Key early metabolic steps in lung carcinogenesis include procarcinogen bioactivation and inactivation. Individual susceptibility to carcinogenesis can therefore be envisioned to be due, in part, to an individual's specific pattern of procarcinogen metabolism. Our general hypothesis is that high phase I and low phase II expression imbalance in carcinogen-metabolizing enzyme levels, identifies individual smokers at particularly high risk for lung cancer. In this proposal, we hypothesize that heritable inter-individual differences in promoter region SNP haplotype and epigenetic CpG methylation features confer upon the individual functionally important differences in gene expression phenotype. Previous studies from this and other laboratories have identified substantial inter-individual variation in phase I and phase II carcinogen metabolism gene expression levels in human lung. We have recently generated preliminary evidence that across individual human subjects, the observed variation of expression phenotype for carcinogen metabolism enzyme expression in human lung may plausibly result from: 1} common promoter region single nucleotide polymorphism (SNP) haplotypes; and/or 2) variation in promoter CpG methylation. In this R21 submission, we propose to explore how each of these two types of regulatory factors impacts on quantitative gene expression at the mRNA level, for the selected index genes CYP1B1 and GSTP1 in the carcinogen metabolism pathway. Functional consequences of these regulatory region features will be verified both in vitro, and from in-situ - derived human lung epithelium. Novel tools employed will include the laboratory's RNA-specific adaptation of real-time quantitative RT-PCR, and the recently developed tag-adaptation of bisulfite genomic sequencing, generating detailed methylation spectra without the need for cloning, each applied to human airway biospecimens. Comparison with tobacco exposure, hormonal and other clinical variables will be made. This proposal is therefore aimed at verifying salient promoter region characteristics that both mechanistically impact on carcinogen metabolism phenotype, and are amenable to genomic DNA based high throughput screening strategies for lung cancer susceptibility.
