During apoptosis of HL-60 and U-937 cells caspases are activated independently of dissipation of mitochondrial electrochemical potential

Collapse of the mitochondrial potential (Delta Psi(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 the y are alive and subsequently after their permeabilization, we explored whet her dissipation of Delta Psi(m) (measured supravitally) is a prerequisite f or the activation of caspases (detected after cell fixation). Apoptosis of HL-60 cells was induced either by TNF-alpha combined with cycloheximide (CI M) or by the DNA topoisomerase I inhibitor camptothecin (CPT) and of U-937 cells by CPT, and Delta Psi(m) was measured using the carbocyanine fluoroch rome 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 consi derable proportion of cells (20-40%) demonstrated PARP cleavage with no evi dence of Delta Psi(m) collapse. Also present in these cultures (3-20%) were cells with collapsed Delta Psi(m) whose PARP was not cleaved. The results provide direct evidence that in HL-60 and U-937 cells treated with TNF-alph a + CHX or CPT the dissipation of Delta Psi(m) is not required for activati on of caspases and these two events are independent of each other. (C) 2000 Academic Press.