Dynamics of caspase activation upon UV induced genotoxic injury

Abstract

Purpose Caspases are common mediators of cell death. Evasion of cell death including apoptosis are considered to be hallmarks of cancer. A deeper understanding of the apoptotic cascade may aid improving cancer therapies. Our aim was to characterize the progression of cell death following UV-induced genotoxic injury in a defined cell culture model. Materials and methods Hela cells were UV-irradiated with doses ranging from 0.1 to 60 mJ/cm2. Cells were counted and colony forming assays were performed with caspase inhibitors. Results In our model of HeLa cells, cells remain >90% viable until 6 hrs after UV radiation (UVR), but more than half of the cells are dead after 12 − 72 hrs after UVR. Within a dose range between 0.1 and 50 mJ/cm2, viability ranges roughly between 20 and 30%. The difference between the lowest dose applied (0.1 mJ/cm2) and the other doses applied is significant, with the exception of the next higher dose of 1 mJ/cm2. The activation of caspases precedes the cell death induction by several hrs. Caspase-9 starts to be activated at 1 hr after UVR followed by caspases 3, 6 and 7 which are fully active at 2 hrs after UVR while caspase-8 is fully active only 3 hrs after UVR. Most caspases are only weakly or not active at 0.1 mJ/cm2 after 3 hrs, but fully active at the same time point with increased radiation doses. PARP-1, a caspase substrate, is cleaved immediately after activation of the caspases. Colony formation activity of the tumor cells decreases exponentially after UVR dropping down to < 0.01% plating efficiency at a dose of 60 mJ/cm2. Interestingly, this drop in plating efficiency cannot be rescued by any of the two caspase inhibitors tested. Conclusions UV-induced cell death in this model involves the activation of apoptosis-related caspases, but this activation seems to be dispensable for the execution of cell death. Further experiments should clarify which mechanisms of cell death are really necessary for the execution of this type of cell death.