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

Estrogen receptor (ER) toxicity has hampered the development of vertebrate cell lines stably expressing substantial levels of recombinant wildtype 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-e pitope-tagged human ER alpha (fER) was translated from a 5'-translation ini tiation site and fused to the neomycin resistance gene, which was translate d from an internal ribosome entry site. One stable HeLa-ER-positive cell li ne (HeLa-ER1) produces 1,300,000 molecules of fER/cell (similar to 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-estrad iol 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 immuno affinity chromatography with anti-FLAG monoclonal antibody. Purified fER ex hibited a distribution constant (K-D) for 17 beta-estradiol of 0.45 nM. Pur ified Hela RR and HeLa fER in crude nuclear extracts exhibit similar K-D va lues for the ERE (0.8 nM and 1 nM, respectively), which are approximately 1 0 times lower than the K-D of 10 nM we determined for purified ER expressed using the baculovirus system. HMG-1 strongly stimulated binding of both cr ude and purified HeLa fER to the ERE (K-D of 0.25 nM). In transfected HeLa cells, HMG-1 exhibited a dose-dependent stimulation of 17 beta-estradiol-de pendent transactivation. At high levels of transfected HMG-1 expression pla smid, transactivation by ER became partially ligand-independent, and transa ctivation by trans-hydroxytamoxifen was increased by more than 25-fold. The se data describe a system in which ER, stably expressed in HeLa cells and e asily purified, exhibits extremely high affinity for the ERE, and suggest t hat intracellular levels of HMG-1 may be limiting for ER action.