Figure 6.

DNA fragmentation and PARP cleavage but not caspase 3 processing are inhibited by GSH in nuclei treated with granzyme B. Nuclei from LYas cells were isolated and treated with 20 units of granzyme B for 1 h at 37°C. Samples were then separated to analyze (A) DNA and (B) PARP and integrity by PFGE and Western blot, respectively. Granzyme-treated samples exhibited both PARP cleavage and generation of a 50-kbp fragment, which was abolished by addition of 10 mM GSH. Although PARP cleavage was not totally suppressed this is probably because of the lack of a GSH-regenerating system (see text). In separate experiments caspase 3 activity and processing (C) were determined by cleavage of a target peptide 30 min following addition of 20 units of granzyme B. Addition of 10 mM GSH was able to suppress the cleavage of DEVD-AFC. Error bars in caspase 3 activity measurements were from replicate wells.