The main objective of this proposal is to analyze the molecular mechanisms by which retinoic acid inhibits mouse skin tumor promoter 12-)-tetradecanoylphorbol-13-acetate (TPA) induction of epidermal ornithine decarboxylase (ODC), a biochemical change associated with skin tumor promotion by TPA. We first demonstrated that the mechanism by which certain retinoids inhibit skin tumor promotion is due to their ability to inhabit it the induction of ODC by TPA. However, the nature of retinoic acid inhibition of ODC induction by TPA remained undefined. Recently, we found that retinoic acid inhibits TPA-caused synthesis of ODC protein. Retinoic acid may inhibit the synthesis of ODC protein at the transcriptional and/or translational levels. In a preliminary experiment, we found retinoic acid inhibits TPA-increased level of ODC RNA. Thus, we hypothesize that TPA-induce ODC activity (peaks at 6 hr and approximately 200-fold above control) is the result of increased synthesis of ODC protein at the transcriptional level and retinoic acid inhibits ODC induction by inhibiting TPA-increased ODC messenger. Specifically, we plan to investigate: 1) a correlation among TPA-induced epidermal ODC activity, ODC protein, rate of ODC synthesis, and ODC mRNA levels, 2) whether retinoic acid inhibition of TPA-induced ODC activity is proportional to the inhibition of ODC protein and ODC mRNA levels, 3) the mechanism of inhibition by retinoic acid of ODC messenger. We will determine whether TPA causes amplification of ODC gene(s) and retinoic acid inhibits this process. Experiments will be performed with CD-1 mice. Total RNA from skin will be isolated by urea extraction and CsC1 gradient centrifugation. Poly(A)+-contining mRNA will be fractionated by affinity chromatography on an agarose poly(U) column. ODC mRNA will be quantitated by solution hybridization using [32p]-labeled single stranded probe and Dot-blot analysis using radiolabeled cDNA probe. Size of ODC mRNA transcripts will be determined by Northern blotting. Mouse skin genomic DNA will be analyzed by Southern blotting. We will analyze both hybridizable and translatable ODC mRNA; translatable ODC mRNA will be determined by translation in a reticulocyte lysate cell-free syftem. Amount of ODC protein will be analyzed by the radioimmunoassay using [3H] difluoromethylornithine-labeled ODC. These data will permit an evaluation of the role of transcription in the regulation of ODC. These data will permit an evaluation of the role of transcription in the regulation of ODC induction by TPA and whether retinoic acid inhibits TPA induction of ODC at the transcriptional level.
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