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All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.

H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.

The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM's role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of gamma-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.