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

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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 (ΔΨm) 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 ≈ 40h, 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 ΔΨm 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, ΔΨm 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 ΔΨm loss by a caspase-9-triggered caspase cascade and the action of AIF.

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