Vitamin A is involved in controlling essential biological processes such as growth, embryonic development, and differentiation. Analogs of vitamin A (retinoids) prevent tumor formation in animals subsequent to administration of chemical carcinogens. Retinoids also induce differentiation of cancer cell in vitro with concomitant loss of their malignant phenotype. In spite of these important actions, we do not understand how retinoids regulate these functions at a molecular level. Our laboratory has found that retinoids can influence the activity of another regulatory system ie. cyclic, by increasing the level of cyclic AMP-dependent protein kinase (cAPK) in melanoma cells. This application describes studies to further elucidate the mechanism of interaction between retinoids and protein kinase as it pertains to the regulation of growth and differentiation in melanoma cells. Specifically we will study the mechanism whereby retinoic acid increases cAPK by determining whether retinoic acid increases the functional level of mRNA for RI (the regulatory subunit of type I protein kinase) and C (the catalytic unit of cAPK). This will be accomplished by in vitro translation and identification and quantitation of R and C in the translation products by a) immunoblots, b) two-dimensional electrophoresis and c) photoaffinity labeling. We will also determine the total level of RI poly A RNA by Northern blot analysis using a specific cDNA probe for RI. Another aim is to study the changes in protein phosphorylation induced by retinoic acid. We will determine the subcellular location of the altered phosphoproteins, the amino acid sites which are being phosphorylated, whether any of the altered phosphoproteins are themselves protein kinases, and the identity of the phosphorylated proteins. This latter objective will be accomplished by two-dimensional electrophoretic separation of the phosphoproteins followed by transfer to nitrocellulose (Western blots) and probing the blots sequentially with a number of antibodies.
The final aim i s to study the relationship between cAPK, protein phosphorylation and the biological effects of retinoic acid on melanoma cells. We will isolate and characterize retinoic acid resistant and cyclic AMP resistant mutants derived from B16 mouse melanoma. We will also determine whether microinjection of C, R, or holo type I cAPK can reproduce the biological effects of retinoic acid treatment of melanoma cells. An understanding of the mechanism of retinoid action may eventually lead to therapeutic applicability in various disease states.
