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 (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent 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 caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH.
Bojes
H K
HK
Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Texas, USA.
Feng
X
X
Kehrer
J P
JP
Cohen
G M
GM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D003374:Coumarins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D006412:Hematopoietic Stem Cells; D007377:Interleukin-3 / Q000378:metabolism*; D034921:Lamin Type B; D034882:Lamins; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects; D009687:Nuclear Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D003374:Coumarins; D007377:Interleukin-3; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D009842:Oligopeptides; D010718:Phosphatidylserines; D011480:Protease Inhibitors; D011506:Proteins; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C103192:butyloxycarbonyl-O-methyl-aspartyl-fluoromethyl ketone; D019328:Buthionine Sulfoximine; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione; D019803:Glutathione Disulfide