Physicochemical and genetic evidence for specific antiestrogen binding sites.

In rat uterus and human breast cancer MCF-7 cell cytosol, the antiestrogens tamoxifen (Tam) and 4-hydroxytamoxifen (OH-Tam) bind to "antiestrogen binding sites" (ABS), which do not bind estradiol (E). Demonstrated in total cytosol by binding studies with radioactive antiestrogens in the presence of a large concentration of E, ABS can be physically separated from E-binding estrogen receptor (ER) by removing the latter with an E-containing bioaffinity adsorbent or with heparin-Sepharose gel. ABS concentration is 10-20% of that of ER; the Kd for Tam and OH-Tam is 1-2 x 10(-9) M, whereas the Kd of OH-Tam binding by ER (approximately equal to 1 x 10(-10) M) is approximately equal to 1/50 that of Tam. Other triphenylethylene antiestrogens compete against Tam for binding to ABS, contrary to steroid hormones. Sucrose gradient ultracentrifugation analyses of total cytosol and of affinity gel effluents show a heterogenous pattern of ABS from 10 to 40 S, unchanged by 0.4 M KCl and limited trypsinization (which however provoke transitions of ER from 8S to 4S forms) and by 20 mM molybdate (which stabilizes the 8S form of ER and prevents large aggregates). Preliminary results suggest that ABS may be associated with particulate components of the cell. RTx6 cells of a clone selected from MCF-7 cells for resistance to the antigrowth effect of Tam have ER in the same concentration and have similar affinity for E and antiestrogens as do unselected MCF-7 cells. However, RTx6 cells have virtually no ABS detectable by binding and gradient ultracentrifugation studies. It is proposed that the double binding of Tam and OH-Tam to ER and ABS in estrogen target cells may be related to the complex double series of estrogenic and "antiestrogenic" activities displayed by nonsteroidal triphenylethylene derivatives.