We investigated the mechanism of the pan-
caspase inhibitor
z-VAD-fmk's augmentation of
TNFalpha-induced L929 cell death and found this mechanism differs from that of
TNFalpha-induced L929 cell death. In the presence of 20 ng/ml
TNFalpha,
z-VAD-fmk initiated apoptosis and
necrosis in the majority of L929 cells as measured by an
agarose gel electrophoresis and
lactate dehydrogenase(LDH)activity based assay. Mitochondrial permeability transition (MPT) inhibitor (
cyclosporine A) effectively inhibited
z-VAD-fmk-augmented cell death. In addition,
z-VAD-fmk plus
TNFalpha increased Bax expression without affecting Bcl-2 and
cytochrome expression. Western-blot analysis showed that
z-VAD-fmk plus
TNFalpha caused persistent JNK activation and ERK inactivation.
Poly(ADP-ribose) polymerase (
PARP) inhibitor (DPQ) effectively reversed the cell death which was augmented by
z-VAD-fmk, and
z-VAD-fmk plus
TNFalpha also caused PARP cleavage to an 85 KDa fragment. These results indicate that in the presence of
TNFalpha,
z-VAD-fmk further augments cell death which requires the mitochondrial permeability transition and the JNK activation. However, we did not detect the changes in
cytochrome c expression and the participation of
caspase-9 in this process, suggesting that there might exist an unknown signal pathway(s) from the mitochondria to the downstream
protein PARP, which is cleaved in a
caspase-independent manner.