Biochimica et Biophysics Acta, 795 (1984) 447-451 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED Elsevier 447 BBA 51737 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA PHORBOL ESTERS STIMULATE PHOSPHATIDYLCHOLINE BIOSYNTHESIS BY TRANSLOCATION OF CTP: PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE FROM CYTOSOL TO MICROSOMES STEVEN L. PELECH, HARRY B. PADDON and DENNIS E. VANCE Department of Biochemistry, University of British Columbia, Vancouver, BC V6T I W5 (Canada) (Received January 30th, 1984) Key wora!s: Phosphatidylcholine synthesis; Phorbol ester; CTP : phosphocholine cytidy&ltransferase; (HeLu cell) Previous studies have demonstrated that 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates phosphati- dylcholine biosynthesis in HeLa cells. The stimulation was apparently caused by an acceleration of the reaction catalyzed by CTP : phosphocholine cytidylyltransferase (CTP : cholinephosphate cytidyltransferase, EC 2.7.7.15) (Paddon, H.B. and Vance, D.E. (1980) Biochim. Biophys. Acta 620,636-640). We now provide evidence that the enzyme activation is due to a translocation of the cytidylyltransferase from the cytosol to the microsomes. The rate of phospho[Me- 3HJcholine conversion into phosphatidylcholine was approx. 3-fold faster in HeLa cells treated with 100 nM TPA. This stimulation correlated with a 2.3-fold activation (P < 0.05) of cytidylyltransferase in homogenates from treated cells. There was a 1.7-fold increase in the enzyme associated with microsomes (P < 0.05) and a corresponding decrease in enzyme recovered from cytosol (P < 0.01). The total amount of enzyme recovered from the homogenates was unchanged. Further evidence for TPA causing an increased association of cytidylyltransferase with cellular membranes was obtained when cells were treated with digitonin. The release of cytidylyltransferase into the medium was inhibited by 4-fold from cells previously treated with TPA. Similar results on phospho[Me-3H]choline incorporation into phosphatidylcholine were found with cells incubated with phorbol-12,13_dibutyrate, a water-soluble tumor-promoting agent. Introduction zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA The underlying mechanism of tumor-promoter action has attracted considerable interest. In vari- ous cell lines from humans [l-3] and other vertebrates [4,5], phorbol esters have been espe- cially effective for evoking cellular transformation. Although there are many phenotypic changes in response to phorbol esters, increased phosphati- dylcholine turnover and induction of ornithine decarboxylase have been commoi~ly described [6-121. Induction of ornithine decarboxylase is essential but not a sufficient factor for skin tumor Abbreviation: TPA, 12-0-tetradecanoyl phorbol 13-acetate. promotion 113,141. A priori, increased phosphati- dylcholine biosynthesis would be expected for membrane biogenesis in faster growing trans- formed cells. The degree of importance of en- hanced phosphatidylcholine synthesis for mainte- nance of transformation still requires assessment. The action of phorbol esters on phosphati- dylcholine metabolism has been well studied in the human cervical carcinoma cell line, HeLa cells. TPA, the most potent of the phobol ester family [4,15], produced a 2-4-fold stimulation of [Me- ‘HIcholine incorporation into phosphatidylcholine of HeLa cells within 20 min [8,16]. This response was not influenced by inhibitors of RNA and protein synthesis, although colchicine enhanced 0005-2760/84/$03.00 0 1984 Elsevier Science Publishers B.V.