Ionizing radiation, ultraviolet light, and certain chemical mutagens induce DNA-protein crosslinks (DPCs). DPCs are by far larger than conventional bulky DNA lesions such as pyrimidine dimers and aromatic base adducts. Since DPCs are extremely bulky, they will strongly block the progression of replication and transcription machineries and hence exert adverse effects on cells. We have previously shown that in E. coli DPCs containing relatively small crosslinked ptoteins (CLPs) (12 kDa) were processed by RecBCD-dependent homologous recombination (HR). Thus, E. coli cells use repair (NER) and damage tolerance (HR) mechanisms to alleviate the genotoxic effects of DPCs. In the present study, we have asked whether mammalian cells use a similar strategy to ensure survival in the face of DPCs. The activity assays with cell extracts showed that the upper size limit of CLPs amenable to mammalian NER was about 8 kDa, which was considerably smaller than that of E. coli. NER-proficient and deficient cells were treated with DPC-inducing agents, and the repair of genomic DPCs was analyzed. However, NER-dependent removal of DPC was not observed. Thus, NER is not involved in the repair of DPCs in mammalian cells. We also found that genomic DPCs were not polyubiqutinated for proteasomal degradation prior to NER. Cnversely, irs1SF (XRCC3) and 51D1 (RAD51D) cells deficient in HR were hypersensitive to DPC-inducing agents, indicating the involvement of the HR-dependent damage tolerance mechanism for DPCs. In keeping with this observation, cells accumulated nuclear RAD51 and γ-H2AX foci following the treatment with DPC-inducing agents. These results show the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells, and demonstrate the conserved role of HR in tolerance to DPCs among species.

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The first report concerning DNA radiolysis was published in the Journal of 'Nature' in 1962 by B. Ekert. It was the oxidation of thymine base, that is, the production of hydroxide.Later, base modifications and strand breaks were progressively studied. The study of water radiolysis started in and has developed since 1970, by which production of ·OH(G=2.70) and e_aq^- (G=2.70) by water molecules was confirmed, which became a key to study the mechanism of DNA radiolysis. Most of DNA damage in cells could be due to oxidation or reduction with these main active species produced by water radiolysis, because the reaction rate constant of DNA bases not only with ·OH but also with e_aq^- was reported as 10¹⁰. However, ·OH contributes to the modification or degradation rate of DNA bases much more than e_aq^-. Hydoxylation at positions 5,6 of pyrimidine bases and at position 8 of purine bases is main reaction, and further oxidation results in base ring breakage. The modification or degradation rate of pyrimidine bases is much higher than that of purine bases (about 4 times higher).
10-20% of ·OH reacts with the deoxyribose group in DNA molecules (the reaction rate constant: 10⁹) and results in strand break. Three processes were presented, that is, extraction of H with ·OH at position 4 and ionic separation, hydrolysis of ester bond at position 3 or 5 after radical formation at position 4, and direct radical formation of position 5. In fact, 30% of strand breaks take place with alkali treatment (Elgaeter et al. , 1976). In addition, crosslinking of DNA with protein is one kind of DNA modification. Sulfur radicals and aromatic ring radicals, easily produced in protein molecules, can bind to DNA base rings.

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Higher-order chromatin structure plays a pivotal role in the function and maintenance of the genome. Ionizing radiation-induced DNA double strand breaks cause detrimental effects on chromatin structure, which result in the activation of DNA damage response and repair. Damaged chromatin is recognized by ATM, and subsequent modification of histones and recruitment of DNA damage checkpoint factors are essential for executing such processes. In this workshop, three topics related to DNA damage response, histone modification, and DNA repair are presented, and a role of chromatin structure on radiation response will be discussed.

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ATM kinase is activated after ionizing radiation (IR) and transduces DNA damage signal by phosphorylating various substrates, including itself. We previously demonstrated that persistent foci of Ser1981-phosphorylated ATM grow to more than 1.6 µm in diameter and the foci growth is quite relevant to G1 checkpoint induction. Moreover, our analysis revealed that the frequency of the grown foci increased as a quadratic function of IR dose. The results led us to draw hypothesis that interaction between two DNA double-strand breaks, which results in chromosome translocation, elicits the foci growth. To test the hypothesis, we performed chromosome analysis (Giemsa staining and WCP-FISH of Chr. #1-6) of the first metaphase 36-48 h after 2 Gy-gamma-irradiation on confluent normal human diploid fibroblasts (S+G2+M<2%) treated with ATM inhibitor, KU55933 (KU). We found that dicentric with fragment (a kind of chromosome translocation) was 4-fold more in KU-treated population than in DMSO-treated one in Giemsa staining. By WCP-FISH analysis, 3-7-fold more chromosome translocation was detected in KU-treated population than in DMSO-treated one. We also performed the similar examination using p53 siRNA to inhibit only G1 checkpoint function of ATM. We found that 2.5-fold more dicentric with fragment was observed in p53-siRNA-treated population than in control-siRNA-treated one. These results implicate that the foci growth-dependent G1 checkpoint is a mechanism in normal human cells, which recognizes chromosome translocation and suppresses propagation of cells with chromosome translocation.

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Recruitment of DNA repair factors and checkpoint proteins is important step for DNA damage response. Chromatin reorganization involving histone modifications, histone variant exchange, histone eviction and nucleosome remodeling is required for DNA repair factors to access DNA damage sites. However, it still remains largely unclear how chromatin reorganization is coupled with the initiation of DNA repair process and the activation of checkpoint machinery immediately after DNA damage. We have already shown that histone H2AX is rapidly evicted from chromatin after induction of DNA damage. Analysis of purified H2AX complex revealed that DNA damage induced the acetylation and ubiquitination of H2AX in addition to the phosphorylation. Interestingly, the eviction of H2AX depends not on phosphorylation, but rather on acetylation by TIP60 histone acetylase, an enzyme involved in DNA repair, and ubiquitination. In addition to these findings, we found that acetylation of H2AX by TIP60 is necessary for the binding of NBS1 and ATM with the damaged chromatin. We will discuss about the crosstalk between H2AX eviction via histone acetylation and checkpoint activation during the DNA damage response.

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We are interested in the mechanisms through which DNA double-strand breaks (DSBs) are repaired by homologous recombination (HR). Understanding how cells maintain genome integrity when challenged with DSBs is of major importance, particularly since the discovery of multiple links between DSB metabolism and genome instability and cancer-predisposition disorders.
We analyzed HR in cells and in single molecule biochemical reactions. Using live cell video-microscopy we analyzed the accumulation of HR proteins in foci at sites of DNA damage. We will report on how the ATPase activity of RAD54 influences the core protein of HR, RAD51. RAD51 is the central catalyst in DSB repair through HR. It promotes DNA homology recognition and strand exchange between broken DNA and the repair template. At the biochemical level we analyzed the interaction of RAD51 with double-stranded DNA in detail. To promote repair, RAD51 polymerizes around single-stranded DNA. This nucleoprotein filament recognizes and invades a homologous duplex DNA segment. After strand exchange, the nucleoprotein filament should disassemble so that the recombination process can be completed. The molecular mechanism of RAD51 filament disassembly is poorly understood. We showed, by combining optical tweezers with single-molecule fluorescence microscopy and microfluidics, that disassembly of human RAD51 nucleoprotein filaments results from the interplay between ATP hydrolysis and the release of the tension stored in the filament. Our integrative single-molecule approach allowed us to dissect the mechanism of this principal homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins.

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A specificity of biological effects with high LET-ionizing radiation like heavy ion particles would be based on the more condensed physical events with the extremely localized dose deposition. The most typical damage is the double strand break (DSB) among various resulting from multiple localized lesions termed clustered DNA damage. There are many studies indicating increase of DSB with increasing LET. However, there are also equal numbers of studies showing the inverse correlation between LET and DSB yields. This inconsistency for the relationship between LET and DSB has been still one of big questions of radiation biology to elucidate the molecular mechanisms of radiobiological effect. The discrepancy suggests the existence of undefined factors for determining the detectable yields of DSB for LET, including used radiations, target molecules (damage), and analyzing procedure etc. In this workshop, we would offer a brief review summarizing the previous and recent data for this matter, and discuss whether DSB increases or decreases with increasing LET.

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Many studies of Monte Carlo track simulation have suggested that the susceptibility of DNA damage to repair strongly depends on the track structure of the radiation. These studies have predicted an increase of the yield of DSB with increasing level of LET. DSB is thought to be produced mainly by the interaction of particle track with DNA (direct effect). We have measured the yield of DSB induced in pUC18 plasmid as a model DNA. In order to focus on the effect of direct energy deposition from radiation track, we prepared hydrated DNA (35 water molecules per nucleotide). In this condition DNA structure is the B-form which is the same conformation with cellular DNA. There is very little balk water in the sample. Thus OH radicals, if produced, hardly diffuse in the sample. TIARA in JAEA-Takasaki and HIMAC in NIRS are used as He, C and Ne ion beam sources. The track structures are different among the ions if they have the same LET values. Hence we have investigated the yield of DSB by systematically changing LET of identical ion. The obtained results show that 1) there is a minimum around 20 keV/µm for He ion irradiation, and the DSB yield increases precipitously toward 120 keV/µm, and then decreases again above the LET value, 2) similarly the yield of DSB increases with increasing level of LET of C ion in 80-500 keV/µm though the trend is not noticeable rather than He ion irradiation, 3) the DSB yield does not significantly depend on LET 300-900 keV/µm for Ne ion irradiation. These results suggest that the DSB yield increases with increasing level of LET, though the trend of increase strongly depends on ion species.

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Through the whole history of radiation biology, it has been convinced that DNA double strand break (DSB) is the most important causation of radiation biological effect. After utilization of various heavy ion particle beams, there have been hundreds of studies to focus the relationship between the yields of DSB and the linear energy transfer of used radiation. However, respective results of those studies showed no uniform tendency of LET-dependent yields of DSB. Therefore, I studied simultaneous parallel processing of different procedures to resolve this problem with LET-DSB relationship mentioned above. For purified DNA molecule targets, both pDEL19, a circular plasmid DNA (4,814 bp) and linear lambda phage DNA (48,502 bp) in buffered solution (10 mM Tris-HCl, pH 7.5) were irradiated by gamma-rays, carbon ion beams and iron ion beams. The LET were 0.2, 13, and 200 keV/µm, respectively. The irradiated pDEL19 were subjected with static field gel electrophoresis and the yield of DSB were estimated by the conformational changes from intact closed circular form to DSB-related linear form. The irradiated lambda phage DNA were separated in pulse field gel electrophoresis and the yield of DSB were determined by number average molecular weight of the released bands derived from fragmentation. Intracellular DSB were estimated by FAR assay of the chromosomal DNA of irradiated cells. Our results with both irradiated purified DNA targets and irradiated cells showed that the yields of DSB decreased with the increase of the LET. I discuss the possible molecular mechanism for the LET-dependent yield of DSB with similar previous studies.

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This oral discusses the data obtained with the increased or decreased tendencies to yields of DNA double strand breaks (DSB) produced by the high linear energy transfer (LET) radiations in order to obtain RBE values.
Our oral focused the indirect action (e.g., OH radicals) as one of reasons relationship to the induction of DSB. Yokoya et al., have reported the induction of DSB induced by the direct action of helium ion irradiation of hydrated plasmid DNA. Induction of DSB increased with increasing LET, increased ionization density of particle beams. Terato et al., have also reported that the plasmid DNA in buffer were irradiated with carbon and iron ions. Effects of these radiations are affected by direct and indirect actions. However, induction of DSB decreased with increasing LET. In the case of experiment system, radiation action of gamma rays and particle beams involve two actions: Direct and indirect actions. Thus, indirect action of radiations may play an important role in the induction of DSB as a function of the LET.

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Purpose
DNA damage mechanism of proton beams may differ at different depths in water or human body. This study was to clarify the difference in production of DNA base damage at two different lineal-energy points of proton beams. In addition, the effect of radical scavenger edaravone was tested whether it can relatively enhance the direct effect at Bragg peak while suppressing the indirect effect at plateau region of proton beams.
Methods
ST-DNA solution and MOLT4 cells were irradiated with 200 MeV proton beams at two different lineal-energy points, i.e., at plateau and at Bragg peak. 200 kV X-rays were used for comparison. Immediately after each irradiation with or without edaravone, ST-DNA solution had been frozen and kept until 8-OHdG was extracted and quantified by HPLC. Using MOLT4 cells, DNA double strand breaks at two lineal-energy points were visualized by immunohistochemical staining of gamma-H2AX foci which were sequentially quantified by image processing.
Results
Production of 8-OHdG in ST-DNA solution was significantly higher after irradiation with X-rays than proton beams of both lineal-energy points. Furthermore, it was higher at plateau than at Bragg peak of proton beams. Edaravone suppressed production of 8-OHdG after every irradiation, and this effect was greater in irradiation with proton beams at plateau than at Bragg peak. Although there was no remarkable difference in gamma-H2AX focus formation after irradiation with proton beams at plateau and Bragg peak, addition of edaravone significantly reduced the focus formation at plateau of proton beams while no significant effect was observed at Bragg peak.
Conclusions
It was demonstrated that the indirect effect measured by 8-OHdG production was relatively higher at plateau region as compared to that at Bragg peak of proton beams. This may indicate that use of edaravone can protect the normal tissue at the plateau region while preserving the direct tumor killing effect at Bragg peak of proton beams.

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[Objective] NAD⁺-dependent protein deacetylase Sirt1 has been attracting attention as a longevity-regulating enzyme. Recent studies have shown that Sirt1 is involved in a myriad of physiological events including DNA repair of radiation-induced double strand brake. Base excision repair is one of DNA repair mechanisms that repairs DNA base damage, which is frequently generated by irradiation or oxidative stress and causes mutations. We investigated in this study (1) if AP endonuclease 1 (APE1), which is a constituent of base excision repair pathway, was a target of deacetylation by Sirt1, (2) how it affected APE1 function and base excision repair.
[Methods and Results] Down-regulation of either APE1 or Sirt1 protein expression by RNAi in Hela cells promoted cell death induced by a DNA alkylating agent, methylmethane sulfonate (MMS), or hydrogen peroxide. Western blot analysis combined with immunoprecipitation revealed that APE1 interacted with Sirt1 in cells and was deacetylated by Sirt1 in vitro and in vivo. It was already reported that the interaction between APE1 and X-ray repair cross-complementing 1 (XRCC1) enhanced AP endonuclease activity of APE1. Resveratrol, a Sirt1 activator, promoted the APE1-XRCC1 interaction, and it was disappeared by Sirt1 RNAi. Furthermore, APE activity assay using XRCC1 immunoprecipitates from Hela cells treated with either nicotinamide, a Sirt1 inhibitor, or resveratrol showed that nicotinamide reduced APE activity and resveratrol increased it.
[Conclusion] Results in this study suggested that (1) Sirt1 interacted with APE1 and deacetylated it; (2) APE1 deacetylation by Sirt1 promoted APE1-XRCC1 interaction and enhanced base excision repair activity, leading to the inhibition of cell death.

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Ionizing radiation induces 8-Oxoguanine (8-Oxo-Gua) which is a typical oxidative DNA adduct that is involved in gene mutations and aging. We have developed a new experimental system as a model of low-dose ionizing radiation exposure, which can be used to determine the mutagenicity of synthetic DNA adducts in the genome of human lymphoblastoid TK6 cells. We first prepared a targeting vector that contained a single 8-Oxo-Gua lesion and introduced it into the genome of a TSCER122 cell line (constructed from TK6 cells), after which it was incubated in selective media for 2 weeks. Next, genomic DNAs were obtained from 8-Oxo-Gua-integrated clones and sequenced around the 8-Oxo-Gua site. As a result, when an unmodified Gua vector (Control) was transfected, the Gua site of all of the 22 clones paired with Cyt, the correct base. Following 8-Oxo-Gua vector transfection, the 8-Oxo-Gua lesions of 5 (15%) or 1 (3%) of the 33 clones formed mispairing with Ade or Thy, respectively. In summary, we developed a new experimental system for determining the mutagenicity of DNA adducts introduced by targeting and found that a single 8-Oxo-Gua lesion induced point mutations in the human genome.

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A DNA site that has no base is called an AP site (APurinic/apyrimidinc site). AP sites arise from both spontaneous depurination/depyrimidination (about 10,000 per cell per day) and removal of a damaged base in the base excision repair (BER) pathway as an intermediate. AP sites are among the major DNA lesions and, if not repaired, inhibit replication and transcription or induce deletional mutations. AP endonuclease is an enzyme that recognizes AP sites and then excises the DNA strand at the site. The resulting nick in DNA is resynthesized and the repair is completed. The accumulation of such damage in DNA is thought to be related to aging, but this is still uncertain. In order to examine the relationship between DNA damage accumulation and aging, we are studying the DNA repair system in the worm C. elegans. C. elegans is very useful for the study of lifespan.
In this study, we focused on the apn-1 gene, which is a putative AP endonuclease in C. elegans. First, we cloned the apn-1 gene from a C. elegans cDNA library and purified APN-1 protein expressed as a GST-fusion protein in E. coli. The purified APN-1 protein showed excision activity toward AP sites in vitro. Second, we used an E. coli mutant that is deficient in AP endonucleases and therefore hypersensitive to DNA-damaging agents that produce AP sites. The E. coli mutant was rescued by expression of C. elegans APN-1. Third, we inactivated the apn-1 gene by feeding RNAi and now we are studying how this affects worms.

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Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, growth retardation, and cancer predisposition. Cells from NBS patients exhibit radiosensitivity, S-phase checkpoint defect, and chromosome instability. The gene product mutated in NBS, NBS1, forms a complex with MRE11 and RAD50 that is involved in the repair of DNA double-strand breaks and DNA damage checkpoints. However, the biological function of NBS1 has not fully been elucidated.
In this study, we tried to find previously unreported proteins that interact with NBS1. To search for NBS1-interacting proteins, we generated 293E derivative cell lines stably expressing FLAG/HA-tagged NBS1 and adopted a tandem affinity purification scheme. Western blotting confirmed that RAD50 and MRE11 were co-precipitated with tagged NBS1. Silver staining of the gel revealed that additional proteins were also precipitated with tagged NBS1. The protein bands were excised from the gel and subject to mass spectrometric analysis. The result of this analysis will be reported.

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Artemis, which is originally discovered as a responsible gene for human radiation-sensitive severe combined immunodeficiency syndrome, is a nuclease required for V(D)J recombination as well as repair of the nonhomologous end-joining pathway of DNA double-strand breaks (DSB) introduced by ionizing radiation (IR).
Artemis cleaves DNA hairpin intermediate during V(D)J recombination, while the precise function and its regulation of Artemis during DSB repair is not well understood.
It is well known that Artemis is phosphorylated at multiple sites by DNA-PKcs and ATM after IR. However, Artemis mutations in these phosphorylation sites only marginally affect their nuclease activities in vitro, and the mutants still complements IR sensitivities and V(D)J recombination defects in Artemis-deficient cells.
We examined nuclear dynamics of human Artemis-GFP fusion protein expressed in human fibroblast cells using laser UVA microirradiation system. We observed the accumulation of Artemis-GFP at damage sites after microirradiation in live cells. By mutational analysis, it has been shown that this accumulation depends on the multiple phosphorylation sites, but not on the nuclease domain. Collectively, these findings suggest that the dynamics of Artemis after IR is regulated by phsphorylation.

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The tumor suppressor p53 is a transcriptional regulator that controls expression of its target genes involved in cell cycle arrest, apoptosis in response to various kinds of stress including DNA damage. The most famous p53 target gene is the cyclin-dependent kinase inhibitor p21, the principal mediator of p53-induced cell cycle arrest. A switch to an apoptotic responses lead an induction of p53-mediated apoptotic target genes like Bax and Puma.
The spindle checkpoint is a guardian of genome instabilities. Mad2 is a key player in the spindle checkpoint mechanism. p31comet serves as a silencer of Mad2-dependent spindle checkpoint.
Here, we report an additional role of p31comet in control of p53. p31comet binds to p53 and p31comet-depleted cells sensitized against DNA damage reagents and undergo p53-dependent apoptosis. Although p53 can induce apoptotic target genes under depletion of p31comet, p53-mediated p21 induction is diminished. Upon depletion of p31comet, p53 becomes highly acetylated at Lys120 within the DNA-binding domain of p53, which is catalyzed by Histone acetylase TIP60. Introduction of site-specific mutantion at Lys 120 residue can suppress apoptosis in p31comet -depleted cells with DNA damage. p31comet appears to determine the cell fate, death or survival, by controlling p53 through acetylation.

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Radiotherapy has been one of the most effective nonsurgical treatments for cancer. However, recurrence frequently occurs through tumor radioresistance and results in failures of clinical outcome. Therefore, the determination of underlying mechanisms of tumor radioresistance may provide novel therapeutic approaches to eradicate radioresistant tumor cells.
We have studied DNA damage response to fractionated radiation (FR), which is the common practice in radiotherapy. We have demonstrated that cells with long-term FR exposure to 0.5 Gy of X-rays twice per day for more than 31 days (31FR cells) conferred radioresistance of tumor cells with cyclinD1 overexpression. CyclinD1 overexpression was also observed in 31FR-31NR cells, 31FR cells cultured without FR for 31 days. Abundant amounts of cyclinD1 suppressed progression of S-phase, resulting in the accumulation of 31FR-31NR cells in S-phase. In addition, 31FR-31NR cells exhibited numerous gamma-H2AX foci and Rad51 foci in S-phase even on 31 days after the cessation of irradiation. Thus, DNA double strand breaks were induced in 31FR-31NR cells and homologous recombination repair (HRR) was activated. To elucidate the role of HRR on the acquired radioresistance of long-term FR cells, we used siRNA targeting Rad51 for suppression of HRR. Radioresistance of long-term FR cells was completely suppressed by down-regulation of Rad51. In conclusion, the radioresistant phenotype of tumor cells acquired by long-term radiation exposure can be controlled by suppressing HRR.

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It is known that genomic lesion introduced by ionizing radiations continuously activates cellular responses against DNA damage even after completion of damage repair of the initial dose, and induces genomic instability or upregulation of mutation rate for a period. This phenomenon is called delayed mutation which is thought as a result of epigenetic damage memory and mutagenic activity at the downstream of the memory.
Our study using the fission yeast Schizosaccharomyces pombe as a model organism has revealed that (i) X-irradiation elevates recombination frequency for about 10 cellular generations after recovery from the cell cycle arrest, (ii) the delayed recombination is possible to occur in trans, or would have a weak relation to the position of the initial damage site and the production of reactive oxygen species induced by the damage, (iii) delayed activation of a recombination repair protein Rad22 is also induced by X-ray in the dividing cells, and (iv) the activation of Rad22 continues for a period similar to that of the delayed recombination. These results suggest that Rad22 has a critical role in the delayed recombination. However, the regulation mechanism of delayed activation of Rad22 is remained to be elucidated.
Now we are challenging to screen regulator of Rad22 with mass spectrometric analysis combined with pull-down assay using Rad22 as bait. We will present a result of screening of the regulator to date with a brief summary of the studies on the delayed mutation and the damage memory.

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It is well established that both MDM2 and MDMX are essential factors for maintaning tumor suppressor p53 in its latent form. We have previously shown that MDMX proteins were rapidly degraded by MDM2 in response to DNA damage, a phenomenon which was a critical event in optimal p53 activation. Recent mouse genetic studies have revealed that the expression level of MDMX critically influences the survival of mice after whole-body ionizing radiation exposure. Many in vivo and in vitro studies have shown that MDM2 functions as a E3 ubiquitin ligase to ubiquitinate and down-regulate P53 protein, and forms a negative feedback loop with. However, it is still controversial whether MDMX has a functional activity in the control of P53 stability. Although our studies suggested that MDMX forms heterodimer with MDM2 and enhances MDM2 E3 ubiquitin ligase activity in cells, the underlying molecular mechanisms of how MDMX regulates p53 are still unclear. In order to investigate further possible molecular mechanisms of the p53 regulation by MDM2 and MDMX, we are trying to develop an in vitro assay system in which ubiquitination studies of p53, MDM2 and MDMX can be performed. Here, we present data validating this assay system and report a novel mechanism by which the MDM2/MDMX heterocomplex regulates the p53 ubiquitination and activation in response to DNA damage.

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Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1, and γ-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. Mono-ubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair, and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.

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Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Using live cell imaging techniques, we have analyzed DSB responses of NHEJ core factors, including DNA-PKcs, XRCC4 and XLF. Based on our previous findings, we have proposed a new model of NHEJ assembly on DSBs, which comprises two critical phases. In the first phase, after the binding of Ku to DSBs, DNA-PKcs, XRCC4/LigIV, and XLF are recruited independently and simultaneously to DSBs. Subsequently, the recruited factors cooperatively interact with each other to form functional NHEJ machinery. In addition, we have demonstrated that Ku-XLF interaction occurs on DSBs, strongly indicating the critical role of the Ku-XLF interaction in the assembly of NHEJ machinery on DSBs. In this study, we performed a detailed analysis of the Ku-XLF interaction. First, we identified a domain in Ku heterodimer that is required for XLF interaction by a gel shift assay using purified Ku and XLF proteins. We confirmed that this domain is required for DSB response of XLF in vivo. Next, we identified an essential region of XLF for DSB accumulation in vivo. We expressed a mutant XLF lacking this region and found that the mutant XLF failed to associate with endogenous Ku. Interestingly, we observed significantly reduced association of the mutant XLF with endogenous XRCC4, although XRCC4 was previously reported to bind to a region distinct from the Ku-interacting region in vitro. These observations indicate that Ku-XLF interaction plays a critical role in cooperative interactions among the NHEJ core factors on DSBs, further supporting our model for DSB recognition in the NHEJ pathway.

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Ionizing radiation causes DNA double strand breaks, which initiate ATM-dependent DNA damage signalling. Activated ATM phosphorylates histone H2AX at serine 139, whose phosphorylation is essential for recruitment of MDC1. MDC1 is another target for ATM, and its phosphorylation recruits RNF8, a ubiquitin ligase, which mediates ubiquitination of histone H2AX. Then, 53BP1 is recruited to the modified chromatin region, and all of the factors form DNA damage checkpoint foci. Although phosphorylation and ubiquitination are important modification for DNA damage foci, there are other major histone modifications, such as acetylation and methylation. Here, we examined a role of these modifications in DNA damage foci formation. Normal human diploid cells were incubated in a special culture condition, in which acetylation or methylation is suppressed. Then, cells were exposed to gamma-rays to investigate the foci of phosphorylated histone H2AX and 53BP1. We found that decreased acetylation of histone H3 and di-methylation of histone H3 at lysine 9 did not affect initial foci formation but they ablated persistent foci formation. These results indicate that chromatin modification is essential for proper formation of DNA damage checkpoint foci in normal human cells exposed to ionizing radiation.

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Autoantibody plays an important role in autoimmune diseases. We have reported that repeated 0.5-Gy gamma-irradiation attenuates the pathology in autoimmune disease models accompanied by suppression of autoantibody production. These observations raise an important question whether gamma-irradiation would suppress the antibody production against external antigen or not. Here, we investigated the effects of gamma-irradiation on antibody production in ovalubmin-immunized mice. Mice were immunized with ovalubmin, and exposed to 0.5-Gy gamma-rays per week. After the immunization with ovalubmin, we observed the increment of total serum IgG and IgE level. We found that 0.5-Gy gamma-irradiation further increased the ability of antibody production. Consistent with these results, higher levels of both ovalubmin-specific IgG and IgE were observed in irradiated mice compared with in non-irradiated mice. Furthermore, the percentage of plasma cells was increased by gamma-irradiation, whereas the percentage of B cell was not changed. Splenocytes-producing cytokines, which are involved in antibody production, were increased in ovalubmin-immunized mice, whereas further increments were observed in irradiated mice. These results indicate that repeated 0.5-Gy gamma-irradiation would enhance antibody production against external antigen rather than internal autoantigen.

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South districts of Belarus are still highly radiocontaminated even after 23 years from the Chernobyl catastrophe in 1986, and consequent environmental changes are stored in the soil, plants and animals. The major radionuclides in the contaminated areas are ¹³⁷Cs and ⁹⁰Sr, and their physical half-lives are 30.2 and 28.9 years, respectively. It is easily predicted that the radionuclides are concentrated by the food chain into the living organisms in the contaminated area, and radionuclides remain in the irradiated organisms not only externally but also internally for long periods.
To simulate the radiocontamination in middle contaminated areas of Belarus (1997, 2005), mice were maintained for 8 months in the radioisotope facility with free access to drinking water containing ¹³⁷CsCl (0Bq/ml, 10Bq/ml and 100Bq/ml).
Mice were assessed the long term low dose rate and low dose internal and external radiation effect by the quantitative measurement of the contaminated radionuclides-induced DNA double-strand breaks by γ-H2AX foci in the organs. The micro nuclei test and the tumorigenicity of mouse lung were also examined under the same condition.
The number of γ-H2AX focus per hepatic cell was 1.4 (0Bq/ml), 9.5(10Bq/ml), 10.7(100Bq/ml), respectively. It was suggested that the genome damage caused internal low dose ¹³⁷Cs radiation were occurring chronically even in low dose contaminated area. But no significant difference was observed in the micro nuclei test. In addition, progress data (by 6 animals in each dose groups) is showing that the number of lung tumors per mouse induced by urethane under the same condition is 34.8 (0 Bq/ml), 41.2 (10Bq/ml), 44.2 (100Bq/ml), and the average of tumor diameter (mm) is 1.70, 1.71, 1.74, respectively. (Supported by JSPS)

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Long term manned space missions are planned to implement within the first two decades of the 21st century. A mission of International Space Station (ISS) is in progress, and a plan to explore Moon as well as to visit the Mars is also underway. In space field, the plan will allow space man to live for long terms to do experiments to examine various space sciences. In space environment, 0.2~0.3 mSv a day of radiation exposure will affect cellular metabolisms. As such missions will inevitably result in a significant space radiation exposure on the astronauts, there is an increasing demand to examine their risk. Evaluation of realistic risk associated with the space mission will be in urgent demand to protect them adequately based on both physical and biological knowledge. Considering cellular metabolisms or homeostasis, which affect their risk, examinations of gene expression change would be important. In this study, a human neuron-like cell line, NB-1 was irradiated with 0.1 mGy, 1.0 mGy, 10 mGy, and 100 mGy in single exposure. Thirty minutes and 2 hours after the treatments, mRNA was isolated from the cells and gene expressions were studied with Quantitative RT-PCR analysis. The results showed that the both up- and downregulated gene expressions were seen among mitochondrial genome, electron transport and TCA cycle genes. On the other hand, beta-oxidation, ion transport, antioxidative enzymes and factors related gene expressions were downregulated. DNA repair, glycolysis, heat shock protein, and apoptosis related gene expressions were also downregulated except for some genes such as p53. The autophagy related gene, and necrosis related gene expressions were also downregulated. These results suggest that the space low-dose irradiation induce intracellular oxidative stress, and may decrease at least partial apoptosis-related gene expression changes, i.e., may increase potential risk of cancer or neuro-degenerative diseases.

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The effects of low-dose gamma rays on the postnatal and prenatal development of mice as well as on the differentiation of neural crest cells were investigated. Pregnant females of C57BL/10JHir mice at 9 days of gestation were whole-body irradiated with a single acute dose of gamma rays. The effect was studied by scoring changes in the postnatal development of mice and in the white spot in the ventrum 22 days after birth. The percentage of pregnancy, birth, survival to day 22 and body weight at day 22 showed no change even in mice irradiated with 0.5 Gy gamma rays. Radiations elicit mid-ventral white spots in offspring 22 days after birth due to the inhibition of neural crest cell differentiation. The frequency of white spots in the mid-ventrum was increased from the dose of 0.25 Gy, and the size of white spots was also increased from the dose of 0.25 Gy. Irradiated 18-day-old embryos were dissected and effects of gamma rays on the number of living embryos, body weight, developmental anomalies, and the differentiation of hair bulb melanocytes were investigated in detail. In irradiated 18-day-old embryos, the litter size was not decreased even in 0.75 Gy. However, the abnormalities in tails were observed at the embryos irradiated with 0.1 Gy gamma rays and its frequency was increased with increasing dose. The hemorrhage at the base of tail and limbs was observed at the control embryos and its frequency was increased with increasing dose. Moreover, the number of hair bulb melanocytes in the dorsal and ventral skins decreased from 0.1 Gy-treated embryos, and gradually decreased with increasing dose. These results suggest that low-dose gamma rays have inhibitory effects on the prenatal development of tail and blood vessels as well as on the neural crest cell differentiation in mice.

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Purpose: To evaluate the effect of manipulating intratumor hypoxia during radiotherapy on lung metastasis, referring to its effect on the sensitivity of quiescent tumor cells.
Materials and Methods: B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. They received gamma-ray irradiation following loading with the acute hypoxia-releasing agent nicotinamide or local hyperthermia at mild temperatures (MTH). Immediately after the irradiation, cells from some tumors were isolated and incubated with a cytokinesis blocker. The sensitivity of quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. That of the total (= P + Q) tumor cell population was determined from BrdU non-treated tumors. In other tumor-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated.
Results: In the total cells, a more marked enhancement in sensitivity was observed with nicotinamide than MTH. In Q cells, MTH combination induced a more marked enhancement than nicotinamide. Both nicotinamide and MTH reduced the size of the hypoxic fraction in the two cell populations, especially nicotinamide in the total cells and MTH in Q cells. Without gamma-ray irradiation, nicotinamide loading tended to decrease the number of lung metastases. With gamma-ray irradiation, nicotinamide loading and MTH, especially the former, reduced the number of metastases more than gamma-ray irradiation only.
Conclusion: Hypoxia manipulation in solid tumors has the potential to influence lung metastases. Notably, acute hypoxia-releasing nicotinamide may be promising for reducing the number of lung metastases.

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Radiotherapy is one of the major therapeutic modalities for the treatment of oral cancer. However, the existence of radioresistant cells would be linked to the failure of tumor radiotherapy. In order to develop more effective tumor radiotherapy, we have to understand the feature of radioresistant cells. For this purpose, clinically relevant radioresistant (CRR) cell line, SAS-R which can continue to proliferate with daily exposure to 2 Gy of X-rays, was obtained from human oral cancer SAS by the exposure to step-wise increase of fractionated X-rays. Recently, many studies suggested that angiogenesis is one of the most important factors for tumor growth and inhibition of angiogenesis is effective to tumor treatment. We observed more number of vessels in SAS-R tumors inoculated in the back of nude mice than SAS tumors. These observations prompted us to examine whether RAD001, a newly-developed angiogenesis inhibitor, is effective for the treatment of CRR tumors or not. Combination of fractionated 2 Gy of X-rays and RAD001 was more effective to reduce the tumor volume than either radiation or RAD001 alone. Interestingly, combination treatment revealed more rapid reduction of SAS-R tumors than SAS tumors. In addition, in vitro study showed that SAS-R secreted higher amount of VEGF than SAS. Our results suggest that radiotherapy with RAD001 is effective for the treatment of radioresistant oral cancer.

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[Purpose] The upregulation of aerobic glycolysis, known as Warburg effect, is one of the metabolic changes prevalent in solid tumors. Warburg effect is characterized by reliance on glycolysis, which is mitochondrial-independent, regardless of oxygen availability. We have recently observed that X-irradiation activated mitochondrial electron transport chain, leading to the activation of apoptosis signaling pathway. In this study, we examined whether the modulation of cellular energy metabolism could enhance radiation-induced cell death.
[Materials and Methods] SCC VII cells derived from mice floor of the mouth squamous carcinoma were used in this study. Two pharmacological inhibitors: 2-deoxy-D-glucose (2DG) as a glycolysis inhibitor and dichroloacetate (DCA) as a pyruvate dehydrogenase kinase (PDK) inhibitor, were used. The effect of these drugs on the radiation-induced reproductive cell death was evaluated by clonogenic assay. Furthermore, in vivo experiment, the effect of 2DG combined with X-irradiation on the tumor growth was examined in SCC VII cells transplanted in C3H/HeJ mice.
[Result] Radiation-induced reproductive cell death was significantly enhanced by 2DG and DCA, respectively. This result suggested that the potential of these drugs as radiosensitizers for tumor cells. We also observed distinct suppression of tumor growth by 2DG combined with X-irradiation in vivo experiment. Since DCA was known to activate pyruvate dehydrogenase by inhibiting PDK and promote the following production of acetyl-CoA for the activation of mitochondrial function, it was suggested that the modification of cellular energy metabolism, i.e., inhibition of glycolysis and activation of mitochondrial energy metabolism, induced the enhancement of the radiation-induced cell death.

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Hypoxia-inducible factor 1 (HIF-1) has been reported to promote tumour radioresistance; therefore, it is recognised as an excellent target during radiation therapy. However, the inhibition of HIF-1 in unsuitable timing can suppress rather than enhance the effect of radiation therapy because its anti-angiogenic effect increases the radioresistant hypoxic fraction. In this study, we imaged changes of HIF-1 activity after treatment with radiation and/or an HIF-1 inhibitor, YC-1, and optimised their combination. Hypoxic tumour cells were reoxygenated 6 h postirradiation, leading to von Hippel-Lindau (VHL)-dependent proteolysis of HIF-1alpha and a resultant decrease in HIF-1 activity. The activity then increased as HIF-1alpha accumulated in the reoxygenated regions 24 h postirradiation. Meanwhile, YC-1 temporarily but significantly suppressed HIF-1 activity, leading to a decrease in microvessel density and an increase in tumour hypoxia. On treatment with YC-1 and then radiation, the YC-1-mediated increase in tumour hypoxia suppressed the effect of radiation therapy, whereas on treatment in the reverse order, YC-1 suppressed the postirradiation upregulation of HIF-1 activity and consequently delayed tumour growth. These results indicate that treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the therapeutic effect of radiation, and the suppression of the postirradiation upregulation of HIF-1 activity is important for the best therapeutic benefit.

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Introduction
As a result of recent biological analyses, the medaka (Oryzias latipes) has gained much attention as an animal model of human disease. In this study, we examined the effects of irradiation on osteoclastogenesis in pharyngeal bones. We also investigated immunochemically the expression of the osteoclast differentiation factors receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG).
Material and Method
We irradiated entire medaka with 15, 30, or 45 Gy of carbon ions or gamma rays. Histochemical assays were performed to determine the effects of irradiation on sections of pharyngeal bone to analyze the histochemical localization of tartrate-resistant acid phosphatase (TRAP) activity, the main marker of osteoclasts and their immediate precursors. Immunostaining assay with RANKL and OPG antibodies was performed.
Result
Early on, both irradiated groups showed high TRAP activity, whereas 5 days after irradiation, no statistical differences were observed between groups that were irradiated at the same dose. Seven days after irradiation, TRAP activity in the gamma ray–irradiated group was stronger than that in the control and carbon ion–irradiated groups. The localization of RANKL was strong in the supporting pharyngeal bone adjacent to the teeth of the gamma ray–irradiated medaka. Expression of OPG was activated in the same parts of the pharyngeal bone in carbon ion–irradiated medaka.
Conclusion
The TRAP activity of osteoclasts increased with gamma ray irradiation; it was lower with carbon ion irradiation than with gamma ray irradiation, although it was slightly increased with carbon ion irradiation than with no irradiation. This suggests that gamma ray irradiation stimulates osteoclast activity, increasing the resorption of pharyngeal bone. Carbon ion irradiation has a different effect on osteoclast activity and might suppress maturation via OPG expression. These observations suggest that carbon ion irradiation induces differential modulation of osteoclast growth factor expression.

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Although chordoma, a rare bone cancer, has been treated with surgery and/or radiation including heavy ions, no in vitro characterizations of chordoma cells are available. The main reason for this is the extremely long doubling time associated with the only recognized cell line, U-CH1. Here, we derived a cell line (designated as U-CH1-N) from U-CH1 and made the in vitro characterization, and results with U-CH1-N were compared with those with HeLa (cervical cancer) and U87-MG (glioblastoma) cells in the following.
1) Cellular doubling time, DNA content
The cell doubling times for HeLa, U87-MG and U-CH1-N were 18 h, 24 h and 3 days respectively. U-CH1-N had the most DNA content among the three, was near tetraploid, and showed an abnormal karyotype.
2) Cellular Radiosensitivity
U-CH1-N and U87-MG were more sensitive to x-irradiation than HeLa cells. Heavy ion irradiation more efficiently killed all three cell lines than x-rays did. Relative biological effectiveness (RBE) at 10% survival for U87-MG and HeLa cells was about 2.5 for 70keV/μm carbon ions and 3 for 200keV/μm iron ions, while for U-CH1-N it was 2.5 for carbon and 4 for iron ions. We proceeded the RBE evaluation with neon ions, argon ions and silicon ions, and the RBE curve of U-CH1-N had a peak value, 5.3 for 150keV/μm silicon ions.
3) Sensitivity to Genotoxic Chemical Agents
Four different chemical agents producing various DNA lesions were used for further characterization of these cells. Although camptothecin, mitomycin C or cisplatin did not reveal strong cytotoxity to U-CH1-N compared with other cells, bleomycin, which produces DNA strand breaks, showed marked cytotoxic effect on U-CH1-N.
Our data provide the first chronological cell survival information using cells of chordoma origin and also help explain the successful chordoma treatment by heavy ions.

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Objectives: Whole genome association study using microsatellite markers has been carried out to search for genes associating with acute adverse effects after radiotherapy for cancer patients.
Materials and Methods: A total of 360 patients with head and neck, lung, esophageous, breast and cervical cancers were recruited in this study. They were divided into following two groups by the severity of the adverse effects graded by NCICTC version 2 within 3 months after completion of radiotherapy; (1) High Grade Group (HGG) who developed grade 3 or more adverse effect and (2) Low Grade Group (LGG) who developed grade 1 or less adverse effect on any of the endpoints. Each group consisted of 180 patients. Genomic DNA was individually extracted from blood cells of these patients. Two-round scanning of microsatellite markers was conducted using two pools of genomic DNA made of 90 non-overlapping patients in each group.
Results and Conclusions:, A total of 21,000 microsatellite markers were scanned using one of the pooled DNA of each group respectively in the first round yielding 3,052 markers with significantly different estimated frequency between the two groups (P-value < 0.05). Reproducibility of these 3052 markers was investigated by the second round scan using the remaining pooled DNA, yielding 159 positive markers (P-value < 0.05). Among them, ten markers locating at the distance within 100 kb from the transcription initiation site of the nearest gene were selected for further individual typing of the all 360 patients. A significant association (P-value = 1.24x10⁵) was displayed by a marker locating at 1,500 bp upstream of Semaphorin 3A gene. Knocking down of this gene expression by siRNA treatment in a human fibroblast culture provided suppression of radiation sensitivity. Semaphorin 3A gene has been reported to encode a secreted protein with a range of functional processes including regulation of axon guidance, cell survival, motility, immune responses, and angiogenesis. It is, thus reasonable to consider this gene to influence adverse effects in several types of cancer patients.

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The effects of high LET carbon-ion (C290, 50keV/um, 6-cm SOBP) irradiation on tumor control and cancer stem cells of HCT116 xenograft tumors were investigated. The tumor growth delay (TGD) was 5 and 28 days after irradiated with15 and 30 Gy X-ray, whereas the TGD was 12 and 82 days after irradiated with 5 or 15 days carbon ion. The tumors were eradicated without relapse when irradiated with 60 Gy X-ray or 30 Gy carbon ion. The relative biological effects (RBE) value of carbon-ion relative to X-rays was 3.82. Carbon-ion irradiation predominantly induced tumor cell cavitation, fiborosis and completely disrupted the duct-like architecture. Tumor-supplying vessels were reduced in carbon-ion irradiated mice compared to those of X-ray irradiated mice. Expression of VEGF, HIF-1a, and cancer stem cell marker CD133, EpCAM was markedly suppressed by 30 Gy carbon-ion irradiation, whereas 30 Gy X-ray increased the expression of these proteins. In conclusion, heavy-ion irradiation can more effectively control xenograft tumors than X-ray through suppression of angiogenesis and disruption of cancer stem cells.

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Researches for biological effect to fast neutrons have been done using NASBEE (Neutron Accelerator System for Biogical Effect Experiment) in NIRS. Neutron beam is produced by 4 MeV deuteron bombarding to thick berylium target. Neutron beam is irradiated to whole body of mice. At the next step, in rder to examine lung cancer of rat, neutron beam was locallized irradiation to rat. The beam is collimated within 4 cm slit width and shielded with iron and polyethylene blocks. Neutron beam specification in the neutron exposured and shielded areas were examined with exposure and absorbed dose rates of neutrons and photons, dose rate profiles, and suvival fraction of colony creation of epithelium cell (new born rat).
The exposured neutron dose rate is 0.05 Gy/min and neutrons are shielded to be decreased to less than 10% on the absorbed dose rate. Gamma rays are produced from the target via the neutron production. In the neutron beam, gamma rays are contaminated to be about 15%. Based on measured microdositry distributions of neutron and photon, RBE value at 2 Gy for endpoint crypt cell survival is obtained to be 3.3.
Now, the locallized neutron irradiation to rat lungs is prepared, (starts at the September 2009). The collimated neutron beam is useful tool to study neutron effect in locallized area.

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Product yields of water radiolysis are inevitably important in understanding detailed mechanism of indirect action of ionizing radiations. For example such information are closely related to track structure, which are still not clarified yet especially for the case of heavy ion beams. We have conducted yield measurement of main products such as hydrated electron, hydroxyl radical (^.OH) and hydrogen peroxide. We have also developed a sensitive method to determine ^.OH yield with a fluorescent probe. In the method, aqueous solution of coumarin-3-carboxylic acid (CCA) is irradiated and ^.OH is scavenged by CCA, resulting in production of fluorescent product 7OH-CCA. Applying this method, ^.OH yield near the Bragg peak has been measured in this study because the Bragg peak is overlapped cancer position in actual therapy.
Beams of C 290 and 135, He 150 and Ar 500 MeV/u were taken for irradiation at the biological irradiation port at HIMAC facility of NIRS. Energy absorber made of PMMA plates was inserted at the upstream of irradiation field to overlap the Bragg peak to sample solutions. 26 and 1.5 mM aqueous solutions of CCA were prepared with ultra-pure water and phosphate buffer (pH 6.8) and used as irradiation sample. After irradiation, samples were analyzed by fluorescence spectrometer connected with HPLC and production of 7OH-CCA was quantified.
It was found that ^.OH yield show minimal value around the Bragg peak for every ion beam and it drastically jumps up several times at just downstream of the Bragg peak. It is already well known that contributions of fragmentation particles become non-negligible for high-energy heavy ions. Thus, further consideration including fragmentations is necessary to separate contributions of different fragmentation particles. Such consideration is being attempted by using simulations with HIBRAC and PHITS.

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The oxygen radicals play a crucial role in biological oxidative damage. It is important to clarify behavior of hydroxyl and superoxide radicals, detection and quantification of these radicals are required. A new spin-trap agent CYPMPO (5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1- pyrroline N-oxide) can spin-trap hydroxyl and superoxide radicals, and then produce hydroxyl and superoxide adducts, respectively. The ESR spectra of these adducts are assigned as different spectra. It is expected that hydroxyl and superoxide radicals can quantified by using CYPMPO, however, furthermore quantitative evaluation of kinetics and reactivity of CYPMPO are required. The oxygen radicals are also produced by water decomposition with irradiation. The behavior of these radicals shows useful information for understanding the indirect effect of irradiation.
In present work, the reactivities of CYPMPO toward a hydrated electron and a hydroxyl radical utilizing a pulse radiolysis system are investigated, and compared with DMPO (5,5-Dimethyl-1-pyrroline N-oxide) results. The oxygen radicals produced by gamma-ray irradiation are also discussed.

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Edaravone or RadiCut^®, 3-methyl-1-phenyl-2-pyrazolin-5-one, is a developed free radical scavenger that has been approved in Japan as a neuroprotective drug since 2001. For its antioxidative property and high reactivity with free radicals, edaravone has been expected as a radioprotector. In the last decade, the reactivity of edaravone with peroxyl radicals or some enzymes which induce oxidizing reactions, as well as the antioxidative properties of edaravone, have been extensively investigated. However, there are only a few reports on the measurements of the reactivity of edaravone towrads OH radical, although OH radical is thought to be a main factor of indirect radiation induced damages. In this work, we measured the reactivity of edaravone with OH radical, and elucidated its reaction mechanism. The reactions of edaravone and its model compound (1,3-dimethyl-2-pyrazolin-5-one) with OH radical or other oxidizing radicals were measured by pulse radiolysis techniques. The result shows that the intermediate radical produced by the reaction of edaravone with OH radical was spectroscopically different from that by the reactions with other radicals. On the other hand, for 1,3-dimethyl-2-pyrazolin-5-one, the intermediate radical by the reaction with OH radical is similar to that by the reaction with other oxidizing radical. All these strongly suggest that the phenyl group of edaravone plays an important role on the reaction with OH radical. Thus, in contrast to the previous reports in which the oxidation by OH radical was assumed to be through electron transfer or hydrogen abstraction from pyrazolin-5-one, the formation of OH adducts is crucial for the reaction of edaravone towards OH radical.

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The health risks incurred when children are exposed to radiation is a problem receiving increased attention. To understand how radiation affects the developing intestine, we examined the intestine of X-irradiated C57BL/6J mice, including neonates, infants, and adults. Intestinal development progressed in three stages: crypt neogenesis pre- and immediately postpartum, crypt fission/branching during infancy, and crypt-villus architectural maturation after weaning. Irradiated adult small intestinal crypts displayed two separate apoptotic waves (as defined by the presence of caspase-3)—the first wave peaked at 3 hr and was followed by a broad wave with a peak persisting from 24 to 48 hr. During the first wave, p53 was expressed, whereas it was not during the second wave. For infant small intestine, the intensity of the first wave was approximately half that of the adult wave, and for the colon the intensity was smaller yet. The second wave was also diminished in infant crypts. In neonates, apoptosis was delayed, peaking at 6 hr for small intestinal crypts and at 24 hr for colonic crypts—apoptotic crypts were absent at 3 hr. Apoptosis of the cells of adult small intestinal crypts was greatest at position 4 but occurred uniformly in infant crypts. Colonic cells at positions 1 and 2 were the most susceptible to radiation, regardless of age. Although no apoptosis occurred at 3 hr post-irradiation in neonates, p53 was induced in small intestinal inter-villi clefts and in colonic crypts. This finding suggests that p53 expression and apoptosis are unrelated during development of the neonatal intestine. Conversely, p21^WAF-1/CIP1 expression was greater in neonatal crypt cells than in those of adults, suggesting efficient cell cycle arrest in neonates. Apparently, the developing intestine exhibits unique apoptotic responses to irradiation that are distinct from those of the adult intestine.

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Deficiencies in DNA mismatch repair (MMR) result in replication errors that cause frameshift mutations and/or point mutations within key tumor suppressor genes or oncogenes. Heterozygous germline mutations in MMR genes are the cause of hereditary nonpolyposis colorectal cancer (HNPCC). In these cancers, mutations in mononucleotide repeat sequences in TGFbRII, BAX, etc. have been frequently reported. Homozygous germline mutations of MMR genes are also manifested by an early onset of childhood T- or B-cell leukemia, but the target gene was not identified yet. Mlh1^-/- mice spontaneously develop T-cell lymphoma and colorectal tumor from 10-week-old and 15-week-old, respectively. In this study, we aimed to determine the age dependency of colorectal tumor induction after exposure to radiation, and to clarify the histological and molecular characteristics of the tumors.
For induction of colorectal tumor, Mlh1^+/+, Mlh1^+/-, and Mlh1^-/- mice were exposed to whole-body X-irradiation at 2 Gy at the age of 2 or 7-week-old, and/or treated with 10% dextran sulfate sodium (DSS) in their drinking water for one week at the age of 10-week-old. Thymectomy was performed at 4 weeks of age to exclude the confounding of T-cell lymphomas. All mice were scarified at the 25-week-old.
The incidence of colorectal tumor in Mlh1^-/- mice was 10% and 0% after irradiation at the age of 2 and 7-week-old, respectively. DSS treatment only resulted in 40%. In the group with combined exposure, tumor induction increased 70% and 50%, respectively. Irradiation at 2-weeks of age increased the number of tumors compared with the irradiation at 7-week-old. This indicates that infant mice are slightly more susceptible to radiation than adults. Mlh1^+/+ mice did not induce any intestine tumor in this study. The pathological and molecular analyses of induced tumors will be shown.

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(Purpose) Previously, we reported that irradiation in young age induced delayed mutation, which were related to dysfunction of p53. In this study, we evaluated the effect of irradiation in young age on lifespan. We analyzed the time-course of the cell numbers and cell cycles of myelocyte and lymphocyte in bone marrow, and also the time-course of cell proliferation of splenocyte. Furthermore, we evaluated the protein expressions of p53 and the related gene. (Materials and Methods) We used p53(+/-) mice and p53(+/+) mice, which were irradiated a whole-body dose of 3 Gy at 8 weeks of age (irradiated group). p53(+/-) mice were sacrificed at 24 and 56 weeks (old mice) of age and p53(+/+) mice at 40 and 72 weeks (old mice) of age. Cell numbers and cell cycles of bone marrow cells from femurs and tibias were analyzed. In p53(+/+) mice, cell proliferation of splenocyte, which was stimulated by Concanavalin A, was measured. From 0 to 24 h after old p53(+/+) mice were irradiated a whole-body dose of 3 Gy, the protein expressions in spleen of p53, p21 and MDM2 in cytoplasm and nucleus were analyzed by Western blot methods. The rate of p53 allele was analyzed by PCR in CD3-CD4+ cells in spleen. (Results) Lifespan was shortened by irradiation at 8 weeks of age. In old mice, the cell number of lymphocyte in bone marrow in irradiated group decreased compared to that in non-irradiated group. In old p53(+/-) mice, cell cycle of lymphocyte was accelerated. The proliferation in splenocyte in p53(+/+) mice decreased with age, and the proliferation in irradiated group was much lower than that in non-irradiated group. In old p53(+/+) mice, the protein expressions of p53, p21 and MDM2 in irradiated group delayed compared to that in non-irradiated group. In old mice, the rate of p53 allele decreased in irradiated group compared to that in non-irradiated group. (Conclusion) We suggested that irradiation accelerated aging, and that, because of these failures of immune system and dysfunction of p53, the lifespan was shortened in irradiated mice.

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Purpose; In order to know the mechanisms of bystander effects through the secreted factor from irradiated cells, we examined the involvement of radicals induced by the conditioned medium from the irradiated cells.
Method; Bystander effects were detected by medium transfer method. The induction of mutations at HPRT locus by bystander effects was examined in CHO cells. Suppressive effects of bystander effects by ascorbic acid were analyzed by the treatments of recipient bystander cells. The radicals induced by conditioned medium in bystander cells were determined by ESR method.
Results; Bystander effects induced mutations at HPRT locus. In the analysis with ascorbic acid, it was effective for the suppression of bystander responses when recipient bystander cells were treated. Slow releasing long-lived radicals (SRLLR) were identified by ESR method in the cells treated with conditioned medium from irradiated cells, and ascorbic acid could relieve the induction of SRLLR, suggesting that the different types of radicals from reactive oxygen species (ROS) are involved in the bystander mutagenesis. These results suggest that inductions of SRLLR play an important role for the bystander mutagenesis in the recipient cells of secreted factors from irradiated cells.

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Because effects of bystander factors on the biology of cultured cells are very limited to compare with tremendous factors for those in directly irradiated cells, bystander systems are better for elucidating the mechanisms even these are still too complex. We have succeed in the direct measurement of slow releasing long-lived radicals (SRLLRs) by ESR in cultured cells. Levels of SRLLRs in irradiated cells have increased to +20% for five hours after irradiation. Suppression of SRLLRs by post-treatment of ascorbate has reduced point mutation frequency in control level. In this study we have measured the SRLLRs in bystander cells induced by medium mediated bystander effect with point mutation frequency. D-MEM(10% fcs) was gamma-ray irradiated in 1 Gy with donor CHO cells in cultur flasks and kept for 24 h. The medium was transferred to other flasks with non-irradiated bystander CHO cells and kept for 24h, then the bystander cells were harvested and put into ESR tube. The ESR spectra of bystander cells were recorded at 77 K. Levels of SRLLRs in bystander cells were increased to +20% in average, and frequency of HPRT- point mutation also increased four times larger than that of control. No increases detected when the medium was irradiated without donor cells. These results indicate that SRLLRs are induced by bystander factor produced by irradiated donor cells. Treatment of ascorbate (1 mM) to bystander cells for 24h from the medium transfer, both levels of SRLLRs and the mutation frequency was reduced with significant difference. The medium with myxothiazol, as an inhibitor of electron transportation in mitochondria, treated to donor cells before irradiation did not increase the levels of SRLLRs. These results strongly indicate that bystander factors are produced with dysfunction of mitochondria in donor cells and affect to produce SRLLRs in bystander cells. SRLLRs might be related to point mutation induction.

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Purpose: Cell-mediated immunity decrease by radiation becomes known, but it is not studied mechanism of effect of humoral immunity in detail. Therefore, in this study, we were aimed at reviewing cell-mediated immunity by radiation, effect to humoral immunity by measuring
Methods: We classified in control group, 0.5Gy group, 1Gy group and 2Gy group. We measured a blood cell count (leukocyte, lymphocyte, monocyte, granulocyte) after 30th since the irradiation day before. We measured by IL-2 Kit, IL-2 Kit, IL-6 Kit, IL-12 Kit of mice serum.
Results: In lymphocytes, meaningful decrease was accepted in 0.5Gy group 24 hours later. In 1Gy group, meaningful decrease was accepted after 3hours, 24 hours and 3days later. In 2Gy group, meaningful decrease was accepted in all assay dates. In IL-2, it was control group comparison with the irradiation day before, it was it with 199.6% in a 0.5Gy crowd in 57.9%, 2Gy group in 425.6%, 1Gy group after irradiation 15th whereas it was 24.6%. In addition, it was it with 170.8% in a 0.5Gy crowd in 158.7%, 2Gy group in 383.8%, 1Gy group after irradiation 30th whereas appearing was 96.6% in control group. In IL-6, meaningful decrease in 0.5Gy group after 7th after 3rd 24 hours later. In 1Gy group, meaningful decrease was accepted after 30days and 7days later. In 2Gy group, meaningful decrease was accepted 24 hours later. In IL-12, meaningful decrease was accepted in 0.5Gy group after 30th. In 2Gy group, meaningful decrease was accepted after 30th after 15th three days later.
Conclusions: In this study, blood corpuscle ingredients such as lymphocytes are affected by effect of radiation early. Production tends to enhance IL-2 by radiation hormesis by radiation of a low dose of 0.5Gy. As for the IL-6, quantity of production decreases by effect of radiation. However, quantity of production of IL-6 tends to increase in 0.5Gy early. IL-12 is tendencies to decrease by radiation, but rather quantity of production tends to increase in low dose radiation. Humoral immunity tended to commit radiation in predominance than the above in the early stage when it was exposed to radiation, and it was accepted that cell-mediated immunity tended to act on predominance with time.

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We investigated the mechanism underlying the radioadaptive response that rescues low-dose preirradiated mice from hematopoietic failure after challenge irradiation and found that myeloid cells were rapidly recovered following the challenge irradiation in the bone marrow of low-dose-preirradiated mice. Cytokine profiles were also indicated that the concentrations of growth factors for myelopoiesis after the challenge irradiation were considerably increased by low-dose preirradiation. Especially, granurocyte colony stimulating factor, G-CSF, was significantly upregulated immediately after challenge irradiation in the low-dose-preirradiated mice. Then we evaluated the effect of administration of G-CSF after challenge irradiation on modifying the survival rate of mice without low-dose preirradiation in this study.

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UVB induces mutation in the skin, increasing the mutant frequency (MF) dose-dependently. We found in our previous study that the mutation induction in the mouse skin epidermis is suppressed and leveled off to a constant MF above a certain dose of UVB. This suppressive response was not detected in the dermis. We named this epidermis-specific suppression of UVB-provoked mutation induction as “mutation induction suppression (MIS)”. To study the mechanism of MIS induction, apoptotic responses in the epidermis exposed to the UVB dose sufficient to induce MIS was examined, using experimental mice. We found that the UVB irradiation induced a total replacement of the epidermal layer by destroying the damaged epidermis by apoptosis and rebuilding a new one through hyperplasia. This apoptotic turnover of the epidermis was p53-independent, although the p53 deficiency delayed the process. The total replacement of the damaged epidermis by a less damaged new epidermal layer could explain the mechanism of MIS phenomenon.

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Purpose: The thermo-enhancement effects of the sesquiterpene lactone parthenolide (PTL), which targets transcription factor NF-κB, with hyperthermia at 40, 42 and 44degreeC on the human lung adenocarcinoma A549 cell line were investigated in vitro. The expressions of p53 and hsp72 proteins following application of PTL, hyperthermia at 44degreeC, and those in combination were examined to investigate whether p53 and hsp72 participated in apoptosis induction via NF-κB signal pathway. The kinetics of apoptosis induction was also determined.
Methods: PTL was dissolved in culture medium to an appropriate final concentration (0.2microM) prior to being used for treatment. Kinetic assessments of the sensitivity of A549 cells to PTL and hyperthermia were carried out using colony forming assays. The accumulation of p53 and hsp72 proteins in cells after the application of hyperthermia, PTL, and those in combination were examined by western blot analysis. The induction of apoptosis in cells was analyzed under a fluorescence microscope, and the percentage of typical apoptotic cells was calculated.
Results: Thermotherapy using a combined treatment with PTL prior to hyperthermia showed synergistic thermo-enhancement effects toward A549 cells. The p53 was increased slightly by PTL. That after combination therapy with PTL and hyperthermia at 44degreeC was a little increase in comparison with hyperthermia alone. The hsp72 after treatment with PTL alone was little increase as same as a control, and also for combination therapy was not induced or reduced significantly as compared with hyperthermia alone. PTL showed remarkable cytotoxicity at S phase of cell cycle on any concentrations as well as hyperthermia. That is, PTL reduced the cells of proliferation phase. PTL alone did not inhibit NF-κB activation in the cells stimulated with TNF-alpha, unexpectedly. While heating alone inhibited NF-κB for early interval time after treatment, but faded for long time of interval. However, the combined treatments with PTL and hyperthermia inhibited NF-κB activation completely.
Conclusions: Thermotherapy of combined treatment with PTL and hyperthermia in A549 cells showed synergistic thermo-enhancement effects. It was suggested that the PTL-induced apoptosis in A549 cells due to suppress the activity of NF-κB directly, and via p53-independent and hsp72-independent manner on NF-κB signaling.

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There is a concern that radiation induces vascular diseases as side effects after radiotherapies. Although radiation-induced atherosclerosis is one of such diseases, the mechanism remains controversial. Atherosclerosis is a multifactorial disease, and apoptosis in vascular smooth muscle cells is one of possible factors. Development of atherosclerosis is mediated by oxidative stresses and oxidized low-density lipoproteins, which can be apoptosis inducers. However, induction or development of atherosclerosis has not been investigated from the viewpoint of radiation-induced apoptosis in vascular smooth muscle cells. Here, using mouse aortic vascular smooth muscle cells, radiation-induced apoptosis in the cells was evaluated under the presence of mediators related to atherosclerosis. Radiation induced apoptosis of vascular smooth muscle cells very slightly. Although H2O2 had no effects on radiation-induced apoptosis, oxidized low-density lipoproteins had synergistic effects on the apoptosis. Mechanisms in radiation-induced apoptosis influenced by oxidized low-density lipoproteins will be discussed with the results of effects of kinase inhibitors on the apoptosis.

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Abstract
Purpose: To clarify the properties of high-energy protons in in situ DNA double strand breaks (DSBs), apoptosis induction, as well as clonogenicity with a comparison to high-energy X-rays.
Materials and methods: Two human tumor cell lines ONS76 and MOLT4 were irradiated with10 MV X-rays or 200 MeV proton beams. In situ DNA double strand breaks were evaluated by immunocytochemical analysis of γ-H2AX foci in both cell lines. Yield of apoptosis was measured by flowcytometry after Annexin-V and PI staining using MOLT4. Standard clonogenic survival assay was performed to obtain relative biological effectiveness of protons to X-rays using ONS76.
Results: Yield of γ-H2AX foci were always higher in proton beam irradiation than in X-ray in both cell lines with factors being 1.14-1.436. γ-H2AX foci formation reached their peak at 30 minutes after irradiation in both cell lines and after this point, they decreased gradually. Proton beams induce apoptosis earlier than X-rays and the final yield of cell death was significantly greater in protons compared to X-ray irradiation. The ratios of apoptosis induced by proton beams and X-rays were 1.01-1.52 at 12 hour after irradiation in MOLT4. RBE of 200MeV proton to 10 MV X-ray at 10% survival and ratio of SF2 in clonogenic survival assay were 1.067 and 1.111, respectively.
Conclusions: Although the proton RBE values in the colony formation assay was within the range previously reported, the induction of DNA double strand breaks and yield of apoptosis were significantly higher in proton irradiation than in X-ray irradiation. Thereby, the underlying mechanisms in cell inactivation may differ between these radiation qualities.

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