During apoptosis of HL-60 and U-937 cells caspases are activated independently of dissipation of mitochondrial electrochemical potential.
Collapse of the mitochondrial potential (DeltaPsi(m)) during apoptosis has been linked with a release of cytochrome c and apoptosis-inducing factor (AIF) and activation of caspases. Using a laser scanning cytometer (LSC), an instrument that allows one to measure the same cells twice, first when they are alive and subsequently after their permeabilization, we explored whether dissipation of DeltaPsi(m) (measured supravitally) is a prerequisite for the activation of caspases (detected after cell fixation). Apoptosis of HL-60 cells was induced either by TNF-alpha combined with cycloheximide (CHX) or by the DNA topoisomerase I inhibitor camptothecin (CPT) and of U-937 cells by CPT, and DeltaPsi(m) was measured using the carbocyanine fluorochrome DiIC(1) (5). The marker of caspase activation was specific cleavage of poly(ADP) ribose polymerase (PARP) detected immunocytochemically. After 30 or 60 min treatment with TNF-alpha + CHX or 60 or 120 min with CPT a considerable proportion of cells (20-40%) demonstrated PARP cleavage with no evidence of DeltaPsi(m) collapse. Also present in these cultures (3-20%) were cells with collapsed DeltaPsi(m) whose PARP was not cleaved. The results provide direct evidence that in HL-60 and U-937 cells treated with TNF-alpha + CHX or CPT the dissipation of DeltaPsi(m) is not required for activation of caspases and these two events are independent of each other.
Li
X
X
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, 10595, USA.
Du
L
L
Darzynkiewicz
Z
Z
D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004563:Electrochemistry; D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020298:U937 Cells
D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D002166:Camptothecin