Role of the AP-1 element and redox factor-1 (Ref-1) in mediating transcriptional induction of DT-diaphorase gene expression by oltipraz: a target for chemoprevention.

The dithiolethione oltipraz is a potent chemopreventive agent in preclinical models, and induces the expression of protective enzymes in the colon mucosa and peripheral mononuclear cells of treated human subjects. We investigated the effects of oltipraz on DT-diaphorase expression in HT29 colon adenocarcinoma cells. Following a 24-hr exposure to 100 microM oltipraz, elevated steady-state levels of mRNA for Jun and Fos family members were observed. A nuclear run-on assay showed induction of c-fos and c-jun transcripts at the end of the exposure, peaking at 12 hr after resuspension of cells in drug-free medium. Gel mobility shift analysis revealed a similar time-course of induced nuclear factor binding to an AP-1 probe. Supershift analysis verified the participation of Jun and Fos in the complexes. The redox coactivator Ref-1, a function of which is to enhance AP-1 binding, was induced 5-fold by oltipraz. Immunodepletion of Ref-1 partially inhibited factor binding to the AP-1 probe. Deletion analysis of the DT-diaphorase promoter in a CAT reporter construct revealed that loss of the AP-1 site accounted for approximately 65% of the induction by oltipraz. Mutation of the AP-1 element in a full-length promoter construct yielded similar results. These data suggest the importance of transcriptional activation mediated by AP-1 in the chemopreventive activity of oltipraz, and indicate that novel chemoprevention structures may be selected based upon agonist activity at this locus.