Oxidant stress impaired DNA-binding of estrogen receptor from human breastcancer

Full-length (67 kDa) immunoreactive estrogen receptor (ER) extracted from a third of untreated ER-positive primary breast tumors appears unable to bin d to its cognate estrogen response element (ERE). We have observed partial reversibility of this ER DNA-binding defect upon treatment of these tumor e xtracts with excess thiol reducing agent (DTT), suggesting that ER DNA-bind ing is subject to redox modulation as is reported for other zinc-finger pro teins and transcriptional activators. Treatment of recombinant ER DNA-bindi ng domain (ER-DBD) or ER-enriched extracts from CHOER and MCF-7 cells with thiol-reacting oxidants (diamide, iodosobenzoate, H2O2) or alkylator (iodoa cetamide) produces a dose-dependent loss in ER DNA-binding capacity. Thiol- specific oxidative loss in ER DNA-binding is fully reversible by DTT reduct ion, unlike the defect caused by thiol-specific alkylation. Circular dichro ism spectrometry shows that both forms of treatment substantially modify ER secondary structure, inducing loss of cc-helical content within the ER-DBD that is reversible after thiol oxidation but not after thiol alkylation. O xidant (H2O2, menadione) exposure of cultured CHOER or MCF-7 cells impairs the ability of endogenous ER to bind DNA and transactivate an ER-responsive reporter gene (ERE-tk-CAT), demonstrating that extracellular redox stress can modulate intracellular ER function. Since these thiol-specific oxidant and alkylator treatments have no significant effect on either recombinant E R ligand-binding or intracellular immunoreactive ER content, our findings s uggest that DNA-binding and transactivation are the most sensitive intracel lular ER functions impaired by oxidant stress in some ER-positive human bre ast tumors. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.