The antiestrogen tamoxifen is widely used for endocrine therapy of breast cancer; however, the mechanisms of estrogen receptor-independent interactions of tamoxifen remain ill defined. Here we examine the effect of tamoxifen on the initial steps of cell signal transduction. To this end, phospholipid metabolism and protein kinase C (PKC) translocation were assessed in CCD986SK human mammary fibroblasts treated with tamoxifen. 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, tamoxifen induced the formation of phosphatidylethanol, demonstrating that tamoxifen activates phospholipase D (PLD). Cellular DG also increased in the presence of ethanol, showing that tamoxifen also activates phospholipase C (PLC). In cells prelabeled with choline and ethanolamine, tamoxifen caused increases in choline, phosphorylcholine, ethanolamine 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 diesters also activated PLD, but estrogen had no influence. Pretreatment of cells with phorbol dibutyrate (PKC down-regulation protocol) blocked phorbol diester-and tamoxifen-induced PLD activity. Exposure of cells to the PKC inhibitor GF 109203X diminished tamoxifeninduced PLD activity. Addition of tamoxifen to cultures elicited selective membrane association of PKC e. We conclude that tamoxifen exerts considerable extra-nuclear influence at the transmembrane signaling level. These events may contribute to effects beyond the scope of estrogen receptordependent actions.

Tamoxifen, a synthetic antiestrogen, is known for its antitumoral action in vivo; however, it is well accepted that many tamoxifen effects are elicited via estrogen receptor‐independent routes. Previously, we reported that tamoxifen induces PKC translocation in fibroblasts. In the present study, we investigated the influence of tamoxifen, and several triphenylethylene derivatives, on protein kinase C (PKC) in MCF‐7 human breast cancer cells. As measured by Western blot analysis, tamoxifen elicited isozyme‐specific membrane association of PKC‐ϵ, which was time‐dependent (as early as 5 min post‐treatment) and dose‐dependent (5.0–20 μM). Tamoxifen did not influence translocation of α, β, γ, δ or ζ PKC isoforms. Structure‐activity relationship studies demonstrated chemical requirements for PKC‐ϵ translocation, with tamoxifen, 3‐OH‐tamoxifen and clomiphene being active. Compounds without the basic amino side chain, such as triphenylethylene, or minus a phenyl group, such as N,N‐dimethyl‐2‐[(4‐phenylmethyl)phenoxy]ethanamine, were not active. In vitro cell growth assays showed a correlation between agent‐induced PKC‐ϵ translocation and inhibition of cell growth. Exposure of cells to clomiphene resulted in apoptosis. Since PKC‐ϵ has been associated with cell differentiation and cellular growth‐related processes, the antiproliferative influence of tamoxifen on MCF‐7 cells may be related to the interaction with PKC‐ϵ. Int. J. Cancer 77:928–932, 1998.© 1998 Wiley‐Liss, Inc.

No abstract

In this study we provide evidence for the involvement of protein kinase C (PKC) in phorbol diester-induced phosphatidylcholine (PC) hydrolysis by the phospholipase D pathway. Rat embryo fibroblasts (REF52) were prelabeled with either tritiated choline or my&tic acid; these compounds are preferentially incorporated into cellular PC. Phorbol diester-induced PC degradation was determined by measuring the release of PHIcholine, and the formation of PHlmyristoyl-containing phosphatidate (PA), diacylglycerol (DG), and phosphatidylethanol (PE). Staurosporine, a PKC inhibitor, blocked from 73 to 90% of the phorbol diester-induced PC hyrolysis. The inhibition of phorbol diester-induced choline release by staurosporine was dose dependent with an approximate ED,, of 150 nM. Pretreatment of cells with phorbol diester inhibited subsequent phorbol diester-induced PC degradation by 78-92s. A close correlation between the EDs0 for phorbol diester-stimulated choline release and the & for phorbol diester binding was demonstrated. Neither forskolin nor dibutyryl CAMP elicited cellular PC degradation. In vitro experiments using phospholipase D from Streptomyces chromofuscus showed that staurosporine did not inhibit and TPA did not stimulate enzyme activity.

No abstract