Antipain-induced suppression of oncogene expression in H-ras-transformed NIH3T3 cells

Antipain (AP; 50 micrograms/ml) inhibits transformation of NIH3T3 cells after transfection with an activated H-ras oncogene. To determine whether AP effects on transformation are associated with alterations in oncogene expression, NIH3T3 cells were cotransfected with an activated H-ras oncogene and the selectable marker gene aph, and gene expression was quantified. Fifty percent of geneticin-resistant colonies which were exposed to AP failed to express the transformed phenotype as determined by their inability to grow in soft agar. Northern blot analysis of the transformed and nontransformed colonies revealed that suppression of H-ras transformation by AP was associated with a decrease in expression of the exogenously transfected H-ras gene by approximately 4-fold. Expression of the endogenous oncogene c-myc was decreased by approximately 2.5-fold, to levels seen in untransfected cells. AP-treated colonies that retained the transformed phenotype had levels of oncogene expression that were similar to untreated ras-transformed colonies. Southern blot analysis revealed no effects of AP on incorporation or copy number of the H-ras gene.