GENERATION OF REACTIVE OXYGEN INTERMEDIATES AFTER TREATMENT OF BLASTSOF ACUTE MYELOBLASTIC-LEUKEMIA WITH CYTOSINE-ARABINOSIDE - ROLE OF BCL-2

Cytosine arabinoside is usually considered to be lethal by incorporati on into DNA followed by chain termination. Recently, we have reported that the radical scavenger N-acetylcysteine (NAG) protects cultured cl onogenic AML blast cells from the lethal affects of Ara-C if given bef ore the drug. This observation provides indirect evidence that toxic r eactive oxygen intermediates (ROI) are generated in AML blast cells fo llowing Ara-C-induced damage to DNA. In the present paper we present e vidence in support of this hypothesis. Using flow cytometry and multip le fluorescent probes for live cell function, we have mapped a sequenc e of discrete stages that occur during Ara-C cytotoxicity. An early ev ent was the increased generation of ROI. Initially this oxidative stre ss was countered by an increase in the cellular content of reduced glu tathione (GSH), but cells then underwent an abrupt transition to a sta te characterized by low GSH and very high ROI generation indicative of collapse of cellular redox balance. Next, the capacity to maintain lo w intracellular ionized calcium was lost, probably due to lipid peroxi dation at membrane sites of calcium regulation. Finally, surface membr ane integrity was lost. Concurrent measurements of clonogenic cell sur vival insured the relevance of these flow cytometry measurements to th e stem cell population. We used OCI/AML-2 cells transfected with bcl-2 to look for the place in this sequence where bcl-2 protein protects c ells against apoptosis; bcl-2 transfectants showed an increase in ROI generation similar to controls, but were able to maintain GSH levels i n the face of this oxidative stress. We conclude that oxidative stress plays a major role in Ara-C toxicity, and that bcl-2 protein protects cells by maintaining cellular redox balance in a reducing state. Thes e studies complement previous work showing how regulators of AML growt h affect the sensitivity of blast cells to Ara-C by changing the conce ntration or stability of bcl-2 protein.