Studies on the mechanism by which phorbol ester tumor promoters inhibit differentiation in friend erythroleukemic cells (cyclic nucleotides, diacylglycerols, protein phosphorylation)
Description
The mechanism of action by which phorbol ester tumor promoters inhibit dimethylsulfoxide (DMSO)-induced differentiation was investigated in Friend erythroleukemic cells (FELC). In most experiments, about 80% of the cells became benzidine-positive when incubated with 1.5% DMSO. The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), inhibited DMSO-induced differentiation in FELC in a dose-responsive manner. In order to investigate the mechanism of action by which TPA inhibits DMSO-induced differentiation, the roles of cyclic nucleotides, calcium, diacylglycerols and protein phosphorylation were examined TPA had no effect on intracellular cyclic AMP levels in incubations ranging from 15 sec to 20 min. Protein phosphorylation experiments revealed that TPA enhanced phosphorylation of a number of different proteins compared to control cells. Comparison to autoradiographs of cells exposed to dibutyrylcyclic AMP or 8-bromo-cyclic GMP revealed that TPA was activating a protein kinase other than cyclic nucleotide dependent protein kinases Diacyglycerols and TPA have recently been demonstrated to activate a calcium dependent, phospholipid dependent protein kinase in vitro. Diacylglycerols were therefore used in the FELC culture in order to study their effect on differentiation. Diacylglycerols were found to inhibit DMSO-induced differentiation in a dose-responsive manner with the order of potency being 1-oleoyl-2-acetylglycerol (OAG) > dicaprylin = dilaurin > diolein. Phospholipase C is an enzyme which releases endogenous diacylglycerols from membrane phospholipids. Phospholipase C was also found to inhibit DMSO-induced differentiation in a dose-responsive manner. These results demonstrate that diacylglycerols can inhibit DMSO-induced differentiation in FELC in a manner analogous to TPA. These findings support the hypothesis that diacylglycerols and phorbol ester tumor promoters can have similar biological effects and suggest that diacylglycerol and TPA may have a common mediator. Protein phosphorylation studies imply that TPA activates a protein kinase that is cyclic nucleotide independent and differs from calcium-calmodulin dependent protein kinases. (Abstract shortened with permission of author.)