Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation

The protease granzyme B (GrB) plays a key role in the cytocidal activity du ring cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induc ed cell death. However, recent studies have provided evidence for caspase-i ndependent pathway(s) during CTL-mediated apoptosis, In this study, we demo nstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA f ragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process D FF45 directly and mediate DNA fragmentation in the absence of caspase-3 act ivity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 a nd GrB share a common cleavage site, which is necessary and sufficient to i nduce DNA fragmentation in target cells during apoptosis. Together, our dat a suggest that CTLs possess alternative mechanism(s) for inducing DNA fragm entation without the requirement for caspases.