ras‐Transformation of MDCK cells alters responses to phorbol ester without altering responses to bradykinin

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Abstract
The results of studies to evaluate the hypothesis that the 21 kDa GTP‐binding protein derived from the ras oncogene is involved in regulation and coupling of hormone receptors to phospholipase activity have thus far been inconsistent. We therefore examined the effect of H‐ras transformation on basal, tumor‐promoting phorbol ester (TPA)‐stimulated, and bradykinin‐mediated phospholipid hydrolysis in Madin Darby canine kidney cells (MDCK) by comparing H‐ras‐transformed MDCK cells (MDCK‐RAS) to two non‐transformed strains of MDCK cells (MDCK‐DI and MDCK‐ATCC). In unstimulated MDCK‐RAS, diacylglycerol (DAG), inositol phosphate accumulation, and choline phosphate release were increased while arachidonic acid and arachidonic acid metabolite (AA) release was not increased, suggesting that ras transformation increased phospholipase C activity. Protein kinase C (PK‐C) activity was decreased, and specific binding of (3H) phorbol ester was reduced in MDCK‐RAS relative to the non‐transformed MDCK cells suggesting that elevated DAG may activate and thereby down‐regulate PK‐C. Consistent with this finding in MDCK‐RAS, TPA‐stimulated AA release and subsequent prostaglandin E2 production were decreased, while TPA‐stimulated choline phosphate release was increased. Bradykinin receptor‐stimulated phospho‐lipid hydrolysis in MDCK‐RAS was similar to that of non‐transformed cells, suggesting that the ras‐derived protein does not directly couple bradykinin receptors to phospholipases in MDCK cells. However, the ability of TPA‐treatment to inhibit bradykinin‐stimulated phosphoinositide hydrolysis and enhance bradykinin‐stimulated AA release was attenuated in MDCK‐RAS. Additionally, in MDCK‐RAS the conversion of arachidonic acid to prostaglandin E, was substantially reduced. We conclude that rus transformation of MDCK cells increases DAG levels, thereby activating and, in turn, down‐regulating PK‐C and certain responses to TPA. Since activation of PK‐C may result in a variety of effects on signal transduction pathways, we propose that increased DAG and altered PK‐C levels associated with rus transformation may account for the inconsistent effects previously observed in studies evaluating the effect of rus transformation on phospholipases and other signal transduction systems.