Singh R K, Ruh M F, Butler W B, Ruh T S
Endocrinology. 1986 Mar;118(3):1087-95. doi: 10.1210/endo-118-3-1087.
We examined the chromatin binding characteristics of estrogen receptor from MCF-7 cells when bound by [3H] estradiol vs. the high affinity antiestrogen [3H]H1285 [4-(N,N-diethylaminoethoxy) 4'-methoxy-alpha-(p-hydroxyphenyl)alpha-ethylstilbene]. Two sublines of MCF-7 cells were used: E-3, which is sensitive to antiestrogens, and RR, which is antiestrogen resistant and was selected for its ability to grow in the presence of tamoxifen. Chromatin was prepared from both E-3 and RR cells, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine hydrochloride (Gdn X HCl). The chromatin acceptor activity unmasked by Gdn X HCl was determined using partially purified (30-fold) activated [3H]estradiol- or [3H] H1285-receptor complexes obtained by KCl step elution from DEAE-cellulose columns. With chromatin from E-3 cells, maximal binding (acceptor activity) for [3H]estradiol-receptor complexes prepared from either type of MCF-7 cells (E-3 or RR) was unmasked by 1 and 6 M Gdn X HCl, whereas [3H]H1285-receptor complexes exhibited maximal binding to 1 and 4 M Gdn X HCl-extracted chromatin subfractions. Chromatin prepared from RR cells was similar to that from E-3 cells in its binding activity for [3H]estradiol-receptor complexes. It differed, however, in that [3H]H1285-receptor complexes showed less chromatin acceptor site binding in general to 1-8 M Gdn X HCl-deproteinized RR chromatin, and the binding peak unmasked by 4 M Gdn X HCl was absent in chromatin from these cells. Receptor binding to chromatin was stable and was competitively inhibited by radioinert estradiol- or H1285-receptor complexes (but not by denatured receptors), demonstrating the saturability and specificity of these acceptor sites. Thus, estrogen receptor binds differently to chromatin depending on whether estradiol or an antiestrogen is bound to it. In addition, the acquisition of antiestrogen resistance by the RR subline of MCF-7 cells appears to result from alterations in the state of its chromatin rather than changes in the receptor itself. Finally, the observation that the chromatin from the resistant cells differs from that of the sensitive cells suggests that antiestrogens may be able to inhibit the growth of MCF-7 and other antiestrogen-sensitive cells not only by antagonizing the stimulatory effect of estrogens, but also by exerting some separate effect of their own.
我们研究了MCF-7细胞中雌激素受体在与[3H]雌二醇结合以及与高亲和力抗雌激素[3H]H1285[4-(N,N-二乙氨基乙氧基)4'-甲氧基-α-(对羟基苯基)α-乙基芪]结合时的染色质结合特性。使用了MCF-7细胞的两个亚系：对抗雌激素敏感的E-3细胞系，以及对他莫昔芬有抗性的RR细胞系，RR细胞系因其在他莫昔芬存在下生长的能力而被挑选出来。从E-3和RR细胞中制备染色质，将其共价连接到纤维素上，并依次用0 - 8M盐酸胍(Gdn X HCl)进行脱蛋白处理。使用通过从DEAE-纤维素柱上进行KCl分步洗脱获得的部分纯化(30倍)的活化[3H]雌二醇或[3H]H1285受体复合物，来测定由Gdn X HCl揭示的染色质受体活性。对于来自E-3细胞的染色质，由任何一种MCF-7细胞类型(E-3或RR)制备的[3H]雌二醇受体复合物的最大结合(受体活性)在1M和6M Gdn X HCl处理下被揭示出来，而[3H]H1285受体复合物对1M和4M Gdn X HCl提取的染色质亚组分表现出最大结合。从RR细胞制备的染色质在其对[3H]雌二醇受体复合物的结合活性方面与来自E-3细胞的染色质相似。然而，不同之处在于，[3H]H1285受体复合物总体上对1 - 8M Gdn X HCl脱蛋白处理的RR染色质显示出较少的染色质受体位点结合，并且在这些细胞的染色质中不存在由4M Gdn X HCl揭示的结合峰。受体与染色质的结合是稳定的，并且被无放射性的雌二醇或H1285受体复合物竞争性抑制(但不被变性受体抑制)，这证明了这些受体位点的饱和性和特异性。因此，雌激素受体与染色质的结合方式取决于它结合的是雌二醇还是抗雌激素。此外，MCF-7细胞的RR亚系获得抗雌激素抗性似乎是由于其染色质状态的改变，而不是受体本身的变化。最后，抗性细胞的染色质与敏感细胞的染色质不同这一观察结果表明，抗雌激素可能不仅通过拮抗雌激素的刺激作用，还通过发挥其自身的一些独立作用来抑制MCF-7和其他抗雌激素敏感细胞的生长。
