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 eects 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 con¯uency, the cells were treated with T 3 (10 712 to 10 77 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 mM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this eect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 mM), an ER antagonist, was combined with E 2 (1 nM) or T 3 (1 mM), eects 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 uorescence 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 [ 3 H]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 (T 3 R)-mediated pathways.

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We have previously shown that presence of estradiol (E2) in the growth medium causes (i) proliferation of T47D breast cancer cells, (ii) elevation of p53 levels, and (iii) hyperphos-phorylation of retinoblastoma protein (pRb). In the present study, we examined the expression of p53, phosphorylation state of pRb and proliferation of T47D cells in the presence of LY117018 (Courtesy of Lilly Research Laboratories), an analog of raloxifene, which is a known selective estrogen receptor modulator (SERM). The cells grown in charcoal-treated serum were treated with 1 nM E2 or different concentrations of LY117018 for 24 h. E2 or LY117018 treatments caused a 2- to 3-fold increase in the level of p53 and hyperphosphorylation of pRb. E2 treatment increased cell proliferation, whereas LY117018 treatment had no such effect but inhibited the E2-dependent cell proliferation. E2 and LY117018 treatments of T47D cells also caused differential effects on intracellular structures. Thus, LY117018 treatment induces changes in the level/activity of p53 and pRb and ultrastructure of T47D cells. Importantly, LY11708 inhibits estrogen-induced cell proliferation while mimicking E2 actions on p53 induction and pRb phosphorylation. The SERM also induced structural alterations in the T47D cells.