Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cells. Differences with anti-estrogen and estrogen

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Abstract

We have examined the binding of two high affinity radiolabeled anti-estrogens, 2-[4-(2-pyrrolidin-1-yl-ethoxy)phenyl]-2-(3-[3H]-4-hydroxyphenyl)-1-phenyl-1-nitroethene ([3H]CI628M) and 1-[4-(2-dimethylaminoethoxy)phenyl]1-(4-hydroxyphenyl)-2-(4-[3H]phenyl)-but-1(Z)-ene ([3H]trans-hydroxytamoxifen) to the estrogen receptor from MCF-7 human breast cancer cells and have used hydrodynamic methods to determine the molecular properties of estrogen and anti-estrogen receptor complexes from these cells. Saturation binding analysis indicates that each compound binds predominantly to a single class of high affinity binding sites with K(d) of 1.3 x 10-10 M for [3H]estra-1,3,5(10)-triene-3, 17β-diol (estradiol (E2)), 1.4 x 10-10 M for [3H]trans-hydroxytamoxifen, and 2.2 x 10-10 M for [3H]CI628M. Marked differences are seen in the sedimentation rate and chromatographic properties of the nuclear estrogen receptor when complexed with anti-estrogen as opposed to the estrogen, estradiol (E2). The nuclar E2 receptor sediments at 4.1 ± 0.03 S on high salt (0.4 M KCl) sucrose gradients; by contrast, receptor complexed with CI628M or trans-hydroxytamoxifen sediments as a 5.5 ± 0.06 S peak. Upon chromatography on Sephadex G-200 columns equilibrated with buffer containing 0.4 M KCl, the E2 nuclear receptor complexes appear to have a Stokes radius of 4.84 ± 0.20 nm, while the nuclear CI628M and trans-hydroxytamoxifen complexes have a Stokes radius of 5.93 ± 0.20 nm. These sedimentation coefficients and Stokes radii correspond to calculated molecular weights of 83,000 for the nuclear E2 receptor complex and 137,000 for the nuclear CI628M and trans-hydroxytamoxifen receptor complexes. By contrast, cytoplasmic estrogen receptors labeled with CI628M, trans-hydroxytamoxifen, or E2 have similar sedimentation coefficients of 4.1 ± 0.03 S and Stokes radii of 4.39 ± 0.30 nm, corresponding to a molecular weight of 76,000. The differences in physical properties of nuclear estrogen and anti-estrogen receptor complexes are obliterated in the presence of 3 M urea where estrogen and anti-estrogen complexes of 81,000 molecular weight (3.9 S, 4.94-nm Stokes radius) are obtained. These data are consistent with the association of the nuclear anti-estrogen receptor complex with an additional protein of ~55,000 molecular weight, an association that can be reversed by 3 M urea. This anti-estrogen-promoted change in receptor association with another cellular component may be an important aspect of the estrogen-antagonist and growth-inhibiting properties of these compounds.

Original languageEnglish (US)
Pages (from-to)8840-8846
Number of pages7
JournalJournal of Biological Chemistry
Volume257
Issue number15
StatePublished - 1982

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Estrogen Receptors
Estrogens
Physical properties
Cells
Breast Neoplasms
Cytoplasmic and Nuclear Receptors
Molecular Weight
Molecular weight
Sedimentation
Association reactions
Urea
Estradiol
Sediments
Estrogen Antagonists
Hydrodynamics
Chromatography
Sucrose
hydroxytamoxifen
CI 628M
Buffers

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cells. Differences with anti-estrogen and estrogen",
abstract = "We have examined the binding of two high affinity radiolabeled anti-estrogens, 2-[4-(2-pyrrolidin-1-yl-ethoxy)phenyl]-2-(3-[3H]-4-hydroxyphenyl)-1-phenyl-1-nitroethene ([3H]CI628M) and 1-[4-(2-dimethylaminoethoxy)phenyl]1-(4-hydroxyphenyl)-2-(4-[3H]phenyl)-but-1(Z)-ene ([3H]trans-hydroxytamoxifen) to the estrogen receptor from MCF-7 human breast cancer cells and have used hydrodynamic methods to determine the molecular properties of estrogen and anti-estrogen receptor complexes from these cells. Saturation binding analysis indicates that each compound binds predominantly to a single class of high affinity binding sites with K(d) of 1.3 x 10-10 M for [3H]estra-1,3,5(10)-triene-3, 17β-diol (estradiol (E2)), 1.4 x 10-10 M for [3H]trans-hydroxytamoxifen, and 2.2 x 10-10 M for [3H]CI628M. Marked differences are seen in the sedimentation rate and chromatographic properties of the nuclear estrogen receptor when complexed with anti-estrogen as opposed to the estrogen, estradiol (E2). The nuclar E2 receptor sediments at 4.1 ± 0.03 S on high salt (0.4 M KCl) sucrose gradients; by contrast, receptor complexed with CI628M or trans-hydroxytamoxifen sediments as a 5.5 ± 0.06 S peak. Upon chromatography on Sephadex G-200 columns equilibrated with buffer containing 0.4 M KCl, the E2 nuclear receptor complexes appear to have a Stokes radius of 4.84 ± 0.20 nm, while the nuclear CI628M and trans-hydroxytamoxifen complexes have a Stokes radius of 5.93 ± 0.20 nm. These sedimentation coefficients and Stokes radii correspond to calculated molecular weights of 83,000 for the nuclear E2 receptor complex and 137,000 for the nuclear CI628M and trans-hydroxytamoxifen receptor complexes. By contrast, cytoplasmic estrogen receptors labeled with CI628M, trans-hydroxytamoxifen, or E2 have similar sedimentation coefficients of 4.1 ± 0.03 S and Stokes radii of 4.39 ± 0.30 nm, corresponding to a molecular weight of 76,000. The differences in physical properties of nuclear estrogen and anti-estrogen receptor complexes are obliterated in the presence of 3 M urea where estrogen and anti-estrogen complexes of 81,000 molecular weight (3.9 S, 4.94-nm Stokes radius) are obtained. These data are consistent with the association of the nuclear anti-estrogen receptor complex with an additional protein of ~55,000 molecular weight, an association that can be reversed by 3 M urea. This anti-estrogen-promoted change in receptor association with another cellular component may be an important aspect of the estrogen-antagonist and growth-inhibiting properties of these compounds.",
author = "Eckert, {R. L.} and Katzenellenbogen, {Benita S}",
year = "1982",
language = "English (US)",
volume = "257",
pages = "8840--8846",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "15",

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TY - JOUR

T1 - Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cells. Differences with anti-estrogen and estrogen

AU - Eckert, R. L.

AU - Katzenellenbogen, Benita S

PY - 1982

Y1 - 1982

N2 - We have examined the binding of two high affinity radiolabeled anti-estrogens, 2-[4-(2-pyrrolidin-1-yl-ethoxy)phenyl]-2-(3-[3H]-4-hydroxyphenyl)-1-phenyl-1-nitroethene ([3H]CI628M) and 1-[4-(2-dimethylaminoethoxy)phenyl]1-(4-hydroxyphenyl)-2-(4-[3H]phenyl)-but-1(Z)-ene ([3H]trans-hydroxytamoxifen) to the estrogen receptor from MCF-7 human breast cancer cells and have used hydrodynamic methods to determine the molecular properties of estrogen and anti-estrogen receptor complexes from these cells. Saturation binding analysis indicates that each compound binds predominantly to a single class of high affinity binding sites with K(d) of 1.3 x 10-10 M for [3H]estra-1,3,5(10)-triene-3, 17β-diol (estradiol (E2)), 1.4 x 10-10 M for [3H]trans-hydroxytamoxifen, and 2.2 x 10-10 M for [3H]CI628M. Marked differences are seen in the sedimentation rate and chromatographic properties of the nuclear estrogen receptor when complexed with anti-estrogen as opposed to the estrogen, estradiol (E2). The nuclar E2 receptor sediments at 4.1 ± 0.03 S on high salt (0.4 M KCl) sucrose gradients; by contrast, receptor complexed with CI628M or trans-hydroxytamoxifen sediments as a 5.5 ± 0.06 S peak. Upon chromatography on Sephadex G-200 columns equilibrated with buffer containing 0.4 M KCl, the E2 nuclear receptor complexes appear to have a Stokes radius of 4.84 ± 0.20 nm, while the nuclear CI628M and trans-hydroxytamoxifen complexes have a Stokes radius of 5.93 ± 0.20 nm. These sedimentation coefficients and Stokes radii correspond to calculated molecular weights of 83,000 for the nuclear E2 receptor complex and 137,000 for the nuclear CI628M and trans-hydroxytamoxifen receptor complexes. By contrast, cytoplasmic estrogen receptors labeled with CI628M, trans-hydroxytamoxifen, or E2 have similar sedimentation coefficients of 4.1 ± 0.03 S and Stokes radii of 4.39 ± 0.30 nm, corresponding to a molecular weight of 76,000. The differences in physical properties of nuclear estrogen and anti-estrogen receptor complexes are obliterated in the presence of 3 M urea where estrogen and anti-estrogen complexes of 81,000 molecular weight (3.9 S, 4.94-nm Stokes radius) are obtained. These data are consistent with the association of the nuclear anti-estrogen receptor complex with an additional protein of ~55,000 molecular weight, an association that can be reversed by 3 M urea. This anti-estrogen-promoted change in receptor association with another cellular component may be an important aspect of the estrogen-antagonist and growth-inhibiting properties of these compounds.

AB - We have examined the binding of two high affinity radiolabeled anti-estrogens, 2-[4-(2-pyrrolidin-1-yl-ethoxy)phenyl]-2-(3-[3H]-4-hydroxyphenyl)-1-phenyl-1-nitroethene ([3H]CI628M) and 1-[4-(2-dimethylaminoethoxy)phenyl]1-(4-hydroxyphenyl)-2-(4-[3H]phenyl)-but-1(Z)-ene ([3H]trans-hydroxytamoxifen) to the estrogen receptor from MCF-7 human breast cancer cells and have used hydrodynamic methods to determine the molecular properties of estrogen and anti-estrogen receptor complexes from these cells. Saturation binding analysis indicates that each compound binds predominantly to a single class of high affinity binding sites with K(d) of 1.3 x 10-10 M for [3H]estra-1,3,5(10)-triene-3, 17β-diol (estradiol (E2)), 1.4 x 10-10 M for [3H]trans-hydroxytamoxifen, and 2.2 x 10-10 M for [3H]CI628M. Marked differences are seen in the sedimentation rate and chromatographic properties of the nuclear estrogen receptor when complexed with anti-estrogen as opposed to the estrogen, estradiol (E2). The nuclar E2 receptor sediments at 4.1 ± 0.03 S on high salt (0.4 M KCl) sucrose gradients; by contrast, receptor complexed with CI628M or trans-hydroxytamoxifen sediments as a 5.5 ± 0.06 S peak. Upon chromatography on Sephadex G-200 columns equilibrated with buffer containing 0.4 M KCl, the E2 nuclear receptor complexes appear to have a Stokes radius of 4.84 ± 0.20 nm, while the nuclear CI628M and trans-hydroxytamoxifen complexes have a Stokes radius of 5.93 ± 0.20 nm. These sedimentation coefficients and Stokes radii correspond to calculated molecular weights of 83,000 for the nuclear E2 receptor complex and 137,000 for the nuclear CI628M and trans-hydroxytamoxifen receptor complexes. By contrast, cytoplasmic estrogen receptors labeled with CI628M, trans-hydroxytamoxifen, or E2 have similar sedimentation coefficients of 4.1 ± 0.03 S and Stokes radii of 4.39 ± 0.30 nm, corresponding to a molecular weight of 76,000. The differences in physical properties of nuclear estrogen and anti-estrogen receptor complexes are obliterated in the presence of 3 M urea where estrogen and anti-estrogen complexes of 81,000 molecular weight (3.9 S, 4.94-nm Stokes radius) are obtained. These data are consistent with the association of the nuclear anti-estrogen receptor complex with an additional protein of ~55,000 molecular weight, an association that can be reversed by 3 M urea. This anti-estrogen-promoted change in receptor association with another cellular component may be an important aspect of the estrogen-antagonist and growth-inhibiting properties of these compounds.

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