Abstract
Polypeptide growth factors that stimulate cell proliferation bind to cell surface receptors and activate intracellular signal transduction pathways. One major signalling pathway, initiated by phosphatidylinositol (PI) turnover, involves activation of protein kinase C. Some polypeptide growth factors, including mitogens that activate protein kinase C, induce a rapid increase in expression of the proto-oncogenes, c-myc and c-fos. In order to characterize the signal transduction pathways responsible for proto-oncogene activation, we treated Swiss 3T3 cells with the tumor promoter phorbol dibutyrate to generate cells deficient in protein kinase C. These cells were then stimulated with platelet extract, bombesin, or epidermal growth factor (EGF) and the levels of c-myc and c-fos mRNA were determined. Platelet extract or bombesin, which stimulate PI turnover, were substantially weaker inducers of c-myc and c-fos mRNA levels in the protein kinase C-depleted cells, although some variability with platelet extract was noted. EGF, which does not stimulate PI turnover in several cell systems, was by contrast a potent inducer of both proto-oncogenes whether or not the cells were deficient in protein kinase C. Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small but significant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways.
Highlights
From the $Department of Applied Biological Sciences, Massachusetts Institute of Technology, Growth factorsthatstimulatethe proliferation of 3T3 murine fibroblastsinclude platelet-derived growth factor
Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small butsignificant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways
We demonstrate that the abiliotfyEGF toinduce cmyc and c-fos mRNA levels is completely independent of proteinkinase C
Summary
Cell Culture-Swiss 3T3 cells were obtained from the American Type Culture Collection. Thecells were washed with cells with phorbol esters causes a disappearance of protein phosphate-buffered saline and scraped in 20 mM Tris, pH 7.5, 1%Triton X-100, 2 mM EDTA, 0.5 mM EGTA, 10 pg/ml aprotinin, 1mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol. These extraction conditions solubilize both cytoplasmic and particulate protein kinase C (21). The area under the peak of the column profile was determined This value was adjusted for the amount of protein loaded onto the columns, Platelet extract (50 units/ml) induced c-myc mRNA 20- to 30-fold in untreated (control) cells. After extraction with phenokchloroform (l:l), the RNA was precipitated in 70% ethanol and 200 mM NaC1, collected by centrifugation, lyophilized, dissolved
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