Differential effects of 16α-hydroxyestrone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells

We studied the effects of 2-methoxyestradiol (2-ME2) and 16alpha-hydroxyestrone (16alpha-OHE1), two metabolites of estradiol (E-2) on DNA synthesis, cell cycle progression and cyclin D1 protein levels in estrogen receptor-positive MCF-7 cells. E-2 and 16alpha-OHE1, stimulated DNA synthesis, and 2-ME2 inhibited the stimulatory effects of these agents. E-2 and 16alpha-OHE1, stimulated the progression of cells from G(1) to S phase and this effect was attenuated by 2-ME2. Western blot analysis showed that E-2 and 16alpha-OHE1, increased cyclin D1 protein level by about fourfold compared with control. 2-ME2 had no significant effect on cyclin D1; however, it prevented the accumulation of cyclin D1 in the presence of E2 and 16alpha-OHE1. Cells transfected with a cyclin D1 reporter gene and treated with E-2 or 16alpha-OHE1, showed 7- and 9.5-fold increase respectively in promoter activity compared with control. This activity was significantly inhibited by 2-ME2. Cyclin D1 transactivation was mediated by the CAMP response element (CRE) region, which binds activating transcription factor 2 (ATF-2). DNA affinity assay showed 2.5- and 3.5-fold increases in ATF-2 binding to CRE in the presence of E-2 and 16alpha-OHE1, respectively. The binding of ATF-2 was inhibited by the presence of 2-ME2. These results show that 2-ME2 can downregulate cyclin D1 and thereby cell cycle progression by a mechanism involving the disruption of ATF-2 binding to cyclin D1 promoter.