Inhibition of MCF-7 Cell Growth by 12-0-Tetradecanoylphorbol-13-acetate and 1,2-Dioctanoyl-sw-glycerol: Distinct Effects on Protein Kinase C Activity1

We have investigated the effects of phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoylsn-glycerol (l)if„)on MCF-7 cell proliferation and protein kinase C activity. l)i< Hmimics the effects of TPA on both cell morphology and proliferation, with an ED»value of 11 ^g/ml for cell growth inhibition. As with TPA and phorbol 12,13-dibutyrati-, DiC'nenhances the degree of phosphorylation of an endogenous M, 28,000 protein in a timeand dosedependent 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 EDsoSobserved are 5 ng/ml and 20 Mg/ml,respectively, for phorbol esters and I)i( „¿ . The M, 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 ki nase 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 paniculate 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, 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. INTRODUCTION Tumor promoter phorbol esters such as TPA3 induce various biochemical and biological effects in cultured cells, including striking stimulatory or inhibitory effects on cell proliferation and differentiation (1,2). In MCF-7 human breast cancer cells, TPA and other active phorbol esters cause growth arrest (3-5) and changes in cell morphology (6). The only currently recog nized mediator of the TPA action is the Ca2+-and phosph oli piddependent protein kinase C (7-9), which most probably repre sents the high affinity phorbol ester receptor in target cells (812). There is increasing evidence that protein kinase C plays a pivotal role in the transmembrane signaling of a wide variety of extracellular stimuli including growth factors, hormones and other biologically active substances (for reviews, see References 7, 13, 14). The physiological activator of protein kinase C is DAG which accumulates transiently as a consequence of the receptor-meReceived 4/25/88; revised 8/11/88; accepted 9/1/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by Institut National de la Santéet de la Recherche Médicale and by Association de la Recherce contre le Cancer. 2To whom requests for reprints should be addressed. 3The abbreviations used are: TPA, 12-0-tetradecanoylphorbol-13-acetate; PDBu, phorbol 12,13-dibutyrate; DAG, 1,2-i/i-diacylglycerol; DiC,, 1,2-dioctanoyl-sn-glycerol; EGTA, ethylene glycol bis (/3-aminoethylether)-JV,A',JV',A''-tetraacetic acid; SDS, sodium dodecyl sulfate; NP-40, nonidet P-40; PBS, phosphate buffered saline; TCA, trichloroacetic acid. dialed inositol phospholipid breakdown (7, 14, 15). TPA can substitute for intracellular messenger DAG to activate protein kinase C (16, 17). As in many other cell types (17, 18) TPA induces a rapid subcellular redistribution of protein kinase C in MCF-7 cells, followed by a progressive disappearance of the enzyme upon prolonged cell treatment (19-22). In order to further define the role of protein kinase C in the inhibition of MCF-7 cell proliferation, we have investigated whether the permeant synthetic DiC8 could mimic the effects of TPA on protein kinase C activity and cell growth. We report that the two activators induce similar changes in cell morphol ogy and inhibit cell proliferation identically. DiCg and TPA similarly increase the degree of phosphorylation of a cytosolic protein with 28,000 molecular weight in intact MCF-7 cells but slightly distinct protein phosphorylation patterns can be dem onstrated by using two-dimensional electrophoresis. Moreover, contrary to TPA and PDBu, DiCg causes only a partial and transient translocation of protein kinase C from the cytosolic to the paniculate compartment. Finally, DiCg is unable to trigger the down-regulation of protein kinase C which occurs at the membrane level after TPA or PDBu stimulation. MATERIALS AND METHODS Chemicals. Histone HI, TPA, phosphatidyl-serine, 1,2-dioleoyl-glycerol and DiC8 were obtained from Sigma. [7-32P]ATP (0.5-3 Ci/mmol) and [32P]phosphoric acid were purchased from Amersham. Acrylamide and bisacrylamide were obtained from Biorad. All other chemicals were from Merck. Cell Culture. MCF-7 cells were grown at 37'C in RPMI 1640 (GIBCO), pH 7.3, supplemented with 2 g/liter of sodium bicarbonate, 2 HIM L-glutamine, 1 ¿iMinsulin, and 5% fetal calf serum (PCS, Seromed). Culture media were changed every 2 days. For cell growth measurement, MCF-7 cells were plated at an initial density of 1-1.S x IO4cells per 35-mm dish. After 48 h (Day 0) the medium was replaced by fresh RPMI-5% PCS and phorbol esters or DiC8 were added at various concentrations. Addition of the permeant diacylglycerol was repeated three times a day whereas phorbol esters were added every 2 or 3 days. Control dishes received the same volume of the solvent acetone (final concentration of 0.1%). Cells were harvested with 0.05% trypsin-0.02% EDTA and cell number was determined by using a Coulter Counter (Coultronics). Protein Phosphorylation. Subconfluent cultures (1 x K)'1cells/dish) were washed twice in a phosphate-free Krebs-Ringer buffer, pH 7.2, containing 20 HIMHEPES, 0.1 % BSA, and 0.2% glucose, and incubated for 2 h at 37'C in 1 ml of the same buffer containing 50 ^Ci ["Piphosphoric acid. Stimuli were then added for a further 30 min period. Cells were washed twice with cold PBS and 10% TCA was added. TCAprecipitated proteins were dissolved in 150 /¿I of electrophoresis sample buffer containing 0.06 M Tris-HCl, pH 6.7, 2% SDS, 8% glycerol, 2% /3-mercaptoethanol and 0.005% bromophenol blue. Samples were then boiled for 5 min at 90°C. Proteins were fractionated by electrophoresis on 4.5 and 12% (w/v) discontinuous SDS-polyacrylamide slab gel as described by Laemmli (23). After protein Fixation by cold trichloroacetic acid and Coomassie blue staining, the gels were dried, then exposed to Hyperfilms-MP for 48-72 h. The autoradiographs were scanned by densitometry and the 32Pincorporated into the proteins was evaluated by measuring the respective peak areas as previously indicated (24). Alternatively, TCA-precipitated proteins were subjected to a two-