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Inhibition of MCF-7 Cell Growth by 12-O-Tetradecanoylphorbol-13-acetate and 1,2-Dioctanoyl-sn-glycerol: Distinct Effects on Protein Kinase C Activity¹

Institut National de la Santé et de la Recherche Médicale, U 168, Department of Endocrinology, CHU Rangueil, Université Paul Sabatier, 31054 Toulouse Cedex, France

We have investigated the effects of phorbol ester 12-O-tetradecanoyl-phorbol-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 µg/ml for cell growth inhibition. As with TPA and phorobol 12,13-dibutyrate, DiC₈ enhances the degree of phosphorylation of an endogenous M_r 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 µg/ml, respectively, for phorbol esters and DiC₈. The M_r 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 M_r 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.

¹ Supported by Institut National de la Santé et de la Recherche Médicale and by Association de la Recherce contre le Cancer.

² To whom requests for reprints should be addressed.

Received 4/25/88. Revised 8/11/88. Accepted 9/ 1/88.

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