Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen

Frederick J. Monsma, Benita S. Katzenellenbogen, Margaret A. Miller, Yvonne S. Ziegler, John A. Katzenellenbogen

Research output: Contribution to journalArticlepeer-review


We have used a covalently attaching antiestrogen, tamoxifen aziridine [TA; (Z)-(l-[4-(2-[N-aziridinyl] ethoxy)phenyl])l,2-diphenyl-l-butene], to analyze the structure and dynamics of the estrogen receptor in MCF-7 human breast cancer cells. The labeling of receptor with [3H]TA is specific, being blocked only by estrogens and antiestrogens, and the labeling is very efficient in that TA labels covalently the same number of receptors that are labeled reversibly by estradiol. In cells exposed to[3H]TA for 1 h, most of the covalently associated radioactivity is found in the 0.6 M KC1 extract of the nuclear fraction; this receptor has an apparent mol wt of 63,000 ± 2000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pi of 5.7 by gel isoelectric focusing in the presence of 8 M urea. The mol wt and pi of cytosol receptor labeled with [3H] TA are identical. In cells labeled with [3H]TA (20 nM) for 1 h and then exposed to a chase of 10-6 M estradiol, [3H]TA-labeled nuclear receptor disappears with a half-life of 4 h. Analysis ofnuclear receptor by sodium dodecyl sulfate-gels during the chase period reveals that this loss reflects a decrease in the 63,000 mol wt species; no significant quantities of lower mol wt TA-labeled fragments are observed in the nuclear, cytosol, or membrane fractions. Affinity labeled receptor interacts with several monoclonal antibodies to MCF-7 estrogen receptor, and it can be purified extensively by immunoadsorbent chromatography. TA has a low affinity (8% that of tamoxifen) for microsomal antiestrogen- binding sites that are distinct from the estrogen receptor, but TA reacts reversibly, rather than covalently, with these sites. The findings of similar mol wt and isoelectric points for soluble cytosol and nuclear extracted receptors under strongly denaturing and disaggregating conditions reveal that nuclear localization of receptor after ligand binding is not associated with major structural alterations in the receptor component labeled by TA. In addition, the receptor, even when occupied by a covalently attached ligand, is rapidly turned over in these cells.

Original languageEnglish (US)
Pages (from-to)143-153
Number of pages11
Issue number1
StatePublished - 1984
Externally publishedYes

ASJC Scopus subject areas

  • Endocrinology


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