Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry
Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2...
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| Veröffentlicht in: | PloS one 2012-08, Vol.7 (8), p.e42487-e42487 |
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| creator | Clerc, Pascaline Carey, Gregory B Mehrabian, Zara Wei, Michael Hwang, Hyehyun Girnun, Geoffrey D Chen, Hegang Martin, Stuart S Polster, Brian M |
| description | Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist. |
| doi_str_mv | 10.1371/journal.pone.0042487 |
| format | Article |
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Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0042487</identifier><identifier>PMID: 22880001</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Anesthesiology ; Aniline Compounds - pharmacology ; Animals ; Apoptosis ; B-cell lymphoma ; Bax protein ; bcl-2 Homologous Antagonist-Killer Protein - metabolism ; Bcl-2 protein ; bcl-2-Associated X Protein - metabolism ; Biochemistry ; Bioenergetics ; Biology ; Biomarkers ; Biphenyl Compounds - pharmacology ; Cancer therapies ; Cancer treatment ; Caspase ; Cell Death - drug effects ; Cell Line ; Cell Line, Tumor ; Cell Respiration - drug effects ; Chemotherapy ; Clinical trials ; Cytochrome ; Cytochrome c ; Cytochromes c - metabolism ; Cytological Techniques - instrumentation ; Cytological Techniques - methods ; Death ; Electron transport ; Electron transport chain ; Embryo fibroblasts ; Embryos ; Energy Metabolism - drug effects ; Enzyme-linked immunosorbent assay ; Epithelial cells ; Fibroblasts ; Humans ; Impairment ; Leukemia ; Lymphocytes B ; Lymphoma ; Lymphoma, B-Cell - pathology ; Lymphomas ; Mammary gland ; Mcl-1 protein ; Medical research ; Medicine ; Mice ; Mitochondria ; Mitochondria - drug effects ; Mitochondria - metabolism ; Models, Biological ; Molecular biology ; Mortality ; Multiple myeloma ; Multiplexing ; Nitrophenols - pharmacology ; Oxygen Consumption - drug effects ; Phenotype ; Phenotypes ; Piperazines - pharmacology ; Proteins ; Proto-Oncogene Proteins c-bcl-2 - metabolism ; Rats ; Reduction ; Respiration ; Respirometry ; Sulfonamides - pharmacology ; Tumor cell lines ; Tumors</subject><ispartof>PloS one, 2012-08, Vol.7 (8), p.e42487-e42487</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>Clerc et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2012 Clerc et al 2012 Clerc et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-25c8cbf28e1bdb01ecf2b31136f794cad77634ec342f539642970cd2fe39f9d93</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411749/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411749/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22880001$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Shirihai, Orian S.</contributor><creatorcontrib>Clerc, Pascaline</creatorcontrib><creatorcontrib>Carey, Gregory B</creatorcontrib><creatorcontrib>Mehrabian, Zara</creatorcontrib><creatorcontrib>Wei, Michael</creatorcontrib><creatorcontrib>Hwang, Hyehyun</creatorcontrib><creatorcontrib>Girnun, Geoffrey D</creatorcontrib><creatorcontrib>Chen, Hegang</creatorcontrib><creatorcontrib>Martin, Stuart S</creatorcontrib><creatorcontrib>Polster, Brian M</creatorcontrib><title>Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.</description><subject>Anesthesiology</subject><subject>Aniline Compounds - pharmacology</subject><subject>Animals</subject><subject>Apoptosis</subject><subject>B-cell lymphoma</subject><subject>Bax protein</subject><subject>bcl-2 Homologous Antagonist-Killer Protein - metabolism</subject><subject>Bcl-2 protein</subject><subject>bcl-2-Associated X Protein - metabolism</subject><subject>Biochemistry</subject><subject>Bioenergetics</subject><subject>Biology</subject><subject>Biomarkers</subject><subject>Biphenyl Compounds - pharmacology</subject><subject>Cancer therapies</subject><subject>Cancer treatment</subject><subject>Caspase</subject><subject>Cell Death - drug effects</subject><subject>Cell Line</subject><subject>Cell Line, Tumor</subject><subject>Cell Respiration - drug effects</subject><subject>Chemotherapy</subject><subject>Clinical trials</subject><subject>Cytochrome</subject><subject>Cytochrome c</subject><subject>Cytochromes c - metabolism</subject><subject>Cytological Techniques - instrumentation</subject><subject>Cytological Techniques - methods</subject><subject>Death</subject><subject>Electron transport</subject><subject>Electron transport chain</subject><subject>Embryo fibroblasts</subject><subject>Embryos</subject><subject>Energy Metabolism - drug effects</subject><subject>Enzyme-linked immunosorbent assay</subject><subject>Epithelial cells</subject><subject>Fibroblasts</subject><subject>Humans</subject><subject>Impairment</subject><subject>Leukemia</subject><subject>Lymphocytes B</subject><subject>Lymphoma</subject><subject>Lymphoma, B-Cell - pathology</subject><subject>Lymphomas</subject><subject>Mammary gland</subject><subject>Mcl-1 protein</subject><subject>Medical research</subject><subject>Medicine</subject><subject>Mice</subject><subject>Mitochondria</subject><subject>Mitochondria - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Clerc, Pascaline</au><au>Carey, Gregory B</au><au>Mehrabian, Zara</au><au>Wei, Michael</au><au>Hwang, Hyehyun</au><au>Girnun, Geoffrey D</au><au>Chen, Hegang</au><au>Martin, Stuart S</au><au>Polster, Brian M</au><au>Shirihai, Orian S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-08-03</date><risdate>2012</risdate><volume>7</volume><issue>8</issue><spage>e42487</spage><epage>e42487</epage><pages>e42487-e42487</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22880001</pmid><doi>10.1371/journal.pone.0042487</doi><tpages>e42487</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2012-08, Vol.7 (8), p.e42487-e42487 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1326226712 |
| source | MEDLINE; Public Library of Science; DOAJ Directory of Open Access Journals; PubMed Central; Free Full-Text Journals in Chemistry; EZB Electronic Journals Library |
| subjects | Anesthesiology Aniline Compounds - pharmacology Animals Apoptosis B-cell lymphoma Bax protein bcl-2 Homologous Antagonist-Killer Protein - metabolism Bcl-2 protein bcl-2-Associated X Protein - metabolism Biochemistry Bioenergetics Biology Biomarkers Biphenyl Compounds - pharmacology Cancer therapies Cancer treatment Caspase Cell Death - drug effects Cell Line Cell Line, Tumor Cell Respiration - drug effects Chemotherapy Clinical trials Cytochrome Cytochrome c Cytochromes c - metabolism Cytological Techniques - instrumentation Cytological Techniques - methods Death Electron transport Electron transport chain Embryo fibroblasts Embryos Energy Metabolism - drug effects Enzyme-linked immunosorbent assay Epithelial cells Fibroblasts Humans Impairment Leukemia Lymphocytes B Lymphoma Lymphoma, B-Cell - pathology Lymphomas Mammary gland Mcl-1 protein Medical research Medicine Mice Mitochondria Mitochondria - drug effects Mitochondria - metabolism Models, Biological Molecular biology Mortality Multiple myeloma Multiplexing Nitrophenols - pharmacology Oxygen Consumption - drug effects Phenotype Phenotypes Piperazines - pharmacology Proteins Proto-Oncogene Proteins c-bcl-2 - metabolism Rats Reduction Respiration Respirometry Sulfonamides - pharmacology Tumor cell lines Tumors |
| title | Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry |
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