Cho Bomsoo, Lee Hansoo, Jeong ShinWu, Bang Yung-Jue, Lee Hyun Joo, Hwang Kyu Sang, Kim Hae-Yeong, Lee Yun-Sil, Kang Gyeong Hoon, Jeoung Doo-Il
Cancer Genomics Division, In2Gen Company, 110-799, Seoul, Republic of Korea.
Biochem Biophys Res Commun. 2003 Jul 18;307(1):52-63. doi: 10.1016/s0006-291x(03)01121-5.
Previously, we reported the identification and characterization of a novel cancer/testis antigen gene, CAGE(4), that was expressed in various histological types of tumors, but not in normal tissues, with the exception of the testis. To date, molecular mechanisms for the expression of CAGE have never been studied. In our expression analysis, we found that some cancer cell lines did not express CAGE. The expression of CAGE could be restored in these cell lines by treatment with 5(')-aza-2(')-deoxycytidine, suggesting that the expression of CAGE is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the 16 CpG sites of the CAGE promoter in various cancer cell lines and tissues revealed a close relationship between the methylation status of the CAGE promoter and the expression of CAGE. The transient transfection experiments displayed that the methylation of CpG sites inhibited the CAGE promoter activity in luciferase reporter assays. The methylation of the CpG sites inhibited the binding of transcription factors, shown by a mobility shift assay. A methylation-specific PCR analysis revealed that hypomethylation of the CAGE promoter was present at frequencies of more than 60% in breast, gastric, and lung cancers, and hepatocellular carcinomas, and at frequencies of less than 40% in prostate, uterine cervical, and laryngeal cancers. Promoter hypomethylation was found in chronic gastritis (19/55, 34.5%) and liver cirrhosis (13/22, 59%), but not in normal prostate, normal colon, or chronic hepatitis. These results suggest that the methylation status of the CpG sites of CAGE determines its expression, that the hypomethylation of CAGE precedes the development of gastric cancer and hepatocellular carcinoma, and that the high frequencies of hypomethylation of CAGE, in various cancers would be valuable as a cancer diagnostic marker.
此前，我们报道了一种新型癌胚抗原基因CAGE(4)的鉴定与特性，该基因在各种组织学类型的肿瘤中表达，但在正常组织中除睾丸外均不表达。迄今为止，尚未对CAGE表达的分子机制进行研究。在我们的表达分析中，我们发现一些癌细胞系不表达CAGE。通过用5(')-氮杂-2(')-脱氧胞苷处理，这些细胞系中CAGE的表达可以恢复，这表明CAGE的表达主要受到高甲基化的抑制。对各种癌细胞系和组织中CAGE启动子的16个CpG位点进行亚硫酸氢盐测序分析，揭示了CAGE启动子的甲基化状态与CAGE表达之间的密切关系。瞬时转染实验表明，在荧光素酶报告基因检测中，CpG位点的甲基化抑制了CAGE启动子活性。迁移率变动分析表明，CpG位点的甲基化抑制了转录因子的结合。甲基化特异性PCR分析显示，CAGE启动子低甲基化在乳腺癌、胃癌、肺癌和肝细胞癌中的频率超过60%，在前列腺癌、子宫颈癌和喉癌中的频率低于40%。在慢性胃炎（19/55，34.5%）和肝硬化（13/22，59%）中发现启动子低甲基化，但在正常前列腺、正常结肠或慢性肝炎中未发现。这些结果表明，CAGE的CpG位点的甲基化状态决定其表达，CAGE的低甲基化先于胃癌和肝细胞癌的发生，并且CAGE在各种癌症中的高频率低甲基化作为癌症诊断标志物将具有重要价值。
