中文摘要
肝癌是台灣最盛行的癌症之一，而肝癌的發展，被認為是多重步驟，關連到基因的變化，例如：致癌基因的活化和腫瘤抑制基因的失活。腫瘤抑制基因p53失活在肝癌發生率約40~50%，並減少肝癌患者術後存活率。當p53失活時會造成染色體的不穩定並誘導下游的目標基因，包括14-3-3σ停止細胞週期進行或PUMA基因調控凋亡之反應。本論文中，利用5株人類肝癌細胞株與10對帶正常和腫瘤配對組織之肝癌檢體來探討14-3-3σ與PUMA在肝癌之表現。
反轉錄聚合酵素連鎖反應定量分析發現14-3-3σmRNA在分化良好或分化不良之肝癌細胞株中均有表現，惟在Mahlavu細胞株幾乎無法偵測14-3-3σmRNA。 西方墨點法分析14-3-3σ蛋白質表現亦証實Mahlavu細胞缺乏14-3-3σ蛋白質表現。反轉錄聚合酵素連鎖反應定量分析肝癌組織發現14-3-3σmRNA在90%肝癌檢体表現量上升。 西方墨點法分析則發現60% 肝癌組織之14-3-3σ蛋白質表現量上升。 免疫組織染色分析發現50% 肝癌組織有14-3-3σ蛋白質表現量上升現象。綜合以上結果，14-3-3σ在肝癌有過度表現之現象。
反轉錄聚合酵素連鎖反應定量和西方墨點法分析PUMA mRNA和蛋白質表現在人類和老鼠肝癌細胞株均為下降現象。反轉錄聚合酵素連鎖反應定量分析發現60% 肝癌組織有PUMA mRNA表現量減少。 西方墨點法分析指出100%肝癌組織有PUMA蛋白質表現減少。免疫組織染色分析70% 肝癌組織PUMA蛋白質表現減少。 綜合以上結果，PUMA在肝癌表現有下降之現象。未來需大規模檢體研究以進一步釐清14-3-3σ/PUMA表現與肝癌表現臨床因子之關聯。

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Taiwan. The development of hepatocellular carcinoma is a multi-step process associated with alterations in genes expression such as activation of oncogenes and inactivation of tumor suppressor genes. Mutation/deletion of tumor suppressor gene p53 occurs in 40-50% HCC. Moreover, patients with p53 inactivation have significantly shorter survival after surgery. Inactivation of p53 leads to chromosome instability and may alter expression of its downstream target genes including 14-3-3s for cell cycle arrest or PUMA for apoptosis induction. In this thesis study, we employed five human hepatoma cell lines and ten surgical HCC samples containing paired normal and tumor tissues to investigate 14-3-3s and PUMA expression during liver carcinogenesis.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that 14-3-3s mRNA expression was detected in well and poorly differentiated hepatoma cells except Mahlavu cells. Western blot analysis further validated such finding that 14-3-3s protein is not detectable in Mahlavu cell. In human surgical HCC tissues, qRT-PCR showed that 14-3-3s mRNA was elevated in 90% of HCC tissues. Western blot analysis indicated that 14-3-3s protein level was increased in 60% of HCC tissues. Finally, immunohistochemical analysis revealed that 14-3-3s was up-regulated in 50% of HCC tissues comparing with their adjacent non-tumor tissues. Together, these results indicated that 14-3-3s expression was up-regulated in HCC.
qRT-PCR and western blot analysis indicated that PUMA mRNA and protein levels were decreased in human and rat hepatoma cells. In human surgical HCC tissues, qRT-PCR showed that PUMA mRNA was reduced in 60% of HCC tissues. Western blot analysis indicated that PUMA protein level was decreased in 100 of HCC tissues. Finally, immunohistochemical analysis revealed that PUMA was down-regulated in 70% of HCC tissues comparing with their adjacent non-tumor tissues. Together, these results indicated that PUMA expression was down-regulated in HCC.
In the future, large-scale analysis using more HCC samples will be required to delineate the correlation of 14-3-3s/PUMA expression with clinical parameters of HCC.

目錄

緒論
肝癌的發………………………………………………………….…….1
肝癌形成的分子機…………………………………………….…....….1
p53………………………………………………………………..…..2
14-3-3σ……………………………………………………..….……3
14-3-3σ蛋白質在細胞週期的角色…………………………...……4
14-3-3σ的外因性的靜止……………………………………...……5
PUMA……………………………………………………….….……7
實驗目的……………………………………………………….………9
材料與方法
人類肝細胞株和老鼠細胞株培養…………………….…………10
人類肝癌檢体………………………………………….…………10
Total RNA的萃取…………………………………….………….10
反轉錄聚合酵素連鎖反應定量分析………………….…………11
西方墨點法……………………………………….………………13
免疫組織染色法…………………………….……………………14
結果
14-3-3σ於肝癌之表現分析……………………………...…………16
PUMA於肝癌之表現分析………………………………..………..16
討論
14-3-3σ於肝癌之表現分析…………………………………………..18
PUMA於肝癌之表現分析……………………………………………20
參考資料…………………………………………………………..…..49






























圖表目錄
圖1：肝癌的病理機轉………………………………………………….22
圖2：p53下游目標基因的功能…………………………………..……23
圖3：14-3-3蛋白質的功能概要…………………………………..……24
圖4：14-3-3σ蛋白質在細胞週期控制的角色……………………..….25
圖5：14-3-3σpromoter CpG island 被不正常甲基化，使14-3-3σ的mRNA的表現下降………………………………………………..…..26
圖6：PUMA之基因結構與蛋白序列……………………………….…27
圖7：p53誘導粒腺體凋亡路徑，基因的概要式圖形…………….….28
圖8：qRT-PCR分析肝癌細胞株14-3-3σmRNA的表現量…………29
圖9：肝癌細胞株中14-3-3σ蛋白質的表現量…………………….…30
圖10：qRT-PCR分析肝癌組織中14-3-3σmRNA的表現量………..31
圖11：在10組正常-腫瘤配對肝癌組織中14-3-3σ蛋白質的表現量.32
圖12：人類肝癌檢體14-3-3σ蛋白免疫組織化學染色……………..33
圖13：人類肝癌檢體14-3-3σ蛋白免疫組織化學染色……………..34
圖14：人類肝癌檢體14-3-3σ蛋白免疫組織化學染色……………..35
圖15：PUMA在老鼠肝癌細胞中蛋白質的表現量………………….36
圖16：qRT-PCR分析肝癌細胞中PUMAmRNA的表現量…………37
圖17：在肝癌細胞中PUMA蛋白質的表現量………………………38
圖18：在肝癌組織中PUMAmRNA的表現量………………………39
圖19：在10組肝癌組織中PUMA蛋白質的表現量………………..40
圖20：人類肝癌檢體PUMA蛋白免疫組織化學染色………………41
圖21：人類肝癌檢體PUMA蛋白免疫組織化學染色………………42
表1：在各類腫瘤中14-3-3σ啟動子CpG甲基化發生頻率………..43
表2：Summary of 14-3-3σexpression in human cancer………………44
表3：14-3-3σ在肝癌檢體免疫組織染色結果………………………..45.
表4：PUMA在肝癌檢體免疫組織染色結果…………………………46
表5：14-3-3σ分別以qRT-PCR，西方墨點法和免疫組織染色法進行組織檢體分析比較……………………………………………………47
表6：PUMA分別以qRT-PCR，西方墨點法和免疫組織染色法進行組織檢體分析比較………………………………..……………………..48

REFERENCES
Alvarez, D., Novac, O., Callejo, M., Ruiz, M. T., Price, G. B., and Zannis-Hadjopoulos, M. (2002). 14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication. J Cell Biochem 87, 194-207.
Aprelikova, O., Pace, A. J., Fang, B., Koller, B. H., and Liu, E. T. (2001). BRCA1 is a selective co-activator of 14-3-3 sigma gene transcription in mouse embryonic stem cells. J Biol Chem 276, 25647-25650.
Bargonetti, J., and Manfredi, J. J. (2002). Multiple roles of the tumor suppressor p53. Curr Opin Oncol 14, 86-91.
Bhatia, K., Siraj, A. K., Hussain, A., Bu, R., and Gutierrez, M. I. (2003). The tumor suppressor gene 14-3-3 sigma is commonly methylated in normal and malignant lymphoid cells. Cancer Epidemiol Biomarkers Prev 12, 165-169.
Chan, T. A., Hermeking, H., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1999). 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature 401, 616-620.
Coleman, W. B. (2003). Mechanisms of human hepatocarcinogenesis. Curr Mol Med 3, 573-588.
Conklin, D. S., Galaktionov, K., and Beach, D. (1995). 14-3-3 proteins associate with cdc25 phosphatases. Proc Natl Acad Sci U S A 92, 7892-7896.
Dhar, S., Squire, J. A., Hande, M. P., Wellinger, R. J., and Pandita, T. K. (2000). Inactivation of 14-3-3sigma influences telomere behavior and ionizing radiation-induced chromosomal instability. Mol Cell Biol 20, 7764-7772.
Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr., Butel, J. S., and Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215-221.
Fantl, W. J., Muslin, A. J., Kikuchi, A., Martin, J. A., MacNicol, A. M., Gross, R. W., and Williams, L. T. (1994). Activation of Raf-1 by 14-3-3 proteins. Nature 371, 612-614.
Feitelson, M. A., Sun, B., Satiroglu Tufan, N. L., Liu, J., Pan, J., and Lian, Z. (2002). Genetic mechanisms of hepatocarcinogenesis. Oncogene 21, 2593-2604.
Ferguson, A. T., Evron, E., Umbricht, C. B., Pandita, T. K., Chan, T. A., Hermeking, H., Marks, J. R., Lambers, A. R., Futreal, P. A., Stampfer, M. R., and Sukumar, S. (2000). High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer. Proc Natl Acad Sci U S A 97, 6049-6054.
Gasco, M., Bell, A. K., Heath, V., Sullivan, A., Smith, P., Hiller, L., Yulug, I., Numico, G., Merlano, M., Farrell, P. J., et al. (2002a). Epigenetic inactivation of 14-3-3 sigma in oral carcinoma: association with p16(INK4a) silencing and human papillomavirus negativity. Cancer Res 62, 2072-2076.
Gasco, M., Sullivan, A., Repellin, C., Brooks, L., Farrell, P. J., Tidy, J. A., Dunne, B., Gusterson, B., Evans, D. J., and Crook, T. (2002b). Coincident inactivation of 14-3-3sigma and p16INK4a is an early event in vulval squamous neoplasia. Oncogene 21, 1876-1881.
Han, J., Flemington, C., Houghton, A. B., Gu, Z., Zambetti, G. P., Lutz, R. J., Zhu, L., and Chittenden, T. (2001). Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals. Proc Natl Acad Sci U S A 98, 11318-11323.
Hemann, M. T., Zilfou, J. T., Zhao, Z., Burgess, D. J., Hannon, G. J., and Lowe, S. W. (2004). Suppression of tumorigenesis by the p53 target PUMA. Proc Natl Acad Sci U S A 101, 9333-9338.
Hermeking, H. (2003). The 14-3-3 cancer connection. Nat Rev Cancer 3, 931-943.
Hermeking, H., Lengauer, C., Polyak, K., He, T. C., Zhang, L., Thiagalingam, S., Kinzler, K. W., and Vogelstein, B. (1997). 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell 1, 3-11.
Hoque, M. O., Begum, S., Sommer, M., Lee, T., Trink, B., Ratovitski, E., and Sidransky, D. (2003). PUMA in head and neck cancer. Cancer Lett 199, 75-81.
Hu, T. H., Huang, C. C., Lin, P. R., Chang, H. W., Ger, L. P., Lin, Y. W., Changchien, C. S., Lee, C. M., and Tai, M. H. (2003). Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 97, 1929-1940.
Ito, Y., Miyoshi, E., Uda, E., Yoshida, H., Uruno, T., Takamura, Y., Miya, A., Kobayashi, K., Matsuzuka, F., Matsuura, N., et al. (2003). 14-3-3 sigma possibly plays a constitutive role in papillary carcinoma, but not in follicular tumor of the thyroid. Cancer Lett 196, 87-91.
Iwata, N., Yamamoto, H., Sasaki, S., Itoh, F., Suzuki, H., Kikuchi, T., Kaneto, H., Iku, S., Ozeki, I., Karino, Y., et al. (2000). Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma. Oncogene 19, 5298-5302.
Jansson, A., Arbman, G., and Sun, X. F. (2004). mRNA and protein expression of PUMA in sporadic colorectal cancer. Oncol Rep 12, 1245-1249.
Kaneuchi, M., Sasaki, M., Tanaka, Y., Shiina, H., Verma, M., Ebina, Y., Nomura, E., Yamamoto, R., Sakuragi, N., and Dahiya, R. (2004). Expression and methylation status of 14-3-3 sigma gene can characterize the different histological features of ovarian cancer. Biochem Biophys Res Commun 316, 1156-1162.
Kensler, T. W., Qian, G. S., Chen, J. G., and Groopman, J. D. (2003). Translational strategies for cancer prevention in liver. Nat Rev Cancer 3, 321-329.
Kopnin, B. P. (2000). Targets of oncogenes and tumor suppressors: key for understanding basic mechanisms of carcinogenesis. Biochemistry (Mosc) 65, 2-27.
Li, C. Q., Robles, A. I., Hanigan, C. L., Hofseth, L. J., Trudel, L. J., Harris, C. C., and Wogan, G. N. (2004). Apoptotic signaling pathways induced by nitric oxide in human lymphoblastoid cells expressing wild-type or mutant p53. Cancer Res 64, 3022-3029.
Ling, G., Ahmadian, A., Persson, A., Unden, A. B., Afink, G., Williams, C., Uhlen, M., Toftgard, R., Lundeberg, J., and Ponten, F. (2001). PATCHED and p53 gene alterations in sporadic and hereditary basal cell cancer. Oncogene 20, 7770-7778.
Liu, F. T., Newland, A. C., and Jia, L. (2003). Bax conformational change is a crucial step for PUMA-mediated apoptosis in human leukemia. Biochem Biophys Res Commun 310, 956-962.
Liu, T. Z., Hu, C. C., Chen, Y. H., Stern, A., and Cheng, J. T. (2000). Differentiation status modulates transcription factor NF-kappaB activity in unstimulated human hepatocellular carcinoma cell lines. Cancer Lett 151, 49-56.
Lodygin, D., Yazdi, A. S., Sander, C. A., Herzinger, T., and Hermeking, H. (2003). Analysis of 14-3-3sigma expression in hyperproliferative skin diseases reveals selective loss associated with CpG-methylation in basal cell carcinoma. Oncogene 22, 5519-5524.
Logsdon, C. D., Simeone, D. M., Binkley, C., Arumugam, T., Greenson, J. K., Giordano, T. J., Misek, D. E., Kuick, R., and Hanash, S. (2003). Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer. Cancer Res 63, 2649-2657.
Miyashita, T., and Reed, J. C. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293-299.
Moreira, J. M., Gromov, P., and Celis, J. E. (2004). Expression of the tumor suppressor protein 14-3-3 sigma is down-regulated in invasive transitional cell carcinomas of the urinary bladder undergoing epithelial-to-mesenchymal transition. Mol Cell Proteomics 3, 410-419.
Nakajima, T., Shimooka, H., Weixa, P., Segawa, A., Motegi, A., Jian, Z., Masuda, N., Ide, M., Sano, T., Oyama, T., et al. (2003). Immunohistochemical demonstration of 14-3-3 sigma protein in normal human tissues and lung cancers, and the preponderance of its strong expression in epithelial cells of squamous cell lineage. Pathol Int 53, 353-360.
Osada, H., Tatematsu, Y., Yatabe, Y., Nakagawa, T., Konishi, H., Harano, T., Tezel, E., Takada, M., and Takahashi, T. (2002). Frequent and histological type-specific inactivation of 14-3-3sigma in human lung cancers. Oncogene 21, 2418-2424.
Parkin, D. M., Pisani, P., and Ferlay, J. (1993). Estimates of the worldwide incidence of eighteen major cancers in 1985. Int J Cancer 54, 594-606.
Rozenfeld-Granot, G., Krishnamurthy, J., Kannan, K., Toren, A., Amariglio, N., Givol, D., and Rechavi, G. (2002). A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1. Oncogene 21, 1469-1476.
Samuel, T., Weber, H. O., Rauch, P., Verdoodt, B., Eppel, J. T., McShea, A., Hermeking, H., and Funk, J. O. (2001). The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax. J Biol Chem 276, 45201-45206.
Sato, N., Maitra, A., Fukushima, N., van Heek, N. T., Matsubayashi, H., Iacobuzio-Donahue, C. A., Rosty, C., and Goggins, M. (2003). Frequent hypomethylation of multiple genes overexpressed in pancreatic ductal adenocarcinoma. Cancer Res 63, 4158-4166.
Uchida, D., Begum, N. M., Almofti, A., Kawamata, H., Yoshida, H., and Sato, M. (2004). Frequent downregulation of 14-3-3 sigma protein and hypermethylation of 14-3-3 sigma gene in salivary gland adenoid cystic carcinoma. Br J Cancer 91, 1131-1138.
Umbricht, C. B., Evron, E., Gabrielson, E., Ferguson, A., Marks, J., and Sukumar, S. (2001). Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer. Oncogene 20, 3348-3353.
Urano, T., Saito, T., Tsukui, T., Fujita, M., Hosoi, T., Muramatsu, M., Ouchi, Y., and Inoue, S. (2002). Efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth. Nature 417, 871-875.
Urano, T., Takahashi, S., Suzuki, T., Fujimura, T., Fujita, M., Kumagai, J., Horie-Inoue, K., Sasano, H., Kitamura, T., Ouchi, Y., and Inoue, S. (2004). 14-3-3sigma is down-regulated in human prostate cancer. Biochem Biophys Res Commun 319, 795-800.
van Hemert, M. J., Steensma, H. Y., and van Heusden, G. P. (2001). 14-3-3 proteins: key regulators of cell division, signalling and apoptosis. Bioessays 23, 936-946.
Villunger, A., Michalak, E. M., Coultas, L., Mullauer, F., Bock, G., Ausserlechner, M. J., Adams, J. M., and Strasser, A. (2003). p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 302, 1036-1038.
Vogelstein, B., Lane, D., and Levine, A. J. (2000). Surfing the p53 network. Nature 408, 307-310.
Vousden, K. H. (2000). p53: death star. Cell 103, 691-694.
Westfall, M. D., Mays, D. J., Sniezek, J. C., and Pietenpol, J. A. (2003). The Delta Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations. Mol Cell Biol 23, 2264-2276.
Wilker, E., and Yaffe, M. B. (2004). 14-3-3 Proteins--a focus on cancer and human disease. J Mol Cell Cardiol 37, 633-642.
Yoshida, K., Monden, M., Nakamura, Y., and Arakawa, H. (2004). Adenovirus-mediated p53AIP1 gene transfer as a new strategy for treatment of p53-resistant tumors. Cancer Sci 95, 91-97.
Yu, J., Wang, Z., Kinzler, K. W., Vogelstein, B., and Zhang, L. (2003). PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci U S A 100, 1931-1936.
Yu, J., and Zhang, L. (2003). No PUMA, no death: implications for p53-dependent apoptosis. Cancer Cell 4, 248-249.
Yu, J., Zhang, L., Hwang, P. M., Kinzler, K. W., and Vogelstein, B. (2001). PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell 7, 673-682.
Zhang, Y., Karas, M., Zhao, H., Yakar, S., and LeRoith, D. (2004). 14-3-3sigma mediation of cell cycle progression is p53-independent in response to insulin-like growth factor-I receptor activation. J Biol Chem 279, 34353-34360.
Zhu, J., Jiang, J., Zhou, W., and Chen, X. (1998). The potential tumor suppressor p73 differentially regulates cellular p53 target genes. Cancer Res 58, 5061-5065.