Liu Wen-bin, Liu Jin-yi, Ao Lin, Zhou Zi-yuan, Zhou Yan-hong, Cui Zhi-hong, Yang Huan, Cao Jia
Department of Hygienic Toxicology, Preventive Medical College, Third Military Medical University, Key Laboratory of Medical Protection for Electromagnetic Radiation, Ministry of Education of China, Chongqing 400038, PR China.
Toxicol Lett. 2009 Aug 25;189(1):5-13. doi: 10.1016/j.toxlet.2009.04.022. Epub 2009 May 3.
3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are typical genotoxic carcinogens that can induce tumors in a variety of human and rodent tissues. However, the epigenetic mechanisms underlying their tumorigenesis are unclear. In this study we used a MCA/DEN-induced multistep lung carcinogenesis rat model to study the evolution of alterations in DNA methylation. Rats were treated with a single dose of MCA and DEN in iodized oil by left intra-bronchial instillation. The animals were killed on days 15, 35, 55, 65 and 75 and samples of various pathological phases during carcinogenesis were obtained on these days. The status of global methylation was analyzed for each sample using a monoclonal antibody specific for 5-methycytosine (5-mC) and quantified by image analysis software. We found that the degree of global methylation was, in general, higher in basal cells compared to luminal cells of normal, precancerous and tumor tissues. The combined 5-mC scores of different types of tissues decreased gradually during the progression of carcinogenesis. We also used methylation-sensitive arbitrarily primed PCR (MS-AP-PCR) to screen a total of eight differentially methylated DNA fragments in both precancerous and tumor tissues isolated using laser capture microdissection (LCM), and observed that both unique hypomethylation and hypermethylation fragments coexist after exposure to genotoxic carcinogens. Remarkably, epigenetic alterations in p16 (CDKN2A), but not in p15 (CDKN2B), were observed, and these correlated with the presence of pathologic lung lesions and loss of p16 protein expression. Moreover, defective expression of p16 in methylated primary tumor cell lines recovered markedly after treated with 5-aza-2'-deoxycytidine (5-aza-dC). These results suggest that DNA methylation alterations are an early event in tumorigenesis and play an important role during MCA/DEN-induced multistep rat lung carcinogenesis.
3-甲基胆蒽（MCA）和二乙基亚硝胺（DEN）是典型的遗传毒性致癌物，可在多种人类和啮齿动物组织中诱发肿瘤。然而，它们致癌作用背后的表观遗传机制尚不清楚。在本研究中，我们使用MCA/DEN诱导的多步骤肺癌发生大鼠模型来研究DNA甲基化改变的演变过程。通过左支气管内滴注碘化油的方式，给大鼠单次注射MCA和DEN。在第15、35、55、65和75天处死动物，并在这些时间点获取致癌过程中不同病理阶段的样本。使用针对5-甲基胞嘧啶（5-mC）的单克隆抗体对每个样本的整体甲基化状态进行分析，并通过图像分析软件进行定量。我们发现，在正常、癌前和肿瘤组织中，基底细胞的整体甲基化程度通常高于管腔细胞。在致癌过程中，不同类型组织的5-mC综合评分逐渐降低。我们还使用甲基化敏感的任意引物PCR（MS-AP-PCR），对通过激光捕获显微切割（LCM）分离的癌前和肿瘤组织中的总共八个差异甲基化DNA片段进行筛选，观察到在暴露于遗传毒性致癌物后，独特的低甲基化和高甲基化片段同时存在。值得注意的是，观察到p16（CDKN2A）存在表观遗传改变，而p15（CDKN2B）未出现，这些改变与肺部病理病变的存在以及p16蛋白表达缺失相关。此外，用5-氮杂-2'-脱氧胞苷（5-aza-dC）处理后，甲基化原发性肿瘤细胞系中p16的缺陷表达明显恢复。这些结果表明，DNA甲基化改变是肿瘤发生的早期事件，并且在MCA/DEN诱导的大鼠多步骤肺癌发生过程中起重要作用。
