The amplification of cellular response to cAMP by retinoic acid (RA) has been described in a number of in vitro systems of differentiation, and is part of a more generalized phenomenon of RA amplification of signal transduction. The mechanism whereby RA amplifies the effect of cAMP is not known. In preliminary studies, the effect of RA and cAMP on uPA (urokinase plasminogen activator) activity and mRNA levels was studied in a mouse mammary tumor cell line (SC115). It was found that cells primed with RA exhibited a 2 to 4-fold amplified response to BrcAMP. This effect was reproducibly observed over more than 150 cell passages. Therefore, SC115 cells represent a stable mammary tumor cell model for the study of the mechanism of RA amplification of molecular signals leading to activation of gene transcription. The study of RA and cAMP interaction in mammary tumor cells is particularly fitting because both retinoids and cAMP analogs are being tested in breast cancer patients. To elucidate the mechanism whereby RA potentiates signal transduction by the cAMP-dependent protein kinase A (PKA) pathway in SC115 mammary tumor cells, studies are proposed that examine the effect of RA on the activity and content of individual PKA isozymes in various subcellular fractions, and then identify RA-dependent changes in protein phosphorylation by PKA. In addition, the effect of RA on PKA mRNA levels will be studied by Northern blot analysis to determine whether PKA is a primary RA target. To corroborate the results of these studies in a human model, an improved technique for the organ culture of human breast cancers will be used. Preliminary findings suggest that synergistic upregulation of uPA by simultaneous treatment with RA and cAMP is prevalent in human breast cancer. Key findings resulting from the study of SC115 cells, including PKA regulation by RA, will be retested using this human model. Finally, the effect of retinoids and cAMP analogs puzzling paradox between the anti-tumor effects of these agents in several experimental systems on one hand, and the association of uPA and malignancy, on the other. The experiments proposed will determine which is the dominant effect of retinoids on SC115 tumor growth and will address interactive regulation of growth by retinoids and cAMP analogs.
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