Repression of endogenous estrogen receptor activity in MCF-7 human breast cancer cells by dominant negative estrogen receptors.
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.
Ince
B A
BA
Department of Cell and Structural Biology, University of Illinois, Urbana 61801, USA.
Schodin
D J
DJ
Shapiro
D J
DJ
Katzenellenbogen
B S
BS
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D000242:Cyclic AMP / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D016368:Frameshift Mutation; D017353:Gene Deletion; D005799:Genes, Dominant; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors* / Q000235:genetics; D013629:Tamoxifen / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D011960:Receptors, Estrogen; D013629:Tamoxifen; D004958:Estradiol; D000242:Cyclic AMP