This is a competitive renewal application to investigate the molecular mechanisms involved in skin cancer chemoprevention by apigenin, a nontoxic and nonmutagenic bioflavonoid which inhibits UV-induced skin carcinogenesis when topically applied to mouse skin. During the previous funding period, the applicant investigated the effect of apigenin treatment on expression of the p53 tumor suppressor gene in mouse keratinocytes. In keratinocyte cell lines with wildtype p53 status, the applicant demonstrated that apigenin is extremely potent in elevating the level of wildtype p53 protein in keratinocytes (27-fold). This level of p53 induction is substantially higher than that induced in keratinocytes by UVB irradiation, for example (5-fold). The applicant further demonstrated that the increased level of p53 protein was due to protein stabilization, accompanied by increased phosphorylation of p53 at Ser15 and subsequent transcriptional activation of p53-responsive genes including p21WAF1. Interestingly, the applicant did not observe any increased accumulation of MDM2 protein in apigenin-treated cells, which is unexpected considering that the MDM2 gene is a downstream target of p53 transcriptional activation and generally responsible for feedback inhibition of p53 by promoting its degradation through ubiquitination. The applicant's results indicate that apigenin treatment of keratinocytes induces many of the same events in the p53 pathway that are normally triggered during the cellular UV DNA damage response, with the exception that the negative feedback MDM2 control loop appears to be absent. The lack of feedback inhibition by MDM2 may prolong the beneficial effects of p53 protein stabilization in apigenin-treated keratinocytes. The hypothesis to be tested in this renewal application is that apigenin's chemopreventive activity is derived from its ability to enhance the response of the normal cellular p53 pathway to UV-induced damage in keratinocytes. The applicant proposes four specific aims to test this hypothesis: (1) Investigate the mechanism(s) by which apigenin treatment induces posttranslational modification of p53, by identifying the p53 phosphorylation sites, the kinases involved in phosphorylation, and whether apigenin induces p53 acetylation in keratinocytes; (2) Having characterized p53 protein post-translational modification induced by apigenin treatment alone, investigate the combined effects of apigenin treatment plus UVB irradiation on p53 protein levels, stabilization, and post-translational modification; (3) Investigate the impact of apigenin treatment on MDM2 gene expression, on interaction of p53 and MDM2 protein, and whether apigenin treatment results in inhibition of MDM2-mediated p53 ubiquitination and degradation; (4) Investigate the combined effects of apigenin treatment plus UVB irradiation on MDM2 gene expression, MDM2/p53 protein interaction, and MDM2/p53 protein interaction, and MDM2-mediated p53 ubiquitination.
Showing the most recent 10 out of 19 publications
