Complementation for the growth response to insulin and MSA occurs at a step distal to hormone‐receptor interaction in mouse embryo fibroblast × melanoma cell hybrids

The PG19 mouse melanoma cell line does not respond to the mitogenic effects of insulin either in serum‐deprived quiescent cultures or when growing in hormone‐supplemented serum‐free medium, while melanoma × mouse embryo fibroblast hybrids respond under both conditions. It has been proposed that some growth effects of insulin are mediated by its binding to receptors for insulin‐like growth factors. To determine whether the PG19 cells do not respond to insulin because they lack receptors for MSA (multiplication‐stimulating activity, an insulin‐like growth factor produced by BRL‐3A cultured rat liver cells), we have examined growth effects and receptor binding of MSA in the melanoma and hybrid cells. MSA stimulates [3H]thymidine incorporation in three melanoma × mouse embryo fibroblast hybrid clones but not in the parental melanoma cells. Thus the pattern of response of the melanoma cells and hybrids to MSA directly parallels their response to insulin. The melanoma cells and hybrids all have high‐affinity specific MSA binding sites (KD = 10–27 nM), indicating that the inability of the melanoma cells to respond to MSA is not attributable to a lack of MSA receptors. Insulin competes very poorly for binding of MSA to hybrid clone 7 but is nevertheless able to stimulate [3H]thymidine incorporation in this clone. This strongly suggests that the effects of insulin on [3H]thymidine incorporation in this clone are not mediated by its binding to MSA receptors. Insulin and MSA stimulate protein synthesis and inhibit protein degradation in the hybrids but not in the parental melanoma cells. These results suggest that both insulin and MSA action are blocked in the melanoma cells at a step distal to the binding of either hormone to its cellular receptors but prior to stimulation of protein synthesis.