BACKGROUND: The apoptotic signalling pathways involved in the delayed type of apoptosis occurring in HeLa Hep2 cells following radiation were investigated. MATERIALS AND METHODS: HeLa Hep2 cells were exposed to 5 Gy of cobalt-60 radiation. The activation of caspase-2, caspase-8, caspase-9 and effector caspase-3 was investigated by caspase assay plates and Western blots. Cleavage of poly (ADP-ribose) polymerase (PARP) was analysed on Western blots. HeLa Hep2 cells were irradiated with or without preincubation with inhibitors of protein synthesis (cycloheximide, CHX) and caspases, followed by TUNEL staining and caspase assay plate evaluation. RESULTS: Initiator caspases-2, -8, -9, and effector caspase-3, were found to be activated and PARP cleaved following irradiation. CHX completely inhibited the caspase activation and the associated apoptosis. Pretreatment with caspase-2 inhibitor indicated that caspase-2 was involved in the execution of the apoptosis. CONCLUSION: Activation of the apoptotic signalling pathways following irradiation of HeLa Hep2 cells includes components from the intrinsic as well as the extrinsic pathways and seems to require de novo protein synthesis.
BACKGROUND: The apoptotic signalling pathways involved in the delayed type of apoptosis occurring in HeLa Hep2 cells following radiation were investigated. MATERIALS AND METHODS: HeLa Hep2 cells were exposed to 5 Gy of cobalt-60 radiation. The activation of caspase-2, caspase-8, caspase-9 and effector caspase-3 was investigated by caspase assay plates and Western blots. Cleavage of poly (ADP-ribose) polymerase (PARP) was analysed on Western blots. HeLa Hep2 cells were irradiated with or without preincubation with inhibitors of protein synthesis (cycloheximide, CHX) and caspases, followed by TUNEL staining and caspase assay plate evaluation. RESULTS: Initiator caspases-2, -8, -9, and effector caspase-3, were found to be activated and PARP cleaved following irradiation. CHX completely inhibited the caspase activation and the associated apoptosis. Pretreatment with caspase-2 inhibitor indicated that caspase-2 was involved in the execution of the apoptosis. CONCLUSION: Activation of the apoptotic signalling pathways following irradiation of HeLa Hep2 cells includes components from the intrinsic as well as the extrinsic pathways and seems to require de novo protein synthesis.