Hypermethylation of the p16(Ink4a) promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines

Primary lung tumors from B6C3F1 mice and mouse lung cell lines were examine d to investigate the role of transcriptional silencing of the p16(Ink4a) tu mor suppressor gene by DNA hypermethylation during mouse lung carcinogenesi s. Hypermethylation (greater than or equal to 50% methylation at two or mor e 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 adenocarcinoma s examined, whereas hypermethylation was not detected in normal B6C3F1, C57 BL/6 and C3H/He mouse lung tissues. Immunohistochemistry performed on the B 6C3F1 lung adenocarcinomas revealed heterogeneous expression of the p16 pro tein within and among the tumors. Laser capture microdissection was employe d to collect cells from immunostained sections of four tumors displaying ar eas of relatively high and low p16 expression. The methylation status of th e microdissected samples was assessed by sodium bisulfite genomic sequencin g. The pattern of p16 expression correlated inversely with the DNA methylat ion 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, th ree mouse lung tumor cell lines (C10, sp6c and CMT64) displaying complete m ethylation at seven promoter CpG sites and no p16(Ink4a) expression were tr eated with the demethylating agent, 5-aza-2'-deoxycytidine. Reexpression of p16(Ink4a) and partial demethylation of the p16(Ink4a) promoter were obser ved in two cell lines (C10 and sp6c) following treatment. These are the fir st reported studies to provide strong evidence that DNA methylation is a me chanism for p16 inactivation in mouse lung tumors.