Hansen E R, Brooks S C
Cancer Res. 1982 May;42(5):1967-74.
Human breast cancer cells (MCF-7, maintained in long-term culture) contain separate estrogen receptors specific for either 17 beta-estradiol or estrone. Utilizing optimum conditions for the protamine sulfate assay, it has been possible to demonstrate both receptors in the 0.6 M KCl extract of nuclei and in the cytosol. Similarly, in the exchange assay, high-affinity low-capacity binding sites for 17 beta-estradiol and estrone have been found in the salt-extracted nuclear residue. Dissociation constants and binding capacities were determined for either receptor in the absence of the other [e.g., estrone receptor (E1R) in the cytosol or nuclear residue from 17 beta-estradiol-stimulated cells] or, when both receptors were present, a saturating amount of the other estrogen (unlabeled) was added to the assay mixture (e.g., the salt-extractable nuclear receptors). Specificity was demonstrated by the inability of estrone to compete with 17 beta-[2,4,6,7-3H]estradiol for the 17 beta-estradiol receptor (E2R) at molar excesses less than 10-fold. Likewise, there was no inhibition of [6,7-3H]estrone binding to its receptor by molar excesses of 17 beta-estradiol below 100-fold. Other steroid hormones were very weak competitors of [6,7-3H]estrone, even at 1000-fold molar excesses. The quantitative relationships of these two estrogen receptors were shown to fluctuate in the various cellular compartments following incubation (37 degrees) of MCF-7 cells with 10(-8) M 17 beta-estradiol. This level of 17 beta-estradiol elicited the translocation of all detectable cytosolic E2R to the nucleus, where, after an incubation of 1 hr, the salt-resistant 17 beta-estradiol disappeared and 40% of the extractable 17 beta-estradiol-binding capacity was lost (processed). Simultaneously, the E1R which remained in the nuclear residue appeared in the nuclear extract, and ultimately this receptor accumulated in the cytosol. The estrone-binding capacity (0.78 pmol/mg DNA) which appeared following the processing of E2R nearly equalled the loss of 17 beta-estradiol binding sites per cell (0.85 pmol/mg DNA). Concentrations of 17 beta-estradiol which elicited the greatest processing of E2R in these incubations brought about the appearance of maximum levels of E1R in MCF-7 cells. Considering these results in the light of data previously reported from this laboratory concerning the metabolic and ligand fate of 17 beta-[3H]estradiol in MCF-7 cells, processing would appear to involve the formation of E1R in the salt-resistant nuclear compartment followed by the accumulation of E1R in the cytosol.
人乳腺癌细胞（长期培养的MCF-7细胞）含有分别对17β-雌二醇或雌酮具有特异性的雌激素受体。利用硫酸鱼精蛋白测定的最佳条件，已能够在细胞核的0.6M KCl提取物和细胞质中检测到这两种受体。同样，在交换试验中，在盐提取的核残余物中发现了对17β-雌二醇和雌酮具有高亲和力、低容量的结合位点。在不存在另一种受体的情况下（例如，细胞质中的雌酮受体（E1R）或来自17β-雌二醇刺激细胞的核残余物）测定了两种受体的解离常数和结合容量，或者当两种受体都存在时，向测定混合物中加入饱和量的另一种雌激素（未标记）（例如，盐可提取的核受体）。雌酮在摩尔过量小于10倍时不能与17β-[2,4,6,7-³H]雌二醇竞争17β-雌二醇受体（E2R），从而证明了特异性。同样，低于100倍摩尔过量的17β-雌二醇对[6,7-³H]雌酮与其受体的结合也没有抑制作用。即使在1000倍摩尔过量时，其他甾体激素对[6,7-³H]雌酮的竞争作用也非常弱。在MCF-7细胞与10⁻⁸M 17β-雌二醇在37℃孵育后，这两种雌激素受体的定量关系在不同细胞区室中显示出波动。这种17β-雌二醇水平促使所有可检测到的细胞质E2R转位至细胞核，在孵育1小时后，耐盐的17β-雌二醇消失，可提取的17β-雌二醇结合容量损失40%（被处理）。同时，留在核残余物中的E1R出现在核提取物中，最终该受体在细胞质中积累。E2R被处理后出现的雌酮结合容量（0.78 pmol/mg DNA）几乎等于每个细胞中17β-雌二醇结合位点的损失量（0.85 pmol/mg DNA）。在这些孵育中引起E2R最大程度处理的17β-雌二醇浓度导致MCF-7细胞中E1R出现最高水平。根据本实验室先前报道的关于MCF-7细胞中17β-[³H]雌二醇的代谢和配体命运的数据来看这些结果，处理过程似乎涉及在耐盐核区室中形成E1R，随后E1R在细胞质中积累。
