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 chromatography, displayed electrophoretic properties similar to those of normal tissues and demonstrated autophosphorylation of the β subunit. Tyrosine kinase activity of tumor preparations was measured by incorporation of 32P from ATP into the synthetic polypeptide substrate glutamic acid80:tyrosine20. The Km (app) for ATP, 15 to 30 µm in tumors from ovariectomized or intact rats, appeared to be increased by 10-7m insulin in vitro, with the calculated Vmax increased by 3- to 5-fold; the Km (app) for glutamic acid80:tyrosine20 was 2 to 3 µm and insulin increased the Vmax 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 dose-related increase in the ability for insulin induction of tyrosine kinase activity in vitro 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 pharmacological 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; (d) progesterone 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 (f) modulation of insulin-induced tyrosine kinase by therapeutic doses of sex steroids may be a potential site for influencing the action of insulin on this neoplasm.

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This work was supported by USPHS Grant CA-16660 and the Animal Tumor Research Facility, University of Rochester Cancer Center (Grant CA-11198).

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