title: Cladribine induces apoptosis in human leukaemia cells by caspase-dependent and -independent pathways acting on mitochondria.
abstract: We have studied the role of caspases and mitochondria in apoptosis induced by 2-chloro-2'-deoxyadenosine (cladribine) in several human leukaemic cell lines. Cladribine treatment induced mitochondrial transmembrane potential (DeltaPsi(m)) loss, phosphatidylserine exposure, caspase activation and development of typical apoptotic morphology in JM1 (pre-B), Jurkat (T) and U937 (promonocytic) cells. Western-blot analysis of cell extracts revealed the activation of at least caspases 3, 6, 8 and 9. Co-treatment with Z-VAD-fmk (benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone), a general caspase inhibitor, significantly prevented cladribine-induced death in JM1 and Jurkat cells for the first approximately 40 h, but not for longer times. Z-VAD-fmk also partly prevented some morphological and biochemical features of apoptosis in U937 cells, but not cell death. Co-incubation with selective caspase inhibitors Ac-DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde), Ac-LEHD-CHO (N-acetyl-Leu-Glu-His-Asp-aldehyde) or Z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone), inhibition of protein synthesis with cycloheximide or cell-cycle arrest with aphidicolin did not prevent cell death. Overexpression of Bcl-2, but not CrmA, efficiently prevented death in Jurkat cells. In all cell lines, death was always preceded by Delta Psi(m) loss and accompanied by the translocation of the protein apoptosis-inducing factor (AIF) from mitochondria to the nucleus. These results suggest that caspases are differentially involved in induction and execution of apoptosis depending on the leukaemic cell lineage. In any case, Delta Psi(m) loss marked the point of no return in apoptosis and may be caused by two different pathways, one caspase-dependent and the other caspase-independent. Execution of apoptosis was always performed after Delta Psi(m) loss by a caspase-9-triggered caspase cascade and the action of AIF.
authors.affiliation: Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
authors.forename: I
authors.initials: I
authors.lastname: Marzo
authors.forename: P
authors.initials: P
authors.lastname: Pérez-Galán
authors.forename: P
authors.initials: P
authors.lastname: Giraldo
authors.forename: D
authors.initials: D
authors.lastname: Rubio-Félix
authors.forename: A
authors.initials: A
authors.lastname: Anel
authors.forename: J
authors.initials: J
authors.lastname: Naval
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017338:Cladribine / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D015843:Serpins; D014764:Viral Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D016590:Aphidicolin; D017338:Cladribine; C074641:interleukin-1beta-converting enzyme inhibitor; D003513:Cycloheximide; D020169:Caspases