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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.

This study was undertaken to clarify the effects of acute or chronic pre-irradiation on the induction of p53-dependent apoptosis by X-rays or heat shock.Having an identical genotype except for p53-status, the human cultured squamous cell carcinoma cells (SAS) were transfected with a mutant p53 gene (SAS/mp53) or neo alone (SAS/neo) as a control. After acute X-irradiation (1 Gy min(-1)), chronic gamma-irradiation (0.001 Gy min(-1)) or heat shock (44 degrees C), the cells were for the incidence of apoptotic bodies and DNA ladders, cellular levels of p53 and bax, and caspase-3 activity.It was found that (1) a challenge treatment with X-rays (5.O Gy) or heat shock (30 min) immediately after chronic pre-irradiation (1.5 Gy) but not acute pre-irradiation (1.5 Gy) resulted in lower levels of apoptosis than those observed after challenge treatment only in SAS/neo cells; (2) a challenge treatment-induced apoptosis was observed 48 h after cessation of chronic pre-irradiation in SAS/neo cells; (3) apoptosis was barely increased in SAS/mp53 cells; and (4) the levels of apoptosis-related proteins after challenge treatments were strongly correlated with the above phenomena.Chronic pre-irradiation at a low dose-rate suppressed induction of p53-dependent apoptosis via bax and caspase-3. These findings suggest that chronic pre-irradiation suppressed p53 function through radiation-induced signalling and/or p53 stability.

To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams.Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with mutated p53 (mp53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV microm(-1)) followed by heating at 44 degrees C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders.It was found that (1) there was no significant difference in cellular sensitivity between SAS/neo and SAS/mp53 cells to LET radiation of30 KeV microm(-1), although the radiosensitivity of SAS/neo cells to X-rays was higher (1.2-fold) than that of SAS/mp53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV microm(-1) in SAS/neo cells, although only additive thermal enhancement was observed in SAS/mp53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/mp53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis.The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.

Purpose : To investigate how DNA-dependent protein kinase (DNA-PK) contributes to p53-dependent signal transduction after heat shock, thermosensitivity and accumulation of p53 and hsp72 after heat shock in lung fibroblasts derived from the SCID mouse were analysed. Materials and methods : Thermosensitivity at 44°C in colony-forming units and Western blot analysis of p53 and hsp72 were analysed. Results : The results indicated that (1) the thermosensitivity at 44°C of SCID cells was higher than that of parental cells and (2) heat-induced accumulation of p53 and hsp72 was abolished and suppressed in SCID cells as compared with that in parental cells respectively. Conclusions : The findings suggest that the catalytic subunit of DNA-PK may play an important role upstream of p53 and hsp72, which are possible determinants of cellular thermosensitivity.

In recent years, contradictory data have been reported about the effects of microgravity on radiation-induced biological responses in space experiments. The aim of the present study was to clarify whether enzymatic repair of DNA double-strand breaks is affected by microgravity using an in vitro enzymatic reaction system.The DNA repair activity of T4 DNA ligase (EC 6.5.1.1) was measured in vitro for a DNA substrate damaged by restriction enzyme digestion during a US Space Shuttle mission (Discovery; STS-91). After the flight, the amount of ligated DNA molecules was measured using an electrophoresis method.Ligated products (closed circular DNA, open circular DNA and multimeric ligated products) were produced by T4 DNA ligase treatment of linear DNA containing double-strand breaks, and they increased with increasing T4 DNA ligase concentration (0-3 units per microg of plasmid DNA). Almost no difference in T4 DNA ligase activity was detected between the space experiments and the control ground experiments.No significant effect of microgravity on ligation of damaged DNA was found during space flight. Therefore, other mechanisms must account for the synergism between radiation and microgravity, if it exists.

Examined the extent of agreement among teachers from China, Indonesia, Japan, the United States, and Thailand who rated videotaped disruptive behaviors. When observing children in solitary activity, the American and Japanese teachers generally gave less extreme ratings than did teachers from the other countries. Weaker differences were found when the same children were observed in groups with their peers. These findings point to the influence of cultural factors in ratings of disruptive behaviors and bring into question the comparability of cross-cultural epidemiological estimates of disruptive behavior disorders.