Nitric oxide induces thymocyte apoptosis via a caspase-1-dependent mechanism

We previously showed that NO induces Apoptosis in thymocytes via a p53-dependent pathway. In the present study, we investigated the role of caspases in this process. The pan-caspase inhibitor, ZVAD-fmk, and the Caspase-1 inhibitor, Ac-YVAD-cho, both inhibited NO-induced thymocyte Apoptosis in a dose-dependent manner, whereas the Caspase-3 inhibitor, Ac-DEVD-cho, had little effect even at concentrations up to 500 microM. ZVAD-fmk and Ac-YVAD-cho were able to inhibit Apoptosis when added up to 12 h, but not 16 h, after treatment with the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Caspase-1 activity was up-regulated at 4 h and 8 h and returned to baseline by 24 h; Caspase-3 activity was not detected. Cytosolic fractions from SNAP-treated thymocytes cleaved the inhibitor of caspase-activated deoxyribonuclease. Such cleavage was completely blocked by Ac-YVAD-cho, but not by Ac-DEVD-cho or DEVD-fmk. Poly(ADP-ribose) polymerase (PARP) was also cleaved in thymocytes 8 h and 12 h after SNAP treatment; addition of Ac-YVAD-cho to the cultures blocked PARP cleavage. Furthermore, SNAP induced Apoptosis in 44% of thymocytes from wild-type mice; thymocytes from Caspase-1 knockout mice were more resistant to NO-induced Apoptosis. These data suggest that NO induces Apoptosis in thymocytes via a caspase-1-dependent but not caspase-3-dependent pathway. Caspase-1 alone can cleave inhibitor of caspase-activated deoxyribonuclease and lead to DNA fragmentation, thus providing a novel pathway for NO-induced thymocyte Apoptosis.