Cytochrome c release upon Fas receptor activation depends on translocation of full-length bid and the induction of the mitochondrial permeability transition.
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.
Tafani
Marco
M
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Karpinich
Natalie O
NO
Hurster
Kathryn A
KA
Pastorino
John G
JG
Schneider
Timothy
T
Russo
Matteo A
MA
Farber
John L
JL
D000199:Actins / Q000378:metabolism; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D003574:Cytochrome c Group / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008291:Malate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D050998:bcl-2 Homologous Antagonist-Killer Protein; D019014:fas Receptor / Q000378:metabolism*
D000199:Actins; C494772:BAK1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D003574:Cytochrome c Group; D008565:Membrane Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D019014:fas Receptor; D008291:Malate Dehydrogenase; D011065:Poly(ADP-ribose) Polymerases