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Binding Analysis of the Estrogen Receptor to Its Specific DNA Target Site in Human Breast Cancer¹

Departments of Molecular Biology [B. D. F., D. R. C.] and Pathology [F. F. P.], Vanderbilt University, Nashville, Tennessee 37232

The estrogen receptor (ER) is a nuclear protein with a hormone- and a DNA-binding domain. We examined the DNA binding of ER in MCF-7 cells and 79 primary breast cancers by gel mobility shift assay using as a probe the estrogen response element (ERE). The mobility shift assay showed saturable, specific binding of ER to ERE in crude, high molar extracts containing 4 mg/ml protein. Nonspecific binding was reduced by increasing concentrations of poly(deoxyinosidylate · deoxycytidylate) and shortening of the ERE probe from 35 to 15 base pairs. In the presence of Mg²⁺ the ER-ERE complex formation was hormone dependent at 22°C but not at 37°C. In the absence of Mg²⁺ estradiol was not necessary for ER-ERE complex formation. Correlation of the mobility shift assay with the hormone-binding (E₂) assay showed agreement in 55 of the 79 tumors. Both assays were positive (E₂+/ERE+) in 35 cases and both were negative (E₂-/ERE-) in 20 cases. In 11 tumors the hormone-binding assay was positive and the mobility shift assay negative (E₂+/ERE-), suggesting an alteration of the DNA-binding domain. In 13 cancers the hormone-binding assay was negative and the mobility shift assay positive (E₂-/ERE+) suggesting an alteration of the hormone-binding domain. By performing both hormone- and DNA-binding assays of ER and the hormone-binding assay of progesterone receptor (PR), we found the following subgroups of breast cancer: (a) E₂+/ERE+/PR+, (b) E₂+/ERE+/PR-, (c) E₂+/ERE-/PR+, (d) E₂+/ERE-/PR-, (e) E₂-/ERE+/PR+, (f) E₂-/ERE+/PR-, (g) E₂-/ERE-/PR-. The simultaneous determination of 17ß-estradiol and ERE binding may provide a better definition of the ER status of individual tumors and prove useful in refining endocrine therapy of patients with breast cancer.

¹ This work was supported by American Cancer Society Institutional Research Grant IN-25-30 (to B. D. F.) and, in part, by American Cancer Society Grant PDT-371 (to F. F. P.).

² To whom requests for reprints should be addressed, at Department of Pathology, Vanderbilt University, Nashville, TN 37232.

Received 10/ 3/89. Accepted 4/23/91.

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