POLYAMINE INHIBITION OF ESTROGEN-RECEPTOR (ER) DNA-BINDING AND LIGAND-BINDING FUNCTIONS

Polyamines are known to inhibit sequence specific DNA-binding activity of several zinc-finger transcription factors, including estrogen rece ptor (ER) binding to its cognate estrogen response element (ERE). The mechanism accounting for this disruption of protein-DNA interaction is unknown, although polyamine induction of DNA conformational changes h as been suggested. To determine if polyamines can directly impair ER a ction, we compared the effects of putrescine (Putr), spermidine (Spd), and spermine (Spm) on ER DNA-binding (ER-ERE complex formation), ER l igand-binding (estradiol), ER structure (circular dichroism and sucros e gradient sedimentation), and the capacity of ER to transactivate an ERE-tk-CAT reporter in transient transfection assays. Polyamine concen trations causing 50% inhibition of ER-ERE formation (IC,, values) were found to be 1 mM for Putr, 4 mM for Spd, and 3 mM for Spm. This loss of ER DNA-binding was associated with a direct and irreversible effect on the ER DNA-binding domain (ER-DBD). Additionally, polyamines were observed to inhibit ER ligand binding with IC50 values of 10 mM for Pu tr, 2 mM for Spd, and <0.1 mM for Spm; and this correlated with a meas ureable change in higher-order ER structure (5S to 3.5S sedimentation) and inhibition of intracellular ER transactivation. These findings su ggest that in ER-positive human breast tumors with increased polyamine (especially Spm) content, ER structure and function may be directly a ltered by tight-ion polyamine complexing that results in loss of ER-me diated gene regulation.