Kim Wan-ju, Vo Quynh N, Shrivastav Meena, Lataxes Tamara A, Brown Kevin D
Department of Biochemistry and Molecular Biology, LSU Health Sciences Center, Louisiana State University, New Orleans, LA 70112, USA.
Oncogene. 2002 May 30;21(24):3864-71. doi: 10.1038/sj.onc.1205485.
Recent findings suggest that DNA alkylating agents trigger cellular responses that overlap those activated after ionizing radiation. Moreover, activation of these responses is dependent upon a functional mismatch repair (MMR) system. These developments led us to test if MMR-deficient cells may be compromised in their ability to activate appropriate cellular signaling pathways after ionizing radiation. An initial experiment to address this notion was to determine the level of radiosensitivity of several MMR-deficient cell lines derived from patients with Hereditary Non-Polyposis Colorectal Cancer (HNPCC). While two of the three HNPCC lines investigated show levels of radiosensitivity consistent with that displayed by normal human fibroblasts, HCT-116 cells display moderate radiosensitivity compared to the other MMR-deficient lines. This increased sensitivity to ionizing radiation correlates with lowered levels of ATM expression in HCT-116. Analysis of genomic DNA from HCT-116 cells determined that these cells possess aberrant methylation of multiple CpG dinucleotides within the proximal promoter region of the ATM gene. The significance of this finding is underscored by our observations that co-culturing HCT-116 cells with the DNA demethylating agent 5-azacytidine reverses promoter methylation, promotes normal levels of ATM expression, and restores normal radiosensitivity. The proximal ATM promoter is a approximately 520 bp region shared with the NPAT gene, and current evidence suggests that this region functions as a bi-directional promoter. We found that, unlike ATM, the methylation status of this intergenic region does not effect the expression of the NPAT gene. In sum, these observations indicate that the ATM gene is a novel target for epigentic silencing through inappropriate methylation of its proximal promoter region.
最近的研究结果表明，DNA烷化剂引发的细胞反应与电离辐射后激活的反应重叠。此外，这些反应的激活依赖于功能正常的错配修复（MMR）系统。这些进展促使我们测试MMR缺陷细胞在电离辐射后激活适当细胞信号通路的能力是否受损。解决这一概念的初步实验是确定几种源自遗传性非息肉病性结直肠癌（HNPCC）患者的MMR缺陷细胞系的放射敏感性水平。虽然所研究的三个HNPCC细胞系中的两个显示出与正常人成纤维细胞一致的放射敏感性水平，但与其他MMR缺陷细胞系相比，HCT-116细胞表现出中等放射敏感性。这种对电离辐射敏感性的增加与HCT-116中ATM表达水平的降低相关。对HCT-116细胞基因组DNA的分析确定，这些细胞在ATM基因近端启动子区域内的多个CpG二核苷酸存在异常甲基化。我们观察到将HCT-116细胞与DNA去甲基化剂5-氮杂胞苷共培养可逆转启动子甲基化、促进ATM表达的正常水平并恢复正常放射敏感性，这突出了这一发现的重要性。ATM近端启动子是与NPAT基因共享的约520bp区域，目前的证据表明该区域作为双向启动子发挥作用。我们发现，与ATM不同，这个基因间区域的甲基化状态不影响NPAT基因的表达。总之，这些观察结果表明，ATM基因是其近端启动子区域通过不适当甲基化导致表观遗传沉默的新靶点。
