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Characterization and Quantitation of Antiestrogen Binding Sites in Estrogen Receptor-positive and -negative Human Breast Cancer Cell Lines¹

Department of Physiology and Biophysics, University of Illinois, and University of Illinois College of Medicine, Urbana, Illinois 61801

Antiestrogens are useful in the treatment of endocrine-responsive breast cancers in humans. In an attempt to understand the mechanisms underlying their estrogen antagonism and antitumor character, we have examined the interaction of antiestrogens with three human breast cancer cell lines that differ markedly in their estrogen receptor content and in their sensitivity to growth suppression by antiestrogens. MCF-7 cells have high levels of estrogen receptor, and their growth is inhibited markedly by antiestrogens; T47D cells contain low levels of estrogen receptor, and their growth is suppressed weakly by antiestrogens; and MDA-MB-231 cells contain no detectable estrogen receptors, and their growth is unaffected by antiestrogens. In addition to binding to the estrogen receptor, antiestrogens are found to be associated with binding sites that are distinct from the estrogen receptor. These estrogen-noncompetible but antiestrogencompetible binding sites are present in the 800 and 12,000 x g supematants of all three breast cancer cells. The antiestrogen binding sites are pelleted upon centrifugation at 100,000 or 180,000 x g and appear to be associated with microsomal membranes, while the majority of the estrogen receptor remains soluble at all centrifugation speeds. Although these cells differ markedly in their estrogen receptor content and sensitivity to growth inhibition by the antiestrogen, tamoxifen, all three cell lines contain similar quantities of estrogen-noncompetible antiestrogen binding sites (MCF-7 cells, 390 ± 50 (S.E.); T47D cells, 360 ± 50; and MDA-MB-231 cells, 260 ± 50 fmol/mg protein) that have a similar affinity (K_d = 2 to 4 nM) for tamoxifen. The affinity of a series of antiestrogens and related compounds for these antiestrogen sites follows the order cis-tamoxifen > -{p-[2-(1-pyrrolidino)ethoxy]phenyl}-4-methoxy-'-nitrostilbene (CI628) > trans-tamoxifen = trans-hydroxytamoxifen > -{p-[2-(1-pyrolidino)ethoxy]phenyl}-4-hydroxy-'-nitrostilbene (CI628M) > 6-hydroxy-2-(p-hydroxyphenyl)benzo(b)thein-3-yl-p-[2(1-pyrrolidinyl)ethoxy]phenyl ketone (LY117018). This order of affinities of different antiestrogens for the antiestrogen binding sites does not parallel their affinity for the estrogen receptor nor the potency of these compounds as antiestrogens.

These findings raise questions about the role of these estrogen-noncompetible sites in mediating directly the estrogen antagonism of antiestrogens in breast cancer cells, and suggest that interaction with the estrogen receptor is most likely the mechanism underlying the growth-inhibitory effects of antiestrogens. It is possible, however, that these antiestrogen binding sites might influence the distribution of antiestrogens and, hence, their accessibility to estrogen receptor in estrogen receptor-positive cells, or they might mediate actions of antiestrogens that are unrelated to estrogen antagonism.

¹ Supported by NIH Grant CA 18119 (USPHS) from the National Cancer Institute [B. S. K.]. A portion of this work was presented at the 64th Annual Endocrine Society Meeting, June 1982 (35).

² To whom requests for reprints should be addressed, at Department of Physiology and Biophysics, 524 Burrill Hall, University of Illinois, 407 South Goodwin Avenue, Urbena, Ill. 61801.

Received 10/20/82. Accepted 3/22/83.