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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.

Over the past several years, current techniques in molecular biology have been used to perform experiments in space, focusing on the nature and effects of space radiation. In the Japanese 'Kibo' facility in the International Space Station (ISS), the Japan Aerospace Exploration Agency (JAXA) has performed five life science experiments since 2009, and two additional experiments are currently in progress. The first life science experiment in space was the 'Rad Gene' project, which utilized two human cultured lymphoblastoid cell lines containing a mutated P53 : gene (m P53 : ) and a parental wild-type P53 : gene (wt P53 : ) respectively. Four parameters were examined: (i) detecting space radiation-induced DSBs by observing γH2AX foci; (ii) observing P53 : -dependent gene expression during space flight; (iii) observing P53 : -dependent gene expression after space flight; and (iv) observing the adaptive response in the two cell lines containing the mutated and wild type P53 : genes after exposure to space radiation. These observations were completed and have been reported, and this paper is a review of these experiments. In addition, recent new information from space-based experiments involving radiation biology is presented here. These experiments involve human cultured cells, silkworm eggs, mouse embryonic stem cells and mouse eggs in various experiments designed by other principal investigators in the ISS/Kibo. The progress of Japanese science groups involved in these space experiments together with JAXA are also discussed here. The Japanese Society for Biological Sciences in Space (JSBSS), the Utilization Committee of Space Environment Science (UCSES) and the Science Council of Japan (ACJ) have supported these new projects and new experimental facilities in ISS/Kibo. Currently, these organizations are proposing new experiments for the ISS through 2024.

Boron neutron capture therapy (BNCT) is a particle radiation therapy that involves the use of a thermal or epithermal neutron beam in combination with a boron ((10)B)-containing compound that specifically accumulates in tumor. (10)B captures neutrons and the resultant fission reaction produces an alpha ((4)He) particle and a recoiled lithium nucleus ((7)Li). These particles have the characteristics of high linear energy transfer (LET) radiation and therefore have marked biological effects. High-LET radiation is a potent inducer of DNA damage, specifically of DNA double-strand breaks (DSBs). The aim of the present study was to clarify the role of DNA ligase IV, a key player in the non-homologous end-joining repair pathway, in the repair of BNCT-induced DSBs. We analyzed the cellular sensitivity of the mouse embryonic fibroblast cell lines Lig4-/- p53-/- and Lig4+/+ p53-/- to irradiation using a thermal neutron beam in the presence or absence of (10)B-para-boronophenylalanine (BPA). The Lig4-/- p53-/- cell line had a higher sensitivity than the Lig4+/+ p53-/-cell line to irradiation with the beam alone or the beam in combination with BPA. In BNCT (with BPA), both cell lines exhibited a reduction of the 50 % survival dose (D 50) by a factor of 1.4 compared with gamma-ray and neutron mixed beam (without BPA). Although it was found that (10)B uptake was higher in the Lig4+/+ p53-/- than in the Lig4-/- p53-/- cell line, the latter showed higher sensitivity than the former, even when compared at an equivalent (10)B concentration. These results indicate that BNCT-induced DNA damage is partially repaired using DNA ligase IV.

5-Fluorouracil (5-FU) is widely used in clinical cancer therapy. It is commonly used either alone or in combination with other drugs and/or radiation for head and neck, and other types of cancers. 5-FU induces DNA double-strand breaks (DSBs). Inhibition of the repair of 5-FU-induced DSBs may improve the therapeutic response in many tumors to this anticancer agent. The aim of the present study was to further our understanding of the pathways which are involved in the repair of 5-FU-induced DSBs. Cell survival after drug treatment was examined with colony forming assays using Chinese hamster lung fibroblast cells or Chinese hamster ovary cell lines which are deficient in DSB repair pathways involving the homologous recombination repair-related genes BRCA2 and XRCC2, and the non-homologous end joining repair-related genes DNA-PKcs and Ku80. It was found that BRCA2 was involved in such repair, and may be effectively targeted to inhibit the repair of 5-FU-induced damage. Observations showed that knockdown of BRCA2 using small interference RNA suppression increased the sensitivity to 5-FU of human oral cancer cell lines (SAS and HSC3). These findings suggest that downregulation of BRCA2 may be useful for sensitizing tumor cells during 5-FU chemotherapy.

Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G(2)/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.

Accepted: 2011-10-22, 資料番号: SA1003629000

On March 11, 2011 eastern Japan was struck by a magnitude 9.0 earthquake and an enormous tsunami, over 13 m in height, which together killed over 20,500 people and resulted in the evacuation of over 320,000 people from the devastated areas. This paper describes the damage sustained by the Fukushima-Daiichi nuclear power plant during this unpredicted major natural disaster and the events that happened in the months after this accident. The events occurring at the Fukushima-Daiichi nuclear power plant, the actions taken to minimize the effects of the damage to the plant and to protect the public, and the points at which the responses proved to be inadequate all offer lessons that will be of value to those planning for and responding to future natural disasters and accidents in Japan and around the world.

Accepted: 2011-08-29, 資料番号: SA1004272000

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.

Accepted: 2010-11-02, 資料番号: SA1003627000

The aim of this study was to examine biological effects of nitric oxide (NO) on radiosensitivity and chromosome aberrations in different phases of the cell cycle in human cancer cells with a wild-type p53 (wtp53) genotype. H1299/wtp53 cells were pre-treated with isosorbide dinitrate (ISDN) at different concentrations or pre-irradiated with a low dose of X-rays, and then exposed to a high dose of X-rays. Cell synchronization was achieved with serum starvation. Cellular radiosensitivity, cell cycle distributions, and chromosome aberrations were assayed with colony-forming assays, flow cytometry and chromosome banding techniques, respectively. After treatment with ISDN at a low concentration or after an exposure to 0.02 Gy of X-rays, radioresistance and a reduction in the number of chromosome aberrations were observed mainly 17.5 h after plating mitotic cells. This radioadaptation effect was observed during a clearly shortened G(2)/M phase and a slightly prolonged S phase. In contrast, in the presence of a high concentration of ISDN, radiosensitization and the enhancement of chromosome aberrations were detected principally 17.5 h after plating mitotic cells, and this radiosensitization was observed during a significantly prolonged G(2)/M phase and a slightly shortened S phase. A range of concentrations of NO induced opposing effects on radiosensitivity and chromosome aberrations in human non-small cell lung cancer cells bearing wtp53 gene status, and these different effects produced by NO depended on the cell cycle phase.

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.

Accepted: 2010-04-21, 資料番号: SA1002706000

The space environment contains two major biologically significant influences; space radiations and microgravity. The 53 kDa tumour suppressor protein (p53) plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity, and the space environment on the gene expression of p53-regulated genes.Space experiments were performed with two human cultured lymphoblastoid cell lines; one line (TSCE5) bears a wild-type p53 gene status, and another line (WTK1) bears a mutated p53 gene status. Under one gravity or microgravity conditions, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station for 8 days without experiencing stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the spaceflight. Gene expression was analysed using an Agilent Technologies 44 k whole human genome microarray DNA chip.p53-dependent up-regulated gene expression was observed for 111, 95, and 328 genes and p53-dependent down-regulated gene expression was found for 177, 16, and 282 genes after exposure to space radiations, to microgravity, and to both, respectively.The data provide the p53-dependent regulated genes by exposure to radiations and/or microgravity during spaceflight. Our expression data revealed genes that might help to advance the basic space radiation biology.

Superb biological effectiveness and dose conformity represent a rationale for heavy-ion therapy, which has thus far achieved good cancer controllability while sparing critical normal organs. Immediately after irradiation, heavy ions produce dense ionization along their trajectories, cause irreparable clustered DNA damage, and alter cellular ultrastructure. These ions, as a consequence, inactivate cells more effectively with less cell-cycle and oxygen dependence than conventional photons. The modes of heavy ion-induced cell death/inactivation include apoptosis, necrosis, autophagy, premature senescence, accelerated differentiation, delayed reproductive cell death of progeny cells, and bystander cell death. This paper briefly reviews the current knowledge of the biological aspects of heavy-ion therapy, with emphasis on the authors' recent findings. The topics include (i) repair mechanisms of heavy ion-induced DNA damage, (ii) superior effects of heavy ions on radioresistant tumors/cells (intratumor quiescent cell population, TP53-mutated and BCL2-overexpressing tumors), (iii) novel capacity of heavy ions in suppressing cancer metastasis and neoangiogenesis, and (iv) potential of heavy ions to induce secondary (especially breast) cancer.

Hyperthermia/γH2AX/ATM/DNA-PK. Heating induces histone H2AX phosphorylation at serine 139 (γH2 AX). Phosphorylated H2AX subsequently forms foci in numerous mammalian cell lines. The aim of this study was to clarify details in the mechanisms involved in the phosphorylation of H2AX after heating. The cell lines used were DNA-PKcs knockout cells, ATM knockout cells, and their parental cell lines. To elucidate mechanisms of induction of phosphorylation of H2AX after heating, ATM/ATR inhibitor (CGK733) and DNA-PK inhibitor (NU7026) were used. The intensity of γH2AX signals was assayed with flow cytometry. The thermal dose-response curve for the fluorescence intensity of γH2AX appearance in DNA-PKcs–/– cells during the heating period was similar to that observed in DNA-PKcs+/+ cells. On the other hand, the slope of thermal dose-response curve for them in ATM–/– cells was lower than that in ATM+/+ cells. Phosphorylation of H2AX after heating was suppressed by a combination of CGK733 and NU7026 in the culture medium in DNA-PKcs–/– cells, ATM–/– cells and in their parental cells. Although the phosphorylation of H2AX after heating was not suppressed by NU7026 in their parental cells, such phosphorylation was suppressed by CGK733 in their parental cells. These results indicate that ATM is the predominant protein which is active in the phosphorylation of histone H2AX after heating.

To clarify the biological effects of space environment, especially space radiations, a proposal of "Rad Gene" was performed as the first life science experiment with two human lymphoblastoid cell lines bearing wild-type p53 gene (wtp53) and mutated p53 gene (mp53) in an International Space Station (ISS) for 133 days. We scheduled four projects: (1) DNA damage induced by space radiations including the high linear energy transfer (LET) particles was detected as a track of γH2AX foci in the nuclei of these frozen cells. (2) To examine the biological effects of microgravity and space radiations on gene and protein expression of p53-dependent regulated genes, these cells were grown under microgravity and 1 gravity in ISS, and on ground for 8 days and analyzed by DNA and protein arrays. (3) p53-Dependent regulated genes were analyzed in the cultured cells after spaceflight at frozen state exposed to space radiations. (4) To clarify the effects of space radiations on the radio-adaptive response, the space flown cells at frozen state were cultured, and then exposed to challenging X-ray-irradiation. All of the radio-adaptive responses of cell killing, apoptosis, chromosomal aberrations and mutations were found only in wtp53 cells, but not in the mp53 cells.

著者人数：11名, Accepted: 2009-05-13, 資料番号: SA1001022000

The aim of this work was to clarify the effect of low dose pre-irradiation on radio- and heat-sensitivity. Wild-type (wt) p53 and mutated (m) p53 cells derived from the human lung cancer H1299 cell line were used. The parental H1299 cell line is p53 -null. Cellular sensitivities were determined with a colony-forming assay. When wt p53 cells were exposed to a low dose X-irradiation, induction of radio- and heat-resistance was observed only in the absence of RITA (an inhibitor of p53-HDM2 interactions), aminoguanidine (an iNOS inhibitor) and c-PTIO (an NO radical scavenger). In contrast, the induced radio- and heat-resistance was not observed under similar conditions in m p53 cells. Moreover, heat-resistance as well as radio-resistance developed when wt p53 cells were treated with ISDN (an NO generating agent) alone. These findings suggest that NO radicals are an initiator of radio- and heat-resistance, and function through the activation of HDM2 and the depression of p53 accumulation.

Accepted: 2009-05-14, 資料番号: SA1001023000

A conditioning mild heat treatment can depress subsequent severe heat-induced biological damage by inducing thermal tolerance. In the work described here, the effect of a conditioning mild heat exposure at 37°C was examined on the formation of gastric mucosa damage induced by a restraint plus water-immersion stress (RWIS) treatment in mice. RWIS treatments induced stomach ulcers in about 42% of the control animals, but a mild conditioning heat treatment at 37°C was found to depress the appearance of ulcers, and only about 9% of the pre-exposed animals developed ulcers. In addition, RWIS treatment induced an apoptosis rate in the stomach of about 13%, but a conditioning mild heat treatment depressed the apoptotic rate to about 8%. Since the heat shock protein (HSP) inducer geranylgeranylacetone (GGA) also efficiently depressed stomach ulcer incidence rates, it is suggested that gastric mucosa damage may be depressed by HSPs induced by a mild conditioning heat treatment through the depression of apoptosis., 著作権者：日本ハイパーサーミア学会, 日本ハイパーサーミア学会及び著作者の許諾を得て登録

The counting and analysis of γH2AX foci is a widely used technique because these foci are considered to be a reliable indicator for the presence of DNA double-strand breaks (DSBs). The most frequently used method to count foci is to examine images and count by eye, but such an approach may not be the most accurate method to use. An alternative method to manual counting is proposed here, and this method is faster, easier, more objective, and can eliminate artifacts which can result from human handling. A dual-step algorithm is described which uses computational methods and consists of two distinct processes : (i) a 5-filter image filtering and processing step, and (ii) a foci counting/analyzing step. The benefits of this 2 stage computational analysis are primarily in the fact that the filtering process compensates for common preparation errors, as well as for image blurring and foci overlap. This method increases the sharpness of the foci edges and converts a 3D focus distance to an optical density, which can then be measured by the counting software. This allows researchers to compare the results of foci counts with increased reliability and objectivity. The features of this method also permit batch microscope images to be processed automatically. This system has a wide potential range of applications, including foci analysis from different types of immunofluorescence staining, and could form the basis for a standardized computational counting method which could eliminate counting subjectivity and provide an increase in counting reliability.

Conventional clinical treatments with X-rays provide an effective modality for widely various human cancers, however, therapeutic results are sometimes poor. Many mutations have been reported to be in the p53 gene in advanced human cancers. The p53 plays a pivotal role in the pathway which controls apoptosis, cell growth and cell proliferation, and mutations or deletions in the p53 gene lead to resistance to cancer therapy. The involvement of the p53 gene in determining the sensitivity of many cell types toward low linear energy transfer (LET) radiation is now well established. In contrast to low LET radiation, high LET radiation has several potential advantages over X-rays, one of which is the fact that its effects may be independent of cellular p53 gene status. It is conceivable that effective future therapeutic strategies may be designed on the basis of genetic and biochemical events involved in cell death. Therefore, the accurate characterization and quantification of the mode of cell death, such as apoptosis and necrosis, has become increasingly important for the further understanding of the biological effectiveness of high LET radiation. This review discusses the mechanisms of p53-independent apoptosis by high LET radiation.

Viable organisms recognize and respond to environmental changes or stresses. When these environmental changes and their responses by organisms are extreme, they can limit viability. However, organisms can adapt to these different stresses by utilizing different possible responses via signal transduction pathways when the stress is not lethal. In particular, prior mild stresses can provide some aid to prepare organisms for subsequent more severe stresses. These adjustments or adaptations for future stresses have been called adaptive responses. These responses are present in bacteria, plants and animals. The following review covers recent research which can help describe or postulate possible mechanisms which may be active in producing adaptive responses to radiation, ultraviolet light, and heat.

H2AX is a histone variant which is present and ubiquitously distributed throughout the genome. An immunocytochemical assay using antibodies capable of recognizing histone H2AX phosphorylated at serine 139 (gammaH2AX) is very sensitive and is a specific indicator for the existence of a DNA double strand break. Although heat stress has been reported to induce the formation of gammaH2AX foci, no gammaH2AX foci formation was observed in several mammalian cell lines after heat shock. Since this was in contrast to earlier reports, the work described here was intended to verify that heat-induced gammaH2AX foci do form in mammalian cell lines other than the cell lines used in earlier reports concerning gammaH2AX foci formation. The cell lines used in this work includes cell lines with differing p53-gene status (H1299, H1299/neo, H1299/mp53 and H1299/wtp53 cells), various cancer cell lines (HeLa, HepG2, U2-OS cells), normal human cells (HEK-293 and AG1522), and cell lines established from other species (MEF normal mouse cells and CHL normal Chinese hamster cells). Exponentially growing cells were exposed to heat shock (42 degrees C for 6 h or 45.5 degrees C for 20 min) or to X-rays (3Gy). The presence of gammaH2AX was examined with immunocytochemistry and flow cytometry. Induction of gammaH2AX foci formation was observed in all of the mammalian cell lines used here after heat-treatment as well as after X-irradiation. However, the intensity of gammaH2AX was different in the different cell lines used. These results confirm that heat can induce gammaH2AX foci formation in many mammalian cell lines.

Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation-induced apoptosis. Human gingival cancer cells (Ca9-22 cells) containing a mutated p53 (mp53) gene were irradiated with X-rays, C-ion (13–100 KeV/µm), or Fe-ion beams (200 KeV/µm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase-3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET-dependent manner. Both RBE curves reached a peak at 100 KeV/µm, and then decreased at values over 100 KeV/µm. When cells were irradiated with high LET radiation, Caspase-3 was cleaved and activated, leading to poly (ADP-ribose) polymerase (PARP) cleavage. In addition, Caspase-9 inhibitor suppressed Caspase-3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase-8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase-3 through Caspase-9, even in the presence of mp53. (Cancer Sci 2008; 99: 1455–1460)

Purpose The aim of this work was to characterize the radioadaptive response at the molecular level. Methods and Materials We used wild-type (wt) p53 and mutated (m) p53- containing cells derived from the human lung cancer H1299 cell line, which is p53 -null. Cellular radiation sensitivities were determined with a colony-forming assay. The accumulations of p53, the human homolog of endogenous murine double minute 2 (Hdm2), and inducible nitric oxide synthase were analyzed with Western blotting. Quantification of chromosomal aberrations was estimated by scoring dicentrics per cell. Results In wt p53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low-dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wt p53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were seen to increase about two- to fourfold after challenging irradiation subsequent to a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wt p53 cells, induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of 5, 5′-(2, 5-Furanidiyl)bis-2-thiophenemethanol (RITA) or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an inducible nitric oxide synthase inhibitor), and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover radioresistance developed when wt p53 cells were treated with isosorbide dinitrate (an NO-generating agent) alone. Conclusions These findings suggest that NO radicals are initiators of the radioadaptive response, acting through the activation of Hdm2 and the depression of p53 accumulations.

Apoptosis plays an important role in eliminating cells from populations when cells have been exposed to UV irradiation and damaged. Studies of cells in culture have provided some details of the mechanisms involved when stress response genes act after exposure to UV irradiation and other environmental stresses. However, little is known about the responses of intact sections of human skin growing in organ culture to UV irradiation. In the work reported here, it was found that the response of organ-cultured human skin after exposure to UV irradiation is different than the response of cultured cells. At wavelengths below 300 nm, the action spectrum obtained from organ-cultured skin samples showed a lower sensitivity than that observed at 300 nm, indicating that the overlying stratum corneum and upper epidermal cell layers had probably caused a selective absorption of incident UV radiation at some wavelengths. At 3 hours after UV irradiation, p53 was phosphorylated at Ser 15 and Ser 46, and accumulated in the cell nuclei, notably after exposure to 280-320 nm wavelengths. Accumulations of Bax, active Caspase-3 and cleaved PARP were detected in apoptotic cells at 24 hours post-exposure, along with a reduction of Bcl-2 levels, notably after exposure to 300-365 nm light. This difference in apoptotic responses may result from the characteristics of the different irradiation wavelengths used, and from details in the skin's structure. The data obtained in this study using an organ-culture system utilized direct measurements of the biological effects of different wavelengths of UV lights.

Hyperthermia is widely used to treat patients with various types of cancers. However, the molecular mechanisms involved in heat-induced cell killing are not yet fully understood. Although protein denaturation is known to be a major type of damage caused by heat treatment, recent work has revealed that DNA double-strand breaks (DSBs) play important roles in heat-induced cell killing. The aim of this review is to specifically examine the formation and repair of DSBs and their role in heat sensitivity and thermotolerance., 著作権者：日本ハイパーサーミア学会, 日本ハイパーサーミア学会及び著作者の許諾を得て登録

Anti-tumor drugs have been utilized in combination with hyperthermia, synergistic effects have been observed, and efforts have been made to explain the basis for these observations. It is thought that the initial extent of DNA damage is enhanced by hyperthermia effects, and this outcome is associated with tissue blood flow. If this is true, then an associated effect would be an additional increase in DNA damage induced by anti-tumor drugs. Another hypothesized mechanism is that DNA repair enzyme activity required for repair of DNA damage is depressed by a heat exposure. A third hypothesis is that more efficient cell killing is induced by the induction of cell death signaling pathways and the depression of cell survival signaling pathways through hyperthermia (Fig. 10.1). In this chapter, the main focus is on the increase in activity of anti-tumor drugs induced by hyperthermia. DNA repair inhibition induced by hyperthermia has been described in other chapters.

Photoreactivation (PR) is an efficient survival mechanism that helps protect cells against the harmful effects of solar-ultraviolet (UV) radiation. The PR mechanism involves photolyase, just one enzyme, and can repair DNA damage, such as cyclobutane-pyrimidine dimers (CPD) induced by near-UV/blue light, a component of sunlight. Although the balance of near-UV/blue light and far-UV light reaching the Earth's surface could be altered by the atmospheric ozone layer's depletion, experiments simulating this environmental change and its possible effects on life have not yet been performed. To quantify the strength of UVB in sunlight reaching the Earth's surface, we measured the number of CPD generated in plasmid DNA after UVB irradiation or exposure to sunlight. To simulate the increase of solar-UV radiation resulting from the ozone layer depletion, Paramecium tetraurelia was exposed to UVB and/or sunlight in clear summer weather. PR recovery after exposure to sunlight was complete at a low dose rate of 0.2 J/m2 x s, but was less efficient when the dose rate was increased by a factor of 2.5 to 0.5 J/m2 x s. It is suggested that solar-UV radiation would not influence the cell growth of P. tetraurelia for the reason of high PR activity even when the ozone concentration was decreased 30% from the present levels.

細胞に温熱処理を施すと X 線の場合と同様に，生体内では細胞の運命を決定するキータンパク質であるがん抑制遺伝子産物 p53 を中心としたシグナルトランスダクションが誘導されることを我々が世界に先駆けて発見した．正常型 p53 遺伝子をもつがん細胞は変異型 p53 がん細胞に比べて温熱で Bax, Caspase-3 を経たアポトーシスが誘導されやすく，温熱に感受性であり，ハイパーサーミア治療に適している．しかし， 変異型 p53 がん細胞に対してもグリセロールや p53C 末端ペプチドを利用した分子シャペロン治療，アポトーシス促進・細胞分裂抑制を標的とした化学物質や siRNA を利用した標的がん治療，p53 遺伝子を利用した遺伝子治療などで有効な治療開発に成功してきており，臨床への応用が期待されている．現在のハイパーサーミア治療においても放射線療法，化学療法，外科療法との併用を行うことによって，より効率的ながん治療を工夫することもできる．いま，その治療増進のしくみを分子生物学的手法で科学している．

It has been suggested that inducible heat shock proteins (HSPs) may function in multiple roles in cytoprotection. However, recent reports have shown that nitric oxide (NO) radicals are an initiator of heat- and radio-resistance, and act through the activation of the human homolog of MDM2 (HDM2), the depression of p53 accumulation, and the induction of NO synthase (iNOS, or alternatively, NOS2) which is observed following a priming irradiation. The aim of this work was to acquire additional information on the roles of p53, HDM2, iNOS, NO radicals, and HSPs on the development of heat- and radio-resistance as following a priming heat treatment. Wild-type (wt) p53 and mutated (m) p53 cells were used. These cells were derived from the H1299 human lung cancer cell line in which p53 is deleted. Cellular sensitivities were determined with a colony-forming assay. In both pre-heated wtp53 cells and in pre-heated mp53 cells, the induction of heat- and radio-resistance was observed in the absence of KNK437 (an inhibitor of HSPs), and in the presence of RITA (an inhibitor of p53-HDM2 interactions), aminoguanidine (an iNOS inhibitor) or c-PTIO (an NO radical scavenger). These findings suggest that following a priming heat treatment, HSPs contribute to heat- and radio-resistance., 著作権者：日本ハイパーサーミア学会, 日本ハイパーサーミア学会及び著作者の許諾を得て登録