Phorbol ester inhibits phosphatidylserine synthesis in human promyelocytic leukaemia HL60 cells. Possible involvement of free radicals and correlation with phosphorylation of nuclear protein 1b

Abstract

Treatment of human promyelocytic leukaemia HL60 cells in conditioned medium with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 4 h resulted in 25-30% inhibition of labelling of phosphatidylserine (PS) with [U-14C]serine. PS labelling was 40% lower, and no inhibitory TPA effect was observed when the experiments were performed in fresh medium. Cycloheximide or puromycin also inhibited PS labelling by 38-44%; their inhibitory effects were non-additive with that of TPA and occurred only in conditioned medium. Catalase (CAT) and superoxide dismutase (SOD), both free-radical scavengers, and H7, a protein kinase C inhibitor, reversed to various extents the inhibitory effect of TPA on PS synthesis. On the other hand, chlorobenzoic acid, a free-radical-generating agent, also inhibited PS synthesis by 22% after 4 h treatment when conditioned medium was used. When ethanolamine was added to cells in conditioned medium to quench PS formation through the exchange of free serine with the ethanolamine moiety of phosphatidylethanolamine (PE), PS labelling was decreased by 33% and the inhibitory TPA effect was significantly decreased. On the other hand, ethanolamine had marginal quenching effect on PS labelling when added to cells in fresh medium. TPA increased the phosphorylation of various proteins in the cells, including protein lb (Mr 80,000; pI 5.5) shown to be localized mainly in the nuclear fraction. Chlorobenzoic acid selectively stimulated the phosphorylation of protein lb, whereas CAT and SOD specifically attenuated the TPA-stimulated phosphorylation of this protein. All these agents affected phosphorylation of protein lb only if conditioned medium was used. The findings suggested that net synthesis of PS through the base-exchange mechanism was stimulated in HL60 cells by cell products present in the conditioned medium. TPA inhibited this stimulated PS synthesis by a mechanism which appeared to involve active oxygen species and protein synthesis and might be related to the phosphorylation of protein lb.