Physicochemical and genetic evidence for specific antiestrogen binding sites.

Abstract

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 concentrationlof 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% gland Nuclear; 17,21-dimethyl-19-nor-4,9-pregna-4,9-diene-3,20-dione) were used. Unlabeled steroids were from Steraloids and Roussel-Uclaf. Tam and OH-Tam were gifts from Imperial Chemical Industries, and CI 628 and nafoxidine-a-(4-pyrrolidinoethoxy)phenyl-4-methoxy-a-nitrostilbene] and 1-{2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]-ethyl}pyrrolidine-were gifts from Parke, Davis and Upjohn, respectively.[3H]Thymidine (28 Ci/mmol) was from Amersham; protease (bovine pancreas), a-chymotrypsin, and trypsin were from Worthington; DNase and RNase II were from Sigma, and heparin-Sepharose CL-6B was from Pharmacia. Rat Uterus. Sprague-Dawley rats (3 mo old) were used during the estrus period. After they were killed, their uteri were homogenized at 0-40C in 4 vol of 20 mM Tris HCl, pH 7.4/1 mM dithiothreitol (TD buffer). The homogenate was centrifuged at 800 x g for 15 min, and the supernatant was recentrifuged at 105,000 x g for 1 hr. The resulting supernatant was referred to as cytosol and contained 6-15 mg of protein per ml. The pellets obtained at 800 X g and 105,000 X g were washed twice with 1 ml of TD buffer and resuspended in 1-3 ml of the same buffer containing 20% calf serum stripped of endogenous hormones by a 30-min incubation at 370C with 5 mg of charcoal per ml.MCF-7 Cells. Gift of M. Rich (Michigan Cancer Foundation, Detroit), MCF-7 cells were grown in an incubator in humidified 5% C02/95% air at 370C in 75-cm2 Nunclon plastic flasks (Nunc) in RPMI 1640 medium supplemented with 2 g of sodium bicarbonate per liter, 2 mM glutamine (pH 7.4 at 230C), and 4% fetal calf serum stripped of endogenous hormones.Tam-Resistant Cells RTx6. Clone RTx6 was isolated from cells growing in the presence of 10 ,uM Tam for 15 days by us-

Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.

“…Since progesterone receptor is an estrogenstimulated protein (15, 19), we examined the effect of phenol red on progesterone receptor levels in MCF-7 cells. Basal progesterone receptor level was 3 times higher in control cells grown in medium containing 30 ,uM phenol red than in control cells grown in phenol red-free MEM (Table 1). Cells treated with 1 nM estradiol exhibited the same high level of progesterone receptor, regardless of whether phenol red was absent or present.…”

“…Effect of Phenol Red on Estrogen Receptor-Negative MDA-MB-231 Breast Cancer Cells. In order to confirm the estrogen receptor-mediated effect of phenol red, we examined the effect of 30 ,LM phenol red on MDA-MB-231 cells, which do not contain estrogen receptors and are reported to be estrogen-and antiestrogen-unresponsive (11,20). Fig.…”

“…To determine the effect of phenol red on cell proliferation, we measured the rate ofproliferation of MCF-7 cells in complete medium ("regular MEM"), in medium lacking phenol red (phenol red-free MEM), and in phenol red-free MEM to which 30 AuM phenol red was added. For each of these media, proliferation rate was determined at four different concentrations of steroid-stripped (treated with dextran-coated charcoal) calf serum, since serum is known to alter the rate of MCF-7 cell proliferation (8,(16)(17)(18) (Fig.…”

Microsomal binding sites for antioestrogens in rat liver. Properties and detergent solubilization

“…Other data, however, suggest that some of the effects of antioestrogens, on growth and cellular proliferation in particular, are considerably more complex than would be anticipated if these effects were mediated solely by the oestrogen receptor (Martin, 1981;Faye et al, 1983;, 1985Reddel et al, 1983Reddel et al, , 1985Sutherland et al, 1983a,b).…”

Microsomal epoxide hydrolase of rat liver is a subunit of theanti-oestrogen-binding site

“…We first reported phenotypic evidence of this by isolating an AEBS-negative cell line Rtx-6 from MCF-7 cells (a human breast cancer cell line) that was resistant to tamoxifen. The Rtx-6 cells were similar to MCF-7 cells in their ER content and functionality, suggesting a correlative link between the lack of AEBS and the insensitivity of these cells to tamoxifen [10] and to a specific AEBS ligand [11,12]. Other studies have reported dose-dependent cytostatic and cytotoxic effects of anti-oestrogens on ER-negative lymphoid cells that contain a high level of microsomal AEBS, thus underlining the importance of AEBS in the mediation of growth control [12][13][14].…”