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CHO-K1 cells, wild type (WT), grow in a defined medium with insulin as the only essential hormone. When starved for insulin, these cells accumulate in G0/G1 stage. Insulin binding to its receptor stimulates DNA synthesis and cell division and induces an increase in abundance of mRNA for c-fos, c-jun, Krox-20, Krox-24 (zif/268), fra-1, jun-B, c-myc, and JE. The kinetics of induction of these genes are similar to that shown with serum induction of 3T3. These genes show maximum stimulation at insulin concentrations of 20, 160, or 320 ng/ml and their expression is inhibited at higher concentrations. The addition of cycloheximide results in superinduction. The WT and insulin-independent mutants show no detectable signal for KC, fos-b, or nur77 and no increase over the basal level of pI-15, probably eliminating these genes as participants in the insulin mitogenic signal. These mutants synthesize DNA in the absence of insulin at rates that vary from 4 to 12 times that of the quiescent (insulin unstimulated) WT and are further inducible by insulin. The mutants have "constitutive" levels of Krox-24 (zif/268), fra-1, jun-B, c-myc, and JE (INS-type 2 genes) mRNAs that vary from mutant to mutant, reaching a maximum of an 8-fold increase for fra-1 and JE over the quiescent WT levels. There were no detectable levels of mRNA for genes c-fos and Krox-20 and no increase in level of mRNA for c-jun (INS-type 1 genes) as compared to the quiescent WT. Thus, although these INS-type 1 and type 2 genes may be involved in the full insulin mitogenic signal, the constitutive up-regulation of only genes in INS-type 2 is sufficient for insulin-independent DNA synthesis and cell division. Analysis of hybrids constructed between WT and mutant 27 indicate that the mutant phenotype is recessive, pointing to the existence of a regulatory gene producing a negative regulator.

proceedings of the national academy of sciences of the united states of america

Analysis of the genes involved in the insulin transmembrane mitogenic signal in Chinese hamster ovary cells, CHO-K1, utilizing insulin-independent mutants.