Purpose: We clarified the radiobiological characteristics of irradiation with 290 MeV/u carbon ions versus gamma-rays based on the responses of quiescent and total tumor cell populations in vivo. Materials/Methods: SCC VII tumor-bearing mice received a continuous administration of BrdU to label all intratumor proliferating (P) cells. Then, they received 290 MeV/u carbon ions or gamma-rays. Right after irradiation, some of them further received intratumor acutely hypoxic fraction-releasing agent nicotinamide (NA) or cramping the tumor-bearing thighs to induce total hypoxia inside the tumors. Immediately or 9-12 hours after the irradiation, the tumors were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (= P+Q) tumor cell population was determined using tumors that were not pretreated with BrdU. Results: The difference in radiosensitivity between total and Q cell populations was markedly reduced with carbon ion beams, especially those with a higher LET value. PLDR by Q cells was efficiently inhibited with the carbon ion beams, again especially with a higher LET value. Carbon ion beam irradiation with a higher LET value could efficiently reduce the dependency of radiosensitivity on the heterogeneity in solid tumors. NA enhanced PLDR and cramping tumor-bearing thighs inhibited PLDR, especially markedly in (P+Q) cells after gamma-ray irradiation.

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The purpose of this study was to compare metastatic capabilities of malignant tumor cells and angiogenesis after irradiation with photon and carbon-ion beams to clarify heavy ion beam-specific biological effects. Several authors have reported that sublethal photon irradiation may potentiate metastatic ability of irradiated cancer cells. We examined the biological properties of highly aggressive HT1080 human fibrosarcoma cells and HUVEC human umbilical vascular endothelial cells to assess their metastatic processes and angiogenesis in vitro. We then assessed the metastatic capabilities of LM8 mouse osteosarcoma irradiated with carbon-ion and photon beam in the syngeneic mice. Our study found that carbon-ion irradiation decreased cancer cell migration, invasion in a dose-dependent manner and strongly inhibited MMP-2 (matrix metalloproteinase-2) activity in vitro. The capillary-like tube structure in three-dimensional culture of endothelial cells was inhibited significantly by carbon-ion irradiation in a dose-dependent manner. In vivo, treatment with carbon-ion reduced the number of lung metastases in a dose-dependent manner. On the other hand, lower X-ray irradiation facilitated cell migration and invasion concomitant with up-regulation of αVβ3 integrin in vitro. These findings provide preclinical rationales that heavy ion radiotherapy may be superior to conventional photon beam therapy in possible preventive effects on metastases of irradiated malignant tumor cells and angiogenesis.

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The p53 tumor suppressor protein functions as a critical component of genotoxic stress response by regulating the gene expression of effectors that control the fate of a cell following DNA damage. In fact, wild-type p53 cells were more sensitive to low-linear energy transfer (LET) radiations than mutated p53 and p53-null cells, because low LET radiations induce p53-dependent apoptosis. In contrast to low LET radiation, high LET radiations such as heavy-ion particles have several potential advantages which are an excellent dose distribution, a higher relative biological effectiveness, a reduction in the oxygen enhancement ratio, less variation in cell cycle-related radiosensitivity, and the existence of less efficient repair of cellular radiation injury. In addition, we have demonstrated that high-LET radiations can have highly lethal effect on radio-resistant tumors, and can induce apoptosis in cancer cells nevertheless of p53 gene status so called as a p53-independent manner. Therefore, we suggested that high LET heavy-ion beams provide an effective tool for any types of p53-pacients. In addition, we proposed that the elucidation of the p53-independent apoptosis-related genes might provide new insights into cancer therapies.

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Bcl-2 overexpression occurs in nearly half of human cancers, and has been associated with radio- and chemoresistance. Here we investigated the potential impact of heavy ions on Bcl-2 overexpressing radioresistant tumors. Whilst Bcl-2 cells (Bcl-2 overexpressing HeLa cells) were more resistant to γ-rays (0.2 keV/µm) and helium ions (16 keV/µm) than Neo cells (neomycin resistant gene-expressing HeLa cells), heavy ions (76-1610 keV/µm) yielded similar survival regardless of Bcl-2 overexpression. Carbon ions (108 keV/µm), which were most effective at reducing the survival among heavy ions tested, decreased the difference in the apoptotic incidence between Bcl-2 and Neo cells, and prolonged G2/M arrest that occurred more extensively in Bcl-2 cells than in Neo cells. These findings indicate that high-LET heavy ions overcome tumor radioresistance caused by Bcl-2 overexpression, which may be potentially accounted for by the enhanced apoptotic response and prolonged G2/M arrest. We further found that preirradiation treatment with the cell-permeable, small-molecule Bcl-2 inhibitor HA14-1 sensitizes Neo cells and Bcl-2 cells, but not normal human fibroblasts, to carbon ions. These results suggest that HA14-1 preferentially sensitizes tumor cells to heavy ions. Thus, Bcl-2 may be an attractive target for improving the efficacy of heavy-ion therapy.

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Improvement in the survival of patients treated with heavy ion therapy may in turn raise a concern about secondary cancer risk. We conducted a series of animal experiments to investigate the carcinogenic effect of carbon ions from the HIMAC synchrotron at NIRS on mammary gland, one of the most susceptible organs to radiation carcinogenesis. First, we compared the susceptibility of four rat strains to mammary cancer induction by the carbon ion SOBP beam and found that only Sprague-Dawley rats showed significantly increased cancer incidence. Using this strain, we next clarified that the dose-effect relationship for carbon ions (0.05–2 Gy) was convex upward, whereas that for 137Cs γ-rays (0.5–2 Gy) was linear, resulting in a dose-dependent relative biologic effectiveness that ranged from 10 to 2 at doses 0.05–1 Gy. Lung metastasis was prominent in carbon ion-irradiated rats. Primary tumors were mostly (~80%) positive for estrogen receptor but lacked H-ras and Tp53 mutations. Using microarrays, we compared the gene expression profiles and genomic copy number changes between carbon ion- and γ-ray-induced cancers; however, we did not observe a marked difference. On the other hand, irradiation of carbon ion mono beam at 1 Gy did not result in significant cancer induction. These results suggest that the carcinogenic effect of carbon ions varies depending on the genetic background and is strong in the SOBP region. Our current data indicate no remarkable molecular and biological difference between carbon ion- and γ-ray-induced mammary cancers except for the metastatic ability.

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DNA double-strand break (DSB) is considered the most critical lesion among various types of radiation-induced DNA damages and most responsible for biological effects of ionizing radiation. Therefore, the elucidation of the molecular mechanism how DSBs are recognized and repaired and how bio-protective responses, such as checkpoints and apoptosis, are activated is one of the central problems in radiation biology. This workshop will introduce recent advances and discuss still unsolved or newly emerged problems, extensively focusing on DSBs.

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DNA-dependent protein kinase (DNA-PK) is considered the "sensor" of DNA double-strand breaks (DSBs) and thought to play an essential role in DSB repair and recombination through non-homologous end-joining (NHEJ) pathway. It is shown that the protein kinase activity of DNA-PK is required for its function and that DNA-PK is capable of phosphorylating a number of proteins in vitro. However, the substrate(s) in vivo and the significance of phosphorylation have remained to be clarified for many years. We have investigated the phosphorylation by DNA-PK of XRCC4, which is thought to join two DNA ends in cooperation with DNA ligase IV. We have so far identified four phosphorylation sites, in addition to two identified by others. Two of the above phosphorylation sites are really phosphorylated in living cells after irradiation in a manner dependent on DNA-PKcs. Further, the mutant lacking these phosphorylation sites exhibited elevated radiosensitivity with reduced DNA repair capability. These results indicated that XRCC4 is one of, at least, the true substrates of DNA-PK in vivo and the phosphorylation is really important in DSB repair. We will also discuss the role of phosphorylation in the assembly/disassembly of the DSB repair machinery based on the analysis of XRCC4-chromatin binding status through biochemical fractionation. Furthermore, we would summarize the current understanding and discuss future directions of this issue.

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Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. We studied the early phase of NHEJ reactions by live cell imaging of NHEJ factors. XLF, XRCC4 and DNA-PKcs were YFP-tagged and expressed in a set of NHEJ deficient and complemented cells. DSBs were introduced into a specific site in a single nucleus by laser microbeam irradiation, and the behavior of YFP-tagged protein was monitored in real time. Although XLF, XRCC4 and DNA-PKcs were reported to biochemically interact with each other, these factors behave independently in the recruitment to DSBs in living cells. We confirmed the essential role of Ku for the damage recognition of these NHEJ factors. Next, we monitored the dynamics of the recruited factors at DSBs by FRAP. We observed that the stability of XLF and XRCC4 at DSBs is defined by the presence of XRCC4 and DNA-PKcs, respectively, indicating that the reported biochemical interactions among XLF, XRCC4 and DNA-PKcs are required for the assembly of the recruited factors at DSBs. A widely prevailing model for the DSB recognition in NHEJ is a sequential assembly of the NHEJ factors on DSBs. Based on our live cell imaging analyses, however, we propose a new model that consists of recruitment and assembly phases. This new model provides good explanations for the published observations that are inconsistent with the traditional model and has broad implications for the mechanism of DSB sensing and functional protein assembly in NHEJ.

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Upon exposure to ionizing radiation, 53BP1 is rapidly redistributed to sites of DNA double-strand breaks and is hyperphosphorylated in an ATM-dependent manner. In experiments using hyper-recombinogenic chicken B cell line DT40, we revealed that there are at least three sub-pathways in repair of X-ray induced DNA damage: 1) the core non-homologous end-joining (core NHEJ) which is dependent on Ku70/Ku80/DNA-PK, 2) Artemis-dependent pathway which includes ATM, and 3) 53BP1-dependent pathway. A cell-line that is deficient for one of these pathways showed elevated X-ray sensitivity in G1 phase. In contrast to the core NHEJ and Artemis-dependent pathways, the 53BP1-dependent pathway was resistant to PI-3 kinase inhibitor wortmannin. To identify proteins involved in the 53BP1-dependent pathway, we established some DT40 cell-lines that are deficient for one of candidate genes expected to be involved in this pathway, and examined the X-ray sensitivity of these cell-lines in the G1 phase. Among these cell lines, Snm1a-defficient, and Snm1b-deficient cell-lines did not show increase in G1-phase X-ray sensitivity. We are now examining some other genes whose products form foci in response to IR. We would like to present proteins which is involved in the 53BP1-dependent repair pathway.

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DNA double-strand breaks (DSBs) are generated in genome DNA by exposure to ionizing radiation (IR) or replication stress. Once DSBs are generated, cells detect DNA damage immediately and repair damaged DNA. Lately, histone H2AX is noticed for role of DNA damage response. H2AX is rapidly phosphorylated following the generation of DSBs and is required for the recruitment of DNA repair-related proteins to DSB sites. Hence, we attempted to identify H2AX-interaction protein by proteomics analysis to clarify the new role of H2AX in DNA damage response. As a result, we identify several candidates and then noticed nucleolin. Nucleolin is nucleolar protein, but functions for the regulation of p53 expression. Therefore, we investigated the role of nucleolin in DNA damage response. Nucleolin localizes in nucleolus without DNA damage, but re-localized to nucleoplasm after DNA damage dependent on replication stress. Further, nucleolin interacted with DNA damage response factors, NBS1 and Replication protein A as well as γ-H2AX. Chromatin immunoprecipitation assay revealed that nucleolin is recruited to DSB sites generated by I-SceI restrication enzyme. Moreover, nucleolin-knockdown cells showed the suppression of ATM-dependent phosphorylation (SMC1, p53) and the decreases in homologous recombination activity by DR-GFP assay. And, DSB-dependent Rad51 focus formation and chromatin association were also suppressed in nucleolin-knockdown cells. Taken together, nucleolin might play important roles in DSB-induced damage response.

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DNA double-strand breaks are repaired through two different pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Yeast Xrs2, a homologue of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Xrs2/Nbs1 contains a conserved FHA domain in the N-terminal end, whose function is largely unknown. In this study, we showed that the FHA domain of Xrs2 plays a critical role in NHEJ, but not in HR. The FHA domain specifically interacts with Lif1, a component of the Ligase-IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1 is phosphorylated in vivo, Serine 383 of Lif1 plays an important role for interaction with Xrs2. Our results suggest that the phosphorylation of Lif1 at Serine 383 is recognized by the FHA domain of Xrs2. The Serine 383 of Lif1 is present in a CK2 (Casein kinase-II) phosphorylation motif. We showed that human CK2 phosphorylates yeast Lif1 protein in vitro and this phosphorylation was largely decreased by the substitution of Serine 383 of Lif1. Interestingly, the interaction between Xrs2 and Lif1 through the FHA domain is conserved for human Nbs1 and Xrcc4, a Lif1 homologue of human. We propose that Nbs1, possibly MRN complex (Mre11-Rad50-Nbs1), is also required for NHEJ in mammalian cells as in yeast. Our results could explain the immuno-deficiency of Nijmigen breakage syndrome (NBS) patients and NBS-variant, which is caused by the dysfunction of Ligase-IV.

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Cerebellar degeneration is the characteristic feature of ataxia telangiectasia (AT). Since the mice deficient for ATM gene do not show the degenerative phenotype, the use of other model animal systems such as that of teleost has been validated. Recently, generation of 'knockout' medakafish was made possible by TILLING. In this method, the animal carrying mutations at specific locus can be identified by high-throughput sequencing of genomic DNA library that had been heavily mutagenized with chemicals. We have created the medakafish that presumably lacks the C-terminal kinase domain of ATM. These fish died during embryogenesis contrary to our expectation. Not only homozygous mutant fish but about half of the fish heterozygous for this mutation showed the developmental anomaly, indicating that the lethality was caused by the dominant negative effect of truncated ATM protein.

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Various types of phenomena for genomic stability have been reported to occur when cultured cells and mice are exposed to radiation. Why are there "interval" and "window" in them? Can the cells respond according to how much irradiation doses? On the other hand, an induction of radioresistance, and a depression of chromosome aberrations, mutations and carcinogenesis have been reported to appear when human cultured cells and mice were treated by challenging exposure following a prior conditioning radiation exposure at low doses or at a low-dose rate. This phenomenon is called the radioadaptive response. The radioadaptive response may express itself only if there is an adequate time interval between the priming irradiation and the challenging irradiation, and over a relatively small range of priming doses. What is the determination factor of the memory of the radiation exposure of the cells? We will discuss these points in this Workshop.

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In 1930, C. P. Oliver carried out a sex linked recessive lethal assay of X-irradiated fruit fly Drosophila, and obtained a linear dose response relationship without a threshold. This was the first proposal of the LNT model. However, he used only mature sperms. Mature sperms lack DNA repair function, and therefore, it is not evident that the LNT model is applicable to cells or individuals that are generally repair proficient. When we irradiated immature sperms before the reduction division with low dose, low dose rate X-rays, mutation frequency was found to be significantly lower than in the sham-irradiated group, and U-shaped dose response was observed. When mei-9[a] mutant flies (homologous to human Xp-f) was used instead of wild type, the dose response became linear, though immature sperms were irradiated. This suggests that the DNA repair function is involved in the formation of U-shaped dose response. Furthermore, we introduced wild type a copy of mei-9[+] gene into mei-9[a] mutant, using Dp(1;2)4FRDup that contains a chromosome fragment including mei-9[+] locus. Flies with white eye color originated from mei-9[a] sperm. These flies showed a linear dose response whereas their red-eyed siblings were carrying mei-9[+] and showed a U-shaped dose response. These findings show that DNA repair function is responsible to the formation of U-shaped dose response. The LNT can be applied to cells without DNA repair function, and cannot be used for estimation of human cancer risks.

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Reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide and hydroxyl radical are generated in cells exposed to ionizing radiation. ROS oxidize nucleic acids, proteins and lipids and act as mediators of ionizing radiation-induced cellular damage. Previous studies have suggested that superoxide dismutases (SODs) play a critical role in protection against ionizing radiation in yeast and mammalian cells. It is of interest to examine whether overexpression of SOD1 (Cu/Zn-SOD) and SOD2 (Mn-SOD) protects cells against cellular effect of gamma rays and to clarify mechanisms underlying the protection by SODs. In this study, human SOD1 and SOD2 genes were cloned into plasmid vector and transfected into human HeLa S3 to construct stable cell lines overexpressing SOD1 and SOD2. Overexpression of SOD2 in mitochondria enhanced the survival following gamma irradiation compared with control HeLa S3, while overexpression of SOD1 in the cytosol did not affect the cell survival. Flow cytometry with fluorescent probe 2',7'-dichlorofluorescein revealed that intracellular ROS levels increased with post-irradiation incubation for up to 24 hr and the prolonged oxidative stress was significantly lowered in SOD-overexpressing HeLa S3. The expression of several genes was up-regulated in HeLa S3 by overexpressing SOD2. The results indicated that excessive amount of Mn-SOD in mitochondria protects HeLa S3 against the lethal effect of ionizing radiation through scavenging ROS generated during post-irradiation incubation and inducing several genes by Mn-SOD, which might play a critical role in protecting cellular effects of radiation. In this work, we also examined whether overexpression of cellular redox-maintaining enzymes such as thioredoxin and glutaredoxin protects HeLa S3 against ionizing radiation.

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Low dose radiation is known to induce anti-oxidative substances, which suppress DNA damages, and activate DNA repair system. Since human being are exposed to multiple environment carcinogens, the effect of radiation may result from the combined exposures with these factors. Little is known, however, if low dose radiation affects carcinogenic responses induced by the other carcinogens. In this study, we examined if the pre-exposure of low dose radiation may affect the mutation induction and T-cell lymphoma development in thymus after exposure to chemical carcinogen, N-ethyl-N-nitrosourea (ENU). B6C3F1 or B6C3F1 (gpt-delta) mice were exposed to X-rays (0.2 or 1.0 Gy per week) for 4 consecutive weeks, and then treated with ENU (200 ppm) in drinking water. Lymphoma incidence and mutation frequency in thymus after the exposures were analyzed. The incidence of lymphomas by single treatment with ENU or X-rays was less than 20%. The incidence of ENU-induced lymphomas was reduced by pre-exposure of 0.2 Gy, in contrast that it was increased by pre-exposure of 1.0 Gy. The gpt-assay also revealed that pre-exposure of 0.2 Gy reduced the frequency of ENU-induced mutation, whereas pre-exposure of 1.0 Gy increased. Especially, the reduction of G to A base substitution and clonally expansion of mutant cells by pre-exposure of 0.2 Gy may contribute to the decrease of lymphoma incidence and mutant frequency. These results suggest that the low dose radiation work on chemical damage (s), showing crosstalk of radiation with chemical response. This study was supported through a grant of LRI by JCIA.

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We have been investigating the cellular adaptive response of hprt mutation in normal human fibroblasts induced by the pre-treatment of low dose (rate) radiations, such as 137Cs gamma rays or 241Am-Be neutrons. Cells were pre-treated with the priming dose (1mGy/8h) of either 137Cs gamma ray or 241Am-Be neutron, following irradiation with the challenging dose (1.5Gy) of 200 kV X rays. For mutation induction at hprt locus detected as 6-thioguanine resistant clones, X-ray-induced mutation frequency in cells pre-treated with 1mGy of 137Cs gamma rays was reduced to around 70% in control cells irradiated with X-ray challenging dose alone. In cells pre-treated with 1 mGy of neutrons derived from 241Am-Be source, it was reduced to around 15%, comparing to the control cells. However, reduced X-ray-induced mutation frequency in cells pre-treated with low-dose neutrons was returned to the control level, when using a specific inhibitor of gap-junction mediated cell-cell communication (40 µM lindane). Furthermore, cells pre-treated with low-fluence proton microbeams accelerated with the Single Particle Irradiation system to Cell (SPICE) were suppressed X-ray-induced mutation induction, assuming that recoiled protons might be responsible to the interaction between fast neutrons and tissue (cells). These results suggest that a possible mechanism of neutron- induced adaptive response is a bystander effect via gap-junction mediated cell-cell communication. As reported at the last year's meeting, X-ray induced mutation frequency was enhanced around 4.0 times higher in carbon-ion pre-treated cells and 1.9 times higher in helium-ion pre-treated cells than that in control cells. There is evidence that radiation-induced adaptive responses depend on radiation quality of giving priming doses.

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For estimating a human health risk from low-dose ionizing radiation (IR), an adaptive response, acquiring a cellular radioresistance to the challenging IR by a pre-exposure to low-dose IR, is a concern. Cellular responses after low-dose exposure, a bystander effect and a genetic instability, are also suggested to be related to the adaptive response. It is important to explore the adaptive response in human cultured cells from not only health risk estimation but also DNA repair studies. We succeeded to detect the adaptive response in human lymphoblastoid cells, providing a reduction in the frequency of mutations induced after IR exposure. For better understanding of those genetic results, we need to elucidate the underlying mechanisms. Since the reduced mutation-frequency can be considered as a result of enhanced DNA- repair efficiency, we first examined the possible enhancement in repair efficiency of DNA double strand breaks (DSBs). The DSBs caused by challenging IR are required to be distinguished from those caused by priming low-dose IR. Therefore, instead of the challenging IR, the cells are infected with a restriction-enzyme I-SceI expression vector for introducing a DSB at a specific site of chromosome. In other words, we could obtain information about DSB repair efficiency under the same condition as genetic measurements. An availability of the I-SceI approach will be discussed here.

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We already reported that conditioning exposures at low doses, or at low dose-rates, lowered radiation-induced p53-dependent apoptosis in human cultured cells in vitro and in the spleens of mice in vivo. In this study, we aim to characterize the p53-dependent radioadaptive response at the molecular level. In wtp53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wtp53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were increased about 2-4 fold after challenging irradiation following a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wtp53 cells, an induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of Pifithrin-α (a p53 inhibitor), RITA or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an iNOS inhibitor) and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover, radioresistance developed when wtp53 cells were treated with ISDN (an NO generating agent) alone. These findings suggest that NO radicals are an initiator of the radioadaptive response acting through the activation of Hdm2 and the depression of p53 accumulation.

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Analysis of the dynamics of the factors involved in DNA repair and DNA damage response is indispensable for comprehensive understandings of the radiation effects on living organisms. However, previous studies have been performed mostly in fixed cells or using cellular extracts obtained from cultured cells. Present workshop will focus on the live or real-time analysis of the process of DNA repair and DNA damage response. Three talks will present updated results that visualize unforeseen processes to be discussed. Future directions of the studies using Living Cell Imaging technology will also be discussed.

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DNA double strand break (DSBs) are major threats to the genomic integrity of cells. Eukaryotic cells have multiple pathways to repair DSBs, such as recombinational repair and end-joining. Several proteins involved in DNA repair, especially recombinational repair, have been shown to form higher order nuclear structures, radiation induced repair foci (RIRF), after induction of DSBs. However, little is known about how RIRF are formed after induction of DSBs. Recent advance in bio-imaging techniques make it possible to study the dynamics of nuclear structures formed by specific proteins in living cells. RAD51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DSBs in yeast and is conserved from yeast to human. RAD51 showed punctuate nuclear localization in human cells, called RAD51 foci, typically during the S phase. To examine the dynamics of RAD51 after induction of DNA damage, we applied local irradiation of cell nuclei with a focused UV-laser (laser-UV-microirradiation). By use of laser-UVA microirradiation the localization of RAD51 at damaged sites containing DSBs could be demonstrated. The accumulation of RAD51 at microirradiated sites was visible 5 - 10 minutes after irradiation, and the number of cells with RAD51 accumulations increases until a plateau is reached 20 - 30 minutes after irradiation. We also studied the dynamics of RAD51 at microirradiated sites by combing lase-UV-maicroirradiation with living cell observation system. In this workshop, we would like to introduce our recent findings obtained by this new system and discuss about the dynamic organization of DNA repair system.

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Analysis of the dynamics of the factors involved in DNA damage response is indispensable for comprehensive understandings of the radiation effects, especially the initial process of recognizing DNA double strand breaks on living organisms. However, previous studies have been performed mostly in fixed cells, which could not allow spatiotemporal analysis. We have established a live cell imaging system, by which the dynamic process of the recruitment of DNA damage checkpoint factors can be visualized. The experimental system uses the EGFP-tagged 53BP1 and MDC1 genes, and fluorescence microscope equipped with time-lapse digital imaging module. Upon irradiation, 53BP1 and MDC1 proteins were recruited to the sites of DNA double strand breaks, and they formed discrete foci. We confirmed the growth of the 53BP1 and MDC1 foci that were observed in fixed cells. In addition, we found that some foci emerged at the sites where previously showed no foci. The delayed foci induction has never been described in the previous literature, suggesting that illegitimate repair causes secondary changes in the higher-order chromatin structure.

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While influence of ionizing radiation on cell has been analyzed by its sensitivity and mutation frequency, molecular mechanisms of the repair process of damaged DNA has been characterized mainly by in vitro analysis using synthesized DNA and purified proteins or cellular extracts. However, even in the most extensively characterized nucleotide excision repair in vitro repair activity attains only a few percentages of that obtained within cell, suggesting that there are effective but still uncovered mechanisms for the repair of DNA damage within cell. Therefore, to understand cellular mechanisms of DNA damage response, its molecular analysis in living cell is absolutely required. To come up with this demand, we have developed various local irradiation systems and analyzed real-time damage response of various proteins and protein complexes in living cell. Real-time visualization of cellular response to DNA damage is especially useful to identify novel proteins and mechanisms. Our recent findings of DNA strand breaks response in human cells will be presented. [References] Hong, Z. et al. Recruitment of mismatch repair proteins to the site of DNA damage in human cells. J. Cell Sci. in press., Hong, Z. et al. A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell. Nucleic Acids Res. 36, 2939-2947, 2008. Prasad, R. et al. HMGB1 is a co-factor in mammalian base excision repair. Mol. Cell, 27, 829-841, 2007, Kanno, S. et al. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses, EMBO J. 26, 2094-2103, 2007, Hashiguchi, K. et al. Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living cells. Nucleic Acids Res. 35, 2913-2923, 2007.

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The goal of radiation research is promote the study of pysical, chemical and biological effects of natural and artificial radiation and to make up the useful information for radiation protection. Most of the artificial radiation exposure is due to the medical use of radiation in diagnosis, which contributed greatly to the welfare of Japanese peoples. Although the development of radio-diagnostic devices has reduced the radiation dose per exposure, the total nation's dose of radiation exposure has increased because of the wide use of these devices. Our society encourages the growing knowledge of the bioeffects of ionizing radiation. As a first step, a textbook for medical education was published in last March by the great contribution of many members of our society. Here, we will try to provide better recognition of the present status of radiation medicine and will discuss the future education required concerning the points of radiation protection and medical utilization of radiation.

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At my university, lectures of basic and clinical radiology are given to medical students at the 4th year of a 6-year MD course. However, only 16 lessons of 90 minutes are allocated to cover all radiological sciences including basic radiological sciences, radiation biology, diagnostic radiology, nuclear medicine and radiation therapy. Therefore, basic radiological science is lectured in only one 90-min lesson. During the lesson, fundamental knowledge about natural radiation, medical radiation exposure and its protection is lectured. The concept of radiation hormesis is also introduced herewith. In the author's lecture, effects of low-dose and high-dose radiation are presented separately. The author thinks that low-dose radiation delivered at radiological examination in the clinic is not hazardous. However, this concept should always be presented to students objectively. Also, the author does not deny the importance of radiation protection. With increasing evidence concerning the non-detrimental effects of low-dose radiation against living organisms, concerns about medical radiation exposure in the clinic appear to be changing. Students appear to be willing to accept the new concept. It seems that this concept should also be taught to diagnostic radiologists. Basic scientists should make further investigation to reveal the truth regarding the effect of low-dose radiation, and if it is clarified that low-dose radiation is not hazardous in the future, it is expected that clinical radiological sciences will make a further progress.

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Most of the artificial radiation exposure is due to the medical use of radiation in diagnosis, which has contributed greatly to the welfare of Japanese people. Recently, clinical radiology has been further applied in a wider medical area, especially for cancer therapy. On the other hand, the number of radiation biology departments has been decreasing. Therefore, the knowledge of the fundamentals of radiology including radiation biology, chemistry and physics is essential for medical school education. Our society has investigated the current status of education on basic radiology and, as a first step, published a textbook for medical education in the last March with the great contributions of many members of the society. Here, a discussion on the future development of basic education on radiation medicine will be held.

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The brightest scientific result of human history may be discovery of atomic energy. However, atomic energy can lead earth life to ruin instantly as have been shown by two A-bomb. At the same time, if it is controlled well, it is a high technology of possibility most to solve the problems, such as the lack of energy, the lack of food and population growth, that the human is facing in the 21st century. We should not do a mistake to choose ruin as by oneself. Therefore, as for the personnel education in atomic science field, it is extremely necessary to pour force into educating a talented person comprising higher morality as well as scientific originality and scientific specialty. In addition, we need to understand that atomic energy is a source of all things in space and radiation biology is a key solving the origin of the life. We have to show to student interesting project of radiological science. In this workshop, I describe my opinion about radiologic education how to bring up a researcher.

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There are several problems that remain unsolved to reach a goal of education of radiological sciences, regardless of basic or technical subject. It will be discussed about the situation and problems of education for radiation physics in our university. Undergraduate school of medicine, dentistry and health sciences at the University of Tokushima provides the education program of radiological sciences. Students in the courses of medicine, dentistry and nursing learn basic radiation physics, which is included in the lecture of introduction to medical radiation, oral and maxillofacial radiology and radiation health. Students learn the structure of atom, the definition, kind and generation of radiation, the interaction between photon and matter, the nature of X-rays and particles, etc. A goal of program of basic radiation physics is that the student can explain a nature of radiation and the technical terms related to radiation biology, radiation protection, radiotherapy and others. Students have some preconceptions. Students believe that radiation is mysterious and produces serious damages in their bodies independently the amount of absorbed dose. Students are lacking in accurate knowledge for the properties of radiation, therefore teachers need to explain in full the subjects. Students have some problems for learning radiation physics: students convince themselves that physics is difficult; demonstration of the phenomena related to radiation effects in class is difficult; many of students have not learned the general physics at a high school. In order to make knowledge enhancement possible in radiation physics field, we need to make teaching materials that help understanding the interaction between radiation and matter. Equipping our graduates with the best of radiation science is still a challenge for the future of medical and radiological education.

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In our school, education of radiological protection for nursing-related students consists of 30 hours lectures and 4 hours training seminars. The lectures cover several aspects that are what is radiation, the physical and biological interaction of radiation with matters, the health effects to humans, risk-based approach and radiological protection. In the training seminars, students learn dose quantity and natural radiation by conducting some measurements of scattered radiation from a mobile X-ray equipment and natural radiation including radon. The key points of radiological education are to understand quantitative concepts such as doses and risk. In particular, risk and benefit are essential to understand how high health effects are from diagnostic radiation and why it must be protected. It should be stressed that risk and benefit are a common way of thinking. To improve biased understanding of radiation and radioactivity that students have before education, the training seminars conducted through measuring natural radiation around human life may be effective. The difficulties we face with are not only to widely cover physical entity of radiation, molecular-cellular level of human biology and epidemiology, but also to touch social issues related with health effects of radiation. The characteristics of radiation education require collaboration and coordination among those who are engaged in education. It is expected that the Japan Radiation Research Society can play a central role as a coordinator.

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In the emergent radiological medical care, establishment of networks, training, and preparation of equipments in various areas are being implemented with an emphasis on a standpoint of life. Needless to say, it is important to familiarize medical drugs assignment and treatment method of the emergent radiation medical care. However, there are a number of controversial objects; the frequency of using these drugs is low and some of them are not approved as domestic pharmaceutical products. Furthermore, only a few chances to touch on the specialized knowledge of this field are provided. In this workshop basic knowledge of the medical drugs used in emergent radiological incident will be learned, and a stream of drug stockpiles for radiation emergencies in the United States will be introduced. We would like to make a report of the recently discussed guideline for the DTPA, which is effective in removing plutonium, americium, and curium. Emergency prescription trainings of the stable iodine agent in Japan will be described through photos and films.

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Radiation accidents rarely occur. Treatment of victims in these accidents is believed to be highly specific for radiation injuries. Indeed, the treatment includes highly advanced medical knowledge and technology but it is not specific for radiation injuries. National Council on Radiation Protection & Measurements (NCRP) published a report entitled "Management of Persons Accidentally Contaminated with Radionuclides" over than 20 years ago. This report is a collection of many of the data and ideas pertinent to accidental contamination into one document intended to aid those called upon to manage contaminated persons. Since then, there are few reports on clinical use of new drugs for treatment of internal contamination. Moreover, there are some medicines effective for the treatment of victims internally contaminated with radionuclides but not approved by the Japanese government, whereas these are licensed in foreign countries. Pentetate calcium trisodium (CaDTPA) and pentetate zinc trisodium (ZnDTPA) are known to be effective for treatment of internal contamination with plutonium, americium, or curium. Contamination with these nuclides can occur through a variety of routes including ingestion, inhalation, and/or wounds. Prussian blue (PB) is effective for elimination of incorporated cesium from body. However, DTPA and PB have not been approved yet. In Japan, thus, treatment of internal contamination has not been discussed. This workshop provides a good opportunity of discussing the treatment.

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This session is to be presented by Dr. Dart, the director of the Rocky Mountain Poison Control Center.

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1. Introduction Since 1960s, diethylene-triamine-penta-acetate (DTPA) has been treated many workers who had an internal contamination of plutonium in the overseas nuclear facilities. In Japan, DTPA has never been treated because an accident needing the DTPA treatment has not occurred. We have prepared the guideline on the DTPA treatment method with physician's review in 2007, referring to an overseas DTPA administration experience and research findings. 2. Characteristics of DTPA There are two types of DTPA treatment; One is Calcium-DTPA (Ca-DTPA) and the other is Zinc-DTPA (Zn-DTPA). It is known that the excretion effect of Ca-DTPA on plutonium is higher than that of Zn-DTPA. DTPA forms stable chelates with plutonium ions into the blood, and most of DTPA is excreted in urine. Although DTPA has the excretion effect on americium or curium in addition to plutonium, its excretion effect on uranium and neptunium cannot be expected. DTPA has been administered to many workers with internal contamination overseas and no serious adverse reactions have been reported. Contraindications of DTPA are not known, but the Ca-DTPA treatment is not recommended for a woman during pregnancy. 3. Administration Methods DTPA is administrated 1g once a day either with slow intravenous injection or by intravenous infusion diluted in saline in principle. DTPA diluted in water or saline can be administered by nebulized inhalation.

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Since 2004, emergency prescription trainings of KI were held every year in Aomori prefecture. It is very improtant for babies and children to be prepared of KI in liqid form, which must be taste good and not be rejected by them. Aomori prefectural drills every year were successful and will be described through photos and films.

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Energy transfer of ionizing radiations to biological systems are fundamental fact to understand the basis of biological effects. Many researchers have studied a lot of radiobiological effects in the view of those points. However, processes in the physical- chemical- and biological-stages at biological molecule and surrounding water by ionizing radiation have not yet well clarified. In this workshop, we are going to discuss how the radiation events proceed in the initial response of biochemical stage and biological stage. Speakers will talk concerning "low- and high-LET radiation, track structure, physical models, mechanism to produce damages" that related to the quality of ionizing radiations and "radical production, DNA cluster damage, damage spectrum, direct- and indirect-action, oxygen effects, simulation studies" as biological endpoints.

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We examined the contribution of indirect action of X-irradiation under oxic condition in cell killing of an NHEJ-defective mutant cell line (a Ku80 mutant of CHO), xrs6. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethyl sulfoxide (DMSO) which suppresses OH radical mediated indirect action without affecting the direct one. Exponentially growing xrs6 cells were exposed to X-rays in the presence or absence of DMSO and their cell survival was determined. The contributions of indirect action for xrs6 cells were 50%. On the other hand, CHO cells were 80%. The percentages of surviving cells in total cells after 2Gy irradiation were 38% and 1% for CHO and xrs6 cells. Furthermore, the percentages of cell killing resulting from indirect action were 47% and 49%, and that from direct action were 14% and 50%, respectively. Cell killing by direct action in xrs6 cells increased 3 (<50/14) time or more than in CHO cells, but showed no increase in the cell killing by indirect action. This phenomenon may suggest that a reparable DSB by NHEJ resulted in lethal damage. The results from our experiments indicates that the cause of high sensitivity of xrs6 cells due to X-rays may be attributed to direct action-induced DSB that was not repaired with NHEJ.

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Indirect action due to radical damage has been recognized to decrease with increasing LET judging from the decreased yield of OH radicals. However our recent experiments showed significant generation of OH radical mediated damage even in the high LET region from the DMSO protection against cell killing and 8-hydroxy-2'-deoxyguanosine (8-OHdG or 8-oxoG) production in exposed cells. Based on these results with the previous reports using OH radical scavengers, we would like to evaluate the contribution of indirect action in the high LET biological effects. In addition, we also discuss about the mechanism of the characteristic reduction of oxygen effect in the high LET region from the viewpoint of unique radical reactions due to densely produced ionizations in water by high LET radiation.

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In a treatment planning for carbon-ion radiotherapy, it is necessary to estimate the biological effect of the carbon-ion beam. The physical dose and the relative biological effectiveness (RBE) are calculated to predict the biological dose equivalent to photon beams in the patient body for optimal treatment planning. So far, we found that the Microdosimetric Kinetic Model (MKM) is very useful in explaining the survival curve of in-vitro cells for high-energetic ion beams. The amorphous track structure model is used in the MKM calculation instead of the microdosimetric stochastic energy deposition. We would like to discuss about using the biophysical model for the biological dose calculation carbon-ion threatment planning.

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The different particles indicate different biological effects even if their LET value is same. This difference in the biological effects by different particles with same LET is considered to be caused by a difference in their pattern of energy deposition in the target materials. However, there are very small evidences that the difference in track structure result to the different biological effects. Thus we investigated the biological effects of the track structure by different particles with the same LET value using visualization of DNA damage induced by hit-ions. CHO-K1 cells attached on ion track detector CR-39 were irradiated with the same LET value (about 437 keV/μm) but different nuclide (12C and 20Ne). DNA strand breaks were immunohistochemically detected with tdt for total breaks, and with γH2AX antibody for double strand breaks only. The fluorescent signal observed in nuclei by immunochemistry was co-localized at the site of etched pit where the ion was traversed on the cell. To evaluate the spatial distribution of DNA damage, the diameter of the area of fluorescent signal was measured. The area of γH2AX induced by C ion and Ne ion were not significantly different, however, the area of DNA strand breaks induced by Ne ion was larger than that induced by C ion. This result indicates that the difference of ion track structure influences the pattern of induction of DNA damage.

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Spatial distribution of Energy deposition by ionizing radiation is thought to strongly affect on the initial DNA damage spectrum and biological consequences. In particular, the serious biological effect observed after irradiation of heavy ions has been suggested to be due to formation of clustered-DNA-damage which may be difficult to be repaired. However, it is difficult to detect such kind of damages experimentally. The purpose of our study is to estimate the yields and the configuration of radiation-induced DNA damages and further to relate the estimated clustered DNA damage with biological consequences. We have developed the Monte Carlo simulation code system to estimate radiation-induced DNA damage spectrum which starts from detailed track structure by considering the direct and indirect actions in cellular environment for X-rays and heavy ions. In our presentation, the calculated DNA damage spectrum for heavy ions as C ions will be shown. Analysis on the contribution of energy deposition pattern, higher-order structure of DNA, direct and indirect action to the DNA damage spectrum will be also shown. The damage spectrum as a function of LET will be discussed with comparison with experimental data on LET-RBE relationship of cell death.

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Clustered DNA damage is densely accumulation of multiple damage localized to the limited region of DNA strand passed through by the beam of ionizing radiation. Since clustered DNA damage is thought to inhibit DNA replication and resist repair activity strongly, we consider that clustered DNA damage is one of major factor deciding the singularity of radiation effect. To confirm this idea, we analyzed the yields of clustered DNA damage in the isolated DNA molecule and the chromosomal DNA in Chinese Hamster Overy cell irradiated with gamma-rays (0.2 keV/μm) and various accelerated ion particle beams including carbon (13 keV/μm), silicon (55 keV/μm), and iron ions (200 keV/μm). Although simultaneous cell surviving tests showed LET-dependent argumentation of the severity, the yields of clustered DNA damage decreased in the irradiated isolated DNA and chromosomal DNA in the irradiated cells with an increase in LET (J. Radiat. Res., 49: 133-136, 2008). The results with these biochemical and intracellular studies suggest that the quantitative factor of clustered DNA damage is not important to the effect of higher LET-radiation. On the other hand, such conventional procedures to analyze clustered DNA damage cannot demonstrate the qualitative aspect of clustered DNA damage. We here refer to our ongoing experimental design to analyze the quality of clustered DNA damage and discuss the implication of clustered DNA damage with radiation effect.

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Fanconi anemia (FA) is clinically characterized by increased occurrence of leukemias and solid tumors, progressive bone marrow failure, and skeletal abnormalities. Altogether 13 genes have been implicated in FA, and their products constitute a common pathway in DNA damage signaling termed FA pathway. The newest member in the FA pathway, FancI, has been identified through a proteomic screen in an effort to identify novel ATM/ATR kinase substrates, or by positional cloning. FancI physically associates with the key factor FancD2, resulting in the D2-I complex formation. Upon DNA damage or S phase stress, FancD2 and FancI are monoubiqutinated in a manner dependent on each other by multi-subunit E3 ubiquitin ligase (called the FA core complex), which comprises of eight FA gene products. We have recently demonstrated that multiple phosphorylation of FancI is critical for FancD2 monobubiquitination following DNA damage, and serves as molecular switch in the FA pathway. Upon monoubiquitintion, FancD2 and FancI are both targeted to chromatin and form colocalizing foci.
To directly test genetic requirements for FancI phosphorylation, we utilized the SDS-PAGE gel containing Phos-tag reagent that selectively retards migation of phosphorylated proteins by binding to phospho-Ser, Thr, or Tyr. We found that the FA core complex and FancD2 are crucial for phosphorylation of FancI. However, the phosphorylation occurs in a manner independent of the ATR activators Rad17-Rad9. Furthermore, in a cell line that expresses the ATR-activation domain of TopBP1 fused with the estrogen receptor (ER-AD, kindly provided by Dr Fernandez-Capetillo), we could not activate FancD2 monoubiquitination by tamoxifen stimulation. Thus these results suggest a unique activation mechanism linking an upstream checkpoint kinase and the FancI phosphorylation that probably involves the FA core complex.

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Seckel syndrome is an autosomal recessive disorder characterized by severe microcephaly, growth retardation and bird-like face. Recently, it was reported that mutations of ATR or PCNT gene caused Seckel syndrome. However, it is considered that other genes are also responsible for Seckel syndrome. Here, we report that novel mutations in MRE11 gene cause Seckel syndrome. We identified MRE11 mutations in 2 Japanese patients. Patient1 had biallelic splice site mutations, and Patient2 had splice site mutation and missense mutation in the MRE11 gene. The expression levels of Mre11, Nbs1 and Rad50 proteins were reduced in both the patients. Also, cells from the patients showed chromosomal instability and X-ray hypersensitivity. Next, we examined the intercellular distribution of Mre11 protein. Immunostaining indicated that Mre11 protein was co-localized with the centrosomal protein gamma-tubulin. Furthermore, the patient's cells showed abnormal centrosome amplification. It is under study about functional roles of Mre11 protein in centrosome.

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Oxidatively damaged bases in DNA cause many types of deleterious effects, such as cell death, cancer induction, and aging. To prevent them, organisms have base excision repair (BER) pathway which removes specifically oxidative bases from DNA. Abnormalities in BER pathway increase the frequency of spontaneous mutations and lead to aging and tumor formation. As the first step in BER, DNA glycosylases recognize and excise oxidative bases in DNA. Many DNA glycosylases have been identified in various species, such as bacteria, yeast, plants and mammals. In the nematode, however, there are little data on DNA glycosylases, while many researches using C.elegans in aging field have extensively been done.
In this study, we identified a C.elegans homologue of E. coli endonuclease III (NTH), named CeNTH. NTH is a well conserved DNA glycosylase that was first identified in E. coli. We showed that the CeNTH enzyme can excise efficiently thymine glycol, 5-formyluracil and 5-hydroxymethyluracil in vitro and the N-terminal region is necessary for this activity . We further compared the characteristics of the Nth-deficient mutant strain (RB877) with those of the wild-type strain (N2).
We are currently looking at more about BER in C.elegans for elucidating the mechanisms and roles of DNA repair systems.

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The ataxia-telangiectasia mutated (ATM) and AT-rad3+ related (ATR) protein family is highly conserved in all eukaryotes. It is well characterized as cell-cycle checkpoint kinases and their disruptions cause instability of nuclear genome. In S. cerevisiae, ATR protein Mec1 regulates an inhibitor of ribonucleotide reductase (RNR) that is involved in dNTP synthesis. Since decreases in dNTP levels preferentially affect mitochondrial DNA (mtDNA) replication in comparison to chromosomal DNA (chrDNA) replication, Mec1 indirectly controls mtDNA copy number. In metazoans, however, the effect of ATR on dNTP pools or mtDNA copy number has not been examined. Therefore, we have examined the involvement of C. elegans ATR checkpoint protein ATL-1 in maintenance of mtDNA.
In C. elegans, both mtDNA and chrDNA copy numbers increased in association with germline proliferation. atl-1 mutants exhibited a roughly equal rate of chrDNA accumulation but a reduced rate of mtDNA accumulation. On the other hand, no reductions were detected in mutants defective for atm-1 and cep-1, which are checkpoint related genes. RNR expression and the ATP/dATP ratio remained unaltered in atl-1 mutants, and inhibition of RNR caused further reductions in mtDNA copy number. These results indicate ATL-1 likely affects mtDNA levels by a novel mechanism independently of RNR and dNTP pools.

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DNA damage induced apoptosis is a critical cellular function for the maintenance of genome stability. The NBS1 protein is a key regulator of DNA damage repair that acts by forming a complex with Rad50/Mre11 and by activating ATM. We found that NBS1 regulates a novel p53 independent apoptotic pathway in response to DNA damage. Experiments using a series of cell lines expressing mutant NBS1 proteins revealed that NBS1 is able to regulate the activation of Bax and Caspase-3 without the FHA, Mre11-binding, or the ATM-interacting domains, whereas the phosphorylation sites of NBS1 were essential for Bax activation. Interestingly, NBS1 regulates a novel Bax activation pathway by disrupting the Ku70-Bax complex which is required for activation of the mitochondrial apoptotic pathway. This dissociation of the Ku70-Bax complex can be mediated by acetylation of Ku70, and NBS1 can function in this process through a protein-protein interaction with Ku70. Thus, NBS1 functions in the prevention of carcinogenesis, not only through the precise repair of damaged DNA by homologous recombination, but also by its role in the elimination of inappropriately repaired cells.

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We investigated the effects of heavy ion beams on chromosomal aberrations in normal and AT cells. Normal, ATM defective and NBS1 defective cells arrested at G0/G1 phase were irradiated at 2 Gy of X-rays, 490 MeV/u silicon, 500 MeV/u and 200 Mev/u iron particles, and then allowed to repair for 24 hours at 37 degrees before subculture. Calyculin-A induced PCC method was employed and whole DNA probes 1 and 3 were used to analyze chromosomal aberrations such as color-junctions, deletions, simple exchanges (incomplete and complete exchanges) and complex exchanges. The percentage of aberrant cells in ATM defective cells was higher when normal, AT and NBS1 defective cells were exposed to heavy ions compared to X-rays. When the frequency of color-junctions (misrejoined breaks) per cell was compared after X-ray exposure, AT cells had around three times higher frequency of color-junctions than normal cells and a little higher frequency of them compared to NBS1 defective cells. However, there was no significant difference in the frequency of color-junctions when cells were exposed to 500 MeV/u iron particles between three cell lines. When the frequency of deletions was compared between AT cells and NBS1 defective cells, AT cells had more deletions than NBS1 cells, especially after X-irradiation. Our results suggest that AT cells and NBS1 defective cells show similar error prone repair under G0/G1 phase but AT cells are less effective in joining dsbs than NBS1 cells, which results in higher frequency of deletions compared to NBS1 cells.

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Nijmegen syndrome is the genetic disorder, which is characterized by high sensitivity to radiation, chromosome instability and aberrant cell cycle checkpoint. NBS1, the gene responsible for Nijmegen syndrome, forms a protein complex with hMRE11 nuclease and hRAD50, and functions in homologous recombination (HR) repair from DNA double strand breaks, which are elicited by ionizing radiation or other stresses. NBS1 also binds to ATM at the C-terminus and disruption of the interaction fails to recruit ATM to damage sites. Both ATM and NBS1 could have pivotal roles in regulations of cell cycle checkpoints. Therefore, the chromosome instability in NBS cells is considered to be due to defects in both DNA repair and cell cycle checkpoints. Thus, DNA repair and cell cycle checkpoint genes are responsible for genome instability. However, it is suggested that genome instability in tumor is related to centrosome aberration. Centrosome is the complex organelles comprising two microtuble-based centrioles surrounded by a protein matrix (pericentriolar material, PCM) and other structural elements, a key regulator for chromosome separation in mitosis. Proper centorosome duplication and spindle formation are crucial for prevention of chromosomal instability. Therefore, the normal function of centrosome is essential for maintenance of genome stability. Recent studies suggest that DNA repair factors are involved in centrosome function. When we examined the localization of NBS1 and ATM by using both protein antibodies, they showed to be accumulated in centrosomes. Moreover, NBS cells showed defect in centrosome amplification, suggesting an indispensable role of NBS1 in centrosome maintenance. We further discuss this novel role of NBS1 in centrosome maintenance.

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Radiation-induced DNA double strand breaks activate ATM-dependent DNA damage checkpoint pathway. It is generally believed that DNA damage-induced change in the higher-order chromatin structure dissociates dimeric or multimeric forms of ATM into a monomer. However, molecular mechanism of the dissociation remains to be elucidated. In the present study, we have hypothesized that a sort of physical process is involved in the dissociation of ATM oligomer, and we examined the possibility by using specific inhibitor for ATM kinase, KU55933. Normal human diploid cells were irradiated with gamma-rays, and activation of ATM molecules was determined by autophosphorylation of ATM at serine 1981 two hours after irradiation. While pretreatment of cells with KU55933 completely suppressed ATM activation, removal of KU55933 one hour after irradiation was found to regain ATM activation. Because delayed activation of ATM is not likely to occur, ATM molecules are highly possible to be activated at the time when DNA damage is introduced to the chromatin. Thus, it is indicated that autophophorylation of ATM is not required for ATM activation. Moreover, we confirmed that NBS1 was not essential for this process. Our findings illuminate the mechanism that disorganization of higher-order chromatin physically dissociates ATM oligomers and it stimulates the cascade of DNA damage signal transduction.

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The fact what cell survival is not dependent on cell cycle with high LET irradiation is strong advantage for the particle cancer therapy compared to conventional x-ray. In order to study the mechanisms of cell cycle independent biological effects in heavy ion exposure, we used DNA repair deficient mutant CHO cell lines, V3.3 (DNA-PKcs) and 51D1 (Rad51D) to analyze cell survival, cycle delay, sister chromatid exchanges. Thought WT CHO cells lacked cell cycle independent cell killing, non-homologous repair deficient cell and homologous recombination repair deficient cell showed clear cell cycle effect in their survival. Cell cycle delay analysis revealed Homologous recombination deficient cells had weak cell cycle arrest in G1/S and G2/M progress. High LET irradiation does not induce sister chromatid exchanges in DNA-PKcs deficient cells. Those results indicate that complex, dirty DNA damages produced by high LET exposure tend not to be an easy target for homologous recombination repair. And this is the reason why cell survival is not dependent on cell cycle with high LET irradiation

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