To identify critical events associated with heat-induced cell killing, we examined foci formation of ␥H2AX (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 ␥H2AX 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°C to 45.5°C, we observed a linear increase with time in the number of ␥H2AX foci. An inflection point at 42.5°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 ␥H2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycledependent pattern of cell killing was the same as the cell cycle pattern of ␥H2AX foci formation. We also found that thermotolerance was due to a depression in the number of ␥H2AX 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.

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Our findings suggest that high-LET radiotherapy is expected to be a valid application for patients carrying mutated p53 cancer cells. We proposed that the elucidation of the p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.

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The aim of this study was to ascertain whether LY294002, an inhibitor of PI-3K, enhances heat sensitivity in human cancer cells regardless of their p53 status. Colony formation assays showed that LY294002 enhanced heat sensitivity in two human lung cancer cell lines; H1299/wildtype p53 (wtp53) and H1299/mutated p53 (mp53) cells. These cell lines have identical genetic backgrounds except for their p53 status. LY294002 suppressed the heat-induced accumulation of heat shock protein 27 (hsp27) and heat shock protein 72 (hsp72) in these cell lines. Heat-induced apoptosis was observed more frequently in H1299/wtp53 cells than in H1299/mp53 cells, and was enhanced by LY294002 in both cell lines. In addition, both the heat-induced phosphorylation of Akt and the accumulation of survivin were suppressed by LY294002. These results suggest that LY294002 inhibits anti-apoptosis signaling through hsp27 and hsp72 as well as cell survival signaling through Akt and survivin. LY294002 appears to be an attractive candidate for a p53-independent heat sensitizer in hyperthermic cancer therapy.