The continuing goal of this proposal is to analyze the mechanism by which retinoic acid (RA) inhibits 12-0 tetradecanylphorbol-13- acetate (TRP)-induced ornithine decarboxylase (ODC)-gene transcription. In this study, we plan to test two hypotheses: 1) TPA-induced ODC-gene transcription is mediated through interaction of PKC- medicated phosphorylated proteins (trans-acting factor) with cis-acting elements in 5'-flanking region of ODC gene and RA inhibits TPA-induced ODC-gene transcription by inhibiting the phosphorylation of those proteins; and 2) RA complexed with cellular RA binding protein (CRABP) is transported to specific nuclear receptors and thus acts as a negative regulator of TPA- induced ODC gene transcription. To test the first hypothesis, we plan to: a) Isolate 5'-flanking region of ODC gene and identify by deletion analysis and CAT assay of TPA-responsive enchaner element (cis-acting); b) synthesize TPA- responsive sequences using oligonucleotide synthesizer, and then to test the enhancer ability; c) determine by DNase I footprinting analysis that TPA-modulated trans-acting factors recognize the TPA- responsive enhancer elements; d) isolate TPA-modulated trans-acting factors by specific sequence DNA affinity chromatography; e) determine that TPA-responsive transcription factors (proteins) are substrates for the purified PKC; f) determine whether RA inhibits in vivo the phosphorylation of trans-action factors. To test the second hypothesis, we plan to: a) identify RA responsive sequences in the 5-flanking region of ODC gene; b) determine by DNase I footprinting assay whether there is negative regulatory factor(s) that recognize RA responsive sequences; and, c) synthesize RA-responsive cis-elements and then purify RA responsive proteins(s) by DNA-affinity chromatography. We will analyze that the negative regulatory factor (protein) is a RA nuclear receptor. We have shown that TPA induces the transcription of ODC gene in two in vitro systems (epidermal cells and human bladder carcinoma cell line T24) which will be used for proposed study. This study will provide information about the mechanism by which RA inhibits the induction of ODC, an enzyme associated with tumor promotion, cell proliferation and differentiation.
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