ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 265, No. 1, August 15, pp. 38-42, 1988 Phorbol Ester Stimulation of Sphingomyelin Synthesis in Human Leukemic HL60 Cells’ ZOLTAN KISS,2 EVA DELI, AND J. F. KU0 Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322 Received December 18, 1987, and in revised form April 26, 1988 Pulse-chase experiments, performed with 14C-labeled choline, were used to study the possible effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) on the terminal step of sphingomyelin (CerPCho) synthesis from phosphatidylcholine in intact human promy- elocytic leukemic HL60 cells. Addition of TPA for the chase period significantly in- creased the rate of CerPCho synthesis; maximal stimulation (104%) required only 3 nM TPA. Treatment of cells with TPA for 6 h also increased the mass of CerPCho by 35%. Sphingosine (25 PM) or H7 (100 PM), inhibitors of protein kinase C (PKC) in vitro, inhibited some, but not all effects of TPA on endogenous protein phosphorylation in intact cells, and failed to inhibit TPA-stimulated synthesis of CerPCho. However, bryostatin, mezerein, 1-oleoyl-2-acetylglycerol, and polymyxin B, previously all shown to stimulate PKC in z)ivo, also stimulated the synthesis of CerPCho. It is suggested that the effect of phorbol ester on CerPCho synthesis is mediated by a subtype of PKC which responds to known activators of enzyme but is not inhibited by H7 or sphingosine. 0 1988 Academic Press, Inc. The biosynthesis of CerPCho,3 a major phospholipid of mammalian cell mem- branes (l), occurs by the transfer of the phosphorylcholine group from PtdCho to ceramide (2-10). Activators of PKC, such as the potent tumor promoter TPA, stimu- late both the synthesis (11-19) and degra- dation (20-26) of PtdCho. On the other hand, metabolism of CerPCho is not known to be regulated by any of the major second messenger systems. During a previous study of PtdCho syn- thesis in human promyelocytic leukemia i This work was supported by USPHS Grants CA-36777, HL-15696 and NS-17608. a To whom correspondence should be addressed at present address: National Institutes of Health, Bldg. 36, Rm. 1D 22, Bethesda, MD 20892. a Abbreviations used: PKC, protein kinase C; TPA, 12-0-tetradecanoylphorbol-13-acetate; CerPCho, sphingomyelin; PtdCho, phosphatidylcholine; H7, 1-(5-isoquinolinylsuIfonyl)-2-methylpiperazine; OAG, m-l-oleoyl-2-acetylglycerol; PMB, polymyxin B. HL60 cells, we found that incorporation of [14C]choline into PtdCho was stimulated by TPA (27,28), and noted that labeling of CerPCho was also increased (unpublished data). In the present study we demon- strate that TPA, and other PKC activa- tors, stimulate the synthesis of CerPCho. MATERIALS AND METHODS Materials. TPA, sphingosine, H7, PtdCho, and CerPCho were purchased from Sigma; [a*P]ortho- phosphate (carrier free) was from ICN Radiochemi- Cal; [methyl-“Clcholine chloride (50 mCi/mmol) was from Amersham. Cell culture. The human promyelocytic leukemia cell line HL60 (29) was cultured continuously in RPM1 1640 medium (GIBCO) supplemented with 20% head-inactivated fetal calf serum, penicillin- streptomycin (50 U/ml and 50 pg/ml, respectively), and glutamine (2 mM). Cells (passages 26-35) were harvested for experiments at a density of 1.5-2.0 X 106/ml. Labeling of CerPCho and PtdCho with [‘%]choline. Cells (2 X 106/ml) were incubated with [methyl-14C] choline (1 &i/ml) for 6 h in the absence or presence 0003-9861/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 38