Caspase inhibition blocks cell death and results in cell cycle arrest in cytokine-deprived hematopoietic cells.
Cytokine deprivation has been classically used to study molecular processes of apoptosis. Following interleukin (IL)-3 withdrawal in FL5.12 cells, Bax undergoes a conformational change that results in its mitochondria targeting, cytochrome c release, activation of caspase-9, and apoptosis. Cells overexpressing Casp9DN (dominant negative caspase-9) or treated with the caspase inhibitor Q-VD-OPh increased viability but failed to increase clonogenic survival. We find that caspase-inhibited cells had a significant fraction of viable cells (herein termed "rescued" cells) that failed to initiate cell division after IL-3 add back. The "rescued" cells had reduced mitochondrial potential, stained for active Bax, and had reduced staining with dihydroethidium, an agent sensitive to superoxide levels. Readdition of IL-3 after deprivation demonstrated that Bax activation was reversed, whereas altered 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and dihydroethidium staining persisted for days. Furthermore, the "rescued" cells were resistant to rotenone, an inhibitor of mitochondrial respiration. The cells were highly sensitive to 2-deoxyglucose, an inhibitor of glycolysis and proposed anti-cancer agent. We conclude that the inhibition of caspase-9 allows cells to retain viability, but cells have prolonged mitochondrial dysfunction and enter a unique nondividing state that shares some properties with malignant cells.
Brown
Nicholas M
NM
Department of Pathology, The University of Iowa Roy J. and Lucille P. Carver College of Medicine, Iowa City, Iowa 52242, USA.
Martin
Sean M
SM
Maurice
Nick
N
Kuwana
Tomomi
T
Knudson
C Michael
CM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology*; D061945:Caspase Inhibitors; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation; D002460:Cell Line; D003574:Cytochrome c Group / Q000502:physiology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006410:Hematopoiesis; D006413:Hematopoietic System / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000172:deficiency / Q000494:pharmacology; D051379:Mice; D011804:Quinolines / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000502:physiology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D007377:Interleukin-3; D011804:Quinolines; D051028:bcl-2-Associated X Protein; C468548:quinoline-val-asp(OMe)-CH2-OPH; C076615:cytochrome c''