Press M F, Xu S H, Wang J D, Greene G L
Department of Pathology, University of Southern California School of Medicine, Los Angeles 90033.
Am J Pathol. 1989 Nov;135(5):857-64.
The estrogen receptor (ER) and progesterone receptor (PR) content of cultured human breast carcinoma cells (MCF-7) was determined by biochemical assay, immunoblot analysis, and immunohistochemical assay under varying conditions of hormonal stimulation. The ER and PR content in cytosolic and nuclear extracts varied with steroid treatment. However, both the amount and distribution of each receptor in these extracts was virtually the same when determined by steroid binding and immunoblot analyses. Two immunocytochemical parameters (staining intensity and proportion of cells stained) correlated with the quantitative analyses of ER and PR, but not with the subcellular distribution. When MCF-7 cells were grown for 4 days in charcoal-stripped serum without phenol red, 93% of total ER was found in the cytosol (10 mM KCl), whereas short-term treatment with 5 nM estradiol resulted in the appearance of 82% of total ER in the nuclear extract (400 mM KCl). With either cell treatment only nuclear staining for ER was observed. Progesterone receptor was virtually undetectable in the same cells by any method. After 4 days of treatment by 5 nM estradiol, PR was strongly induced (50-fold) in MCF-7 cells as determined by all three methods. As observed for ER, 95% of total induced PR was found in the cytosol in the absence of a progestin. Short-term treatment with 5 nM ORG 2058, a synthetic progestin, resulted in the appearance of 42% of total PR in the nuclear extract. However, only strong nuclear staining for PR was observed in either the presence or absence of a progestin. These findings are consistent with the current view of ER and PR as nuclear receptors present in at least two forms. One of these, the unoccupied form of the receptor, is easily removed from the nucleus by hypotonic buffers during the cell homogenization process and appears in the cytosolic extract. The other form of the receptor, the steroid-occupied form, is more tightly bound to nuclear components and is removed from nuclei only under more vigorous extraction conditions.
在不同激素刺激条件下，通过生化测定、免疫印迹分析和免疫组织化学测定，确定培养的人乳腺癌细胞（MCF - 7）中的雌激素受体（ER）和孕激素受体（PR）含量。胞质和核提取物中的ER和PR含量随类固醇处理而变化。然而，通过类固醇结合和免疫印迹分析确定时，这些提取物中每种受体的数量和分布实际上是相同的。两个免疫细胞化学参数（染色强度和染色细胞比例）与ER和PR的定量分析相关，但与亚细胞分布无关。当MCF - 7细胞在不含酚红的活性炭处理血清中培养4天时，发现总ER的93%存在于胞质溶胶（10 mM KCl）中，而用5 nM雌二醇短期处理导致82%的总ER出现在核提取物（400 mM KCl）中。无论哪种细胞处理方式，仅观察到ER的核染色。用任何方法在相同细胞中几乎检测不到孕激素受体。用5 nM雌二醇处理4天后，通过所有三种方法测定，MCF - 7细胞中的PR被强烈诱导（50倍）。如对ER的观察，在没有孕激素的情况下，总诱导PR的95%存在于胞质溶胶中。用5 nM合成孕激素ORG 2058短期处理导致42%的总PR出现在核提取物中。然而，无论是否存在孕激素，仅观察到PR的强核染色。这些发现与目前将ER和PR视为至少以两种形式存在的核受体的观点一致。其中一种，受体的未占据形式，在细胞匀浆过程中很容易被低渗缓冲液从细胞核中去除，并出现在胞质提取物中。受体的另一种形式，类固醇占据形式，与核成分结合更紧密，仅在更剧烈的提取条件下才从细胞核中去除。
