It has been suggested that alterations in estradiol (E2) metabolism, resulting in increased production of 16α-hydroxyestrone (16α-OHE1), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16α-OHE1 on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G1 synchronized cells were treated with 1 to 25 nM 16α-OHE1 for 24 and 48 h. [3H]Thymidine incorporation assay showed that 16α-OHE1 caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E2 caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16α-OHE1 in stimulating cell growth. When G1 synchronized cells were treated with 10 nM 16α-OHE1 for 24 h, 62 ± 3% of cells were in S phase compared with 14 ± 3% and 52 ± 2% of cells in the control and E2-treated groups respectively. In order to explore the role of 16α-OHE1 in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16α-OHE1 resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB 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 compared with control. These results show that 16α-OHE1 is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.
All Science Journal Classification (ASJC) codes
- Molecular Biology