Effects of the N-RAS oncogene on differentiation of CO25 myoblast cells

Abstract

Effects of a mutant N-ras oncogene (under transcriptional control of the glucocorticoid- inducible MMTV-LTR promotor) on the differen- tiation of a CO25 myoblast cell line have been investigated using various techniques. Scanning electron microscopy demonstrated that CO25 cells were more rounded in shape with numerous microvilli on their surface after induction of the N-ras oncogene by dexamethasone. After 10 days, they lost their anchorage dependency and formed foci. In contrast, the control cells formed long branched myotubes with a few microvilli on their surface in the absence of dexamethasone. Distribution of F-actin was investigated by fluoresence micros- copy using rhodamine-phalloidin. There was a dramatic change in actin organization during transformation of the cells. Stress fibers were replaced by a diffuse bright fluorescence in ras-induced cells. Transcription levels of the cellular oncogenes, c-myc, p53, c- Ki-ras and a control B-actin gene, were examined by Northern blotting. During differentiation of CO25 cells, mRNA levels of these genes dec- lined up to 2-3 days and returned to the initial levels in 4 days. Following induction of the N-ras oncogene, the level of c-myc mRNA first decreased in 2 days and then increased in 3 and 4 days. The level of B-actin mRNA was lower during transformation, and there were no changes in the mRNA levels of p53 and c-Ki-ras genes.