The objective of this proposal is to understand the molecular mechanism(s) through which retinoic acid (RA), cAMP and laminin interact to control the growth and differentiation of F9 teratocarcinoma cells. RA inhibits the expression of the proto- oncogene c-myc in F9 stem cells. We will determine the mechanism(s) for this regulation. We will also investigate the function of c-myc in F9 growth and differentiation by transfecting stem cells with a retrovirus containing an expressible c-myc gene. Thus we will determine whether continued high expression of c-myc can prevent the effects of RA and cAMP on growth and differentiation. F9 cells grown on either gelatin or laminin-coated dishes can be induced to differentiate into parietal endoderm-like cells by cholera toxin + 1-methyl, 3- isobutylxanthine (CT/MIX) in the absence of RA. Laminin potentiates the differentiation of this cAMP-induced pathway. Thus we will characterize this cAMP-induced pathway by determining whether it leads to irreversible differentiation, regulates differentiation-specific genes at the transcriptional level and by exploring the relationship between the RA and cAMP pathways through the isolation of RA and cAMP resistant mutants. We will also study the mechanism by which laminin potentiates the cAMP-induced differentiation pathway. We will establish the kinetic properties of the laminin enhanced cAMP- induced differentiation pathway. Subsequently, we will determine if the effects of laminin are mediated through specific cell surface receptors, and whether the interaction of laminin with its receptor increases cAMP-dependent protein kinase. Through the use of butyrate, we will determine whether the failure of previous investigators to detect the cAMP-induced pathway was due to the metabolism of dibutyryl cAMP to butyrate which in turn blocked the cAMP pathway. Retinoids are being used experimentally to manage some malignancies. However rational approaches to use of retinoids and cAMP elevating agents requires an understanding of the molecular mechanisms by which these compounds interact. The studies outlined in this proposal will provide new insights into these mechanisms.
