RELATIONSHIPS BETWEEN THE MITOCHONDRIAL PERMEABILITY TRANSITION AND OXIDATIVE STRESS DURING ARA-C TOXICITY

The mitochondrial permeability transition and oxidative stress seem to be critical alterations in cellular physiology that take place during programmed cell death. Failure to undergo apoptosis is associated wit h drug resistance in acute myeloid leukemia and other cancers. Therefo re, it is important to establish causal relationships between the phys iological changes that take place in apoptosis, because these are pote ntial targets for novel treatment strategies to overcome this form of drug resistance. We describe the use of multilaser flow cytometry meth ods to make correlated measurements of mitochondrial membrane potentia l (MMP), the generation of reactive oxygen intermediates, the cellular content of reduced glutathione (GSH), intracellular calcium, and expo sure of phosphatidylserine on the cell surface. Using these combined m ethods, we have mapped a ''death sequence'' that occurs after treatmen t of leukemic blasts with clinically relevant concentrations of 1-beta -D-arabinofuranosylcytosine (ara-C). Dual labeling of MMP and cellular glutathione content showed that loss of MMP, indicative of the permea bility transition, took place in cells that were depleted of glutathio ne. The loss of MMP coincided with phosphatidylserine exposure and pre ceded a state of high reactive oxygen generation. Finally, there was a n increase in intracellular calcium. These results demonstrate that th e mitochondrial permeability transition takes place during ara-C toxic ity but suggest that this occurs downstream of the loss of GSH. Thus, oxidative stress after ara-C-induced toxicity seems to be a biphasic p henomenon, with the permeability transition occurring after a depletio n of GSH and preceding a state of high reactive oxygen generation.