Background: GSTP1-1 is a phase II enzyme playing an important role in the detoxification of oxidized carcinogens, including polycyclic aromatic hydrocarbons (PAH), as well as other compounds. Animal data suggest that the anticarcinogenesis effects of several candidate chemopreventive agents may operate through GSTP1 induction. Previous human studies suggest that there are wide inter-individual differences of GSTP1 expression that may be relevant to the development or biology of cancer. We explored the interaction of inter-individual sequence variation in the GSTP1 promoter and the effect of candidate phytochemicals in regulating GSTP1 expression. Methods: Individual SNPs and resulting haplotypes along 2 kb of the GSTP1 promoter were explored in PBMCs from 40 patients from a lung cancer case-control study. Ten previously-unreported polymorphisms found in the promoter with strong linkage disequilibrium were statistically evaluated, resulting in a frequency distribution of haplotypes. The three most frequently-observed haplotypes [HAP1 (43%), HAP2 (36%) and HAP3 (8%)] were then replicated using a site-directed mutagenesis strategy, placed into a luciferase reporter construct, transfected into normal human bronchial epithelial (NHBE) cells, and then treated with different concentrations of a panel of six plant-derived putative antioxidant compounds, cigarette smoke extract (CSE) and the oxidant hydrogen peroxide. Results: HAP3 had a significantly higher (1.8-fold) basal activity. This reporter was sensitive to increasing concentrations of sulforaphane, BITC and EGCG (but not the other phytochemical compounds); HAP3 promoter activity was decreased to the same levels as HAP2 and HAP1. To confirm this HAP 3 functionality, the native promoter regulating native mRNA transcript levels was examined in PBMCs and lung epithelial cells. Quantitative RNA-specific real-time RT-PCR experiments revealed that HAP3 is again associated with higher basal mRNA expression levels in unexposed cells, compared with the other two haplotypes [18-fold for homozygote (HAP3-HAP3), and 2-fold for heterozygotes (HAP3-HAP1, HAP3-HAP2)]. After exposure to sulforaphane, BITC or EGCG, HAP3-containing PBMCs return to basal GSTP1 mRNA expression levels. The same phenomenon was shown at the protein level for PBMCs [10-fold for homozygote (HAP3-HAP3), and 7-fold for heterozygotes (HAP3-HAP1, HAP3-HAP2) when unexposed cells] Perspectives: Further investigations on haplotype-specific responses to various combinations of CSE, hydrogen peroxide, and candidate antioxidant chemopreventive agents are underway to evaluate the actual impact on DNA damage. This study suggests the existence of vulnerable and protective promoter haplotypes for this anticarcinogenesis gene, and haplotype-specific interaction with individual chemopreventive agents.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]