Epigenetic silencing of cell cycle regulatory genes during 3-methylcholanthrene and diethylnitrosamine-induced multistep rat lung cancer.

The rat lung cancers induced by 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are considered to be a good model for illustrating genetic alterations in human lung precancerous and cancerous lesions. Recently, we had reported that the model can also be used to investigate the step-by-step dynamic changes in DNA methylation during lung carcinogenesis. In this study, we have used the same animal model to further study the evolution of methylation alterations of cell cycle regulatory genes CDKN1B (p27) and CDKN1C (p57). Our results showed epigenetic alterations in p27 and p57. Promoter hypermethylation of p27 was detected in one sample of carcinoma in situ (CIS) and two samples of infiltrating carcinoma, all three of which lacked expression of the p27 protein. Methylation of the p57 promoter correlated with the loss of protein expression in lung pathologic lesions, with a gradual increase in methylation frequency from 0 sample in the normal epithelium and hyperplasia, to 11.1% in squamous metaplasia, 18.9% in dysplasia, 26.7% in CIS, and finally 36.0% in infiltrating carcinoma samples. Immunohistochemical analysis showed that p27 and p57 protein expression decreased as lung carcinogenesis progressed. Moreover, weak expression of p27 and p57 in methylated primary tumor cell lines increased markedly after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), confirming that methylation was indeed responsible for the gene downregulation. These results suggest that the progression of rat lung carcinogenesis induced by MCA/DEN is associated with dynamic changes in promoter hypermethylation of cell cycle regulatory genes, including p27 and p57, accounting for their defective expression.