Haplotype-environment interactions that regulate the human GSTP1 promoter.

B98

Phase II detoxification of carcinogens is reported to mediate some of the anti-carcinogenesis effects of candidate chemopreventive agents. We explored the interaction between sequence variation in the GSTP1 gene promoter and candidate chemopreventive exposure in regulating human GSTP1 expression. Polymorphisms along 1.8 kb of the GSTP1 promoter were identified in leukocytes (PBMCs) from 40 Caucasian subjects. Ten promoter polymorphisms (nine previously unreported) displayed strong linkage disequilibrium, yielding identification of three frequently-observed haplotypes [HAP1 (43%), HAP2 (36%), and HAP3 (8%)]. The existence of these haplotypes has now been experimentally confirmed by direct sequencing of representative homozygotes, and by cloning and sequencing representative heterozygotes. Each haplotype was reproduced from a common template by iterative site-directed mutagenesis, cloned into luciferase reporter constructs, and then transfected into normal human bronchial epithelial (NHBE) cells. Basal HAP3 reporter activity was significantly elevated (1.8-fold), but decreased to the same levels as HAP2 and HAP1 with increasing concentrations of sulforaphane, BITC, and EGCG. To confirm native HAP3 functionality, we quantitated mRNA expression in uncultured PBMCs and in laser microdissected normal lung epithelial cells (MNLECs) from the same patients. Basal mRNA expression was higher in HAP3 individuals [1.8-fold (PBMCs) and 4-fold (MNLECs) for HAP3 heterozygotes; 2.3-fold (PBMCs), and 15-fold (MNLECs) for the HAP3 homozygote], than in the other genotypes. PBMC GSTP1 mRNA expression correlated to MNLEC expression (R² = 0.77). After culture and in vitro exposure to sulforaphane, BITC, or EGCG, the elevated GSTP1 mRNA expression of PBMCs from HAP3 individuals decreased to common expression levels. Elevated HAP3 function was confirmed at the protein level in PBMCs (5-fold higher for HAP3 heterozygotes, 7.6-fold for the HAP3 homozygote). These data suggest a potentially protective GSTP1 promoter haplotype, and unpredicted inhibitory chemopreventive agent-haplotype interactions.