The induction of neoplastic transformation by chemical and physical carcinogens are be modeled in in vitro culture systems. In one of the most widely used system, the IOTI/2 assay system, the frequency of transformation can be enhanced by tumor promoters such as TPA, and inhibited by retinoids. This applicant seeks to continue studies into the mechanism of action of retinoids in inhibiting the carcinogenic process. Although retinoids are being widely used clinically, their mechanisms of action is not understood. We have recently shown that retinoids strongly induce gap junctional cell communication between normal or between neoplastic cells. This is associated with enhanced growth control of these cells. We propose that growth regulatory signals are transmitted via gap junctions. We have identified the major structural protein in 10T1/2 cells as connexin43 and find that retinoids increase cellular levels of this protein, as measured on Western blots and the message for this protein determined by Northern blotting. In contrast to retinoid effects on homologous communication, communication between normal and transformed cells is inhibited by retinoids. We propose to examine the role of retinoids in controlling the assembly of connexins into functional junctions in normal and neoplastic cells and the role of protein phosphorylation in junctional conductance. Modification of connexin phosphorylation by agents which up regulate (cAMP), or down regulate (TPA) communication will be examined. To determine the validity of this model system to the cancer chemopreventive action of retinoids in humans, we propose to examine retinoid effects on gap junctional communication in an in vitro human skin system. Communication will be measured by dye injection and electrochemical means. Induction of junctional complexes will be probed by immunofluorescence and electron microscopy. Retinoid effects in pre-neoplastic human skin cells will also be studied. These studies will aid our understanding of growth control processes in general, their aberration in neoplasia, and the mechanism of action of chemopreventive agents which are in extensive clinical use.
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