Carcinogenesis, Vol. 21, No. 9, 1691-1700,
September 2000
© 2000 Oxford University Press
Carcinogenesis |
Hypermethylation of the p16 Ink4a promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines
1 Laboratory of Molecular Carcinogenesis,
2 Laboratory of Experimental Pathology and
3 Division of Extramural Research and Training, Chemical Exposures and Molecular Biology Branch, National Institute of Environmental Health Sciences, PO Box 12233, Mail Drop D4-04, Research Triangle Park, NC 27709, USA and
4 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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 (
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. Re-expression 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.
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