Comparative bioenergetic assessment of transformed cells using a cell energy budget platform

A V Zhdanov, C Favre, L O'Flaherty, J Adam, R O'Connor, P J Pollard, D B Papkovsky

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)1135-42
Number of pages8
JournalIntegrative Biology
Volume3
Issue number11
DOIs
Publication statusPublished - Nov 2011

Keywords

  • Adenosine Triphosphate
  • Animals
  • Carbon Dioxide
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone
  • Cell Respiration
  • Citric Acid Cycle
  • Embryo, Mammalian
  • Energy Metabolism
  • Extracellular Space
  • Fibroblasts
  • Fumarate Hydratase
  • Gene Deletion
  • Gene Knockout Techniques
  • Glycolysis
  • HeLa Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Lactic Acid
  • Mice
  • Mitochondrial Proteins
  • Nucleotide Transport Proteins
  • Oligomycins
  • Oxidative Phosphorylation
  • Oxygen Consumption
  • RNA Interference
  • RNA, Small Interfering

Fingerprint Dive into the research topics of 'Comparative bioenergetic assessment of transformed cells using a cell energy budget platform'. Together they form a unique fingerprint.

Cite this