10194768

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4

Estrogen receptor (ER) toxicity has hampered the development of vertebrate cell lines stably expressing substantial levels of recombinant wild-type ER. To isolate clonal lines of HeLa cells stably expressing epitope-tagged ER, we used a construction encoding a single bicistronic mRNA, in which FLAG-epitope-tagged human ER alpha (fER) was translated from a 5'-translation initiation site and fused to the neomycin resistance gene, which was translated from an internal ribosome entry site. One stable HeLa-ER-positive cell line (HeLa-ER1) produces 1,300,000 molecules of fER/cell (approximately 20-fold more ER than MCF-7 cells). The HeLa fER is biologically active in vivo, as judged by rapid death of the cells in the presence of either 17 beta-estradiol or trans-hydroxytamoxifen and the ability of the cell line to activate a transfected estrogen response element (ERE)-containing reporter gene. The FLAG-tagged ER was purified to near homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified fER exhibited a distribution constant (KD) for 17 beta-estradiol of 0.45 nM. Purified HeLa fER and HeLa fER in crude nuclear extracts exhibit similar KD values for the ERE (0.8 nM and 1 nM, respectively), which are approximately 10 times lower than the KD of 10 nM we determined for purified ER expressed using the baculovirus system. HMG-1 strongly stimulated binding of both crude and purified HeLa fER to the ERE (KD of 0.25 nM). In transfected HeLa cells, HMG-1 exhibited a dose-dependent stimulation of 17 beta-estradiol-dependent transactivation. At high levels of transfected HMG-1 expression plasmid, transactivation by ER became partially ligand-independent, and transactivation by trans-hydroxytamoxifen was increased by more than 25-fold. These data describe a system in which ER, stably expressed in HeLa cells and easily purified, exhibits extremely high affinity for the ERE, and suggest that intracellular levels of HMG-1 may be limiting for ER action.

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0888-8809

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632-43

pubmed-meshheading:10194768-Animals, pubmed-meshheading:10194768-Binding Sites, pubmed-meshheading:10194768-Breast Neoplasms, pubmed-meshheading:10194768-Carrier Proteins, pubmed-meshheading:10194768-Cell Extracts, pubmed-meshheading:10194768-Epitopes, pubmed-meshheading:10194768-Estradiol, pubmed-meshheading:10194768-Estrogen Antagonists, pubmed-meshheading:10194768-Estrogens, pubmed-meshheading:10194768-Female, pubmed-meshheading:10194768-HMGB1 Protein, pubmed-meshheading:10194768-HeLa Cells, pubmed-meshheading:10194768-High Mobility Group Proteins, pubmed-meshheading:10194768-Humans, pubmed-meshheading:10194768-Receptors, Estrogen, pubmed-meshheading:10194768-Recombinant Proteins, pubmed-meshheading:10194768-Response Elements, pubmed-meshheading:10194768-Tamoxifen, pubmed-meshheading:10194768-Transcriptional Activation, pubmed-meshheading:10194768-Transfection, pubmed-meshheading:10194768-Tumor Cells, Cultured

HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells.

Journal Article, Research Support, U.S. Gov't, P.H.S.