TAMOXIFEN ACTIVATES CELLULAR PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D AND ELICITS PROTEIN-KINASE-C TRANSLOCATION

The antiestrogen tamoxifen is widely used for endocrine therapy of bre ast cancer; however, the mechanisms of estrogen receptor-independent i nteractions of tamoxifen remain ill defined, Here we examine the effec t of tamoxifen on the initial steps of cell signal transduction. To th is end, phospholipid metabolism and protein kinase C (PKC) translocati on were assessed in CCD986SK human mammary fibroblasts treated with ta moxifen, The addition of tamoxifen resulted in dose-dependent and time -dependent increases in the cellular second messengers phosphatidate ( PA) and diacylglycerol (DG), On addition of ethanol to the medium, tam oxifen induced the formation of phosphatidylethanol, demonstrating tha t tamoxifen activates phospholipase D (PLD). Cellular DG also increase d in the presence of ethanol, showing that tamoxifen also activates ph ospholipase C (PLC), In cells prelabeled with choline and ethanolamine , tamoxifen caused increases in choline, phosphorylcholine, ethanolami ne and phosphorylethanolamine. Structure-activity relationship studies for activation of PLD revealed that tamoxifen was the most effective, whereas 4-hydroxy tamoxifen was nearly devoid of activity. Phorbol di esters also activated PLD, but estrogen had no influence, Pretreatment of cells with phorbol dibutyrate (PKC down-regulation protocol) block ed phorbol diester- and tamoxifen-induced PLD activity, Exposure of ce lls to the PKC inhibitor GF 109203X diminished tamoxifen-induced PLD a ctivity. Addition of tamoxifen to cultures elicited selective membrane association of PKC epsilon. We conclude that tamoxifen exerts conside rable extra-nuclear influence at the transmembrane signaling level. Th ese events may contribute to effects beyond the scope of estrogen rece ptor-dependent actions. (C) 1997 Wiley-Liss, Inc.