title: Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.
abstract: The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.
authors: Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.
authors: A V
authors: AV
authors: Zhdanov
authors: C
authors: C
authors: Favre
authors: L
authors: L
authors: O'Flaherty
authors: J
authors: J
authors: Adam
authors: R
authors: R
authors: O'Connor
authors: P J
authors: PJ
authors: Pollard
authors: D B
authors: DB
authors: Papkovsky
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D002952:Citric Acid Cycle / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005649:Fumarate Hydratase / Q000172:deficiency / Q000235:genetics; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D006019:Glycolysis / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D019344:Lactic Acid / Q000378:metabolism; D051379:Mice; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000172:deficiency / Q000235:genetics; D033704:Nucleotide Transport Proteins / Q000235:genetics; D009840:Oligomycins / Q000494:pharmacology; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects / Q000502:physiology; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D033704:Nucleotide Transport Proteins; D009840:Oligomycins; D034741:RNA, Small Interfering; C483480:SLC25A33 protein, human; D002245:Carbon Dioxide; D019344:Lactic Acid; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D005649:Fumarate Hydratase