Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry

• Published in: PloS one 2012-08, Vol.7 (8), p.e42487-e42487
• Main Authors: Clerc, Pascaline, Carey, Gregory B, Mehrabian, Zara, Wei, Michael, Hwang, Hyehyun, Girnun, Geoffrey D, Chen, Hegang, Martin, Stuart S, Polster, Brian M
• Format: Article
• Language: English
• 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
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0042487

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.