To determine whether selenite has estrogen-like activities, the effects of this compound on estrogen receptor-alpha (ER-alpha) and other estrogen-regulated genes were measured in the human breast cancer cell line MCF-7. Treatment of cells with 1 uM of sodium selenite resulted in a 40% decrease in the amount of estrogen receptor-alpha and in a parallel decrease of 40% in ER-alpha mRNA. Progesterone receptor concentration increased 2.6-fold and pS2 mRNA increased 2.4-fold after selenite treatment. The induction of progesterone receptor and pS2 was blocked by the anti-estrogen ICI-182,780. In transient co-transfection experiments of Wild-type ER-alpha and an estrogen response element-reporter construct, selenite stimulated CAT activity. In binding assays, selenite blocked the binding of estradiol to ER-alpha (K(i) = 23 +/- 17 nM, n = 3) suggesting that this compound interacts with the hormone binding domain of the receptor. To determine whether interaction of selenite with the hormone binding domain results in receptor activation, COS-1 cells were transiently co-transfected with the chimeric receptors GAL-ER, which contains the hormone binding domain of ER-alpha and the DNA binding domain of the transcription factor GAL4, and a GAL4-responsive CAT reporter gene. Treatment of cells with estradiol or selenite resulted in a three- to five-fold increase in CAT activity. The effects of selenite on the chimeric receptor were blocked by the antiestrogen, suggesting that selenite activates ER-alpha through an interaction with the hormone binding domain of the receptor. Transfection assays with ER-alpha mutants identified C381, C447, H524, and N532 as interaction sites of selenite with the hormone binding domain.
Copyright 2000 Wiley-Liss, Inc.