Effects of Diabetes and Sex Steroid Hormones on Insulin Receptor Tyrosine Kinase Activity in R3230AC Mammary Adenocarcinomas1

Insulin binding and receptor tyrosine kinase activity were investigated in the insulin-responsive R3230AC mammary adenocarcinoma. Insulin receptors, partially purified by wheat germ agglutinin-agarose Chroma tograph), displayed electrophoretic properties similar to those of normal tissues and demonstrated autophosphorylation of the ft subunit. Tyrosine kinase activity of tumor preparations was measured by incorporation of 32P from ATP into the synthetic polypeptide substrate glutamic adds,,:! yrosine»!,. The A',,,(app) for ATP, 15 to 30 nM in tumors from ovariectomized or intact rats, appeared to be increased by 10~7M insulin in vitro, with the calculated ¥„»» increased by 3to 5-fold; the Kr, (app) for glutamic acidso^yrosine^o was 2 to 3 nM and insulin increased the Vm.« by 25 to 50%. The effects of diabetes and insulin treatment and of various doses of estradiol, progesterone, estradiol plus progesterone, or tamoxifen on insulin binding, basal tyrosine kinase activity, and insulin inducible tyrosine kinase activity in vitro were studied in tumors from treated animals. Preparations from diabetic rats had elevated insulin binding and basal tyrosine kinase activity and displayed a striking doserelated increase in the ability for insulin induction of tyrosine kinase activity in vitm compared to intact animals; these effects of diabetes were prevented by administration of insulin. Over comparable doses, insulin growth factor 1 added in vitro induced tyrosine kinase activity minimally versus that seen for insulin. Treatment of rats with pharma cological doses of sex steroid hormones produced changes in insulin binding capacity and/or basal tyrosine kinase activity and, depending on dose, usually resulted in increased basal kinase activity relative to insulin binding. The insulin-inducible increase in tyrosine kinase activity in vitro was not altered by treatment with estradiol or estradiol plus progesterone in vivo, whereas high doses of progesterone attenuated the response. A consistent finding with increasing doses of sex steroids was an increase in the half-maximum dose or 50% maximum induction dose for insulin, implying reduced responsiveness. Tamoxifen administered to intact rats increased insulin binding and blunted the insulin-induced increase in tyrosine kinase in vitro; these effects were not seen in ovariectomized rats. We conclude that: (a) insulin receptors from R3230AC mammary tumors display properties similar to those of nonneoplastic tissues, including induction of tyrosine kinase activity by insulin in vitro; (b) tumor preparations from diabetic rats showed enhanced induction of tyrosine kinase to insulin versus intact or insulin-treated diabetic rats; (c) coordinate regulation of insulin binding and basal tyrosine kinase activity was not observed for tumors from sex steroid-treated rats; (</) progester one at therapeutic doses blunted the insulin-induced increase in tyrosine kinase activity; (e) pharmacological doses of these sex steroids increased the 50% maximum induction dose for insulin induction of tyrosine kinase activity; and (/) modulation of insulin-induced tyrosine kinase by thera peutic doses of sex steroids may be a potential site for influencing the action of insulin on this neoplasm.