臺灣博碩士論文加值系統

Tazarotene-induced gene 1 (TIG1) 又稱為 retinoic acid receptor responder gene 1 (RARRES1)，可轉譯出含有 2 個異構物之穿膜蛋白，此蛋白含有 1 個 Latexin 功能區，此功能區可能具有抑制羧基胜肽酶 A (carboxypeptidase A) 之活性，過去之研究顯示 TIG1 蛋白具有抑制前列腺癌細胞侵襲及生長之活性。TIG1 在許多正常組織中皆有表現，但在癌細胞中卻經常呈現低量表現或不表現。此基因之啟動區含有 CpG 島，但在癌細胞中 TIG1 基因之啟動區卻常呈現高度甲基化，所以基因甲基化在抑制 TIG1 之表現上扮演ㄧ個重要角色。本實驗室先前之研究結果顯示原位大腸直腸腺癌中 TIG1 蛋白常呈現低量或不表現，因此本研究進ㄧ步探討大腸癌細胞是否經由啟動區高度甲基化來調節 TIG1 之活化與表現。CC-M2 和 HCT116 大腸癌細胞之 TIG1 啟動區為高度甲基化，首先分析去甲基藥物 5-aza-2’-deoxycytidine (5-aza-dC)、all trans retinoic acid (atRA) 或 Trichostatin (TSA) 對 CC-M2 和 HCT116 大腸癌細胞生長之影響，經由 WST-1 或 MTT 方法證明單獨以 5-aza-dC 或 TSA 處理會抑制細胞生長，而兩者合併有加乘之抑制作用，然而 atRA 對細胞生長則無明顯抑制作用。此外，進一步以即時定量聚合酶連鎖反應，發現 5-aza-dC (30 μM) 之單獨處理 3 天，可誘導 TIG1 和 RARβ2 mRNA 之表現達 2 ~ 3 倍，合併 atRA (10 μM) 之處理只對 RARβ2 表現具有協同促進作用 (synergistic effect)。單獨以 5-aza-dC 處理可降低 CC-M2 細胞中 TIG1 啟動區之甲基化程度，而 5-aza-dC 和 TSA (100 or 165 nM) 之合併處理可同時降低 2 株大腸癌細胞啟動區之甲基化程度。後續分析 12 例大腸直腸腺癌組織之 TIG1 啟動區甲基化與蛋白表現之相關性，結果發現在 4 例無 TIG1 表現之癌組織中有3 例組織其 TIG1 啟動區呈現高度甲基化，然而在 8 例有TIG1 蛋白表現之組織中仍有 5 例其啟動區呈現高度甲基化。最後，自 HCT116 細胞中已成功建立誘導 TIG1A 和 TIG1B 表現之單源細胞株，未來將利用此單株細胞分析 TIG1A 和 TIG1B 蛋白之生物活性。總結，大腸癌細胞中 TIG1 啟動區之高度甲基化可能在抑制 TIG1 表現上扮演ㄧ個重要角色，而 TIG1 之表現可經由 5-aza-dC 處理以抑制其基因甲基化而達到 TIG1 表現之活化效果，TIG1 之增加表現是否直接與參與 5-aza-dC 對細胞之生長抑制則需未來進ㄧ步之研究。

Tazarotene-induced gene 1 (TIG1), also known as retinoic acid receptor responder gene 1 (RARRES1), encodes transmembrane proteins with two isoforms. The proteins contain a latexin domain that may function as the carboxypeptidase A inhibitor. TIG1 protein exhibits activities to suppress invasion and growth of prostate cancer cells. The TIG1 is expressed in several normal tissues, and its expression is frequently down-regulated in cancer cells. The TIG1 promoter contains CpG island that is frequently hypermethylated in cancer cells, and the methylation plays an important role in TIG1 silencing. Previous results from our laboratory demonstrated the down-regulation of TIG1 expression in primary colorectal adenocarcinomas. This study investigated expression and activities of TIG1 as well as its regulation by promoter hypermethylation in colon cancer cells. Role of TIG1 promoter hypermethylation on TIG1 expression in HCT116 and CC-M2 colon cancer cells treated with 5-aza-deoxycytidine (5-aza-dC), trichostatin A (TSA) or all-trans retinoic acid (atRA) was first analyzed. The compound 5-aza-dC (30 μM) or TSA (100 or 165 nM) alone inhibited cell growth, and the combination led to an additive effect on cell growth, determined by the WST-1 or MTT assay. However, atRA (10 μM) had no effect on cell growth mediated by 5-aza-dC or TSA . Treatment with 5-aza-dC (30 μM) for 3 days induced TIG1 and RARβ2 mRNA expression by 2- to 3- fold, and the addition of atRA had synergistic effect only for RARβ2 expression, determined by real-time RT-PCR. The 5-aza-dC decreased TIG1 promoter hypermethylation only in CC-M2 cells, and the effect was enhanced by combination with 5-aza-dC and TSA in CC-M2 and HCT116 cells. TIG1 promoter hypermethylation and protein expression from 12 primary colorectal adenocarcinomas were then analyzed. Among 4 tumors without TIG1 expression, these (75%) had hypermethylation at the TIG1 promoter, in contrast to 5 out of 8 (60%) tumors that were stained positive for TIG1 protein. Finally, inducible TIG1-A or TIG1-B stable clones have been established using the GeneSwitch system. Activities of TIG1-A and TIG1-B protein will be investigated further. In conclusion, TIG1 promoter hypermethylation may play an important role in TIG1 silencing in colon cancer cells. That 5-aza-dC suppressed TIG1 methylation and inhibited cell growth correlated with induction of TIG1 expression.

第一章 緒論 1
一、抑癌基因異常表現之機轉 1
二、維甲酸化合物之作用機轉與調控基因之功能 4
三、TIG1 6
四、本論文研究方向 9
第二章 材料與方法 10
ㄧ、細胞培養 10
二、TSA及5-aza-dC之細胞毒性分析 11
三、細胞中全核醣核酸 (total RNA) 的萃取 12
四、互補去氧核醣核酸 (cDNA) 的製備 12
五、即時定量聚合酶連鎖反應 (real-time quantitative polymerase
chain reaction, QPCR) 13
六、細胞存活試驗分析 14
七、細胞核去氧核醣核酸 (Genomic DNA extraction) 之純化 16
八、Genomic DNA 之 Sodium Bisulfite 處理 17
九、甲基化專一性聚合酶連鎖反應 (Methylation specific PCR,
MSP) 19
十、免疫組織學染色 (immunohistochemical staining) 20
十一、臘塊包埋之大腸癌組織 DNA 之萃取 21
十二、TIG1 表現載體之構築 21
十三、大量質體製備及純化 22
十四、轉染作用 (Transfection) 及誘導性 TIG1 單源細胞株之建立 23
十五、蛋白質之萃取 25
十六、蛋白質濃度測定 26
十七、蛋白質膠體電泳 (SDS-PAGE) 分析 26
十八、西方墨點法 (Western blot) 27
第三章 結果 29
ㄧ、人類大腸癌細胞之 TIG1 基因表現及甲基化之分析 29
二、5-aza-dC 及 TSA 對大腸癌細胞株生長毒性之分析 30
三、去甲基藥物 5-aza-dC 及 乙醯化抑制劑 TSA 對大腸癌細胞株
中TIG1 及 RARβ2 基因表現的影響 31
四、5-aza-dC、TSA 及 atRA 對大腸癌細胞株生長之分析 31
五、5-aza-dC、TSA 及 atRA 對大腸癌細胞株 mRNA 表現之分析 32
六、atRA 對 TIG1 啟動區為非甲基化之 HT-29 大腸癌細胞之生
長及 mRNA 表現之分析 33
七、大腸癌組織中 TIG1 表現量與 TIG1 基因啟動區甲基化之分析 33
八、誘導性 TIG1A 及 TIG1B 融合蛋白表現之確認 34
九、誘導性 TIGA 及 TIG1B 單源細胞株之建立與確認 35
十、利用誘導性 TIG1 轉殖單株分析 TIG1A 及 TIG1B 之分泌
及對細胞生長的影響 36
第四章 討論 38
第五章 參考文獻 42

Aagaard, A., Listwan, P., Cowieson, N., Huber, T., Ravasi, T., Wells, C. A., Flanagan, J. U., Kellie, S., Hume, D. A., Kobe, B., and Martin, J. L. (2005). An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: relationship to cystatins and the tumor suppressor TIG1. Structure 13, 309-317.

Barlow, D. P. (1995). Gametic imprinting in mammals. Science 270, 1610-1613.

Bastien, J., and Rochette-Egly, C. (2004). Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene 328, 1-16.

Baylin, S. B., and Herman, J. G. (2000). DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 16, 168-174.

Berger, S. L. (2002). Histone modifications in transcriptional regulation. Curr Opin Genet Dev 12, 142-148.

Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev 16, 6-21.

Bird, A. P., and Wolffe, A. P. (1999). Methylation-induced repression--belts, braces, and chromatin. Cell 99, 451-454.

Chambon, P. (1996). A decade of molecular biology of retinoic acid receptors. Faseb J 10, 940-954.

Denissenko, M. F., Chen, J. X., Tang, M. S., and Pfeifer, G. P. (1997). Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc Natl Acad Sci U S A 94, 3893-3898.

Dilworth, F. J., and Chambon, P. (2001). Nuclear receptors coordinate the activities of chromatin remodeling complexes and coactivators to facilitate initiation of transcription. Oncogene 20, 3047-3054.

Douer, D., and Koeffler, H. P. (1982a). Retinoic acid enhances growth of human early erythroid progenitor cells in vitro. J Clin Invest 69, 1039-1041.
Douer, D., and Koeffler, H. P. (1982b). Retinoic acid. Inhibition of the clonal growth of human myeloid leukemia cells. J Clin Invest 69, 277-283.

Fischle, W., Wang, Y., and Allis, C. D. (2003). Histone and chromatin cross-talk. Curr Opin Cell Biol 15, 172-183.

Giguere, V. (1994). Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr Rev 15, 61-79.

Glass, C. K., and Rosenfeld, M. G. (2000). The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14, 121-141.

Goto, T., and Monk, M. (1998). Regulation of X-chromosome inactivation in development in mice and humans. Microbiol Mol Biol Rev 62, 362-378.

Hake, S. B., Xiao, A., and Allis, C. D. (2004). Linking the epigenetic 'language' of covalent histone modifications to cancer. Br J Cancer 90, 761-769.

Herman, J. G., and Baylin, S. B. (2003). Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349, 2042-2054.

Jing, C., El-Ghany, M. A., Beesley, C., Foster, C. S., Rudland, P. S., Smith, P., and Ke, Y. (2002). Tazarotene-induced gene 1 (TIG1) expression in prostate carcinomas and its relationship to tumorigenicity. J Natl Cancer Inst 94, 482-490.

Jones, P. A., and Baylin, S. B. (2002). The fundamental role of epigenetic events in cancer. Nat Rev Genet 3, 415-428.

Kang, G. H., Shim, Y. H., Jung, H. Y., Kim, W. H., Ro, J. Y., and Rhyu, M. G. (2001). CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res 61, 2847-2851.

Kouzarides, T. (2002). Histone methylation in transcriptional control. Curr Opin Genet Dev 12, 198-209.

Kwong, J., Lo, K. W., Chow, L. S., Chan, F. L., To, K. F., and Huang, D. P. (2005). Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma. Int J Cancer 113, 386-392.
Lotan, R., Lotan, D., and Sacks, P. G. (1990). Inhibition of tumor cell growth by retinoids. Methods Enzymol 190, 100-110.

McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999). Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20, 321-344.

Mizuiri, H., Yoshida, K., Toge, T., Oue, N., Aung, P. P., Noguchi, T., and Yasui, W. (2005). DNA methylation of genes linked to retinoid signaling in squamous cell carcinoma of the esophagus: DNA methylation of CRBP1 and TIG1 is associated with tumor stage. Cancer Sci 96, 571-577.

Morriss-Kay, G. (1992). Retinoic acid receptors in normal growth and development. Cancer Surv 14, 181-193.

Nagpal, S., Patel, S., Asano, A. T., Johnson, A. T., Duvic, M., and Chandraratna, R. A. (1996). Tazarotene-induced gene 1 (TIG1), a novel retinoic acid receptor-responsive gene in skin. J Invest Dermatol 106, 269-274.

Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997). Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373-380.

Nagy, L., Thomazy, V. A., Heyman, R. A., and Davies, P. J. (1998). Retinoid-induced apoptosis in normal and neoplastic tissues. Cell Death Differ 5, 11-19.

Nakao, M. (2001). Epigenetics: interaction of DNA methylation and chromatin. Gene 278, 25-31.

Ng, H. H., and Bird, A. (1999). DNA methylation and chromatin modification. Curr Opin Genet Dev 9, 158-163.

Pallares, I., Bonet, R., Garcia-Castellanos, R., Ventura, S., Aviles, F. X., Vendrell, J., and Gomis-Ruth, F. X. (2005). Structure of human carboxypeptidase A4 with its endogenous protein inhibitor, latexin. Proc Natl Acad Sci U S A 102, 3978-3983.

Takai, N., Kawamata, N., Walsh, C. S., Gery, S., Desmond, J. C., Whittaker, S., Said, J. W., Popoviciu, L. M., Jones, P. A., Miyakawa, I., and Koeffler, H. P. (2005). Discovery of epigenetically masked tumor suppressor genes in endometrial cancer. Mol Cancer Res 3, 261-269.

Ueki, T., Walter, K. M., Skinner, H., Jaffee, E., Hruban, R. H., and Goggins, M. (2002). Aberrant CpG island methylation in cancer cell lines arises in the primary cancers from which they were derived. Oncogene 21, 2114-2117.

Wu, C. C., Shyu, R. Y., Chou, J. M., Jao, S. W., Chao, P. C., Kang, J. C., Wu, S. T., Huang, S. L., and Jiang, S. Y. (2006). RARRES1 expression is significantly related to tumour differentiation and staging in colorectal adenocarcinoma. Eur J Cancer 42, 557-565.

Youssef, E. M., Chen, X. Q., Higuchi, E., Kondo, Y., Garcia-Manero, G., Lotan, R., and Issa, J. P. (2004). Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers. Cancer Res 64, 2411-2417.

Zhang, J., Liu, L., and Pfeifer, G. P. (2004). Methylation of the retinoid response gene TIG1 in prostate cancer correlates with methylation of the retinoic acid receptor beta gene. Oncogene 23, 2241-2249.

Zirn, B., Samans, B., Spangenberg, C., Graf, N., Eilers, M., and Gessler, M. (2005). All-trans retinoic acid treatment of Wilms tumor cells reverses expression of genes associated with high risk and relapse in vivo. Oncogene 24, 5246-5251.

鄭智勇 (1988) 腫瘤標幟在國人結腸直腸腫瘤表現之研究, 國防醫學院醫學科學研究所博士論文