Sensitization to CD95 ligand-induced apoptosis in human glioma cells by hyperthermia involves enhanced cytochrome c release

CD95L-induced apoptosis involves caspase activation and is facilitated when RNA and protein synthesis are inhibited. Here, we report that hyperthermia sensitizes malignant glioma cells to CD95L- and APO2L-induced apoptosis in the absence, but not in the presence, of inhibitors of RNA and protein syn thesis. Hyperthermia does not alter CD95 expression at the cell surface and does not modulate the morphology of CD95-mediated cell death on electron m icroscopy, Bcl-2 gene transfer inhibits apoptosis and abrogates the sensiti zation mediated by hyperthermia, Hyperthermia does not overcome resistance to apoptosis conferred by the viral caspase inhibitor, crm-A, indicating th e absolute requirement for the activation of crm-A-sensitive caspases, prob ably caspase 8, for apoptosis, CD95L-evoked DEVD-amc-cleaving caspase activ ity is enhanced by hyperthermia, suggesting that hyperthermia operates up-s tream of caspase processing to promote apoptosis, There is no uniformly enh anced processing of three caspase 3 substrate, poly-ADP ribose polymerase ( PARP), protein kinase C (PKC) delta and DNA fragmentation factor (DFF) 45, Yet, hyperthermia promotes CD95L-evoked DNA fragmentation, Interestingly, h yperthermia enhances the CD95L-evoked release of cytochrome c in the absenc e, but not in the presence, of CHX. In contrast, the reduction of the mitoc hondrial membrane potential is enhanced by hyperthermia both in the absence and presence of CHX, and enhanced cytochrome c release is not associated w ith significantly enhanced caspase 9 processing. The potentiation of cytoch rome c release at hyperthermic conditions in the absence of CHX is abrogate d by Bcl-2, Thus, either hyperthermia or inhibition of protein synthesis by CHX potentiate cytotoxic cytokine-induced apoptosis, These pathways show n o synergy, but rather redundance, indicating that CHX may function to promo te apoptosis in response to cytotoxic cytokines by inhibiting the synthesis of specific proteins whose synthesis, function or degradation is temperatu re-sensitive.