ANTIESTROGEN BINDING IN ANTIESTROGEN GROWTH-RESISTANT ESTROGEN-RESPONSIVE CLONAL VARIANTS OF MCF-7 HUMAN-BREAST CANCER-CELLS

  • 1 January 1984
    • journal article
    • research article
    • Vol. 44  (11) , 5038-5045
Abstract
Although antiestrogen therapy is effective in the treatment of hormone-responsive breast tumors, .apprx. 40% of the patients with estrogen receptor-positive tumors fail to respond to antiestrogens. To better understand the mechanisms by which antiestrogens inhibit the growth of hormone-dependent breast cancers, the physicochemical properties and binding characteristics of the estrogen receptors with estradiol and antiestrogens and the occurrence of estrogen-noncompetible antiestrogen binding sites in 2 estrogen-sensitive but tamoxifen-growth-resistant estrogen receptor-positive MCF-7 cell variant clones, R3-98 and R27 was investigated. In the variant cells, estradiol (10-8 M) significantly stimulates cell proliferation as in the parent MCF-7 cells, but the antiestrogen tamoxifen (10-6 M) has no significant effect on growth of the variant cells, whereas antiestrogen strongly inhibits proliferation of the parent MCF-7 cells. All 3 cell types contain high concentrations of estrogen receptor (150 to 250 fmol/mg protein) and competition binding analysis shows that the relative binding affinity of a series of compounds for estrogen receptor is similar among the 3 cell types with the affinity of trans-hydroxytamoxifen > estradiol > .alpha.-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-.alpha.''-nitrostilbene > tamoxifen. Salt-extracted nuclear receptor complexes prepared from the 3 cell types showed similar sedimentation behavior on 0.4 M KCl-containing sucrose gradients with [3H]estradiol-labeled receptor complexes sedimenting at 4.2S [Svedberg], whereas receptors complexed with either of the antiestrogens trans-[3H]-hydroxytamoxifen or [3H].alpha.-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-.alpha.''-nitrostilbene sediment at 5.5S. In all 3 cell types, the nuclear receptor forms react with an estrogen receptor monoclonal antibody, D547Sp.gamma., to form complexes which sediment at 8.5S. The nuclear estrogen receptors from the parental MCF-7 and the 2 variant cells, when convalently labeled with [3H]-tamoxifen aziridine in intact cells and then salt extracted have identical MW of .apprx. 62,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The covalently labeled nuclear and cytosol receptors in these 3 cell lines also show identical migration in 8 M urea polyacrylamide isoelectric focusing gels consistent with a predominant receptor species of isoelectric point .apprx. 5.7. In addition to binding to estrogen receptors in these cells, the antiestrogen tamoxifen binds to estrogen-noncompetible triphenylethylene-specific binding sites present in the 12,000 .times. g [gravity] cell supernatant. The 3 cell types contain similar quantities of these binding sites (350-550 fmol/mg protein) and these sites have a similar high affinity (Kd = 3 to 4 nM) for tamoxifen. Therefore, by the criteria examined, the estrogen receptors amd estrogen-noncompetible triphenylethylene-binding sites of the variant cells appear similar to those in the parent MCF-7 cell line. Alteration in antiestrogen responsiveness of the variant cells must occur at steps beyond the initial interaction of ligand with intracellular estrogen receptors or triphenylethylene-binding sites.