Characterization and quantitation of antiestrogen binding sites in estrogen receptor-positive and -negative human breast cancer cell lines

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 antiestrogen-competible binding sites are present in the 800 and 12,000 χ g supernatants of all three breast cancer cells. The antiestrogen binding sites are pelleted upon centrifugation at 100,000 or 180,000 χ 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 > α-{p-[2-(1 -pyrrolidino)ethoxy]phenyl}-4-methoxy-α= nitrostilbene (CI628) > trans-tamoxifen = trans-hydroxytamoxifen > α-{p-[2-(1 -pyrolidino)ethoxy]phenyl}-4-hydroxy-α’-nitrostilbene (CI628M) > 6-hydroxy-2-(p-hydroxyphenyl)benzo(b)thien-3-yl-p-[2(1-pyrrolidinyl ethyl]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 antiestro-.