Novel Therapeutic Strategy Based on Systematic Regulation of Metastasis-related Genes Expression in Human Transitional Cell Carcinoma of the Urinary Bladder

目的：探讨转移相关基因（基质金属蛋白酶（MMP）、金属蛋白酶组织抑制剂（TIMP）和e -钙粘蛋白）启动子区DNA甲基化在移行细胞癌（TCC）中的作用。实验设计：采用T24、EJ-1、HT-1197、rt4253j - p、253J-BV、UM-UC-14、NS和KMBC-2 9株人TCC细胞株。在推荐的培养基中培养正常肾近端小管细胞系RPTEC和正常人脐静脉内皮细胞系HUVEC。在高知医学院通过手术切除获得22例日本TCC患者的22例TCC和匹配的邻近正常组织。通过甲基化特异性PCR （MSP）方法（E-cadherin）和亚硫酸氢盐限制性结合分析（COBRA）方法（MMP2,MMP9,TIMP1,TIMP2）鉴定了这些tcc中转移相关基因启动子区域的DNA甲基化。用5-aza-脱氧胞苷（5-aza）和trichostatin A （TSA）处理也检测了基因再表达的诱导。结果：RT-PCR在T24和EJ-1中未检测到E-cadherin RNA的表达，在HT-1197和kmb -2中未检测到TIMP2的表达。在这些细胞系中检测到每个基因的DNA甲基化。其他细胞系和手术标本的转移相关基因DNA甲基化无差异。5-aza-脱氧胞苷（5-aza）和/或trichostatin A （TSA）处理可诱导E-cadherin基因在T24和EJ-1中重新表达，但TIMP2基因在HT1197和kmb -2中未表达。结论：启动子区DNA甲基化可能参与了e -钙粘蛋白RNA表达的调控。然而，启动子区域DNA甲基化在其他转移相关基因（MMP和TIMP）中的作用目前尚不清楚。

Purpose : To assess the role of DNA methylation of metastasis-related genes (matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP) and E-cadherin) promoter region in transitional cell carcinoma (TCC).Experimental Design : The following 9 human TCC cell lines were used :T24, EJ-1,HT-1197,RT4,253J-P,253J-BV, UM-UC-14,NS and KMBC-2. Normal renal proximal tubule cell line RPTEC and normal human umbilical vein endothelial cell line HUVEC was cultured in the recommended growth medium. 22 TCCs and matched adjacent normal tissues were obtained from 22 Japanese TCC patients by surgical resection at the Kochi Medical School. The DNA methylation of metastasis-related genes promoter region in these TCCs was identified by methylation specific PCR (MSP) method (E-cadherin) and combined bisulfite restriction analysis (COBRA) method (MMP2,MMP9,TIMP1,TIMP2). The induction of gene re-expression was also examined by treatment with 5-aza-deoxycytidine (5-aza) and trichostatin A (TSA).Results : E-cadherin RNA expression was not detected by RT-PCR in T24 and EJ-1, and also TIMP2 in HT-1197 and KMBC-2. Each gene DNA methylation was detected in these cell lines. There was no difference of metastasis-related genes DNA methylation in other cell lines and surgical specimens. E-cadherin gene re-expression was induced by treatment with 5-aza-deoxycytidine (5-aza) and/or trichostatin A (TSA) in T24 and EJ-1, but TIMP2 gene was not in HT1197 and KMBC-2.Conclusions : DNA methylation of the promoter region may be involved in the regulation of E-cadherin RNA expression. However, the role of DNA methylation of the promoter region in other metastasis-related genes (MMP and TIMP) could not be clarified in present study.