Beta2-microglobulin induces caspase-dependent apoptosis in the CCRF-HSB-2 human leukemia cell line independently of the caspase-3, -8 and -9 pathways but through increased reactive oxygen species.
Exogenous beta(2)-microglobulin (beta(2)m) induces significant apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line as detected by DNA fragmentation, DAPI staining and annexin V binding assay. beta(2)m treatment induced the release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria, but no change in mitochondrial membrane potential (DeltaPsim) was observed during apoptosis, suggesting that cytochrome c may be released through a mechanism independent of mitochondrial permeability transition (MPT) pore formation. Moreover, the beta(2)m-induced release of cytochrome c and AIF from the mitochondria in CCRF-HSB-2 cells was caspase-independent, since Z-VAD-fmk, a general inhibitor of caspases, did not block the release of these factors. However, Z-VAD-fmk treatment significantly blocked beta(2)m-induced apoptosis, while Western blot analysis revealed that caspases-1, -2, -3, -6, -7, -8 and -9 are not activated during beta(2)m-induced apoptosis in these cells. These results collectively indicate that a post-mitochondrial caspase-dependent mechanism is involved in beta(2)m-induced apoptosis. Moreover, beta(2)m significantly enhanced the production of reactive oxygen species (ROS) during 12-48 hr treatment, and beta(2)m-induced apoptosis was almost totally inhibited in cells pre-treated with the antioxidant N-acetylcysteine (NAC), providing evidence that beta(2)m-induced apoptosis in CCRF-HSB-2 cells is ROS-dependent. Therefore, these results reveal that beta(2)m-induced apoptosis in CCRF-HSB-2 cells may occur through an unknown caspase-dependent and ROS-dependent mechanism(s) that is associated with cytochrome c and AIF release from mitochondria, but is independent of the caspase -3, -8 and -9 pathways.
Gordon
John
J
Department of Pharmacology and Toxicology and Indiana University Cancer Center, Indianapolis 46202, USA.
Wu
Ching-Huang
CH
Rastegar
Mojgan
M
Safa
Ahmad R
AR
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005420:Flavoproteins / Q000378:metabolism; D005434:Flow Cytometry; D006680:HLA Antigens / Q000378:metabolism; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000378:metabolism / Q000473:pathology*; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000494:pharmacology*
C494829:AIFM1 protein, human; D017304:Annexin A5; D051033:Apoptosis Inducing Factor; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D006680:HLA Antigens; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D001613:beta 2-Microglobulin; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases