Hypermethylation of the p16Ink4a promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines
Hypermethylation of the p16Ink4a promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines
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DOI：
10.1093/carcin/21.9.1691
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发表时间：
2000-09-01
期刊：
影响因子：
4.7
通讯作者：
Devereux, TR
中科院分区：
文献类型：
--
作者：
Patel, AC;Anna, CH;Devereux, TR
我们研究了来自B6C3F1小鼠和小鼠肺细胞系的原发性肺肿瘤，以研究p16（Ink4a）肿瘤抑制基因在小鼠肺癌发生过程中DNA高甲基化转录沉默的作用。在17例（70%）B6C3F1原发性小鼠肺腺癌中，有12例（70%）检测到p16（Ink4a）启动子区域的高甲基化（在检测的两个或多个CpG位点上甲基化大于或等于50%），而在正常的B6C3F1、C57BL/6和C3H/He小鼠肺组织中未检测到高甲基化。对B6C3F1肺腺癌进行的免疫组化显示p16蛋白在肿瘤内和肿瘤间的异质表达。采用激光捕获显微解剖从四个肿瘤的免疫染色切片上收集细胞，显示p16相对高和低表达区域。通过亚硫酸氢钠基因组测序评估微解剖样品的甲基化状态。在显示可变p16表达的11个微解剖区域中，有9个（82%）的p16表达模式与启动子CpG位点的DNA甲基化模式呈负相关。为了进一步证明高甲基化与p16（Ink4a）基因表达缺失有关，我们用去甲基化剂5-aza-2'-脱氧胞苷处理了3种小鼠肺肿瘤细胞系（C10、sp6c和CMT64），它们在7个启动子CpG位点显示完全甲基化，而p16（Ink4a）没有表达。在两种细胞系（C10和sp6c）中观察到p16（Ink4a）的重表达和p16（Ink4a）启动子的部分去甲基化。这些是首次报道的研究，提供了强有力的证据，证明DNA甲基化是小鼠肺肿瘤中p16失活的机制。
Primary lung tumors from B6C3F1 mice and mouse lung cell lines were examined to investigate the role of transcriptional silencing of the p16(Ink4a) tumor suppressor gene by DNA hypermethylation during mouse lung carcinogenesis. Hypermethylation (greater than or equal to 50% methylation at two or more of the CpG sites examined) of the p16(Ink4a) promoter region was detected in DNA from 12 of 17 (70%) of the B6C3F1 primary mouse lung adenocarcinomas examined, whereas hypermethylation was not detected in normal B6C3F1, C57BL/6 and C3H/He mouse lung tissues. Immunohistochemistry performed on the B6C3F1 lung adenocarcinomas revealed heterogeneous expression of the p16 protein within and among the tumors. Laser capture microdissection was employed to collect cells from immunostained sections of four tumors displaying areas of relatively high and low p16 expression. The methylation status of the microdissected samples was assessed by sodium bisulfite genomic sequencing. The pattern of p16 expression correlated inversely with the DNA methylation pattern at promoter CpG sites in nine of 11 (82%) of the microdissected areas displaying variable p16 expression. To provide further evidence that hypermethylation is involved in the loss of p16(Ink4a) gene expression, three mouse lung tumor cell lines (C10, sp6c and CMT64) displaying complete methylation at seven promoter CpG sites and no p16(Ink4a) expression were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Reexpression of p16(Ink4a) and partial demethylation of the p16(Ink4a) promoter were observed in two cell lines (C10 and sp6c) following treatment. These are the first reported studies to provide strong evidence that DNA methylation is a mechanism for p16 inactivation in mouse lung tumors.