Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells

M. A. Miller, Marc E Lippman, B. S. Katzenellenbogen

Research output: Contribution to journalArticle

38 Citations (Scopus)

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 > estrogen > α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene > 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]α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene sediment at 5.5S. In all 3 cell types, the nuclear receptor forms react with an estrogen receptor monoclonal antibody, D547Spγ, 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. 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 x g cell supernatant. The three cell types contain similar quantities of these binding sites (350 to 550 fmol/mg protein), and these sites have a similar high affinity (K(d) = 3 to 4 nM) for tamoxifen. Therefore, by the criteria examined, the estrogen receptors and estrogen-noncompetible triphenylethylene-binding sites of the variant cells appear similar to those in the parent MCF-7 cell line. These findings suggest that the 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.

Original languageEnglish
Pages (from-to)5038-5045
Number of pages8
JournalCancer Research
Volume44
Issue number11
StatePublished - Dec 1 1984
Externally publishedYes

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Estrogen Receptor Modulators
Estrogens
Estrogen Receptors
Breast Neoplasms
Growth
Tamoxifen
MCF-7 Cells
Binding Sites
Cytoplasmic and Nuclear Receptors
Estradiol
Salts
Estradiol Receptors
Cell Line
Isoelectric Point
Isoelectric Focusing
Protein Binding
Sodium Dodecyl Sulfate
Growth Hormone
Sucrose
Urea

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells. / Miller, M. A.; Lippman, Marc E; Katzenellenbogen, B. S.

In: Cancer Research, Vol. 44, No. 11, 01.12.1984, p. 5038-5045.

Research output: Contribution to journalArticle

Miller, M. A. ; Lippman, Marc E ; Katzenellenbogen, B. S. / Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells. In: Cancer Research. 1984 ; Vol. 44, No. 11. pp. 5038-5045.
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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 > estrogen > α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene > 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]α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene sediment at 5.5S. In all 3 cell types, the nuclear receptor forms react with an estrogen receptor monoclonal antibody, D547Spγ, 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. 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 x g cell supernatant. The three cell types contain similar quantities of these binding sites (350 to 550 fmol/mg protein), and these sites have a similar high affinity (K(d) = 3 to 4 nM) for tamoxifen. Therefore, by the criteria examined, the estrogen receptors and estrogen-noncompetible triphenylethylene-binding sites of the variant cells appear similar to those in the parent MCF-7 cell line. These findings suggest that the 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.",
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N2 - 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 > estrogen > α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene > 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]α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene sediment at 5.5S. In all 3 cell types, the nuclear receptor forms react with an estrogen receptor monoclonal antibody, D547Spγ, 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. 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 x g cell supernatant. The three cell types contain similar quantities of these binding sites (350 to 550 fmol/mg protein), and these sites have a similar high affinity (K(d) = 3 to 4 nM) for tamoxifen. Therefore, by the criteria examined, the estrogen receptors and estrogen-noncompetible triphenylethylene-binding sites of the variant cells appear similar to those in the parent MCF-7 cell line. These findings suggest that the 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.

AB - 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 > estrogen > α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene > 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]α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α'-nitrostilbene sediment at 5.5S. In all 3 cell types, the nuclear receptor forms react with an estrogen receptor monoclonal antibody, D547Spγ, 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. 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 x g cell supernatant. The three cell types contain similar quantities of these binding sites (350 to 550 fmol/mg protein), and these sites have a similar high affinity (K(d) = 3 to 4 nM) for tamoxifen. Therefore, by the criteria examined, the estrogen receptors and estrogen-noncompetible triphenylethylene-binding sites of the variant cells appear similar to those in the parent MCF-7 cell line. These findings suggest that the 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.

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