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Activation of Estrogen Receptor- by the Anion Nitrite

Departments of ¹ Biochemistry and Molecular and Cellular Biology, ² Oncology, and ³ Human Sciences, Georgetown University, Washington, District of Columbia

Requests for reprints: Mary Beth Martin, Lombardi Comprehensive Cancer Center, Research Building, 3970 Reservoir Road NW, Washington, DC 20007. Phone: 202-687-3768; E-mail: martinmb{at}georgetown.edu.

Key Words: breast cancer • steroid hormones and receptors • environmental carcinogenesis/toxicology • molecular modeling

In this study, the ability of nitrite and nitrate to mimic the effects of estradiol on growth and gene expression was measured in the human breast cancer cell line MCF-7. Similar to estradiol, treatment of MCF-7 cells with either 1 µmol/L nitrite or 1 µmol/L nitrate resulted in 4-fold increase in cell growth and 2.3-fold to 3-fold increase in progesterone receptor (PgR), pS2, and cathepsin D mRNAs that were blocked by the antiestrogen ICI 182,780. The anions also recruited estrogen receptor- (ER) to the pS2 promoter and activated exogenously expressed ER when tested in transient cotransfection assays. To determine whether nitrite or nitrate was the active anion, diphenyleneiodonium was used to inhibit oxidation/reduction reactions in the cell. The ability of diphenyleneiodonium to block the effects of nitrate, but not nitrite, on the induction of PgR mRNA and the activation of exogenously expressed ER suggests that nitrite is the active anion. Concentrations of nitrite, as low as 100 nmol/L, induced a significant increase in PgR mRNA, suggesting that physiologically and environmentally relevant doses of the anion activate ER. Nitrite activated the chimeric receptor Gal-ER containing the DNA-binding domain of GAL-4 and the ligand-binding domain of ER and blocked the binding of estradiol to the receptor, suggesting that the anion activates ER through the ligand-binding domain. Mutational analysis identified the amino acids Cys381, His516, Lys520, Lys529, Asn532, and His547 as important for nitrite activation of the receptor. [Cancer Res 2008;68(10):3950–8]