Heat Induces Histone γH2AX Formation under Hypoxia

: Tumor hypoxia is a negative prognostic and predictive factor for radiotherapy, and hyperthermia therapy is clinically useful for overcoming radioresistance in hypoxic tumors. However, the mechanism for the hyperthermia-induced cell death observed in hypoxic tumors remains unknown. We aimed to clarify the relationship between heat sensitivity and heat-induced DNA double-strand breaks (DSBs), reflecting DNA damage, in tumor cells under hypoxia. HeLa human cervical epithelial adenocarcinoma cells were subjected to heat treatment or X-ray irradiation under hypoxia or normoxia. Control cells were left untreated. The formation of DSBs was evaluated by immunocytochemistry for histone γ H2AX foci, given that one histone γ H2AX focus reflects one DSB. Cell survival was evaluated by colony-formation assays. The colony-formation assays revealed that hypoxic cells showed greater radioresistance, as expected, but only slightly higher heat resistance than normoxic cells. Under normoxia, heat-treated or X-ray-irradiated cells showed larger amounts of γ H2AX foci formation than control cells, reflecting increased DSB formation and more DNA damage. Under hypoxia, heat-treated cells showed a less remarkable decrease in γ H2AX foci formation than X-ray-irradiated cells, reflecting sustained levels of DSB formation and DNA damage. The present findings indicate that heat treatment can induce DNA damage via DSB formation reflected by γ H2AX foci formation under hypoxia. The findings provide further support for an important role of heat-induced DSB damage in cell killing in hypoxic tumors that show radioresistance. Hyperthermia therapy can be beneficial for the prognosis of cancer patients through increased DNA damage leading to tumor cell death.