The focus of this project is to better elucidate the possible involvement of transmembrane signal transmission systems in the regulation of cell growth and as possible targets in the process of tumor initiation and tumor promotion. Phospholipase D-mediated hydrolysis of phospholipids may represent an important element in transmembrane signaling. It was determined that activators of protein kinase C (PKC), adenine and guanine nucleotides, and sphingosine all act to stimulate phospholipase D-catalyzed hydrolysis of phosphatidylethanolamine (PtdEtn). Studies performed with both intact cells and isolated membranes established that the prominent lipid peroxidation product 4-hydroxynonenal (HNE) significantly inhibited phospholipase D activity. In contrast, HNE stimulated a phospholipase C activity to enhance PtdEtn hydrolysis and increase DAG levels in NIH 3T3 cells. Selective oxidative modification of the regulatory domain of PKC by the oxidant tumor promoter m-periodate was found to generate a constitutively active kinase no longer regulated by Ca2+ or phospholipids. Oxidation of the catalytic domain resulted in the formation of an inactive PKC, and this inactivation may play a role in the process of down regulation. Activation of PKC, in turn, leads to the activation and subsequent redistribution of Raf-1 protein kinase (Raf-1PK) to the nucleus. Cerebral ischemia in Mongolian gerbils also was found to cause a redistribution of B-raf PK to the nucleus of hippocampal neurons. Together, the above results indicate a possible interrelationship between oxygen free radicals, lipid peroxidation products, and transmembrane signaling systems to modulate cell growth regulation, carcinogenesis, and tumor promotion. Immunohistochemical evidence, kinase activity measurements, and Western blot analysis all indicate that PKC is significantly elevated in multiple drug-resistant MCF-7/ADR cells. High levels of PKC activity are found in the nucleus of MCF-7/ADR cells, and immunoblot analysis with PKC isotype-specific antisera indicate that nuclei isolated from MCF-7/ADR cells contain markedly elevated levels of a modified form of PKCalpha. These results suggest that an increase in a form of PKCalpha may play a role in modulating nuclear events to influence the development of multidrug resistance in MCF-7 cells.
