Analysis of phospholipid metabolism in murine keratinocytes transformed by the v-ras oncogene: relationship of phosphatidylinositol turnover and cytokine stimulation to the transformed phenotype

Abstract

Introduction of a v-ras^Ha oncogene into cultured mouse keratinocytes by transduction with a defective retrovirus is sufficient to transform keratinocytes to the benign phenotype. Transduced keratinocytes overexpress TGFα and hyperproliferate in culture medium with 0.05 mM Ca²⁺. Whereas normal keratinocytes respond to elevated medium Ca²⁺ by cessation of proliferation and induction of terminal differentiation, v-ras^Ha keratinocytes are not induced to differentiate by Ca²⁺. We now demonstrate that v-ras^Ha keratinocytes have elevated basal levels of phosphatidylinositol, inositol phosphates and diacylglycerols in 0.05 mM Ca²⁺ medium. Basal turnover of phosphatidylcholine is not altered by the ras^Ha oncogene. The generation of inositol phosphates is even further stimulated in v-ras^Ha cells by an increase in extracellular Ca²⁺ or by exposure to aluminum fluoride. Thus, the v-ras^Ha gene product does not stimulate the inositol phospholipid pathway maximally and additional phosphatidylinositol is available for turnover in response to inducers of phospholipase C activity. TGFα and medium conditioned by v-ras^Ha keratinocytes, both of which stimulate proliferation of normal cells in 0.05 mM Ca²⁺, transiently increased phosphatidylinositol turnover in normal keratinocytes but did not inhibit Ca²⁺-induced terminal differentiation. In contrast, sustained elevation in basal phosphatidylinositol metabolism was produced by aluminum fluoride. Combined exposure to aluminum fluoride and exogenous TGFα caused hyperproliferation, resistance to Ca²⁺-induced differentiation and morphological changes identical to those of v-ras^Ha keratinocytes. These results provide a link between the biological consequences of v-ras^Ha gene expression and biochemical changes which are known to alter the keratinocyte phenotype.