BRCA2 protects mammalian cells from heat shock.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 G2/M arrest. CONCLUSION: BRCA2 has a protecting role against heat-induced cell death. BRCA2 might be a potential molecular target for hyperthermic cancer therapy.

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR. CONCLUSIONS: The 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.

Life science experiments performed in space in the ISS/Kibo facility and future research plans.

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.

DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.

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.

The BRCA2 gene is a potential molecular target during 5-fluorouracil therapy in human oral cancer cells.

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.

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair.

PURPOSE: From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. MATERIALS AND METHODS: DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. RESULTS: Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37 °C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. CONCLUSION: It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

Therapeutic strategies for head and neck cancer based on p53 status.

Squamous cell carcinomas of the head and neck (HNSCC) are one of the most common types of cancers worldwide, and despite advances in treatment, they still represent a clinical challenge. Inactivation of one or more components in the p53 signaling pathway is an extremely common event in human neoplasia, including HNSCC. The loss of p53 function is responsible for increased aggressiveness in cancers, while tumor chemoresistance and radioresistance can depend on deleted p53 expression, or on the expression of mutated-p53 proteins. Thus, consideration and manipulation of the p53 status during HNSCC cancer therapy should be considered. This review discusses the p53 signaling pathways activated by various cellular stresses, including exposure to cancer therapies. The recognition of the p53 status in cancer cells is a significant factor and could provide valuable assistance during the selection of an effective therapeutic approach.

Depression of p53-independent Akt survival signals in human oral cancer cells bearing mutated p53 gene after exposure to high-LET radiation.

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.

Expression of p53-regulated proteins in human cultured lymphoblastoid TSCE5 and WTK1 cell lines during spaceflight.

The aim of this study was to determine the biological effects of space radiations, microgravity, and the interaction of them on the expression of p53-regulated proteins. 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 1 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 the stress during launching and landing because the cells were frozen during these periods. Ground control samples were simultaneously cultured for 8 days in the CBEF on the ground for 8 days. After spaceflight, protein expression was analyzed using a Panorama(TM) Ab MicroArray protein chips. It was found that p53-dependent up-regulated proteins in response to space radiations and space environment were MeCP2 (methyl CpG binding protein 2), and Notch1 (Notch homolog 1), respectively. On the other hand, p53-dependent down-regulated proteins were TGF-ß, TWEAKR (tumor necrosis factor-like weak inducer of apoptosis receptor), phosho-Pyk2 (Proline-rich tyrosine kinase 2), and 14-3-3θ/τ which were affected by microgravity, and DR4 (death receptor 4), PRMT1 (protein arginine methyltransferase 1) and ROCK-2 (Rho-associated, coiled-coil containing protein kinase 2) in response to space radiations. ROCK-2 was also suppressed in response to the space environment. The data provides the p53-dependent regulated proteins by exposure to space radiations and/or microgravity during spaceflight. Our expression data revealed proteins that might help to advance the basic space radiation biology.

The disaster at Japan's Fukushima-Daiichi nuclear power plant after the March 11, 2011 earthquake and tsunami, and the resulting spread of radioisotope contamination.

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.

Nitric oxide radical-induced radioadaptation and radiosensitization are G2/M phase-dependent.

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.

FANCD1/BRCA2 plays predominant role in the repair of DNA damage induced by ACNU or TMZ.

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.

siRNA targeted for NBS1 enhances heat sensitivity in human anaplastic thyroid carcinoma cells.

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.

Induction of multinucleation in oral squamous cell carcinoma tissue with mutated p53 surviving boron neutron capture therapy.

PURPOSE: To clarify the role of p53 in boron neutron capture therapy (BNCT) for oral squamous cell carcinoma (SCC), the effect of BNCT on oral SCC xenografts with either wild-type or mutant-type p53 was examined. MATERIALS AND METHODS: Oral SCC cells expressing either wild-type (SAS/neo) or mutant-type p53 (SAS/mp53) were used to produce nude mouse tumours. Tumour-bearing mice received boronophenylalanine (BPA) at a dose of 250 mg/kg and tumours were exposed to neutron irradiation. RESULTS: After BNCT, the growth of SAS/neo and SAS/mp53 tumours was suppressed remarkably and all tumours became undetectable within two weeks. However, three of six SAS/mp53 tumours showed regrowth in two months. Histological examination of BNCT-treated tumours revealed chromosomal condensation, micronucleation, nuclear segmentation and intra- and intercelluar vacuolation. Notably, multinucleated giant cells appeared in SAS/mp53 tumours early after BNCT, suggesting mitotic catastrophe. In SAS/mp53 tumours treated with BNCT, a rapid decrease in phosphorylated cell division cycle 2 (cdc2) and a high level of cyclin B1, required for premature mitosis, were observed. CONCLUSION: These results indicate that BNCT suppressed oral SCC xenografts in nude mice efficiently, but cells survived in mutant-type p53 tumours. BNCT induces multinucleation which represents prestage of apoptosis or necrosis in oral SCC with mutant-type p53, but it may be also associated with the recurrence of BNCT-treated tumours.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage.

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.

DNA damage induced by alkylating agents and repair pathways.

The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O(6)-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O(6)-methylguanine-DNA methyltransferase, and O(6)MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O(6)MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine.

Effect of rapamycin, an mTOR inhibitor, on radiation sensitivity of lung cancer cells having different p53 gene status.

Activation to a large extent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and mutations in the p53 gene are involved in lung cancer therapeutic resistance. The mammalian target of rapamycin (mTOR) acts as a downstream effector for Akt. Activation of the Akt/mTOR signal is a contributing factor to decreased radiation sensitivity. The purpose of this study was to examine whether the effect of rapamycin on radiation sensitivity is affected by cellular p53 gene status. Cellular radiation sensitivity was evaluated by using two human non-small cell lung cancer (NSCLC) cell lines with the same genetic background except for their p53 gene status (H1299/wtp53 and H1299/mp53). The cells were treated with rapamycin and/or radiation. Cell viability, cell proliferation, apoptosis, cell cycle and Akt/mTOR signaling activity were explored. Rapamycin synergistically enhanced the cytotoxicity of radiation, promoting the induction of apoptosis. Moreover, the combined treatment augmented the cytostatic effects of radiation regardless of cellular p53 gene status. Rapamycin in combination with radiation increased G1 arrest and suppressed progression to S phase in both cell lines. Furthermore, the combined treatment conduced to a prominent p53-independent down-regulation of the mTOR signal and pro-survival molecule, cyclin D1. Rapamycin can enhance the effect of radiation through the repression of pro-survival signals and the reduction in the apoptotic threshold. Taken together, inhibition of the mTOR signal may be a promising strategy for radiosensitization with no relevance to p53 gene status from the aspects of cell lethality and cell growth depression.

p53-dependent adaptive responses in human cells exposed to space radiations.

PURPOSE: It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. METHODS AND MATERIALS: Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. RESULTS: In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. CONCLUSION: These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

The expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines during spaceflight.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.