Mitogen-potentiating action and binding characteristics of insulin and insulin-like growth factors in Chinese hamster fibroblasts

alpha-Thrombin alone is able to stimulate DNA synthesis reinitiation of G0-arrested Chinese hamster lung fibroblasts (CC139) as well as continued growth of these cells in serum-free medium. Although insulin at high concentrations (1-10 micrograms/ml) is not intrinsically mitogenic for these cells, it potently enhances the growth-promoting action of thrombin. The generation time of CC139 cells in the defined medium, transferrin, alpha-thrombin, insulin, is around 15 h. To determine whether this effect of insulin is mediated via putative receptors for the insulin-like growth factors (IGFs) on these cells, we examined the abilities of two IGFs, Multiplication-Stimulating Activity (MSA) and IGF-I, to potentiate the thrombin-induced reinitiation of DNA synthesis. Both IGFs were found to be as effective as insulin for this biological effect; however, much lower concentrations were required to elicit half-maximal response, 100 ng/ml of MSA and 30 ng/ml of IGF-I. Detailed binding studies using 125I-labelled insulin, MSA, and IGF-I revealed that CC139 cells specifically bind all three polypeptides with IC50 values for the corresponding ligands of 1-2 ng/ml, 80-100 ng/ml, and 30-40 ng/ml, respectively. 125I-MSA binding was insulin-insensitive, whereas insulin did compete with 125I-IGF-I for binding to CC139 cells. These results indicate that CC139 cells possess at least two types of IGF receptors, an insulin-insensitive IGF receptor with high affinity for MSA which apparently mediates its biological effect, and an insulin-sensitive IGF-I receptor. Insulin appears to exert its mitogen-potentiating activity in CC139 fibroblasts by interacting with the IGF-I receptor.