Effects of Arsenite on Estrogen Receptor-  Expression and Activity in MCF-7 Breast Cancer Cells

Abstract

To determine whether arsenite 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 mu M arsenite resulted in a 60% decrease in the amount of ER alpha and in a parallel decrease of 40% in ER alpha messenger RNA. Progesterone receptor concentration increased 22-fold after arsenite treatment, pS2 messenger RNA also increased 2,1-fold after treatment. The induction of progesterone receptor and pS2 was blocked by the antiestrogen ICI-182,780. In transient cotransfection experiments of wild-type ER alpha and an estrogen response element-reporter construct, arsenite stimulated chloramphenicol acetyltransferase (CAT) activity. In growth assays, arsenite significantly stimulated the proliferation of MCF-7 cells compared with cells grown in estrogen-depleted medium. Addition of an antiestrogen blocked growth stimulation by arsenite. In binding assays, arsenite blocked the binding of estradiol to ER alpha (K-i = 5 +/- 0.5 nM; n = 3), suggesting that the compound interacts with the hormone-binding domain of the receptor. To determine whether interaction of arsenite with the hormone-binding domain results in receptor activation, COS-1 cells were transiently cotransfected 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 arsenite resulted in a 4-fold increase in CAT activity. The effects of arsenite on the chimeric receptor were blocked by the antiestrogen, suggesting that arsenite 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 arsenite with the hormone-binding domain.