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Title:Dominant negative mutants of the human estrogen receptor
Author(s):Ince, Basil Avery
Doctoral Committee Chair(s):Katzenellenbogen, Benita S.
Department / Program:Biology
Discipline:Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Genetics
Biology, Cell
Abstract:We have characterized three human estrogen receptor (ER) mutants which, at low concentrations, are capable of blocking the intracellular activity of wild type ER. The mutants, a truncated ER (ER1-530), a point mutant (L540Q), and a frameshift (S554fs), were generated by random chemical mutagenesis of the ER hormone binding domain and screened first for low transcriptional activity in a yeast selection system. When co-expressed with wild type ER in transient co-transfection assays using ER-deficient Chinese hamster ovary cells, each of the mutants effectively suppresses the ability of wild type ER to activate transcription of an estrogen-regulated reporter plasmid. Of the three mutants, S554fs is the most potent ER inhibitor. A fourth ER mutant, V364E, is also found to be a strong dominant negative inhibitor of wild type ER transcriptional activity although, alone, it exhibits transcriptional superactivity at high levels of estradiol ($\rm10\sp{-8}\ M\ E\sb2).$ We next demonstrate that co-treatment with IBMX/CT (agents which elevate intracellular cAMP) and one of the three ligands (E$\sb2$, TOT or ICI) results in the unexpected recovery of strong receptor activation of the L540Q and S554fs receptors, the magnitude of which is dependent upon promoter- and cell-contexts. Unlike L540Q and S554fs, the transcriptionally inactive ER1-530 is not activated by any combination of ligands and IBMX/CT. These phenomena may provide a partial explanation of the ability of some estrogen-dependent human breast tumors to resist antiestrogen therapies currently employed. Lastly, given the previous findings, we have directly investigated the ability of the ER mutants to block endogenous ER-mediated transcription in a Michigan Cancer Foundation (MCF-7) human breast cancer cell line. S554fs and L540Q prove to be strong repressors of both E$\sb2$- and TOT stimulated transcription, and the effectiveness of neither ER mutant is compromised by the presence of elevated levels of intracellular cAMP, despite the cAMP-enhanced transcriptional activity of endogenous wild type ER. In summary, the data seem to suggest that S554fs and L540Q are reasonable candidates for studies designed to inhibit the estrogen- and tamoxifen-stimulated growth of human breast cancer cells.
Issue Date:1995
Type:Text
Language:English
URI:http://hdl.handle.net/2142/20701
Rights Information:Copyright 1995 Ince, Basil Avery
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9522123
OCLC Identifier:(UMI)AAI9522123


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