Inhibition of MCF-7 cell growth by 12-O-tetradecanoylphorbol-13-acetate and 1,2-dioctanoyl-sn-glycerol: distinct effects on protein kinase C activity.

We have investigated the effects of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol (DiC8) on MCF-7 cell proliferation and protein kinase C activity. DiC8 mimics the effects of TPA on both cell morphology and proliferation, with an ED50 value of 11 micrograms/ml for cell growth inhibition. As with TPA and phorbol 12,13-dibutyrate, DiC8 enhances the degree of phosphorylation of an endogenous Mr 28,000 protein in a time- and dose-dependent manner. The effect is measurable upon 5 min of cell treatment with each protein kinase C activator and reaches a maximum at 30 min. The ED50s observed are 5 ng/ml and 20 micrograms/ml, respectively, for phorbol esters and DiC8. The Mr 28,000 protein is found in the cytosolic fraction and is phosphorylated on serine residues by both TPA and DiC8. Further characterization of the phosphorylated proteins using a highly resolutive two-dimensional electrophoresis demonstrates that the two-protein kinase C activators lead to slightly distinct protein phosphorylation patterns with an extra set of proteins phosphorylated under TPA but not DiC8 stimulation. Contrary to TPA, DiC8 induces only a partial and transient translocation of protein kinase C activity from the cytosolic to the particulate compartment. Moreover, no down-regulation of protein kinase C is observed after prolonged treatment of MCF-7 cells with DiC8, while only 10% of the initial protein kinase C level remains present in cells treated with TPA for 48 h. However, this remainder enzymatic activity is sufficient to induce the phosphorylation of the Mr 28,000 protein at its maximal level. In conclusion, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation but suggest that the two activators TPA and DiC8 could induce distinct molecular events with regard to the enzyme recruitment and activity as well as to its further processing.