Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells
Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells
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DOI：
10.1038/sj.onc.1205136
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发表时间：
2002-01-24
期刊：
影响因子：
8
通讯作者：
Moudgil, V
中科院分区：
文献类型：
--
作者：
Dinda, S;Sanchez, A;Moudgil, V
T47D细胞是一种雌激素敏感的人导管癌细胞系，表达可检测水平的雌激素受体（ER）。我们之前的研究表明，雌二醇（E-2）处理T47D细胞会导致p53水平升高，并伴随视网膜母细胞瘤蛋白（pRb）的磷酸化。在本研究中，我们分析了p53的表达和pRb的磷酸化状态，并比较了E-2和三碘甲状腺原氨酸（T-3）对这些现象的影响。细胞在含有木炭处理血清的培养基中生长，以消耗内源性类固醇的水平。融合后，将细胞用T-3 (10 （12) ~ 10(-7) M）处理24小时，用Western分析法检测p53和pRb的存在。E-2处理细胞导致p53水平增加2-3倍。T-3在培养基中的存在导致p53水平以浓度依赖性的方式逐渐增加。在上述条件下，在E-2和T-3存在的情况下，pRb被磷酸化（SDS-PAGE检测到为上升）。在生长培养基中添加T-3 （1 muM）可在4 h内提高T47D细胞的增殖速度并诱导pRb的过度磷酸化；当内质网拮抗剂ICI 164 384 (ICI) （1 muM）与E-2 （1 muM）或T-3 （1 muM）联合使用时，激素对细胞增殖和pRb过度磷酸化的影响被阻断。使用激光扫描共聚焦荧光显微镜进行免疫标记，对p53进行Western分析和细胞定位，结果显示激素处理细胞中p53丰度的ici敏感性增加。类固醇结合分析显示T-3对[H-3]E-2结合缺乏竞争。这些结果表明，T-3通过ER和T-3受体（T3R）介导的途径共同调节T47D细胞周期进程和增殖，提高p53水平并引起pRb的过度磷酸化。
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.