究發現,P0 蛋白和Grap2 and cyclin D interacting protein (GCIP)有很強的交互作用,同時將P0 過度表現於人類乳癌及肝癌細胞後會使得細胞增生能力提升,顯示P0 應具有和腫瘤生成有關的功能。GCIP 全名為Grap2 and cyclin D interacting protein,在我們先前的研究中指出GCIP 蛋白表現量和乳癌和肝癌細胞癌化程度呈現負相關,同時也發現P0 和其交互作用的GCIP 蛋白會分別影響腫瘤生成及抑制的功能。我們推測P0 和其交互作用的GCIP 蛋白在細胞癌化的過程當中皆為重要的調節因子,因此藉由過度表現P0 和GCIP 蛋白的方式進一步觀察EMT 現象的產生是否有何改變。實驗結果發現過度表現P0 的前期乳癌細胞MCF-7 中,調控cell adhesion 及維持表皮細胞正常型態的重要因子E-Cadherin表現量明顯下降,同時伴隨其上游的轉錄因子Snail1 表現量上升,顯示癌細胞較有能力開始轉換成間葉細胞 (mesenchyme)型態,進行EMT 現象來助於腫瘤細胞惡化。由於過去有文獻指出GCIP 存在的細胞中會抑制其細胞週期調控蛋白cyclin D1的表現,我們也進一步證實GCIP 會藉由調節cyclin D1 promoter 的活性使得cyclin D1 表現量下降。另一方面,以暫時性轉染 (transient transfection)方式將GCIP 送入具有高度侵入 (invasive)及轉移 (metastatic)能力的後期乳癌細胞MDA-MB-435S 細胞中,同時我們也建立MDA-MB-435S 細胞的GCIP 穩定表現系統 (stable clone)。
結果我們發現vimentin 和N-Cadherin 這兩種和癌化呈現正相關的蛋白表現量有降低的趨勢,顯示EMT 現象的發生可能較為趨緩而有能力抑制腫瘤惡化。同時我們發
現有GCIP 表現的MDA-MB-435S 細胞移行以及侵入能力也有下降的情形。另外,我們也探討了GCIP 在MDA-MB-435S 細胞中基因表現靜默 (gene silencing)的情形應與甲基化 (DNA methylation)有關。在本實驗結果中,藉由了解P0 和GCIP 與EMT現象間的關係進一步確立二者在腫瘤惡化過程中扮演的角色;我們認為P0 和GCIP分別擁有促進及抑制乳癌癌化過程之能力。
Most cancer deaths are ascribed the failure treatment of migrating tumor cells.
Investigation of the mechanisms of tumor progression has become the major cynosure in cancer research. EMT (Epithelial-mesenchymal transition), the conversion of an epithelial cell to a mesenchymal cell was defined structural feature of organ development in embryogenesis, and its occurrence during tumor progression was demonstrated to be the major mechanism responsible for mediating invasiveness and metastasis of cancer cells nowadays. In our previous studies, the ribosomal protein P0, an essential component of the eukaryotic ribosomal stalk, was found to interact with the helix–loop–helix protein human Grap2 and cyclin D interacting protein (GCIP). Also, we found that overexpression of P0 in breast cancer and hepatocellular cancer cell lines promoted cell growth and colony formation. This increase of cell proliferating is apparently due to presence of increased level of free form P0 executed its tumorigenetic function by inhibiting GCIP-mediated reduction of Rb phosphorylation and enhancing the expression of cyclin D1. On the other hand, higher GCIP expression was observed in breast cancer patients with lower histological grade, mucinous carcinomas, and better clinical outcome during follow-up. Thus, our preliminary studies indicated P0 is a newly identified oncogenic protein and GCIP is a newly identified tumor suppressor of breast cancer cells.
In order to gain full understanding the mechanisms of tumor progression of P0 and GCIP, in this study, we first characterized the relationships between P0 and GCIP with EMT in human breast cancer cell. Our preliminary data of this study revealed that lower expression of EMT hallmark, E-Cadherin, was observed in a stable P0-overexpressing MCF-7, indicating that a significant correlation between high P0 and low E-Cadherin protein levels and higher expression of snail1 in breast cancer cell lines. Since a recent study reported that GCIP could inhibit the expression of cyclin D1, a key molecule involved in cell cycle and tumorigenesis in hepatocellular cancer cell, we investigate whether GCIP could inhibit cyclin D1 expression through the transcriptional repression of cyclin D1 promoter. To further confirm that GCIP is tumor suppressor, GCIP was transient transfected and stable expressed in malignant MDA-MB-435S. The stable GCIP-overexpression in MDA-MB-435S cell downregulated the expression of mesenchymal markers vimentin and N-Cadherin, suggesting a significant correlation of between high GCIP and low vimentin and N-Cadherin protein levels as well.
Furthermore, we also found that decreased the cell proliferation rate, migration capacity and invasion capability of the stable GCIP- overexpressing MDA-MB-435S. The investigation whether GCIP is inactivated by DNA methylation in malignant breast cancer cell is undertaken. In conclusion, we suggested that P0 promotes tumorigenesis and GCIP plays a tumor suppressor in breast cancer furthermore could provide the information for the diagnosis/prognosis and future target of antimetastasis therapy in breast cancer patients.
目錄
中文摘要…… I
英文摘要…… III
誌謝…… V
目錄…… VI
圖表目錄…… IX
縮寫表…… XII
第一章、緒論…… 1
一. 癌症與細胞生長的關係(Cell Growth and Its Abnormalities in Cancer)……1
二. 癌化過程簡介 (The Procedures of Tumor Progression) …… 6
三.Epithelial-mesenchymal transition; EMT 簡介(The Introduction of EMT)……7
四. P0 蛋白簡介 (The Introduction of P0 Protein) …… 9
五. GCIP (Grap2 and Cyclin D Interacting Protein)(The Introduction of GCIP)簡介……10
六. 實驗目標 (Aim) …… 11
第二章、材料與方法……14
第三章、實驗結果……40
一. 利用西方墨點法分析乳癌病患臨床檢體內P0 及GCIP 之表現量。……40
二. 利用西方墨點法分析不同時期人類乳癌細胞內P0 及GCIP 之表現量。……40
三. 分析穩定過度表現P0 蛋白的第二期乳癌細胞MCF-7 中EMT 現象之變化。……41
四. 利用螢光免疫染色法觀察穩定過度表現P0 蛋白的第二期乳癌細胞MCF-7 (P0-B8)細胞株內 E-cadherin 表現量。……43
五. P0 對於第二期乳癌細胞MCF-7 移行及侵入能力之影響。 ……44
六. 利用西方墨點法及Luciferase Assay 探討GCIP 對於cyclin D1 表現量之調控。……44
七. 利用RT-PCR 與西方墨點法分析暫時轉染GCIP 對於EMT 現象之影響。……46
八. 暫時轉染GCIP 對於第四期乳癌細胞MDA-MB-435S 移行及侵入能力之影響。……47
九. 建立GCIP 於MDA-MB-435S 穩定表現之細胞株。……47
十. 利用西方墨點法分析穩定過度表現GCIP 蛋白的第四期乳癌細胞 MDA-MB-435S 和EMT 現象之變化。……48
十一. 利用相位差顯微鏡觀察穩定過度表現GCIP 蛋白的第四期乳癌細胞MDA-MB-435S 之細胞型態。……49
十二. 利用螢光顯微鏡觀察穩定過度表現GCIP 蛋白的第四期乳癌細胞MDA-MB-435S 內肌動蛋白及肌動蛋白應力纖維之活動情形。……50
十三. 觀察穩定過度表現GCIP 蛋白的第四期乳癌細胞MDA-MB-435S 之細胞生長速率。……50
十四. 利用Boyden chamber 觀察穩定過度表現GCIP 蛋白的第四期乳癌細胞MDA-MB-435S 之移行能力。……51
十五. 利用CHEMICONTM 96-well cell Invasion Assay 觀察穩定過度表現GCIP 蛋白的第四期乳癌細胞MDA-MB-435S之侵入能力。……52
十六. 探討MDA-MB-435S 細胞中GCIP gene silencing 和表型遺傳調控(epigenetic regulation)甲基化 (DNA methylation)的關係。……52
第四章、實驗討論……54
第五章、實驗圖表……57
附錄 ……76
參考文獻 ……81
自述 ……85
1. Tilkin, A.F., et al. Primary proliferative T cell response to wild-type p53 protein in patients with breast cancer. Eur J Immunol 25, 1765-1769 (1995).
2. Sirvent, J.J., et al. p53 in breast cancer. Its relation to histological grade, lymph-node status, hormone receptors, cell-proliferation fraction (ki-67) and c-erbB-2. Immunohistochemical study of 153 cases. Histol Histopathol 10, 531-539 (1995).
3. Zwijsen, R.M., et al. Cyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit. Mol Cell Biol 16, 2554-2560 (1996).
4. Ruas, M., et al. CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms. Mol Cell Biol 27, 4273-4282 (2007).
5. Reddy, H.K., et al. Cyclin-dependent kinase 4 expression is essential for neu-induced breast tumorigenesis. Cancer Res 65, 10174-10178 (2005).
6. Hui, R., et al. Constitutive overexpression of cyclin D1 but not cyclin E confers acute resistance to antiestrogens in T-47D breast cancer cells. Cancer Res 62, 6916-6923 (2002).
7. Mohamood, A.S., et al. Estrogen receptor, growth factor receptor and protooncogene protein activities and possible signal transduction crosstalk inestrogen dependent and independent breast cancer cell lines. J Submicrosc Cytol Pathol 29, 1-17 (1997).
8. Megha, T., et al. p53 mutation in breast cancer. Correlation with cell kinetics and cell of origin. J Clin Pathol 55, 461-466 (2002).
9. Seitz, S., et al. Identification of microsatellite instability and mismatch repair gene mutations in breast cancer cell lines. Genes Chromosomes Cancer 37, 29-35 (2003).
10. Engebraaten, O. & Fodstad, O. Site-specific experimental metastasis patterns of two human breast cancer cell lines in nude rats. Int J Cancer 82, 219-225 (1999).
11. Bitran, J.D., et al. Her2/neu overexpression is associated with treatment failure in women with high-risk stage II and stage IIIA breast cancer (>10 involved lymph nodes) treated with high-dose chemotherapy and autologous hematopoietic progenitor cell support following standard-dose adjuvant chemotherapy. Clin Cancer Res 2, 1509-1513 (1996).
12. Fong, S., et al. Id-1 as a molecular target in therapy for breast cancer cell invasion and metastasis. Proc Natl Acad Sci U S A 100, 13543-13548 (2003).
13. Liu, X., et al. Difference of cell cycle arrests induced by lidamycin in human breast cancer cells. Anticancer Drugs 17, 173-179 (2006).
14. Caldani, C., Far, D.F., Birtwisle-Peyrottes, I., Ettore, F. & Rostagno, P. Cell cycle expression of p53 protein, c-Myc gene product and tyrosine-phosphorylation level determined by image analysis in human breast cancer cells. Anal Quant Cytol Histol 18, 233-240 (1996).
15. Hlobilkova, A., et al. Tumour suppressor PTEN regulates cell cycle and protein kinase B/Akt pathway in breast cancer cells. Anticancer Res 26, 1015-1022 (2006).
16. Pukrop, T., et al. Wnt 5a signaling is critical for macrophage-induced invasion of breast cancer cell lines. Proc Natl Acad Sci U S A 103, 5454-5459 (2006).
17. Caldon, C.E., Daly, R.J., Sutherland, R.L. & Musgrove, E.A. Cell cycle control in breast cancer cells. J Cell Biochem 97, 261-274 (2006).
18. Runnebaum, I.B., Nagarajan, M., Bowman, M., Soto, D. & Sukumar, S. Mutations in p53 as potential molecular markers for human breast cancer. Proc Natl Acad Sci U S A 88, 10657-10661 (1991).
19. Moll, U.M., Ostermeyer, A.G., Ahomadegbe, J.C., Mathieu, M.C. & Riou, G. p53 mediated tumor cell response to chemotherapeutic DNA damage: a preliminary study in matched pairs of breast cancer biopsies. Hum Pathol 26, 1293-1301(1995).
20. Somasundaram, K. Breast cancer gene 1 (BRCA1): role in cell cycle regulation and DNA repair--perhaps through transcription. J Cell Biochem 88, 1084-1091 (2003).
21. Veeck, J., et al. Epigenetic inactivation of the Secreted frizzled-related protein-5 (SFRP5) gene in human breast cancer is associated with unfavorable prognosis. Carcinogenesis (2008).
22. Sirchia, S.M., et al. Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor beta2 promoter in breast cancer cells. Oncogene 19, 1556-1563 (2000).
23. Steeg, P.S. Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer 3, 55-63 (2003).
24. Koten, J.W. & Den Otter, W. The transition of benign to malignant in epithelial and mesenchymal tumours. Anticancer Res 11, 567-568 (1991).
25. Sukumar, S. & Barbacid, M. Specific patterns of oncogene activation in transplacentally induced tumors. Proc Natl Acad Sci U S A 87, 718-722 (1990).
26. Thompson, E.W., Newgreen, D.F. & Tarin, D. Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition? Cancer Res 65, 5991-5995; discussion 5995 (2005).
27. Thiery, J.P. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2, 442-454 (2002).
28. Liu, Y.N., et al. Regulatory mechanisms controlling human E-cadherin gene expression. Oncogene 24, 8277-8290 (2005).
29. Hennig, G., et al. Progression of carcinoma cells is associated with alterations in chromatin structure and factor binding at the E-cadherin promoter in vivo. Oncogene 11, 475-484 (1995).
30. Thiery, J.P. & Sleeman, J.P. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7, 131-142 (2006).
31. Acloque, H., Thiery, J.P. & Nieto, M.A. The physiology and pathology of the EMT. Meeting on the epithelial-mesenchymal transition. EMBO Rep 9, 322-326 (2008).
32. De Craene, B., et al. The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res 65, 6237-6244 (2005).
33. Hotz, B., et al. Epithelial to mesenchymal transition: expression of the regulators snail, slug, and twist in pancreatic cancer. Clin Cancer Res 13, 4769-4776 (2007).
34. Sarrio, D., et al. Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res 68, 989-997 (2008).
35. Toyoshima, M., et al. Inhibition of tumor growth and metastasis by depletion of vesicular sorting protein Hrs: its regulatory role on E-cadherin and beta-catenin. Cancer Res 67, 5162-5171 (2007).
36. Lee, J.M., Dedhar, S., Kalluri, R. & Thompson, E.W. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 172, 973-981 (2006).
37. Uchiumi, T., Hori, K., Nomura, T. & Hachimori, A. Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding. J Biol Chem 274, 27578-27582 (1999).
38. Barnard, G.F., et al. Increased expression of human ribosomal phosphoprotein P0 messenger RNA in hepatocellular carcinoma and colon carcinoma. Cancer Res 52, 3067-3072 (1992).
39. Kondoh, N., et al. Identification and characterization of genes associated with human hepatocellular carcinogenesis. Cancer Res 59, 4990-4996 (1999).
40. Xia, C., et al. GCIP, a novel human grap2 and cyclin D interacting protein, regulates E2F-mediated transcriptional activity. J Biol Chem 275, 20942-20948 (2000).
41. Li, N., Sokal, I., Bronson, J.D., Palczewski, K. & Baehr, W. Identification of functional regions of guanylate cyclase-activating protein 1 (GCAP1) using GCAP1/GCIP chimeras. Biol Chem 382, 1179-1188 (2001).
42. Ma, W., et al. Expression of GCIP in transgenic mice decreases susceptibility to chemical hepatocarcinogenesis. Oncogene 25, 4207-4216 (2006).
43. Ma, W., et al. GCIP/CCNDBP1, a helix-loop-helix protein, suppresses tumorigenesis. J Cell Biochem 100, 1376-1386 (2007).
44. Sonnenberg-Riethmacher, E., Wustefeld, T., Miehe, M., Trautwein, C. & Riethmacher, D. Maid (GCIP) is involved in cell cycle control of hepatocytes. Hepatology 45, 404-411 (2007).
45. Chang, T.W., et al. Ribosomal phosphoprotein P0 interacts with GCIP and overexpression of P0 is associated with cellular proliferation in breast and liver carcinoma cells. Oncogene 27, 332-338 (2008).
46. Laurance, M.E., et al. Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells. Nucleic Acids Res 29, 652-661 (2001).
47. Christman, J.K. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21, 5483-5495 (2002).
48. 葉彥秀,與GCIP交互作用的蛋白之探討。 國立成功大學生物科技研究所碩士論文,2003。
49. 鄭嘉華,探討GCIP蛋白在人類惡性乳癌細胞MDA-MB-231中所扮演的角色。 國立成功大學生物科技研究所碩士論文,2006。
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