Department of Hygiene Toxicology, Preventive Medical College, Third Military Medical University, Key Laboratory of Medical Protection for Electromagnetic Radiation, Ministry of Education of China, Chongqing 400038, P.R. China.
Toxicol Appl Pharmacol. 2011 Feb 15;251(1):70-8. doi: 10.1016/j.taap.2010.12.002. Epub 2010 Dec 14.
To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.
为了评估基因毒性致癌物 3-甲基胆蒽（MCA）和二乙基亚硝胺（DEN）诱导的肺多步致癌作用中细胞黏附相关基因甲基化改变的意义，从 MCA/DEN 诱导的大鼠肺癌发生模型中微分离组织样本，采用甲基化特异性 PCR 评估 CADM1、TIMP3、E-钙黏蛋白和 N-钙黏蛋白的 DNA 甲基化状态。免疫组织化学用于确定 CADM1、TIMP3、N-钙黏蛋白和 DNA 甲基转移酶（DNMTs）1、3a 和 3b 的蛋白表达。在任何组织中均未检测到 E-钙黏蛋白的高甲基化。CADM1、TIMP3 和 N-钙黏蛋白的高甲基化与它们在病理病变进展过程中蛋白表达的丧失相关。至少一个基因的 DNA 甲基化的发生率和平均甲基化基因数量随着组织学进展而增加。DNMT1 和 DNMT3a 蛋白表达在肺癌发生过程中逐渐增加，而 DNMT3b 的过表达仅在几个样本中发现。此外，DNMT1 蛋白表达水平与 CADM1 甲基化相关，DNMT3a 蛋白表达水平与 CADM1、TIMP3 和 N-钙黏蛋白甲基化相关。致癌过程中平均甲基化基因数量与 DNMT1 和 DNMT3a 蛋白表达水平显著相关。此外，在 CADM1 甲基化的原发性肿瘤细胞系中用 DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷处理后，CADM1 的 mRNA 表达显著增加。我们的研究结果表明，细胞黏附相关基因沉默与 MCA/DEN 诱导的大鼠肺癌发生进展的动态变化相关，其原因是异常甲基化的积累。我们认为 DNMT1 和 DNMT3a 蛋白过表达可能是这种异常 DNA 甲基化的原因。
