Abstract
In Jurkat cells Bid was cleaved upon activation of the Fas receptor with an anti-Fas antibody. The caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-CH(2)F (IETD) prevented the cleavage of Bid and the loss of viability. The nuclear enzyme poly(ADP-ribose)polymerase (PARP) was also cleaved upon the activation of caspases, and IETD similarly prevented PARP cleavage. The PARP inhibitor 3-aminobenzamide (3-AB) restored the cell killing in the presence of IETD, an effect that occurred without restoration of the cleavage of Bid or PARP. In the presence of 3-AB and IETD, translocation occurred of full-length Bid to the mitochondria. The induction of the mitochondrial permeability transition (MPT) was documented by the cyclosporin A (CyA) sensitivity of the release of cytochrome c, the release of malate dehydrogenase from the mitochondrial matrix, the loss of the mitochondrial membrane potential, and the pronounced swelling of these organelles, as assessed by electron microscopy. In addition to preventing all evidence of the MPT, CyA prevented the loss of cell viability, without effect on the cleavage of either Bid or PARP. The prevention of PARP cleavage by inhibition of caspase-3 resulted in a 10-fold activation of the enzyme and a resultant depletion of NAD and ATP. The PARP inhibitor 3-AB prevented the loss of NAD and ATP. Depletion of ATP by metabolic inhibitors similarly prevented the cell killing. It is concluded that the cleaving of PARP in Fas-mediated apoptosis allowed expression of an energy-dependent cell death program that included the translocation of full-length Bid to the mitochondria with induction of the MPT.
Highlights
The participation of mitochondria in the pathogenesis of apoptosis is generally held to be initiated by the interaction of these organelles with one or more of the proapoptotic members of the Bcl-2 family of proteins
Activated caspase-8 cleaves Bid to generate truncated Bid (tBid) that translocates to the mitochondria. tBid induces release of cytochrome c by a mechanism that is independent of the induction of the mitochondrial permeability transition [4, 5, 10, 11]
The data presented below argue that upon activation of the Fas receptor full-length Bid translocates to the mitochondria, where it produces the release of cytochrome c as a result of induction of the mitochondrial permeability transition (MPT)
Summary
Bak is envisioned to undergo allosteric activation as a result of a conformational change induced by its interaction with tBid. In the present study, we have utilized Jurkat cells, a human T-cell leukemia/lymphoma cell line, to re-examine the role of Bid in the cell killing that occurs subsequent to activation of the Fas receptor. Jurkat cells are very sensitive to activation of the Fas receptor. Caspase inhibitors readily prevent the resulting apoptosis. Jurkat cells do not contain Bax. The data presented below argue that upon activation of the Fas receptor full-length Bid translocates to the mitochondria, where it produces the release of cytochrome c as a result of induction of the MPT. The ability of caspase inhibitors to prevent the killing of Jurkat cells cannot be attributed to an inhibition of Bid processing. The likely basis of their anti-apoptotic effect is the prevention of PARP cleavage and the resultant activation of the enzyme
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