Differential effects of diacylglycerols and 12-O-tetradecanoylphorbol-13-acetate on protein phosphorylation and cell proliferation of tumor promoter-dependent leukemia cell line A65T

The Growth of A65T cells, a mouse thymic leukemia cell line, was stricktly dependent on the presence of tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and mezerein. In the presence of the tumor promoters, A65T cells proliferated rapidly in a concentration dependent manner. When the cells were cultured with a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) or 1, 2-dicaprylin, cell proliferation was not stimulated. In addition, bihourly cumulative addition of OAG or 1, 2-dicaprylin again failed to induce A65T cell proliferation. Phospholipase C(PLC) induced a concentration dependent stimulation of A65T cell proliferation, though the effect was slight compared with that of the tumor promoters. Protein kinase C activity was detected both in cytosol and particulate fractions of A65T cells. When the cells were incubated in the TPA-free medium for 5 h, the protein kinase C activity in the cytosol fraction increased whereas a the activity in the particulate fraction decreased. Treatment of the cells with the tumor promoters mentioned above resulted in the increased phosphorylation of proteins with apparent mol;. wts of 27 000 and 68 000. The effects were concentration dependent and the half maximal phosphorylation was observed either with 3.6 nM TPA, 4.5 ng/ml teleocidin or 0.33 μM mezerein, respectively. On the other hand, a half maximal effect on cell proliferation was observed either with 0.14 nM TPA, 47 pg/ml teleocidin or 6.3 nM mezerein, respectively. At these concentrations, these tumor promoters never induced the detectable stimulations of the protein phosphorylation. A synthetic diacylglycerol 1, 2-dicaprylin failed to stimulate the phosphorylation of 27- and 68-kd proteins, but stimulated the phosphorylation of proteins with apparent mol. wts of 100 000 and 54 000. The effect was concentration-dependent and a half maximal stimulation was observed with 35 μg/ml 1, 2-dicaprylin. Neither TPA, teleocidin nor mezerein stimulated the phosphorylation of these 100- and 54-kd proteins. OAG and PLC failed to induce any detectable alterations in the protein phosphorylation in A65T cells. Both OAG and 1, 2-dicaprylin, which we used, actually activated partially purified mouse brain protein kinase C in a concentration dependent manner. These results clearly demonstrate that in A65T cells TPA and diacylglycerol mutually stimulate the phosphorylation of the completely different set of endogenous proteins, and also suggest that the cell-proliferating effects of the tumor promoters do not appear to be mediated through the phosphorylation of 27-and 68-kd proteins.