{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/20701"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/20701","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Dominant negative mutants of the human estrogen receptor","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.","abstract_html":"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 (<span class=\"etd-inline-math\">\\rm10\\sp{-8} M E\\sb2).</span> 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.","abstract_has_math":true,"creators":["Ince, Basil Avery"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biology","degree_department":null,"school":null,"contributors":["Katzenellenbogen, Benita S."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T12:46:50Z","date_published":"2011-05-07T12:46:50Z","updated_at":"2026-07-22T22:25:16Z","subjects":["Biology, Genetics","Biology, Cell"],"languages":["eng"],"rights":["Copyright 1995 Ince, Basil Avery"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9522123","(UMI)AAI9522123"],"render_values":[{"text":"AAI9522123","href":null,"code":true},{"text":"(UMI)AAI9522123","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/20701","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Katzenellenbogen, Benita S."]},{"key":"dc:creator","label":"Author","values":["Ince, Basil Avery"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T12:46:50Z","10000-01-01","1995"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology, Genetics","Biology, Cell"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1995 Ince, Basil Avery"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9522123","(UMI)AAI9522123","http://hdl.handle.net/2142/20701"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","Made available in DSpace on 2011-05-07T12:46:50Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9522123.pdf: 5444852 bytes, checksum: fa01552950d27160955a2cd734d0bb8f (MD5) Previous issue date: 1995","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:45:40Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:20:17-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only"]},{"key":"dc:title","label":"Title","values":["Dominant negative mutants of the human estrogen receptor"]}]}],"canonical_facts":{"dc:contributor":["Katzenellenbogen, Benita S."],"dc:creator":["Ince, Basil Avery"],"dc:date":["2011-05-07T12:46:50Z","10000-01-01","1995"],"dc:description":["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.","Made available in DSpace on 2011-05-07T12:46:50Z (GMT). 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