Shao Genze, Berenguer Jessica, Borczuk Alain C, Powell Charles A, Hei Tom K, Zhao Yongliang
Center for Radiological Research and Department of Pathology, Columbia University, New York, New York 10032, USA.
Cancer Res. 2006 May 1;66(9):4566-73. doi: 10.1158/0008-5472.CAN-05-2130.
Gene silencing by CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. It has been shown previously that Betaig-h3 gene, which encodes an extracellular matrix protein involved in cell adhesion and tumorigenesis, is down-regulated or silenced in a variety of human cancer cell lines. To unravel the underlying molecular mechanism(s) for this phenomenon, DNA methylation patterns of Betaig-h3 CpG island were examined in normal, immortalized, and cancer cell lines derived from lung, prostate, mammary, and kidney. A good correlation was observed between promoter hypermethylation and lost expression of Betaig-h3 gene, which was supported by the data that demethylation of promoter by 5-aza-2'-deoxycytidine reactivated Betaig-h3 and restored its expression in Betaig-h3-silenced tumor cell lines. This result was further substantiated by a luciferase reporter assay, showing the restoration of promoter activities and increased response to transforming growth factor-beta treatment in Betaig-h3-negative 293T cells when transfected with unmethylated Betaig-h3 promoter. In contrast, activity of Betaig-h3 promoter was completely inactivated by in vitro methylation. Furthermore, CpG methylation of Betaig-h3 promoter was also shown in primary lung tumors that expressed decreased level of Betaig-h3 protein. These results suggest that promoter methylation plays a critical role in promoter silencing of the Betaig-h3 gene in human tumor cells.
启动子区域的CpG岛甲基化导致基因沉默是人类癌症中肿瘤抑制基因失活的机制之一。先前已表明，编码参与细胞黏附和肿瘤发生的细胞外基质蛋白的Betaig-h3基因，在多种人类癌细胞系中表达下调或沉默。为了阐明这一现象的潜在分子机制，研究人员检测了来自肺、前列腺、乳腺和肾脏的正常、永生化及癌细胞系中Betaig-h3 CpG岛的DNA甲基化模式。研究发现启动子高甲基化与Betaig-h3基因表达缺失之间存在良好的相关性，5-氮杂-2'-脱氧胞苷对启动子进行去甲基化可使Betaig-h3重新激活并恢复其在Betaig-h3沉默肿瘤细胞系中的表达，这一数据支持了上述发现。荧光素酶报告基因检测进一步证实了这一结果，该检测表明，用未甲基化的Betaig-h3启动子转染Betaig-h3阴性的293T细胞时，启动子活性得以恢复，且对转化生长因子-β处理的反应增强。相反，体外甲基化可使Betaig-h3启动子的活性完全丧失。此外，在表达Betaig-h3蛋白水平降低的原发性肺肿瘤中也发现了Betaig-h3启动子的CpG甲基化。这些结果表明，启动子甲基化在人类肿瘤细胞中Betaig-h3基因的启动子沉默中起关键作用。
