title: Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells.
abstract: Although antiestrogen therapy is effective in the treatment of hormone-responsive breast tumors, approximately 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, we have investigated the physicochemical properties and binding characteristics of the estrogen receptors with estradiol and antiestrogens and the occurrence of estrogen-noncompetible antiestrogen binding sites in two estrogen-sensitive but tamoxifen-growth-resistant estrogen receptor-positive MCF-7 cell variant clones, R3-98 and R27. 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 three 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 three cell types with the affinity of trans-hydroxytamoxifen greater than estradiol greater than alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilben e greater than tamoxifen. Salt-extracted nuclear receptor complexes prepared from the three cell types showed similar sedimentation behavior on 0.4 M KCl-containing sucrose gradients with [3H]estradiol-labeled receptor complexes sedimenting at 4.2S, whereas receptors complexed with either of the antiestrogens trans-[3H]-hydroxytamoxifen or [3H]alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrosti lbene 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 two variant cells, when covalently labeled with [3H]-tamoxifen aziridine in intact cells and then salt extracted have identical molecular weights of approximately 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 approximately 5.7.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002999:Clone Cells; D003600:Cytosol / Q000378:metabolism; D004351:Drug Resistance; D004958:Estradiol / Q000378:metabolism; D004965:Estrogen Antagonists / Q000378:metabolism* / Q000633:toxicity; D005260:Female; D006801:Humans; D008970:Molecular Weight; D011955:Receptors, Drug; D011959:Receptors, Estradiol / Q000302:isolation & purification / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000302:isolation & purification / Q000378:metabolism*; D013629:Tamoxifen / Q000378:metabolism
chemical_list: D004965:Estrogen Antagonists; D011955:Receptors, Drug; D011959:Receptors, Estradiol; D011960:Receptors, Estrogen; D013629:Tamoxifen; D004958:Estradiol