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Heat shock induces DNA double-strand breaks (DSBs) in mammalian cells. Mammalian cells are capable of repairing DSBs by utilising the homologous recombination (HR) pathway. Breast cancer susceptibility gene 2 (BRCA2) is known to regulate the HR pathway. Here, we investigate the role of BRCA2 in repairing DNA damage induced by heat shock.Chinese hamster lung fibroblast cell lines and human tongue squamous cell carcinoma SAS cells were used. RAD51 foci formation assay was used as an HR indicator. Heat sensitivity was analysed with colony forming assays. Phosphorylated histone H2AX (γH2AX) intensity, which correlates with the number of DSBs, was analysed with flow cytometry.RAD51 foci appeared with heat shock, and the number of cells with RAD51 foci was maximal at about 4 h after heat shock. Heat-induced RAD51 foci co-localised with γH2AX foci. BRCA2-deficient cells were sensitive to heat when compared to their parental wild-type cells. Heat-induced γH2AX was higher in BRCA2-deficient cells compared to parental cells. In SAS cells, cells transfected with BRCA2-siRNA were more sensitive to heat than cells transfected with negative control siRNA. Apoptotic bodies increased in number more rapidly in BRCA2-siRNA transfected cells than in cells transfected with negative control siRNA when cells were observed at 48 h after a heat treatment. In addition, cells deficient in BRCA2 were incapable of activating heat-induced GBRCA2 has a protecting role against heat-induced cell death. BRCA2 might be a potential molecular target for hyperthermic cancer therapy.

Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock.B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock.Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the GThe HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

Nijmegen breakage syndrome 1 (NBS1) plays an important role as a key protein in the repair of radiation-induced DNA double strand breaks (DSBs), and the work described here was designed to examine the effect of NBS1 on heat sensitivity for human anaplastic thyroid carcinoma 8305c cells. Cellular heat sensitivity was evaluated with colony formation assays. Apoptosis was detected and quantified with terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay and Hoechst33342 staining assay. Heat-induced DSBs were measured with flow cytometry using γH2AX antibodies. The transfection of NBS1-siRNA into cells specifically inhibited the expression of NBS1, and enhanced heat sensitivity and the frequency of apoptosis through caspase pathway. In addition, more frequent γH2AX foci were observed in the NBS1-siRNA transfected cells than in control cells transfected with scrambled siRNA at 24 h after heat treatment with a pan-caspase inhibitor. These results suggest that heat sensitisation might result from NBS1-siRNA mediated suppression of heat-induced DSB repair, indicating that NBS1-siRNA could potentially function as a heat sensitiser for cancer patients.

To examine p53-dependency in hyperthermic cancer therapy, heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinoma (HNSCC) tumours were analysed with different status of p53 into nude mice. The tumour tissue from HNSCC cell line (SAS) transfected with mutant p53 gene (SAS/mp53) or control vector containing neo gene (SAS/neo) was transplanted into the subcutaneous tissue of the thigh of nude mice using a trocar. Hyperthermia was performed at 42 degrees C when the mean diameter of tumour was 5-6mm. The diameter of tumours was measured using vernier calipers and tumour weight (TW) and the relative tumour weight (RW) was calculated. Tumour regrowth delay was evaluated when the RW reached 5-fold against the control group. The accumulation of p53 and Bax proteins was examined by an immunohistochemical technique. Apoptotic cells in the sections were detected by staining of DNA ends using an immunohistochemical technique. SAS/mp53 tumours showed more heat-resistance than SAS/neo tumours. The p53-positively staining cells were observed in untreated SAS/mp53 tumours, but not in untreated SAS/neo tumours. After heat treatment, the accumulation of p53 and Bax proteins was observed in SAS/neo tumours, but little in SAS/mp53 tumours. The incidence of apoptotic cells induced by heat treatment was very low in SAS/mp53 tumours compared with SAS/neo tumours. In conclusion, the heat-induced growth inhibition of a transplanted HNSCC may be correlated with the induction of p53-dependent Bax-mediated apoptosis. Thus, p53 status appears to be one of the useful parameters for the predictive assays in hyperthermic cancer therapy.

To elucidate p53-dependency on combined treatment with radiation and hyperthermia, growth inhibition and apoptosis were analysed using transplantable human tumour. Human head and neck squamous cell carcinoma (HNSCC) cells carrying different p53 genes were transplanted into the thigh of nude mice. When the mean diameter of tumour reached 5-6 mm, the tumours were exposed to X-rays (2 Gy) or Carbon-ion (C-) beams (1 Gy) followed by heating at 42 degrees C for 20 min. Tumour growth inhibition was evaluated by measuring the diameters of tumour. The induction of apoptosis and accumulation of apoptosis-related proteins were also analysed by immunohistochemical staining. Synergistic enhancement of tumour growth inhibition by hyperthermia was observed in wild-type p53 tumours treated with X-rays or C-beams but not in mutant p53 tumours. The incidence of apoptotic cells and activated-caspase-3-positive cells after combined treatment with them were significantly high in wild-type p53 tumours compared with that in mutant p53 tumours. The hyperthermic enhancement of tumour growth inhibition by X-ray- or C-beam-irradiation was p53-dependent, suggesting that it might be highly correlated with p53-dependent apoptosis.

A tumour suppressor gene product, p53, is well known to be activated by genotoxic stress such as radiation and DNA damaging agents. Recently, it has been found that non-genotoxic physiological stresses such as heat, cold and low pH also activate p53, which regulates the expression of downstream genes. p53 was found to contribute to heat sensitivity through Bax-mediated apoptosis via activation of caspase-3 in the human cancer cells. This study reviews heat-induced p53-dependent signal transduction and heat sensitivity via a p53-regulated pathway for apoptosis in human cancer cells with identical genetic backgrounds except for p53 status. Furthermore, based on recent reports, it is proposed that p53 status could be a useful indicator in predictive assays for hyperthermic cancer therapy. Hyperthermia treatment based on such predictive assays might improve the outcome and efficiency of cancer therapy in the future. It is further proposed that manipulation of mp53 by glycerol as a chemical chaperone may provide a new cancer therapy for patients with mp53-tumours.

The effects of the combination of cis-diamminedichloroplatinum(II) (CCDP) and hyperthermia on tumour growth were examined using transplantable human oesophageal cancer (ESO-2), having a histological type of moderately differentiated squamous cell carcinoma, in nude mice. Eighteen days after the inoculation of the tumour fragment into the subcutaneous tissue of the right hind foot, the treatment of CDDP and/or hyperthermia was performed and the antitumour effect was evaluated 21 days after the treatment. The combination of 4 mg/kg of CDDP and 43 degrees C heating for 30 min effectively depressed tumour growth in comparison with the individual treatment. The mean relative tumour weight of the combination group at 3 weeks after the treatment was 15% of that of the control group without treatment. On the other hand, pre-heating at 42 degrees C for 30 min did not influence the inhibition of tumour growth by CDDP alone or the concentration of CDDP in tumour. When pre-heating at 42 degrees C for 30 min was performed at 6 or 12 h prior to the combined treatments of 2 mg/kg of CDDP and 43 degrees C hyperthermia for 30 min, however, tumour growth depression by CDDP-hyperthermia was diminished. When pre-heating was performed 4 days prior to CDDP-hyperthermia, however, tumour growth depression occurred. These results showed that thermotolerance of tumour cells induced by pre-heating diminished hyperthermic potentiation for cytotoxicity of CDDP, though the thermotolerance did not affect the cytotoxicity of CDDP.