Fluorescence based oxygen uptake analysis in the study of metabolic responses to apoptosis induction.
Mitochondrial activity has been shown to be centrally involved in the progression of apoptosis. The electron transport chain is a major player in this process and oxygen uptake analysis provides detailed information on its activity. Here we examined the ability of a fluorescence based oxygen uptake assay to inform on cellular responses to apoptosis induction. HL60 cells treated with camptothecin and UV light were used as a model and the ability of the assay to detect dose and time dependent decreases in respiratory activity analysed. The data obtained were compared to more specific markers of apoptosis including annexin V binding, and caspase-3 activity. Reductions in oxygen uptake rates were seen at lower doses than increases in annexin V binding or mitochondrial membrane potential depolarisation. These reductions were observed earlier than detectable caspase-3 activity and were unaffected by pre-treatment with the caspase-3 inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (zVADfmk).
Hynes
James
J
Biochemistry Department, University College Cork, Lee Maltings, Cork, Ireland.
O'Riordan
Tomás C
TC
Curtin
James
J
Cotter
Thomas G
TG
Papkovsky
Dmitri B
DB
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000493:pharmacokinetics; D017209:Apoptosis; D002166:Camptothecin / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis; D019069:Cell Respiration; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D005456:Fluorescent Dyes; D018922:HL-60 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000187:drug effects; D010100:Oxygen / Q000032:analysis*; D014466:Ultraviolet Rays
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D010100:Oxygen; D002166:Camptothecin