title: HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells.
abstract: 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.
authors: Department of Biochemistry, University of Illinois, Urbana 61801, USA.
authors: C C
authors: CC
authors: Zhang
authors: S
authors: S
authors: Krieg
authors: D J
authors: DJ
authors: Shapiro
mesh_terms: D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D000939:Epitopes; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D004967:Estrogens / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D024243:HMGB1 Protein; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011960:Receptors, Estrogen / Q000819:agonists / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects / Q000502:physiology*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D002457:Cell Extracts; D000939:Epitopes; D004965:Estrogen Antagonists; D004967:Estrogens; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol