Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione

The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (6 murine prolymphocytic cell line), following interl eukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis, In the present study, the induc tion of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthio nine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dep endent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous ca spase substrate poly(adenosine diphosphate ribose)polymerase (PARP), Apopto sis induced by IL-3 deprivation but not BSO also caused lamin B-1 cleavage, suggesting activation of caspase-6, Despite a more profound depletion of G SH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoro ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not a fter IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z- VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the clea vage and activation of caspase-3, the breakdown of lamin B1 and partially m itigated PARP degradation. However, the externalization of phosphatidylseri ne, the fall in mitochondrial membrane potential and subsequent apoptotic c ell death still occurred. These results suggest that IL-3 withdrawal may me diate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH.