Hafner F, Holler E, von Angerer E
Institut für Pharmazie, Universität Regensburg, Germany.
J Steroid Biochem Mol Biol. 1996 Jul;58(4):385-93. doi: 10.1016/0960-0760(96)00054-4.
The proliferation of mammary carcinoma cells can be stimulated by estrogens and various growth factors such as EGF and IGF-I. Steroid hormones and growth factors are understood to exert their effects via different receptors and signal transduction pathways. Recently, it has been shown that growth factors can utilize the unliganded estrogen receptor (ER) as a transcription factor. This study was aimed at identifying the growth factors that can act via the estrogen receptor, and finding new estrogen antagonists that block this activity. Originally, a transcription assay was used in which HeLa cells had been transiently co-transfected with the expression vector for the human ER and a reporter plasmid EREwtc luc. EGF and, to a lesser extent, insulin stimulated the expression of the reporter gene in the absence of estradiol (E2), whereas IGF-I was inactive. The stimulatory effect of E2 and insulin was suppressed when the ER was blocked by the pure antiestrogen ICI 182,780. In ER-positive MCF-7 cells, transfected transiently with the reporter plasmid, EGF had no stimulatory effect on luciferase expression. IGF-I stimulated the transcription to about 50% of the E2 value. Similar activity was found for insulin. The effect of both growth factors was only partly reversed by the addition of a pure antiestrogen. The combination of E2 and IGF-I or insulin led to a synergistic activation of transcription. Because transiently transfected cells do not allow one to study the influence of chromatin structure on gene expression, an MCF-7 subline (MCF-7/2a) was established, in which the reporter construct had been integrated in the genome. IGF-I stimulated luciferase expression in these cells, but showed no overadditive effect with E2. The effects of both agents were completely suppressed by the pure antiestrogen ICI 182,780. These data suggest the existence of an ER-independent mechanism for the activation of the reporter gene in transiently transfected cells, but not in stable transfectants.
雌激素以及多种生长因子（如表皮生长因子和胰岛素样生长因子-I）均可刺激乳腺癌细胞的增殖。已知类固醇激素和生长因子通过不同的受体和信号转导途径发挥作用。最近研究表明，生长因子可利用未结合配体的雌激素受体（ER）作为转录因子。本研究旨在确定可通过雌激素受体发挥作用的生长因子，并寻找能阻断该活性的新型雌激素拮抗剂。最初，采用转录分析方法，将人ER的表达载体与报告质粒EREwtc luc瞬时共转染至HeLa细胞。在无雌二醇（E2）的情况下，表皮生长因子以及程度稍弱的胰岛素可刺激报告基因的表达，而胰岛素样生长因子-I则无活性。当ER被纯抗雌激素ICI 182,780阻断时，E2和胰岛素的刺激作用受到抑制。在瞬时转染报告质粒的雌激素受体阳性MCF-7细胞中，表皮生长因子对荧光素酶表达无刺激作用。胰岛素样生长因子-I可将转录刺激至约E2值的50%。胰岛素也有类似活性。添加纯抗雌激素只能部分逆转这两种生长因子的作用。E2与胰岛素样生长因子-I或胰岛素联合使用可导致转录的协同激活。由于瞬时转染细胞无法用于研究染色质结构对基因表达的影响，因此构建了一个MCF-7亚系（MCF-7/2a），其中报告构建体已整合到基因组中。胰岛素样生长因子-I可刺激这些细胞中的荧光素酶表达，但与E2无叠加效应。这两种药物的作用均被纯抗雌激素ICI 182,780完全抑制。这些数据表明，在瞬时转染细胞中存在一种不依赖雌激素受体的报告基因激活机制，但在稳定转染细胞中不存在。
