Pink J J, Jiang S Y, Fritsch M, Jordan V C
Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
Cancer Res. 1995 Jun 15;55(12):2583-90.
Long-term growth of estrogen-responsive human breast cancer cell lines in estrogen-free media leads inevitably to the development of estrogen-independent growth. We have identified and characterized a unique subclone of the MCF-7 human breast cancer cell line, named MCF-7:2A, which grows maximally in the absence of endogenous estrogens but whose growth is inhibited by the antiestrogens 4-hydroxytamoxifen and ICI 164,384. The MCF-7:2A cells express high levels of estrogen receptor (ER; 477 fmol/mg protein), which can be reduced by growth in 10 nM 17 beta-estradiol (201 fmol/mg protein). Basal progesterone receptor synthesis is very low in the 2A cells (< 1 fmol/mg protein) but can be dramatically increased by 10 nM 17 beta-estradiol (384 fmol/mg protein). Clearly, the pathways that control growth and estrogen-regulated genes such as the progesterone receptor are now dissociated in these cells. MCF-7:2A cells also possess two unique characteristics. First, the MCF-7:2A cells constitutively activate an ER-responsive luciferase reporter construct in the absence of any estrogens, and this activation can be blocked by either 4-hydroxytamoxifen or ICI 164,384. This constitutive activity is not observed in the parental MCF-7 cells. Second, they express an 80-kDa protein that cross-reacts with three distinct antibodies to the ER. The MCF-7:2A cells were subjected to an additional round of limiting dilution subcloning, and 10 independent clones were all shown to express both the 66- and 80-kDa ERs as observed in the MCF-7:2A line. This confirms that both ERs are being expressed in each cell and are not the result of a mixed population of cells. While numerous ER variants have been reported previously, no ER has until now been described that is larger than the wild-type 66-kDa ER. The MCF-7:2A cells provide a unique model to use in the study of ER action and the development of estrogen-independent growth in human breast cancer cells.
雌激素反应性人乳腺癌细胞系在无雌激素培养基中的长期生长不可避免地导致雌激素非依赖性生长的发展。我们已鉴定并表征了MCF-7人乳腺癌细胞系的一个独特亚克隆，命名为MCF-7:2A，其在无内源性雌激素的情况下生长最佳，但其生长受到抗雌激素4-羟基他莫昔芬和ICI 164,384的抑制。MCF-7:2A细胞表达高水平的雌激素受体（ER；477 fmol/mg蛋白），在10 nM 17β-雌二醇中生长可使其降低（201 fmol/mg蛋白）。2A细胞中基础孕酮受体合成非常低（<1 fmol/mg蛋白），但可被10 nM 17β-雌二醇显著增加（384 fmol/mg蛋白）。显然，在这些细胞中，控制生长和雌激素调节基因（如孕酮受体）的途径现在已分离。MCF-7:2A细胞还具有两个独特特征。首先，MCF-7:2A细胞在无任何雌激素的情况下组成性激活ER反应性荧光素酶报告构建体，且这种激活可被4-羟基他莫昔芬或ICI 164,384阻断。在亲本MCF-7细胞中未观察到这种组成性活性。其次，它们表达一种80 kDa的蛋白，该蛋白与三种针对ER的不同抗体发生交叉反应。对MCF-7:2A细胞进行了另一轮有限稀释亚克隆，10个独立克隆均显示如在MCF-7:2A系中观察到的那样表达66 kDa和80 kDa的ER。这证实每个细胞中都表达两种ER，而不是细胞混合群体的结果。虽然先前已报道了许多ER变体，但迄今为止尚未描述大于野生型66 kDa ER的ER。MCF-7:2A细胞为研究ER作用和人乳腺癌细胞中雌激素非依赖性生长的发展提供了一个独特的模型。
