Dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/MDA6 increases the susceptibility of human leukemia cells (U937) to 1-beta-D-arabinofuranosylcytosine-mediated mitochondrial dysfunction and apoptosis.
The effects of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the apoptotic response of U937 monocytic leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) were examined. After a 6-h exposure to 1 microM ara-C, cells stably transfected with a p21WAF1/CIP1 antisense construct were significantly more sensitive to the induction of classic apoptotic morphology, DNA fragmentation, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and underphosphorylation of the retinoblastoma protein (pRb) than their empty-vector counterparts. Enhanced susceptibility of antisense-expressing cells to ara-C was accompanied by a corresponding reduction in clonogenic and suspension culture growth. The increased sensitivity of these cells to ara-C-mediated lethality could not be attributed to cytokinetic perturbations, nor did ara-CTP formation or (ara-C)DNA incorporation differ significantly between the cell lines. Moreover, synchronization of p21 antisense-expressing cells in S-phase by aphidicolin block resulted in a further increase in ara-C-mediated apoptosis, suggesting enhanced drug sensitivity of the S-phase cell fraction. After exposure to ara-C, p21 antisense-expressing cells displayed a greater decline in mitochondrial membrane potential (deltapsi(m)) and generation of reactive oxygen species than their empty-vector counterparts, as well as early potentiation (e.g., within 2-4 h) of cytochrome c release into the cytosolic S-100 fraction. Lastly, ara-C-mediated increases in mitogen-activated protein kinase activity over basal levels were attenuated in p21 antisense-expressing cells. Collectively, these findings indicate that dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 increases the susceptibility of U937 human leukemia cells to ara-C-related lethality, and this phenomenon occurs as a relatively early event that is independent of cell cycle or pharmacodynamic factors and is associated with mitochondrial perturbations implicated in activation of the apoptotic protease cascade.
Wang
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Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0230, USA.
Van Tuyle
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Conrad
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Fisher
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PB
Dent
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Grant
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D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D003561:Cytarabine / Q000494:pharmacology*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D016376:Oligonucleotides, Antisense / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D020298:U937 Cells
D000964:Antimetabolites, Antineoplastic; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016376:Oligonucleotides, Antisense; D003561:Cytarabine; D018844:Cyclin-Dependent Kinases