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 synthesis.
Hyperthermia does not alter CD95 expression at the cell surface and does not modulate the morphology of CD95-mediated cell death on electron microscopy. Bcl-2 gene transfer inhibits apoptosis and abrogates the sensitization mediated by
hyperthermia.
Hyperthermia does not overcome resistance to apoptosis conferred by the viral
caspase inhibitor, crm-A, indicating the absolute requirement for the activation of crm-A-sensitive
caspases, probably
caspase 8, for apoptosis. CD95L-evoked
DEVD-amc-cleaving
caspase activity is enhanced by
hyperthermia, suggesting that
hyperthermia operates upstream of
caspase processing to promote apoptosis. There is no uniformly enhanced processing of three
caspase 3 substrates,
poly-ADP ribose polymerase (PARP),
protein kinase C (PKC) delta and DNA fragmentation factor (DFF) 45. Yet,
hyperthermia promotes CD95L-evoked DNA fragmentation. Interestingly,
hyperthermia enhances the CD95L-evoked release of
cytochrome c in the absence, but not in the presence, of CHX. In contrast, the reduction of the mitochondrial membrane potential is enhanced by
hyperthermia both in the absence and presence of CHX, and enhanced
cytochrome c release is not associated with significantly enhanced
caspase 9 processing. The potentiation of
cytochrome c release at hyperthermic conditions in the absence of CHX is abrogated by Bcl-2. Thus, either
hyperthermia or inhibition of
protein synthesis by CHX potentiate cytotoxic
cytokine-induced apoptosis. These pathways show no synergy, but rather redundance, indicating that CHX may function to promote apoptosis in response to cytotoxic
cytokines by inhibiting the synthesis of specific
proteins whose synthesis, function or degradation is temperature-sensitive.