The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis.
Ectopic overexpression of Apaf-1 (2.5-fold) in human acute myelogenous leukemia HL-60 cells (HL-60/Apaf-1 cells) induced apoptosis and sensitized HL-60/Apaf-1 cells to etoposide- and paclitaxel-induced apoptosis (C. Perkins et al., Cancer Res., 58: 4561-4566, 1998). In this report, we demonstrate that in HL-60/Apaf-1 cells, the activity of caspase-9 and -3 induced by Apaf-1 overexpression was associated with a significant increase (5-fold) in the cytosolic accumulation of cytochrome c (cyt c), loss of mitochondrial membrane potential (deltapsim), and an increase in the reactive oxygen species. These were also associated with the processing of procaspase-8 and Bid (cytosolic, proapoptotic BH3 domain containing protein). Transient transfection of Apaf-1 into the Apaf-1-containing mouse embryogenic fibroblasts (MEFs; Apaf-1+/- MEFs) or Apaf-1-/- MEFs also induced the processing of procaspase-9 and procaspase-8, Bid cleavage, and apoptosis. These events were secondary to the activity of the downstream caspases induced by Apaf-1. This conclusion is supported by the observation that in HL-60/Apaf-1 cells, ectopic expression of dominant negative caspase-9, its inhibitory short isoform caspase-9b, or XIAP or treatment with the caspase inhibitor zVAD (50 microM) inhibited Apaf-1-induced caspase-8 and Bid cleavage, mitochondrial deltapsim, release of cyt c, and apoptosis. In contrast, a transient transfection of dominant negative caspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk inhibited the processing of procaspase-8 and Bid but did not inhibit the cytosolic accumulation of cyt c in either the untreated HL-60/Apaf-1 cells or the etoposide-treated HL-60/Apaf-1 and HL-60/neo cells. These results indicate that Apaf-1 overexpression lowers the apoptotic threshold by activating caspase-9 and caspase-3. This triggers the mitochondrial deltapsim and cyt c release into the cytosol through a predominant mechanism other than cleavage of caspase-8 and/or Bid. This mechanism may involve a cytosolic mitochondrial permeability transition factor, which may be processed and activated by the downstream effector caspases, thereby completing an amplifying feedback loop, which triggers the mitochondrial events during apoptosis.
Perkins
C L
CL
Division of Clinical and Translational Research, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Florida 33136, USA.
Fang
G
G
Kim
C N
CN
Bhalla
K N
KN
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism / Q000502:physiology; D003574:Cytochrome c Group / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D018922:HL-60 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D017239:Paclitaxel / Q000494:pharmacology*; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology
C506592:APAF1 protein, human; D000972:Antineoplastic Agents, Phytogenic; C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C494815:Bid protein, mouse; D002352:Carrier Proteins; D003574:Cytochrome c Group; D004792:Enzyme Precursors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005047:Etoposide; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D017239:Paclitaxel