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Biological Activity and Receptor Binding of a Strongly Interacting Estrogen in Human Breast Cancer Cells¹

Departments of Physiology and Biophysics [G. C. A. R., B. S. K.] and Chemistry [R. D. B., J. A. K.], University of Illinois, and University of Illinois College of Medicine [G. C. A. R., B. S. K.], Urbana, Illinois 61801

A substantial proportion of human breast cancers contain estrogen receptors, and it is believed that the growth of some of these tumors and their synthesis of specific proteins are stimulated by estrogens. Since natural estrogens, such as estradiol, react reversibly with estrogen receptors, it was of interest to determine the biological consequences that would result from very strong, possibly irreversible interaction of an estradiol-based ligand with the estrogen receptor of breast cancer cells. For these studies, we have examined the receptor interactions and biological character of 11ß-chloromethylestradiol (CME) and 11ß-bromomethylestradiol (BME) as potential estradiol-based affinity labeling ligands in MCF-7 human breast cancer cells which contain high levels of estrogen receptors.

The apparent relative binding affinities of CME and BME for MCF-7 estrogen receptor measured by competitive binding assay are 230 and 15%, respectively, whereas the affinity of estradiol is considered 100%. Incubation of receptor preparations from MCF-7 cells or rat uteri with CME at 21° results in a concentration- and time-dependent decrease in receptor content measured by exchange assays with [³H]estradiol. This may be due to covalent attachment of CME to receptor and is termed "inactivation." Inactivation of 80 to 85% of the receptors occurs within 30 min at 21° by exposure to 5 or 20 nM CME, with 2 nM giving 20 to 40% inactivation. This receptor inactivation is prevented by preincubation with 2000 nM estradiol, indicating that the interaction of CME is occurring at the estradiol binding site on the receptor. MCF-7 cells incubated with 20 nM CME show a rapid loss of cytosol receptor sites and no accumulation of receptors detectable by exchange assay in the nucleus, while 20 nM estradiol shows nuclear localization of receptor. BME, in contrast, inactivates only a portion (approximately 40%) of estrogen receptors.

CME and BME both behave as estrogen agonists. They stimulate the proliferation of MCF-7 cells and increase cellular progesterone receptor content and plasminogen activator activity. CME is at least as potent as estradiol on a molar basis in increasing all of these activities, while BME shows a biopotency of only 1% of that of estradiol or CME. Hence, although CME reacts very strongly and apparently irreversibly with estrogen receptors in MCF-7 cells, it still behaves as a potent estrogen agonist.

¹ Reported, in part, at the Sixth Annual San Antonio Breast Cancer Conference, November 1983 (34).

² Recipient of support from a fellowship from the Max Kade Foundation. On leave from the First Surgical University Clinic, University of Vienna, Austria.

³ Recipient of support from NIH Grants CA31870 and CA18119. To whom requests for reprints should be addressed, at Department of Physiology and Biophysics, 524 Burrill Hall, University of Illinois, 407 South Goodwin Avenue, Urbana, IL 61801.

⁴ Recipient of support from NIH Grant AM 15556.

Received 11/ 2/83. Accepted 2/27/84.

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