Tumor promoter phorbol esters, and cellular regulators which elevate intracellular diacylglycerol, induce protein kinase C association to the plasma membrane and thereby influence various cellular functions including cell adhesion and exocytosis. These Ca2+-regulated functions play key roles in hematogenous metastasis. Our original observations showing a strong correlation between the levels of membrane-bound protein kinase C activity and metastatic ability in three well characterized phenotypically stable sublines of B16 murine melanoma exhibiting low (F1), intermediate (BL6) and high (F10) metastatic behaviour prompted the present proposal designed to investigate the role of membrane-bound protein kinase C in hematogenous metastasis. The reasons why membrane-bound protein kinase C activity is low in the F1 subline compared F10 will be tested. Based on observations that 12-0 tetradecanoylphorbol-13-acetate (TPA) (1 h)-treatment stimulates membrane-association of protein kinase C and increases metastasis whereas TPA (24 h) treatment inactivates protein kinase C and decreases metastasis, this study will investigate the effects of physiological regulators and pharmacological agents, which modulate membrane association of protein kinase C, on hematogenous metastasis. Metastasis will be evaluated by counting the number of pulmonary nodules appearing 3 weeks after tail vein injection of treated cells. These cells will also be tested for their in vitro attachment to endothelial monolayers and basement membranes and their early (3 h) in vivo pulmonary retention to assess alterations in cell adhesion mediated by protein kinase C. The study will identify the membrane target proteins phosphorylated by protein kinase C and examine whether their affinity for endothelium and basement membranes is altered. Finally, the mechanism of protein kinase C inactivation with prolonged TPA treatment will be addressed with specific attention placed on the roles of proteolytic inactivation by calpain (Ca2+-activated protease) and oxidative inactivation by reactive oxygen species. Certain agents including anticancer drugs that are known to generate oxygen free radicals will be tested for their ability to inactivate protein kinase C and inhibit metastasis. This study also will evaluate the additional role of tumor promoters in the hematogenous spread of cancer.
