The applicants have investigated the role of PKC in the regulation of apoptosis and proliferation in two groups of colon cancer cell lines. Autonomous proliferation of the first group, EGFR ligand-dependent (ELD) cells, is dependent on autocrine EGFR stimulation, while autonomous proliferation of the second group, EGFR ligand-independent (ELI) cells, is independent of this autocrine loop. ELD cells show a unique bimodal proliferative response to phorbol ester. Approximately half of the proliferative effect of phorbol ester is dose-dependently inhibited by the competitive EGFR antagonist, MAb 225. The trough between the two proliferative peaks is due to apoptosis. The apoptotic trough is reversed by addition of nonselective or selective global PKC inhibitors but not by a selective inhibitor of Ca+2 dependent PKCs (Go 6976). All three PKC antagonists inhibit PMA-induced proliferation. These data suggest that the proliferative and apoptotic effects of phorbol ester in ELD cells are related to the activity of specific PKC isoforms and are mediated by EGRF ligand-dependent and independent mechanisms. Furthermore, these data suggest that one or more Type I (Ca+2 -dependent PKCs) are involved in proliferative effects and one or more Type II (Ca+2-independent PKCs) are involved in apoptotic effects. In contrast, ELI cells do not show any proliferative or apoptotic response to phorbol ester. However, their autonomous proliferation is inhibited by all three PKC inhibitors at a concentration that is 5-10 fold lower than that required to inhibit autonomous proliferation of ELD cells. These data suggest that one or more Ca+2 dependent PKCs are critical for the autonomous proliferation ELI cells. The clear distinctions in the effects of phorbol esters and PKC antagonists on proliferation and apoptosis between ELD and ELI cell lines provides us with a unique opportunity to elucidate the roles and mechanisms of action of specific PKC isoforms in proliferative and apoptotic signaling in colon cancer.
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