臺灣博碩士論文加值系統

由前人的研究中發現：在各種不同細胞中的tumor suppressor and tumor –related genes經過不正常的epigenetic modification後易趨向形成癌細胞。DNA methylation為最主要的epigenetic modification，它在調控基因表現中扮演一很重要的角色。當一基因的啟動子區的CpG island有高度甲基化時， methyl-CpG binding protein(MBDs)與其它使基因不表現的蛋白(ex：HDAC)所形成complex會結合於5-methylcytosine上使DNA與nucleosome纏繞緊密並在結構空間上阻擋了transcription factor的結合造成基因的不表現 。許多證據指出MBD2基因的表現量與tumorgenesis相關，並發現MBD2基因的表現為癌症發展過程中所必需。文獻指出攝護癌細胞中GSTP1、RASSF1A、RAR2、COX-2的基因表現主要受到DNA methylation所控制，因此我假設MBD2會參與此DNA methylation的機制來調控GSTP1、RASSF1A、RAR2、COX-2基因表現。利用si-RNA技術抑制MBD2的表現，反向觀察MBD2與GSTP1、RASSF1A、RAR2、COX-2基因表現之間的關係。結果顯示，MBD2會影響GSTP1、RAR2的表現；當細胞中MBD2表現量減少時，GSTP1、RAR2的表現量有回復的情形。因此，推測MBD2有參與調控GSTP1、RAR2經由DNA的 methylation 調控基因表現的機制。

Abnormal epigenetic modification of tumor suppressor and tumorgenesis related genes may contribute to the process of cancer formation. DNA methylation is one of the major epigenetic modification, and it plays an important role to regulate gene expression. Aberrant DNA methylation may cause human malignancies, including prostate cancer. Repression of transcription accompanying CpG islands hypermethylation is mediated by methyl-CpG binding domain (MBD) proteins. Previous reports indicated that MBD2 bound to hypermethylated promoter of GSTP1 gene in LNCaP cells. In addition, CpG islands in GSTP1, RASSF1A, COX-2 were reported to be hypermethylated in DU 145 cells. Therefore, in this study I use methyl DNA-binding domain protein 2 antisense oligonucleotide (si-MBD2) to investigate whether the MBD2 participated in the mechanism of hypermethyaltion to repress GSTP1, RASSF1A, RARb2 or to influence COX-2 gene expression in DU 145 cells. Data revealed that MBD2 and MBD3 have higher expression than other MBDs in DU145 cells. Besides, GSTP1, RASSF1A, RARb2 , COX-2 gene expression in different prostate cancer cell lines is lower than that in normal prostate cells. DU 145 cells express lower GSTP1, RASSF1A, RARb2, COX-2 comparing with normal prostate cells. This result is confirm the previous data reported. Using si-MBD2 to repress MBD2 expression, the GSTP1 and RARb2 gene expressions were somehow restored. However, decreasing the expression level of MBD2 does not increase RASSF1A and COX-2 expression. Hence, MBD2 has greater effect on GSTP1 and RARb2 expression than it has on RASSF1A and COX-2 expression.

第一章 背景簡介……………………………………………………...…1
1.1 攝護腺癌的流行病學…………………………………………....1
1.2 攝護腺癌的分期與病徵………………………………………....2
1.3 攝護腺癌的早期診斷…………………………………………....3
1.4 異常epigenetic modification導致癌症的形成…………………3
1.5 methyl CpG binding domain 2(MBD2)在癌症形成中扮演重要角色………………………………………………………………5
1.6 高度甲基化應用於早期診斷攝護腺癌的生物指標……………6
1.7 攝護腺癌的治療方式……………………………………………8
第二章 研究目標……………………………………………………….10
第三章 材料與方法.................................................................................12
3.1 實驗材料………………………………………………………12
3.2 實驗方法………………………………………………………13
第四章 實驗結果……………………………………………………….18
4.1 MBD於不同的人類攝護腺癌細胞株中的表現情形………...18
4.2在不同人類攝護腺細胞株中GSTP1 mRNA表現的差異……18
4.3在不同人類攝護腺細胞株中RASSF1A mRNA表現的差異 ..19
4.4在不同人類攝護腺細胞株中RARβ2 mRNA表現的差異…..19
4.5 在不同人類攝護腺細胞株中COX-2 mRNA表現的差異…...19
4.6 設計MBD2 siRNA抑制MBD2表現……………………...…20
4.7 觀察MBD2 knockdown的DU-145細胞中GSTP1 mRNA的表現情形……………………………………………………...20
4.8 觀察MBD2 knockdown的DU-145細胞中RASSF1A mRNA的表現情形…………………………………………………...21
4.9 觀察MBD2 knockdown的DU-145細胞中RARβ2 mRNA的表現情形……………………………………………………...22
4.10觀察MBD2 knockdown的DU-145細胞中COX-2 mRNA的表現情形……………………………………………………...22
第五章 討論………………………………………………………….…23
5.1 MBD2於人類攝護腺細胞系中的表現……………………….23
5.2 GSTP1在DU-145細胞與si-MBD2 treated DU-145細胞中的表現……………………………………………………………….24
5.3 RASSF1A在DU-145細胞與si-MBD2 treated DU-145細胞中的表現…………………………………………………………….25
5.4 RARβ2在DU-145細胞與si-MBD2 treated DU-145細胞中的表現…………………………………………………….………26
5.5 COX-2在DU-145細胞與si-MBD2 treated DU-145細胞中的表現…………………………………………………...…………..27
5.6 結論……………………………………………………………28
參考文獻…………………………………….…………………………..29
Figure……………………………………………………………………37
Table……………………………………………………………………..50
附圖……………………………………………………………….……..51

Agathanggelou, A., Cooper, W. N., and Latif, F. (2005). Role of the Ras-association domain family 1 tumor suppressor gene in human cancers. Cancer Res 65, 3497-3508.

Arnadottir, M., Laxdal, T., and Halldorsdottir, B. (2005). 2,8-dihydroxyadeninuria: are there no cases in Scandinavia? Scand J Urol Nephrol 39, 82-86.

Bakker, J., Lin, X., and Nelson, W. G. (2002). Methyl-CpG binding domain protein 2 represses transcription from hypermethylated pi-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells. J Biol Chem 277, 22573-22580.

Ballestar, E., Paz, M. F., Valle, L., Wei, S., Fraga, M. F., Espada, J., Cigudosa, J. C., Huang, T. H., and Esteller, M. (2003). Methyl-CpG binding proteins identify novel sites of epigenetic inactivation in human cancer. EMBO J 22, 6335-6345.

Berger, J., and Bird, A. (2005). Role of MBD2 in gene regulation and tumorigenesis. Biochem Soc Trans 33, 1537-1540.

Billard, L. M., Magdinier, F., Lenoir, G. M., Frappart, L., and Dante, R. (2002). MeCP2 and MBD2 expression during normal and pathological growth of the human mammary gland. Oncogene 21, 2704-2712.
Bird, A. P. (1986). CpG-rich islands and the function of DNA methylation. Nature 321, 209-213.

Campbell, P. M., Bovenzi, V., and Szyf, M. (2004). Methylated DNA-binding protein 2 antisense inhibitors suppress tumourigenesis of human cancer cell lines in vitro and in vivo. Carcinogenesis 25, 499-507.
Chen, H., Toyooka, S., Gazdar, A. F., and Hsieh, J. T. (2003). Epigenetic regulation of a novel tumor suppressor gene (hDAB2IP) in prostate cancer cell lines. J Biol Chem 278, 3121-3130.

Costello, J. F., and Plass, C. (2001). Methylation matters. J Med Genet 38, 285-303.

Di Croce, L., Raker, V. A., Corsaro, M., Fazi, F., Fanelli, M., Faretta, M., Fuks, F., Lo Coco, F., Kouzarides, T., Nervi, C., Nervi, C., Minucci, S., Giuseppe Pelicci, P. (2002). Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 295, 1079-1082.

Esteller, M. (2002). CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 21, 5427-5440.

Esteller, M., Corn, P. G., Urena, J. M., Gabrielson, E., Baylin, S. B., and Herman, J. G. (1998). Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res 58, 4515-4518.

Felsenfeld, G., and Groudine, M. (2003). Controlling the double helix. Nature 421, 448-453.

Frankel, S., Smith, G. D., Donovan, J., and Neal, D. (2003). Screening for prostate cancer. Lancet 361, 1122-1128.

Fujita, H., Koshida, K., Keller, E. T., Takahashi, Y., Yoshimito, T., Namiki, M., and Mizokami, A. (2002). Cyclooxygenase-2 promotes prostate cancer progression. Prostate 53, 232-240.

Gambert, S. R. (2001). Screening for prostate cancer. Int Urol Nephrol 33, 249-257.

Gowher, H., Liebert, K., Hermann, A., Xu, G., and Jeltsch, A. (2005). Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L. J Biol Chem 280, 13341-13348.

Gupta, S., Adhami, V. M., Subbarayan, M., MacLennan, G. T., Lewin, J. S., Hafeli, U. O., Fu, P., and Mukhtar, H. (2004). Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model. Cancer Res 64, 3334-3343.

Gupta, S., Srivastava, M., Ahmad, N., Bostwick, D. G., and Mukhtar, H. (2000). Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma. Prostate 42, 73-78.

Hendrich, B., and Bird, A. (1998). Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol 18, 6538-6547.

Jackson-Grusby, L., Laird, P. W., Magge, S. N., Moeller, B. J., and Jaenisch, R. (1997). Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase. Proc Natl Acad Sci U S A 94, 4681-4685.

Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R. C., Ghafoor, A., Feuer, E. J., and Thun, M. J. (2005). Cancer statistics, 2005. CA Cancer J Clin 55, 10-30.

Jemal, A., Siegel, R., Ward, E., Murray, T., Xu, J., Smigal, C., and Thun, M. J. (2006). Cancer statistics, 2006. CA Cancer J Clin 56, 106-130.

Jeronimo, C., Henrique, R., Hoque, M. O., Ribeiro, F. R., Oliveira, J., Fonseca, D., Teixeira, M. R., Lopes, C., and Sidransky, D. (2004). Quantitative RARbeta2 hypermethylation: a promising prostate cancer marker. Clin Cancer Res 10, 4010-4014.

Jones, P. A., and Taylor, S. M. (1980). Cellular differentiation, cytidine analogs and DNA methylation. Cell 20, 85-93.

Juttermann, R., Li, E., and Jaenisch, R. (1994). Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc Natl Acad Sci U S A 91, 11797-11801.

Kawamoto, K., Okino, S. T., Place, R. F., Urakami, S., Hirata, H., Kikuno, N., Kawakami, T., Tanaka, Y., Pookot, D., Chen, Z., Majid, S., Enokida, H., Nakagawa, M., Dahiya, R. (2007). Epigenetic modifications of RASSF1A gene through chromatin remodeling in prostate cancer. Clin Cancer Res 13, 2541-2548.

Kirschenbaum, A., Klausner, A. P., Lee, R., Unger, P., Yao, S., Liu, X. H., and Levine, A. C. (2000). Expression of cyclooxygenase-1 and cyclooxygenase-2 in the human prostate. Urology 56, 671-676.

Le Guezennec, X., Vermeulen, M., Brinkman, A. B., Hoeijmakers, W. A., Cohen, A., Lasonder, E., and Stunnenberg, H. G. (2006). MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties. Mol Cell Biol 26, 843-851.

Lee, M. G., Kim, H. Y., Byun, D. S., Lee, S. J., Lee, C. H., Kim, J. I., Chang, S. G., and Chi, S. G. (2001). Frequent epigenetic inactivation of RASSF1A in human bladder carcinoma. Cancer Res 61, 6688-6692.

Lee, W. H., Morton, R. A., Epstein, J. I., Brooks, J. D., Campbell, P. A., Bova, G. S., Hsieh, W. S., Isaacs, W. B., and Nelson, W. G. (1994). Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci U S A 91, 11733-11737.

Li, L. C., Carroll, P. R., and Dahiya, R. (2005). Epigenetic changes in prostate cancer: implication for diagnosis and treatment. J Natl Cancer Inst 97, 103-115.

Lin, X., Asgari, K., Putzi, M. J., Gage, W. R., Yu, X., Cornblatt, B. S., Kumar, A., Piantadosi, S., DeWeese, T. L., De Marzo, A. M., and Nelson, W. G. (2001). Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide. Cancer Res 61, 8611-8616.

Litvinov, I. V., De Marzo, A. M., and Isaacs, J. T. (2003). Is the Achilles' heel for prostate cancer therapy a gain of function in androgen receptor signaling? J Clin Endocrinol Metab 88, 2972-2982.

Lotan, R., Xu, X. C., Lippman, S. M., Ro, J. Y., Lee, J. S., Lee, J. J., and Hong, W. K. (1995). Suppression of retinoic acid receptor-beta in premalignant oral lesions and its up-regulation by isotretinoin. N Engl J Med 332, 1405-1410.

Lu, Q., Kaplan, M., Ray, D., Ray, D., Zacharek, S., Gutsch, D., and Richardson, B. (2002). Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus. Arthritis Rheum 46, 1282-1291.

Madaan, S., Abel, P. D., Chaudhary, K. S., Hewitt, R., Stott, M. A., Stamp, G. W., and Lalani, E. N. (2000). Cytoplasmic induction and over-expression of cyclooxygenase-2 in human prostate cancer: implications for prevention and treatment. BJU Int 86, 736-741.

Magdinier, F., and Wolffe, A. P. (2001). Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia. Proc Natl Acad Sci U S A 98, 4990-4995.

Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M. K., Warholm, M., and Jornvall, H. (1985). Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc Natl Acad Sci U S A 82, 7202-7206.

Maruyama, R., Toyooka, S., Toyooka, K. O., Virmani, A. K., Zochbauer-Muller, S., Farinas, A. J., Minna, J. D., McConnell, J., Frenkel, E. P., and Gazdar, A. F. (2002). Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features. Clin Cancer Res 8, 514-519.

Millar, D. S., Ow, K. K., Paul, C. L., Russell, P. J., Molloy, P. L., and Clark, S. J. (1999). Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer. Oncogene 18, 1313-1324.

Miyamoto, H., Altuwaijri, S., Cai, Y., Messing, E. M., and Chang, C. (2005). Inhibition of the Akt, cyclooxygenase-2, and matrix metalloproteinase-9 pathways in combination with androgen deprivation therapy: potential therapeutic approaches for prostate cancer. Mol Carcinog 44, 1-10.

Ng, H. H., Zhang, Y., Hendrich, B., Johnson, C. A., Turner, B. M., Erdjument-Bromage, H., Tempst, P., Reinberg, D., and Bird, A. (1999). MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet 23, 58-61.

Okano, M., Xie, S., and Li, E. (1998). Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 19, 219-220.

Perry, A. S., Foley, R., Woodson, K., and Lawler, M. (2006). The emerging roles of DNA methylation in the clinical management of prostate cancer. Endocr Relat Cancer 13, 357-377.

Pruthi, R. S., Derksen, E., and Gaston, K. (2003). Cyclooxygenase-2 as a potential target in the prevention and treatment of genitourinary tumors: a review. J Urol 169, 2352-2359.

Pulukuri, S. M., and Rao, J. S. (2006). CpG island promoter methylation and silencing of 14-3-3sigma gene expression in LNCaP and Tramp-C1 prostate cancer cell lines is associated with methyl-CpG-binding protein MBD2. Oncogene 25, 4559-4572.

Qiu, H., Zhang, W., El-Naggar, A. K., Lippman, S. M., Lin, P., Lotan, R., and Xu, X. C. (1999). Loss of retinoic acid receptor-beta expression is an early event during esophageal carcinogenesis. Am J Pathol 155, 1519-1523.

Saito, M., and Ishikawa, F. (2002). The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2. J Biol Chem 277, 35434-35439.

Sansom, O. J., Berger, J., Bishop, S. M., Hendrich, B., Bird, A., and Clarke, A. R. (2003). Deficiency of Mbd2 suppresses intestinal tumorigenesis. Nat Genet 34, 145-147.

Sato, M., Horio, Y., Sekido, Y., Minna, J. D., Shimokata, K., and Hasegawa, Y. (2002). The expression of DNA methyltransferases and methyl-CpG-binding proteins is not associated with the methylation status of p14(ARF), p16(INK4a) and RASSF1A in human lung cancer cell lines. Oncogene 21, 4822-4829.

Scanlan, M. J., Welt, S., Gordon, C. M., Chen, Y. T., Gure, A. O., Stockert, E., Jungbluth, A. A., Ritter, G., Jager, D., Jager, E., Knuth, A., Old, L.J. (2002). Cancer-related serological recognition of human colon cancer: identification of potential diagnostic and immunotherapeutic targets. Cancer Res 62, 4041-4047.

Shukeir, N., Pakneshan, P., Chen, G., Szyf, M., and Rabbani, S. A. (2006). Alteration of the methylation status of tumor-promoting genes decreases prostate cancer cell invasiveness and tumorigenesis in vitro and in vivo. Cancer Res 66, 9202-9210.

Slack, A., Bovenzi, V., Bigey, P., Ivanov, M. A., Ramchandani, S., Bhattacharya, S., tenOever, B., Lamrihi, B., Scherman, D., and Szyf, M. (2002). Antisense MBD2 gene therapy inhibits tumorigenesis. J Gene Med 4, 381-389.

Song, J. Z., Stirzaker, C., Harrison, J., Melki, J. R., and Clark, S. J. (2002). Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells. Oncogene 21, 1048-1061.

Stirzaker, C., Song, J. Z., Davidson, B., and Clark, S. J. (2004). Transcriptional gene silencing promotes DNA hypermethylation through a sequential change in chromatin modifications in cancer cells. Cancer Res 64, 3871-3877.

Szyf, M. (1994). DNA methylation properties: consequences for pharmacology. Trends Pharmacol Sci 15, 233-238.

Tchou, J. C., Lin, X., Freije, D., Isaacs, W. B., Brooks, J. D., Rashid, A., De Marzo, A. M., Kanai, Y., Hirohashi, S., and Nelson, W. G. (2000). GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas. Int J Oncol 16, 663-676.

Wade, P. A. (2001). Methyl CpG-binding proteins and transcriptional repression. Bioessays 23, 1131-1137.

Walker, C., and Nettesheim, P. (1986). In vitro transformation of primary rat tracheal epithelial cells by 5-azacytidine. Cancer Res 46, 6433-6437.

Wolffe, A. P., and Matzke, M. A. (1999). Epigenetics: regulation through repression. Science 286, 481-486.

Xu, X. C., Sneige, N., Liu, X., Nandagiri, R., Lee, J. J., Lukmanji, F., Hortobagyi, G., Lippman, S. M., Dhingra, K., and Lotan, R. (1997a). Progressive decrease in nuclear retinoic acid receptor beta messenger RNA level during breast carcinogenesis. Cancer Res 57, 4992-4996.

Xu, X. C., Sozzi, G., Lee, J. S., Lee, J. J., Pastorino, U., Pilotti, S., Kurie, J. M., Hong, W. K., and Lotan, R. (1997b). Suppression of retinoic acid receptor beta in non-small-cell lung cancer in vivo: implications for lung cancer development. J Natl Cancer Inst 89, 624-629.

Yegnasubramanian, S., Kowalski, J., Gonzalgo, M. L., Zahurak, M., Piantadosi, S., Walsh, P. C., Bova, G. S., De Marzo, A. M., Isaacs, W. B., and Nelson, W. G. (2004). Hypermethylation of CpG islands in primary and metastatic human prostate cancer. Cancer Res 64, 1975-1986.

Yoshimura, R., Sano, H., Masuda, C., Kawamura, M., Tsubouchi, Y., Chargui, J., Yoshimura, N., Hla, T., and Wada, S. (2000). Expression of cyclooxygenase-2 in prostate carcinoma. Cancer 89, 589-596.

Zha, S., Gage, W. R., Sauvageot, J., Saria, E. A., Putzi, M. J., Ewing, C. M., Faith, D. A., Nelson, W. G., De Marzo, A. M., and Isaacs, W. B. (2001). Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. Cancer Res 61, 8617-8623.

Zhang, J., Ren, H., Yuan, P., Lang, W., Zhang, L., and Mao, L. (2006). Down-regulation of hepatoma-derived growth factor inhibits anchorage-independent growth and invasion of non-small cell lung cancer cells. Cancer Res 66, 18-23.

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