The molecular mechanism of action of the tumor promoting phorbol esters will be investigated by determining the normal physiological function of the phorbol ester receptor protein in cells and the effect of tumor promoters on receptor function. Phorbol esters elicit numerous changes in cellular morphology, growth, and differentiation. This is generally assumed to occur via binding reversibly and with high affinity and specificity to a cellular receptor protein which has protein kinase activity tightly associated with it. Both membrane bound and soluble cytosolic receptors exist in cells although the latter requires calcium and phospholipid to function. This and other data suggest that as part of its normal function in cells the soluble cytosolic receptor becomes associated with cell membranes and that phorbol esters may directly affect this aspect of its function. The research plan involves analysis of changes in the localization of the receptor in stimulated cells by quantitation of phorbol ester binding activity, ultraviolet light microscopic observation of cells stained with fluorecently labeled receptor specific antibodies, and viewing of cells stained with receptor specific antibodies covalently bound to ferritin using a trasmission electron microscope. The antibodies will be produced by the monoclonal hybridoma method using as antigen receptor protein purified from mouse brain. Initial biochemical experiments will use as a model system primary mouse spleen cells stimulated with lectins, phorbol esters, and lymphokines. These results will be applied to a study of primary mouse epidermal keratinocytes and C3H 10T-1/2 fibroblasts in which growth and morphological controls are affected by calcium, polypeptide growth factors, and phorbol esters. Ultimately the investigation will be extended to mouse epidermis, in vivo, so that the effects of in vivo treatments known to affect the carcinogenic process in epidermis can be compared to the results obtained with cells in culture. These studies as well as a biochemical investigation of interactions of the receptor with other cellular proteins will lead to the development of a cell free system in which the actions of phorbol esters can be observed. These studies will help characterize the biological action of phorbol esters and aid in understanding the biological processes of tumor promotion and chemical carcinogenesis.
