title: Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry.
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.
authors.affiliation: Department of Anesthesiology and the Shock, Trauma and Anesthesiology Research Center, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Pascaline
authors.initials: P
authors.lastname: Clerc
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Carey
authors.forename: Zara
authors.initials: Z
authors.lastname: Mehrabian
authors.forename: Michael
authors.initials: M
authors.lastname: Wei
authors.forename: Hyehyun
authors.initials: H
authors.lastname: Hwang
authors.forename: Geoffrey D
authors.initials: GD
authors.lastname: Girnun
authors.forename: Hegang
authors.initials: H
authors.lastname: Chen
authors.forename: Stuart S
authors.initials: SS
authors.lastname: Martin
authors.forename: Brian M
authors.initials: BM
authors.lastname: Polster
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D019069:Cell Respiration / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D003584:Cytological Techniques / Q000295:instrumentation* / Q000379:methods*; D004734:Energy Metabolism / Q000187:drug effects; D006801:Humans; D016393:Lymphoma, B-Cell / Q000473:pathology; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D009596:Nitrophenols / Q000494:pharmacology*; D010101:Oxygen Consumption / Q000187:drug effects; D010641:Phenotype; D010879:Piperazines / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C501332:ABT-737; D000814:Aniline Compounds; D001713:Biphenyl Compounds; D009596:Nitrophenols; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; C528561:navitoclax