Vitamin A is essential for the maintenance of epithelial differentiation. The most biologically active form of vitamin A, retinoic acid (RA), has been shown to induce certain tumor cells to differentiate and lose their malignant phenotype. However, the molecular mechanisms involved in this response are poorly understood. Our laboratory has shown that RA increases the amount of mRNA and protein for protein kinase C-alpha (PKCalpha) in mouse B16 melanoma cells. These changes occur prior to growth arrest and differentiation. Recently, we have established two clones (via transfection with a PKCalpha expression vector) which overexpress PKCalpha compared to wildtype cells or clones transfected with only the gene for neomycin resistance. These PKCalpha clones mimic the phenotype of wild type cells treated with RA,ie decreased monolayer growth rate, decreased colony formation in soft agarose and increased melanin production. In animal experiments, the PKC overexpressing clones had a longer latency time and significantly smaller tumors than either wild type or neomycin resistant clones. Thus the induction of PKCalpha by RA appears to play an integral role in the differentiation pathway. In light of these results, this proposal address four important questions. 1) How does RA induce PKCalpha? Since we have demonstrated that this induction requires new protein synthesis, we hypothesize that the retinoic acid receptor-beta (RARbeta) must first be induced by RA, and it is this receptor which mediates the induction of PKC. This will be addressed by transfecting RARbeta in both sense and antisense orientation and determining the effect on PKC induction. 2) How does increased PKC lead to differentiation? We will determine whether the increased PKC is enzymatically active in vivo by measuring phosphorylation of the MARCKS protein. If the protein is enzymatically active then we will determine whether inhibition of this activity will inhibit differentiation. Also we will determine the subcellular location of the increased PKC using a combined cell fractionation biochemical and immuno-fluorescence/immuno-gold EM approach. 3) Are the biological effects induced by increased PKCalpha specific for this isoform or can similar results be obtained by overexpression of the beta & gamma isoforms? This will be addressed by transfecting B16 cells with the appropriate isoform and examining the phenotype. 4) Is the role of PKC unique to Bl differentiation or do other RA-responsive melanoma cells exhibit the same sequence of events?This information might suggest ways to improve cyto-differentiation therapy.
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