TEMPORAL RELATIONSHIPS BETWEEN DE-NOVO PROTEIN-SYNTHESIS, CALPAIN ANDCASPASE 3-LIKE PROTEASE ACTIVATION, AND DNA FRAGMENTATION DURING APOPTOSIS IN SEPTOHIPPOCAMPAL CULTURES

Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade, However, the influence of ups tream events on calpain/caspase activation and the role of calpain/cas pase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurospor ine (0.5 mu M), calpain and caspase 3-like proteases processed a-spect rin to their signature proteolytic fragments prior to endonuclease-med iated DNA fragmentation (not evident until 6 hr), indicating that endo nuclease activation is downstream from calpain/ caspase activation, Cy cloheximide, a general protein synthesis inhibitor, completely prevent ed processing of alpha-spectrin by calpains and caspase 3-like proteas es, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains an d caspase 3-like proteases, Calpain inhibitor II and the pan-caspase i nhibitor Z-D-DCB each inhibited their respective protease-specific pro cessing of alpha-spectrin and attenuated endonuclease DNA fragmentatio n and cell death. Thus, activation of calpains and caspase 3-like prot eases is an early event in staurosporine-induced apoptosis, and synthe sis of, as yet, unknown protein(s) is necessary for their activation. (C) 1998 Wiley-Liss, Inc.