Cladribine induces apoptosis in human leukaemia cells by caspase-dependentand -independent pathways acting on mitochondria

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 ( AT.) loss, phosphatidylserine exposure, caspase activation and development of typical apoptotic morphology in JM1 (pre-B), Jurkat (T) and U937 (promon ocytic) cells. Western-blot analysis of cell extracts revealed the activati on of at least caspases 3, 6, 8 and 9. Co-treatment with Z-VAD-fmk (benzylo xy-carbonyl-Val-Ala-Asp-fluoromethylketone), a general caspase inhibitor, s ignificantly prevented cladribine-induced death in JM1 and Jurkat cells for the first approximate to 40 h, but not for longer times. Z-VAD-fmk also pa rtly prevented some morphological and biochemical features of apoptosis in U937 cells, but not cell death. Coincubation with selective caspase inhibit ors Ac-DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde), Ac-LEHD-CHO (N-acetyl- Leu-Glu-His-Asp-aldehyde) or Z-IETD-fmk (benzyloxy-carbonyl-Ile-Glu-Thr-Asp -fluoromethylketone), inhibition of protein synthesis with cycloheximide or cell-cycle arrest with aphidicolin did not prevent cell death. Overexpress ion of Bcl-2, but not CrmA, efficiently prevented death in Jurkat cells. In all cell lines, death was always preceded by AT. loss and accompanied by t he translocation of the protein apoptosis-inducing factor (AIF) from mitoch ondria to the nucleus. These results suggest that caspases are differential ly involved in induction and execution of apoptosis depending on the leukae mic cell lineage. In any case, Delta Psi (m) loss marked the point of no re turn in apoptosis and may be caused by two different pathways, one caspase- dependent and the other caspase-independent. Execution of apoptosis was alw ays performed after Delta Psi (m) loss by a caspase-9-triggered caspase cas cade and the action of AIF.