Loss of expression of the death-associated
protein (
DAP)-kinase gene by aberrant promoter methylation may play an important role in
cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the
DAP-kinase gene in
adenocarcinomas induced in mice by chronic exposure to mainstream cigarette
smoke, the tobacco
carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and
vinyl carbamate, and the occupational
carcinogen methylene chloride. The timing for inactivation was also determined in alveolar
hyperplasias that arise in
lung cancer induced in the A/J mouse by NNK. The
DAP-kinase gene was not expressed in three of five NNK-induced lung
tumor-derived cell lines or in a spontaneously arising lung
tumor-derived cell line. Treatment with
5-aza-2'-deoxycytidine restored expression; dense methylation throughout the
DAP-kinase CpG island detected by
bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the
DAP-kinase gene in 43% of
tumors associated with cigarette
smoke, a frequency similar to those reported in human
non-small cell lung cancer. In addition,
DAP-kinase methylation was detected in 52%, 60%, and 50% of
tumors associated with NNK,
vinyl carbamate, and
methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced
hyperplasias and
adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for
tumor development in the mouse lung.
Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of
adenocarcinoma development. This study is the first to use a murine model of cigarette
smoke-induced
lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development of this disease in humans.