Ince B A, Schodin D J, Shapiro D J, Katzenellenbogen B S
Department of Cell and Structural Biology, University of Illinois, Urbana 61801, USA.
Endocrinology. 1995 Aug;136(8):3194-9. doi: 10.1210/endo.136.8.7628351.
We have investigated the ability of several transcriptionally inactive estrogen receptor (ER) mutants to block endogenous ER-mediated transcription in MCF-7 human breast cancer cells. In transient transfections of MCF-7 cells, two of the mutants, a frame-shifted ER (S554fs) and a point-mutated ER (L540Q), strongly inhibit the ability of endogenous wild-type ER to activate transcription of estrogen-regulated reporter plasmids. A third mutant, ER1-530, which is missing 65 residues from its carboxy-terminus, is a weaker repressor of estradiol-stimulated transcription. When an estrogen response element (ERE)-thymidine kinase-chloramphenicol acetyltransferase reporter gene is used, S554fs, L540Q, and ER1-530 suppress the transcriptional activity of endogenous MCF-7 ER by 87%, 97%, and 62%, respectively. The magnitude of dominant negative repression is promoter specific; when an ERE-pS2-chloramphenicol acetyltransferase reporter is employed, inhibition of endogenous ER activity by equivalent amounts of S554fs, L540Q, and ER1-530 ranges from 85-97%. Dose-response studies show the S554fs mutant to be the most potent of the three ER mutants as a repressor of estrogen action in these cells. In addition, elevated levels of intracellular cAMP, achieved by the addition of 3-isobutyl-1-methylxanthine plus cholera toxin to cells, fail to compromise the effectiveness of these mutants as dominant negative ERs despite the cAMP-enhanced transcriptional activity of ER. The mutants are also powerful repressors of the agonist activity of trans-hydroxytamoxifen-stimulated ER transcription. The dominant negative activity of the three mutants is lost when the A/B domain of these receptors is deleted, implying an important role for this N-terminal region of the ER in the ability of these mutants to inhibit endogenous wild-type ER activity. All in all, the data suggest that S554fs in particular is a reasonable candidate for studies designed to use a dominant negative ER to inhibit the estrogen- and tamoxifen-stimulated growth of human breast cancer cells.
我们研究了几种转录失活的雌激素受体（ER）突变体在MCF-7人乳腺癌细胞中阻断内源性ER介导转录的能力。在MCF-7细胞的瞬时转染中，其中两个突变体，一个移码ER（S554fs）和一个点突变ER（L540Q），强烈抑制内源性野生型ER激活雌激素调节报告质粒转录的能力。第三个突变体ER1-530，其羧基末端缺失65个残基，是雌二醇刺激转录的较弱抑制剂。当使用雌激素反应元件（ERE）-胸苷激酶-氯霉素乙酰转移酶报告基因时，S554fs、L540Q和ER1-530分别将内源性MCF-7 ER的转录活性抑制87%、97%和62%。显性负抑制的程度具有启动子特异性；当使用ERE-pS2-氯霉素乙酰转移酶报告基因时，等量的S554fs、L540Q和ER1-530对内源性ER活性的抑制范围为85%-97%。剂量反应研究表明，在这些细胞中，作为雌激素作用的抑制剂，S554fs突变体是这三种ER突变体中最有效的。此外，通过向细胞中添加3-异丁基-1-甲基黄嘌呤和霍乱毒素使细胞内cAMP水平升高，尽管cAMP增强了ER的转录活性，但并未损害这些突变体作为显性负性ER的有效性。这些突变体也是反式羟基他莫昔芬刺激的ER转录激动剂活性的强大抑制剂。当这些受体的A/B结构域被删除时，这三个突变体的显性负性活性丧失，这意味着ER的这个N端区域在这些突变体抑制内源性野生型ER活性的能力中起重要作用。总而言之，数据表明，特别是S554fs是旨在使用显性负性ER抑制人乳腺癌细胞雌激素和他莫昔芬刺激生长的研究的合理候选者。
