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Estrogen Receptor Binding to Nuclei from Normal and Neoplastic Rat Mammary Tissues in Vitro¹

Departments of Biochemistry and Cancer Center, University of Rochester Medical Center, Rochester, New York 14642

To investigate the role of hormones in regulating growth of neoplastic mammary cells, we established a heterologous assay for studying interactions of partially purified calf uterine [³H]estradiol-charged estrogen receptor ([³H]ER) with rat tumor nuclei in vitro. This system displays saturable high affinity binding of [³H]ER which is time and salt dependent. Optimal assay conditions required for the heterologous system were identical to those we reported for the homologous calf nuclear binding system. Specificity of [³H]ER binding was demonstrated; 10-fold excess unlabeled estrogen-charged ER (EcR) competed for >90% of the [³H]ER binding sites and binding of [³H]estradiol (not complexed with ER) was <1% of [³H]ER binding. Binding of [³H]ER displayed tissue specificity in decreasing order: R3230AC mammary tumor > lactating mammary gland = liver > kidney > lung. Scatchard analysis of saturation data provided estimates of binding affinity to nuclei from R3230AC mammary tumors [K_d, 2.0 ± 0.3 (SE) nM); the number of binding sites per nucleus for R3230AC tumors was 95,000 ± 13,800. [³H]ER binding to nuclei isolated from R3230AC rat mammary tumors grown in intact rats was 40% higher than that observed in tumors from ovariectomized animals. Results of administration of individual pharmacological doses of either progesterone or an estrogen to ovariectomized rats did not restore nuclear ER binding levels in R3230AC tumors to those detected in tumors from intact rats. These results suggest that the physiological levels of endogenous hormones produced by the ovaries are important in regulating the number of ER binding sites in nuclei from these mammary tumors.

¹ The work was supported by Grant BC-475 from the American Cancer Society. Presented in part at the Seventy-seventh annual meeting of the American Association for Cancer Research, Los Angeles, CA, May 1986.

² Supported by Cancer Research Training Grant T32CA09363.

³ Supported by American Cancer Society Faculty Research Award FRA220.

⁴ To whom requests for reprints should be addressed, at Department of Biochemistry, Box 607, The University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642.

Received 10/27/86. Revised 2/27/87. Accepted 3/ 6/87.

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