The phospholipase D (PLD)-mediated synthesis of phosphatidylethanol (PtdEtOH) and the hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) were examined in drug-sensitive and multidrug-resistant lines of MCF-7 human breast carcinoma cells. In drug-sensitive (MCF-7/WT) cells, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) failed to enhance either the synthesis of PtdEtOH or the hydrolysis of either phospholipid. In the drug-resistant (MCF-7/MDR) cells, 100 nM PMA greatly enhanced both the synthesis of PtdEtOH (approximately 21-fold) and the hydrolysis of PtdEtn (approximately 29-fold), but had no effect on the hydrolysis of PtdCho. The PLD activators sphingosine and H2O2 were found to elicit only a slight (1.28-1.4-fold) stimulatory effect on PtdCho hydrolysis in both the MCF-7/WT and MCF-7/MDR cell types, and had only a small effect on PtdEtn hydrolysis in the MCF-7/WT cells as well. However, these agents significantly (approximately 2.6-3.5-fold) stimulated PtdEtn hydrolysis in the MCF-7/MDR cells. These data indicate that MCF-7/MDR cells contain a PtdEtn-specific PLD activity which can be selectively stimulated by PMA, sphingosine and H2O2.
DNA synthesis was measured 16 h after stimulation of Swiss 3T3 fibroblasts in the resting phase with various growth factors (platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid and thrombin). When extracellular Ca2+ was chelated by EGTA, or when the influx of Ca2+ from outside to inside the cell was blocked by cobalt, DNA synthesis was completely inhibited. As there was no effect whatsoever on DNA synthesis when Ca2+ was chelated, or when the influx of Ca2+ was blocked up to the first 4 h after growth stimulation, it was concluded that, at an early stage, Ca2+ influx from outside to inside the cell is not related to the transition from the G1 to the S phase. A Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) had no effect on DNA synthesis. However, cyclosporin A and FK-506, which are inhibitors of Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin), markedly inhibited DNA synthesis stimulated by all of the growth factors. These results indicate that calcineurin plays a role, not only in activation of T-cells of the immune system in the initial phase, but also in DNA synthesis in fibroblasts. It was concluded that Ca2+ influx from outside to inside the cell during the mid-to-late G1 phase, followed by calcineurin activation, is essential as a mechanism of growth signal transduction.
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