Dominant negative mutants of the human estrogen receptor
Ince, Basil Avery
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Permalink
https://hdl.handle.net/2142/20701
Description
Title
Dominant negative mutants of the human estrogen receptor
Author(s)
Ince, Basil Avery
Issue Date
1995
Doctoral Committee Chair(s)
Katzenellenbogen, Benita S.
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Genetics
Biology, Cell
Language
eng
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.
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