Analysis of phospholipid metabolism in murine keratinocytes transformed by the v-ras oncogene: relationship of phosphatidylinositol turnover and cytokine stimulation to the transformed phenotype.
Introduction of a v-rasHa oncogene into cultured mouse keratinocytes by transduction with a defective retrovirus is sufficient to transform keratinocytes to the benign phenotype. Transduced keratinocytes overexpress TGF alpha 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-rasHa keratinocytes are not induced to differentiate by Ca2+. We now demonstrate that v-rasHa 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 rasHa oncogene. The generation of inositol phosphates is even further stimulated in v-rasHa cells by an increase in extracellular Ca2+ or by exposure to aluminum fluoride. Thus, the v-rasHa 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. TGF alpha and medium conditioned by v-rasHa 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 Ca(2+)-induced terminal differentiation. In contrast, sustained elevation in basal phosphatidylinositol metabolism was produced by aluminum fluoride. Combined exposure to aluminum fluoride and exogenous TGF alpha caused hyperproliferation, resistance to Ca(2+)-induced differentiation and morphological changes identical to those of v-rasHa keratinocytes. These results provide a link between the biological consequences of v-rasHa gene expression and biochemical changes which are known to alter the keratinocyte phenotype.
Lee
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E
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, MD 20892.
Punnonen
K
K
Cheng
C
C
Glick
A
A
Dlugosz
A
A
Yuspa
S H
SH
D000535:Aluminum / Q000633:toxicity; D017607:Aluminum Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D005459:Fluorides / Q000633:toxicity; D011905:Genes, ras; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010641:Phenotype; D010716:Phosphatidylinositols / Q000378:metabolism*; D014162:Transfection
D017607:Aluminum Compounds; D016207:Cytokines; D004075:Diglycerides; D010716:Phosphatidylinositols; D000535:Aluminum; D002794:Choline; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride