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The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis.
Cancer Res. 2000 Mar 15; 60(6):1645-53.CR

Abstract

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.

Authors+Show Affiliations

Division of Clinical and Translational Research, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Florida 33136, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

10749135

Citation

Perkins, C L., et al. "The Role of Apaf-1, Caspase-9, and Bid Proteins in Etoposide- or Paclitaxel-induced Mitochondrial Events During Apoptosis." Cancer Research, vol. 60, no. 6, 2000, pp. 1645-53.
Perkins CL, Fang G, Kim CN, et al. The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis. Cancer Res. 2000;60(6):1645-53.
Perkins, C. L., Fang, G., Kim, C. N., & Bhalla, K. N. (2000). The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis. Cancer Research, 60(6), 1645-53.
Perkins CL, et al. The Role of Apaf-1, Caspase-9, and Bid Proteins in Etoposide- or Paclitaxel-induced Mitochondrial Events During Apoptosis. Cancer Res. 2000 Mar 15;60(6):1645-53. PubMed PMID: 10749135.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis. AU - Perkins,C L, AU - Fang,G, AU - Kim,C N, AU - Bhalla,K N, PY - 2000/4/5/pubmed PY - 2000/5/8/medline PY - 2000/4/5/entrez SP - 1645 EP - 53 JF - Cancer research JO - Cancer Res VL - 60 IS - 6 N2 - 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. SN - 0008-5472 UR - https://www.unboundmedicine.com/medline/citation/10749135/The_role_of_Apaf_1_caspase_9_and_bid_proteins_in_etoposide__or_paclitaxel_induced_mitochondrial_events_during_apoptosis_ L2 - http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=10749135 DB - PRIME DP - Unbound Medicine ER -