Regulation of cell cycle and cyclins by 16 alpha-hydroxyestrone in MCF-7 breast cancer cells

It has been suggested that alterations in estradiol (E-2) metabolism, resul ting in increased production of 16 alpha -hydroxyestrone (16 alpha -OHE1), is associated with an increased risk of breast cancer. In the present study , we examined the effects of 16 alpha -OHE1, on DNA synthesis, cell cycle p rogression, and the expression of cell cycle regulatory genes in MCF-7 brea st cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16 al pha -OHE1, for 24 and 48 h. [H-3]Thymidine incorporation assay showed that 16 alpha -OHE1, caused an 8-fold increase in DNA synthesis compared with th at of control cells, whereas E-2 caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16 alph a -OHE1 in stimulating cell growth. When G(1) synchronized cells were treat ed with 10 nM 16 alpha -OHE1 for 24 h, 62 +/- 3% of cells were in S phase c ompared with 14 +/- 3% and 52 +/- 2% of cells in the control and E-2-treate d groups respectively. In order to explore the role of 16 alpha -OHE1, in c ell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cy clin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16 alpha -OHE1, resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, resp ectively, at the protein level. There was also a significant increase in pR B phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation com pared with control. These results show that 16 alpha -OHE, is a potent estr ogen capable of accelerating cell cycle kinetics and stimulating the expres sion of cell cycle regulatory proteins.