Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells

T47D cells represent an estrogen-responsive human ductal carcinoma cell line which expresses detectable levels of estrogen receptor (ER). We have previously shown that estradiol (E-2) treatment of T47D cells causes an increase in the level of p53 and a concomitant phosphorylation of retinoblastoma protein (pRb). In the present study, we have analysed the expression of p53 and phosphorylation state of pRb and compared the effects of E-2 and triiodothyronine (T-3) on these phenomena. Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids. Upon confluency, the cells were treated with T-3 (10 (12) to 10(-7) M) for 24 h and the presence of p53 and pRb was detected by Western analysis. E-2 treatment of cells caused a 2-3-fold increase in the level of p53. Presence of T-3 in the medium caused a gradual increase in the level of p53 in a concentration-dependent manner. Under the above conditions, pRb was phosphorylated (detected as an upshift during SDS-PAGE) in the presence of E-2 and T-3. Supplementation of growth medium with T-3 (1 muM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this effect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 muM), an ER antagonist, was combined with E-2 (1 muM) or T-3 (1 muM), effects of hormones on cell proliferation and hyperphosphorylation of pRb were blocked. Western analysis of p53 was supplemented with its cytolocalization by immuno-labeling using laser scanning confocal fluorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells. Steroid binding analysis revealed lack of competition by T-3 for the [H-3]E-2 binding. These results indicate that T-3 regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T-3 receptor (T3R)-mediated pathways.