These results indicate a strong link between reduced DSBRC and risk for methylation in sputum. Our studies transcend from a functional assay for DNA repair to specific genotypes, and finally demonstrate an activity deficit of the MRE11A gene that plays a critical role in recognition of double-strand break DNA damage and activation of the ATM gene (32
). The mechanism underlying this association could in part, be mediated by the genes that are recruited to sites of DSBs, and the resultant modification of chromatin to facilitate repair. One of the earliest responses to DSB damage is phosphorylation by ataxia telangiectasia mutated kinase (ATM) of the histone H2AX which then facilitates accumulation of repair/signaling proteins and also SWI/SNF complexes that have been implicated in transcriptional silencing to chromatin regions distal to a DSB (33
). A recent study demonstrated activation of H2AX by ATM in the A549 lung tumor-derived cell line by tobacco smoke (35
Another key contributing factor for aberrant de novo
methylation during DNA damage is the rapid recruitment of DNMT1 to sites of DNA damage (36
). Le Gac et al.
) found that in cells treated with doxorubicin which induces DSBs, DNMT1 is recruited by activated p53 and binds to functional Sp1 sites within promoters of the survivin, cdc2, and cdc25 genes. Moreover, the transcriptional repressor HDAC1 and the repressive chromatin mark H3K9me2 were also found at these promoters following DNA damage (37
). Subsequent in vitro
studies showed that following DSB DNA damage induced by doxorubicin, DNMT1 complexed with p53 was recruited to the survivin gene promoter followed by de novo
methylation and gene silencing (39
). Cuozzo et al.
) provides even stronger support for a mechanistic link between DNA damage and methylation. In that study, a recombinant plasmid containing a 1-SCE1 restriction site within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes was introduced into Hela, or mouse embryonic stem cells. The restriction endonuclease 1-Sce1 was added to the cell to induce a DSB in the GFP gene at this site. Rapid gene silencing associated with homologous recombination and DNA methylation of the recombinant gene was seen and could be blocked by treatment with the demethylating agent, 5-aza-deoxycytidine. Chromatin immunoprecipitation revealed that DNMT1 was bound specifically to the homologous GFP DNA. Together, these in vitro
studies strongly support a direct mechanistic link between DNA damage and induction of de novo
methylation by DNMT1. Our population-based studies now provide for the first time, an in vivo
association between DRC and gene promoter methylation, both through a functional assay and genetic variants in genes within the double-strand break repair pathway. Thus, in the absence of efficient repair, the recruitment of p53, DNMT1, and transcriptional repressors to many genes such as p16 that also contain Sp1 sites within its promoter could lead to de novo
methylation and gene silencing.
The identification of DSBRC and specific genes within this pathway as a critical determinant for gene promoter hypermethylation has important implications for basic and translational science. Our study substantiates that DNA damage that has long been recognized as an initiating event for mutagenesis, is also likely a major factor in initiating aberrant promoter hypermethylation. Other DNA damage response pathways such as apoptosis, nucleotide and base excision repair may also contribute to the induction of aberrant promoter hypermethylation. A major priority for our research is to replicate the provocative findings in this study along with our emerging methylation gene panel in a prospective population-based study. Genetic variants associated with promoter hypermethylation could be used to identify young smokers who would be most susceptible to induction of preneoplasia, and thus, should receive chemoprevention. In addition, the integration of these genetic variants with detection of gene promoter hypermethylation in sputum in long-term heavy smokers could lead to the first diagnostic test for incident lung cancer and impact long-term survival from this fatal disease.