Evidence for the involvement of double-strand breaks in heat-induced cell killing.

To identify critical events associated with heat-induced cell killing, we examined foci formation of gammaH2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of gammaH2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5 degrees C to 45.5 degrees C, we observed a linear increase with time in the number of gammaH2AX foci. An inflection point at 42.5 degrees C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of gammaH2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of gammaH2AX foci formation. We also found that thermotolerance was due to a depression in the number of gammaH2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.