The quiescent state is thought to be a characteristic property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). We demonstrate that quiescent HSCs adhere to the hypoxic endosteal niche through the Tie2/ Angiopoietin-1, mpl/thrombopoietin and/or N-cadherin.
During BM transplantation or after treatment with myeloablative agents, the quiescent HSCs enter the cell cycle and proliferate to supply progenitors of hematopoietic cells. Reactive oxygen species (ROS)induce the exit of HSCs from the niche, resulting in the exhaustion of HSCs. To reduce the generation of ROS from mitochondria, HSCs gain the energy from glycolytic pathway rather than from oxidative phosphrylation under the regulation of HIF1alpha. I will present the change of HSC behavior and metabolism in mutant mice with HIF1alpha or VHL and discuss about the similarities between normal stem cells and cancer stem cells from the aspect of cell metabolism.

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Direct reprogramming of somatic cells into pluripotent stem cells has been reported by several combinations of transcription factors (Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28) and chemical compounds in mouse and human. They are similar to ES cells in morphology, proliferation and differentiation potential. The patient-specific iPS cells will contribute to elucidation of pathogenesis, drug discovery, and toxicology study. The iPS cells are also useful for study of radiation effect on human cells, especially on the cell of limited availability. While the use in basic research, studies of regeneration medicine by iPS cells are advancing all over the world. It has been reported that in vitro differentiation of mouse iPS cells into hematopoietic cells, neuron, and cardiomyocytes. Treatment of humanized sickle cell anemia mouse model with mouse iPS cells was already reported. However, there are several problems that remain to be solved before human iPS cells can undergo clinical trials. It will be necessary to analyze their character and differentiation potential. It is also important to guarantee the quality and the safety of differentiated cells for transplantation. In this meeting, I would like to discuss current progress and difficulties on iPS research based on the latest knowledge.

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Undifferentiated embryonic stem cells proliferate under a unique cell cycle program characterized by truncated G1 phase. Molecular basis for this unique cell cycle regulation and how it is related to their pluripotency are still elusive. In mouse embryonic stem cells, Cdc6, ASK(activation subunit for Cdc7), CyclinA1 and CyclinB2 proteins are highly overexpressed, and their protein levels decrease upon induction of differentiation. This is mainly caused by highly abundant Emi1 protein which inhibits ubiquitin ligase APC, since downregulation of Emi1 reduces the levels of ASK, CyclinA and CyclinB in ES cells. Emi1 expression is regulated by E2F, which is constitutively active in ES cells. We propose that a positive feedback loop involving active E2F and Emi1-mediated activation of Cdk activities may be a part of the system which permits unique ES cell cycle. Indeed, Emi1 depletion causes increased levels of CycinD2 and unphosphorylated forms of Rb, the hallmarks of G1 phase, Experiments with the Fucci (fluorescent cell cycle indicator) ES cells also indicate that depletion of Emi1 leads to appearance of cells with G1 features. Currently, effects of manipulation of the Emi1 protein level on the pluripoitency or differentiation processes are being examined. A possibility to manipulate pluripotency and differentiation potential by regulating cell cycle will be discussed.

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Whether cell senescence is involved in the aging of neural precursor cells (NPCs) remains unknown, although it is obvious that the cells accumulate genetic mutations and lose their self-renewal capability (stem cell exhaustion) with age. Using a serum-induced mouse oligodendrocyte precursor cell (OPC) senescence model and DNA microarray, we identified esophageal cancer-related gene 4 (Ecrg4), which was originally found as a gene upregulated in non-tumor cells surrounding the tumor, with implications for the senescence-like state in the aging brain. We show that Ecrg4 is upregulated in senescent OPCs and that its overexpression in OPCs induces senescence by the acceleration of the proteasome-dependent degradation of Cyclins D1 and D3, whereas knockdown of Ecrg4 by a specific short hairpin RNA (shRNA) prevented these phenotypes. We demonstrate that senescent OPCs secrete Ecrg4 and that recombinant Ecrg4 induces OPC senescence in culture. Moreover, Ecrg4 expression increases in both OPCs and NPCs in aged brain, accompanied by the expression of senescence-associated beta-galactosidase activity, indicating the cells' entrance into senescence. These results suggest Ecrg4 is a novel factor linking neural-cell senescence and aging as well as tumorigenesis.

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Japanese Abstract only

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Japanese Abstract only

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Japanese Abstract only

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The space environment contains two major biologically significant influences: space radiations and microgravity. Space radiation environment consists of space radiations such as high linear energy transfer (LET) particles with low dose-rate and low dose even inside of the International Space Station (ISS). Space radiations have been reported to induce DNA damage, mutations, chromosomal aberrations, abnormal differentiation and cataracts. We expect that the data from these proposals will be useful for designing physiological protection against the serious effects of space radiations during long-term stay in space. We want to introduce the aims and results about space experiments (LOH, Rad Gene, Rad Silk and Neuro Rad) from 2008-2010 in the newly established experimental area 'Kibo' facility of the ISS.

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The pressurize module of the Japanese spacelab "KIBO" was connected to ISS in June 2008. Then, the samples of "Rad Gene/LOH", which are the first life science experiments in KIBO, were launched to KIBO on November 2008 and the experiments were started in February, 2009. Following that, several space experiments were carried out. Among them, four space radiation experiments have been planned and completed in KIBO, that is Rad Gene, LOH, Rad Silk and Neuro Rad. These themes are carried out with the Cell Biology Experiment Fascility (CBEF) installed in KIBO, and the experiment samples have already been recovered on the ground. In this presentation, I shall report the summaries of the experiments, preparation activities on ground, and results of on-board activity.

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For human beings, environments in space are extremely different from those on earth. It is, therefore, important to elucidate biological influences of space environmental factors for promoting a utilization of space environments. Here, we report the results of experiments for estimating the space-radiation effects by using human lymphoblastoid TK6 cells, which were preserved as a frozen state in the Kibo module of International Space Station (134 days in space, 72 mSv). In our post-flight assay, a radiation-sensitive methodology, LOH (Loss of Heterozygosity) analysis, was applied for detecting and characterizing the mutants at both levels of gene and chromosome, which could be considered to be caused by low-dose space radiation. In addition, the recovered cells were exposed to challenging X-rays (2 Gy) and the obtained mutants were also analyzed by the LOH methodology for elucidating the possibility that the radiation damage accumulated during the space flight could express the radioadaptation when the cell were incubated after the recovery (suppression of X-ray induced mutation). Furthermore, we examined an enhanced efficiency of repair of chromosomal DNA double-strand breaks created by the I-SceI vector infection of the recovered cells. The observed results support the possibility that the cells exposed to space radiation exhibit both mutation-induction and radioadaptation.

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The space environment contains two major biologically significant influences; space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of these space experiments was to clarify the biological effects of space radiations and/or microgravity on the gene and protein expression of p53-dependent regulated genes and proteins. Space experiments were performed with two human cultured lymphoblastoid cell lines; one cells line (TSCE5) and another cells line (WTK1) bear a wild-type p53 gene and a mutated p53 gene, respectively. Under one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) in the International Space Station for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. The ground control samples were cultured for 8 days in the CBEF on ground. The expression of genes and proteins was analyzed by using DNA chip and protein chip, respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discuss from the viewpoint of the functions of the up- and down-regulated genes and proteins in these samples. It is expected that data from the space experiments will be helpful in designing physical and biological protection from the deleterious effects of space radiations during long term stays in space.

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A physiological dosimetry for space radiations was performed with Bio Passive dosimeter for life science experiment in space (PADLES) in ISS for 133 days. For biological dosimetry, we applied two human cultured lymphoblastoid cell lines bearing wild-type p53 gene (wtp53) and mutated p53 gene (mp53) at frozen state. We detected DNA damage induced by space radiations including the high linear energy transfer (LET) particles as a track of γH2AX positive foci in the nuclei. From the frequency of the track of γH2AX, we speculated that total dose equivalent of high LET radiations was calculated to be about 0.7 mSv per day as 200 keV/μm Fe-ions of a relatively high energy component among space radiation species. We also estimated radio-adaptive response in space-flown samples by analysis of cell death, apoptosis, chromosomal aberrations and mutations. We irradiated with X-rays at high doses to space and the ground control samples. We found all of the radio-adaptive responses investigated here only in space-flown wtp53 cells, but not mp53 cells and the ground control cells with wtp53 and mp53 genes. These results suggested that the space-flown cells were exposed to space radiations with the range of 20-100 mSv as a "window" for radio-adaptive response.

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The exposure of the silkworm eggs to space by a space shuttle for 9 days induced abnormality in fusion of integument, but exhibited no morphological mutation related to cosmic radiation (1997). On the basis of this result, we have established the simple biological method for detecting the effects of cosmic rays on the silkworm eggs in a ground experiment using HIMAC. The exposure of heterozygous eggs of the black-striped strain(P^S/p) to heavy ion particles such as Fe, C and Ne resulted in somatic mutations appearing as white spots on the black integument during larval stage. A few spots were detected in the irradiations of C or Ne ion beams, whereas many tiny spots in Fe irradiation. Incident rate of the mutation increased dependently on irradiation dose and LET. The detection sensitivity was at least above 40mGy of Fe ion particles. As flight experiment, we loaded the eggs(P^S/p3) on the International Space Station (ISS) for about 3 months to examine the effect of the long-term exposure of cosmic rays. Bio PADLES estimated the total cosmic radiation of 25-27mGy in the ISS for about 3 months, and there was no mutation in the first generation. However, the larvae of the next generation exhibited two types of mutation with a few or with many tiny white spots, suggesting that heavy ion beams will hit reproductive cells during embryonic development

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Space environmental condition composed of micro gravity and space radiation, of which dose is at least one hundred times more compared with those on the earth. Long-term manned space mission inevitably receive space radiation exposure and microgravity might give a synergistic effect on those space radiation effect. Human SK-N-SH cell is a slow growing neuron like cells, of which p53 status is normal. We send the cells to International Space Station, Japan Experimental Module (JEM), namely "Kibo" by STS131(19A) and cells were cultured at 37˚C with 1G or μG condition for 14 and 28 days at CBEF. Then cells were fixed and stored at -80˚C. The cells were returned by STS132 (ULF4). The frozen samples were transported to "Tsukuba Space center", and then brought them to Kagoshima University. Now, gene expression analysis was started using the sample. Mitochondrial impairment will be also examined using the same samples.

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Radiotherapy is an indispensible modality in cancer therapy. The mechanism of cell killing induced by ionizing radiation has been the focus of interest for decades. Based on a flurry of studies, it has been widely accepted that ionizing radiation can induce apoptosis as well as necrosis. Recently, other modes of cell killing have been also reported. The mechanisms and signal transduction pathways involved in these modes are important topics for future research for they would serve in optimizing the radiotherapeutic outcomes. Here, current data on apoptosis, autophagy, mitotic catastrophe, and senescence-like growth arrest induced by ionizing radiation will be discussed for better understanding of cell death modes and their implications.

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Ionizing radiation (IR) affects living systems at the molecular, cellular, tissue, and individual levels. A "cell" is the smallest unit that can be viewed in terms of "life and death", and the events associated with IR could significantly affect the higher dimensions of living systems. In classic radiobiology, quantitative issues regarding cell death have been discussed based on the target theory or linear quadratic (LQ) model, but qualitative factors, including cell death modes, have not been thoroughly examined. Meanwhile, the molecular mechanisms underlying apoptosis have been greatly advanced and details on the mechanisms that trigger and execute apoptosis have been clarified. Furthermore, it was determined that there are different types of apoptotic and non-apoptotic cell death. Therefore, it is important to elucidate the molecular mechanisms and biologically characterize each mode of cell death in order to better understand the effects of IR on living systems and tumor responses to radiotherapy. In this symposium, the complete picture of cell death modes will be delineated and discussed in terms of apoptosis, mitotic catastrophe, senescence-like growth arrest, and autophagy.

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Radiotherapy is an indispensible modality in cancer therapy. The molecular mechanism of cell death induced by ionizing radiation has been the focus of interest for decades. Based on a flurry of studies, it has been widely accepted that ionizing radiation can induce apoptosis as well as necrosis. Recently, other various modes of cell killing have been also reported. The mechanisms and signal transduction pathways involved in apoptosis are important topics for future research for they would serve in optimizing the radiotherapeutic outcomes. Here, we will show current our data on radiation-induced apoptosis and its enhancement by peroxides and the underlying mechanism and implication will be discussed.

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It is well established that ionizing radiation causes cell death, however, its mechanism has not been fully understood yet. We have reported that non-apoptotic cell death is the major cause of cell death induced by radiation, particularly in attached cells. In normal human diploid cells, residual DNA double-strand breaks persistently activate ATM-dependent G1 checkpoint, thereby persistent G1 arrest leads to senescence-like growth arrest (SLGA). SLGA is also detectable in human cancer cells. Since G1 checkpoint is abrogated in most cancer cases, cells proceed to G2 phase with DNA damage. In addition, G2 arrest is prematurely abandoned in cancer cells, so that cells go into mitosis with DNA damage. As a result, cells initiate division with fragmented chromosomes and chromosome bridges, which result in abnormal mitosis, called mitotic catastrophe. These cells could not accomplish cell division and form multiple micronucleated cells in the next G1 phase. Other cells, which do not show cell division, also stay in G1 phase. We found that these cells stayed in G1 phase induced SLGA. Thus, it become evident that SLGA is induced by ionizing radiation, irrespective of activation of cell cycle checkpoint. In most tissues, SLGA is the major mode of cell death induced by radiation, and therefore, to understand its mechanism could contribute to improve the efficacy of radiotherapy.

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Apoptosis, a form of physiological cell death, has attracted considerable attention over the 20 years and a significant part of the molecular mechanisms involved has already been unveiled. However, there is considerable evidence to suggest that physiological cell death is not confined to apoptosis and that other mechanisms may also operate. Recently, we found such non-apoptotic cell death mechanisms. One of these mechanisms is the so-called 'autophagic cell death', which is a process that is dependent on autophagy, and another is the 'permeability transition-mediated necrosis', which is a process that is dependent on mitochondrial permeability increase. In this meeting, by describing some of the recent data concerning the novel macroautophagy process we discovered, I will discuss radiation-induced autophagy and cell death.

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Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents and ionizing radiation. We characterized the dynamics and regulation of mitotic catastrophe induced by DNA-damaging agents and anti-microtubules agents by using time-lapse microscopy. Cells entering mitosis arrested at metaphase without chromosome segregation and subsequently collapsed. We found that the long-term metaphase arrest (more than 10 hours) is a critical factor for induction of the mitotic catastrophe. In this symposium, we would like to propose the molecular mechanism of mitotic catastrophe and to discuss why this mechanism is specific for cancer cells.

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The aim of this symposium is to introduce cutting-edge researches in the field of radiation-induced leukemia and myelodysplastic syndrome (MDS), a hematopoietic disorder closely related to leukemia. Acute and chronic leukemia among atomic bomb survivors increased soon after bombing and gradually returned to the base line within 30 years. MDS, by contrast, began to increase 30 years after the bombing and high frequencies of MDS continues to be high even today among A-bomb survivors. Molecular mechanisms making this difference have been elucidated by recent studies. On the other hand, genome researches revealed a population harboring high risk for radiation-induced leukemia and lymphoma. Future direction of researches in this field, particularly identification of high-risk individuals will be discussed.

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Leukemia is the first malignancy that appeared in A-bomb survivors due to the effect of radiation exposure. Among several types of leukemia, the excessive mortality risk by radiation exposure was observed in chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myelogeneous leukemia (AML). However, there was no morphological difference in leukemia cells between patients of A-bomb survivors and those of a general population. The excess mortality risk peaked in the 1950's and decreased afterwards, but the risk did not end at all. In a recent report of risk analysis of leukemia by Radiation Effects Research Foundation (RERF) based on death information during 1950-2000, it was observed that an excess risk of AML existed in young victims. The treatment for leukemia has been improved dramatically in recent years. If an analysis is done based on incidence data, more excess risk is expected. Myelodysplastic syndrome (MDS) is a disorder of a hematopoietic stem cell characterized in the morphological abnormality of the blood cells. The disease came to be considered to be a category of hematological malignancies in 2000. About 25-30% of MDS may progress to leukemia (mainly, AML). Therefore, MDS is usually recognized as pre-Leukemia. Although chromosomal abnormality is closely related to MDS and AML, there are a lot of differences in clinical characteristics between the both diseases. Therefore, it cannot simply extrapolate the leukemia risk to MDS risk in A-bomb survivors. Nagasaki University and RERF have jointly studied a detailed epidemiologic study of MDS from several years ago. In the recent analysis, we confirmed a distance and a dose related risk increase of MDS in A-bomb survivors. MDS is a disease of the elderly. Because A-bomb survivors become old, it is feared that the number of MDS will increase in the future. In the symposium, I will explain leukemia and MDS in A-bomb survivors, regarding the similarity and the difference of the clinical characteristics and of the radiation risk pattern, based on existing findings and the latest study results.

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Leukemias were the earliest delayed effect of radiation exposure seen in atomic bomb survivors in Hiroshima and Nagasaki. Acute and chronic leukemias appeared after a minimum latency period of 2-3 years, reached a maximum after 6-7 years and decreased slowly with time. In contrast, the incidence of myelodysplastic syndrome (MDS) continued to increase with time, and are still high, even now, more than 60 years after exposure. MDS is a clonal disorder of hematopoietic stem cells characterized by ineffective and inadequate hematopoiesis and a tendency to progress into acute myeloid leukemia (AML). Chromosomal translocasions that are detected half of AML patients are rare in MDS patients, and MDS are considered to develop as a result of the accumulation of gene mutations. We found that point mutations of AML1/RUNX1 gene, which encodes a critical transcriptional factor in hematopoiesis, are frequently detected in patients with MDS and MDS following AML (MDS/AML), and clarified that the AML1 mutation is one of the pivotal gene abnormalities to develop MDS. In particular, radiation-associated MDS/AML patients among the atomic bomb survivors of Hiroshima and residents around Semiparatinsk Nuclear Test Site showed high frequency of the mutations. Furthermore, AML1 mutations were also seen frequently in therapy-related MDS/AML patients who had a history of radio- and/or chemo-therapy. These results indicate that AML1 mutations might be a useful biomarker that differentiates radio- and/or chemo-induced MDS/AML from spontaneous MDS/AML. The mutations are distributed throughout the full length of AML1, resulting in the various structures of AML1 mutants. Although most of the mutants lose their trans-activation potential, they show divergent biochemical features and different oncogenic abilities reading to MDS/AML. These results suggest that the AML1 mutation is one of the master genetic abnormalities for the development of MDS/AML, and the detection of AML1 mutation will be useful for early diagnosis of radiation-associated and/or chemotherapy-induced MDS/AML.

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Monosomy 7 and interstitial deletions in the long arm of chromosome 7 (-7/7q-) is a chromosomal abnormality found in 10 to 30% of myeloid disorders including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). -7/7q- is more frequently found in radiation- or chemotherapy-related AML/MDS, whose prognosis is known to be poor. Using an array CGH technology, we identified three candidate myeloid tumor suppressors (Samd9=Kasumi, Samd9L=Titan, LOC253012=Miki) from a common microdeletion cluster in 7q21.3 subband. Among them, Kasumi and Titan locate adjacent each other and share a common gene structure with 60% amino acid identity, while mouse has Titan gene alone. Because Titan-deficient mice develop AML at a high frequency, deletion of Kasumi and/or Titan is predisposal to human AML/MDS. Titan protein possibly participates in the degradation of endocytosed cytokine receptors; In Titan-downregulated cells, cytokine receptors were accumulated in intracellular vesicles, where sustained activation of cytokine signaling was seen after cytokine stimulation. Another candidate -7/7q- responsible gene, Miki, locates to mitotic spindles and centrosomes. Miki protein would contribute to the maturation of centrosomes toward mitosis because it was found that downregulation of Miki by RNA interference caused smaller mitotic centrosomes and induced abnormalities in mitosis and nuclear morphology, similar to myelodysplasia. We are now focusing on the isolation of genetical or epi-genetical gene alterations that occurs collaboratively with -7/7q- and are studying to elucidate the molecular mechanism of -7/7q- AML/MDS. This would lead us to the development of -7/7q- targeted drugs.

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Exposure to anti-cancer drug or irradiation in cell produces DNA damage. The cellular response to DNA damage induces cell cycle arrest, DNA repair, or apoptosis, called DNA damage response. The central player in DNA damage response is ATM. DNA damage activates ATM and transduces the signal to downstream effectors. Not only outer side genotoxic stresses, cells constantly suffer DNA damage caused by such as free oxygen radicals or unscheduled replication induced by aberrant oncogene activation. Cellular DNA in cells under constant DNA damaging stress always carries the risk of mutation. We revealed DNA damage response pathway is activated in cells in MDS. Activation of DNA damage response pathway induces cell cycle arrest and/or apoptosis to cells in MDS stage, which is consistent with clinically observed ineffective hematopoiesis. This activation of DNA damage response works as a cancer barrier to prevent leukemic transformation. Then, disruption of DNA damage check point directly connects to evolution into overt leukemia. Somatic mutations of ATM have been reported in various hematological malignancies. Not only somatic mutation, we reported rare ATM gene SNPs increases the risk of Hodgkin disease and infantile leukemia development. Especially, our experiment suggested that loss of ATM function increases the susceptibility of MLL gene translocation by etoposide treatment. These observations propose that disruption of ATM dependent genome stability surveillance systems is indispensable for development of lymphoid malignancy. Recently it has been elucidated that DNA damage related molecules regulate of cellular homeostasis other than classical DNA damage. We revealed that ATM are involved in transcriptional regulation, and control cellular differentiation, especially adipocyte differentiation. In this symposium, we would like to present cutting edge of DNA damage related molecule and cellular homeostasis associated with cancer development.

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Ionizing radiation causes DNA double strand breaks, which disorganize higher-order chromatin architecture. ATM, a critical player in DNA damage checkpoint, is now recognized as a molecular sensor for detecting chromatin structural abnormality. Upon activation, ATM elicits DNA damage signal transduction leading to several modifications of histones, such as phosphorylation, ubiquitination and sumoylation, which further promote alterations in high-order chromatin structure. The aim of this international symposium is to uncover the effects of radiation on genome architecture at a molecular level and to understand a starring role of chromatin structure in triggering DNA damage response.

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DNA-Pk is the essential component of nonhomologous end joining (NHEJ) pathway for DNA-double strand break (DSB) repair. While NHEJ is predominately active in G1, it is not clear how interplay between NHEJ and homologous recombination (HR) in S or G2 phases of the cell cycle. We have tested the hypotheses that DNA-Pk plays an active role in modulating the interplay between NHEJ and HR in S phase of the cell cycle. I'll first discuss the role of Ku in modulating DNA end resection; subsequently, I'll discuss a potential role of phosphorylation status of DNA-PKcs at T2609 cluster in modulating HR. We have studied the inhibition of DNA end resection in vitro mediated by human Exo1 in the presence of Ku70/80, DNA-PKcs, Mre11 or MRN complex using purified proteins. Our results showed that Ku blocks DNA end resection (5'-3') mediated by Exo1 if Ku is bound to dsDNA. Such inhibition cannot be reversed by the addition of Mre11 or MRN complex. Further more, when DSB ends are persistently occupied by Ku, DNA end resection is blocked and subsequently HR is attenuated. In addition, when DNA-PKcs phosphorylation at T2609 cluster is blocked, it affects both NHEJ and HR. Hamster cells with T2609 S/T to A mutations (6A) are highly sensitive to MMC, CPT and UV in addition to IR. Further more, 6A mutant is sensitive to IR in S phase of the cell cycle compares with the null mutation and defective in I-sceI mediated HR. Taken together, our results support the hypothesis that Ku is recruited to DSBs, stabilizing the ends and interplay between HR and NHEJ in S phase of the cell cycle. In addition, phosphorylation status of DNA-PKcs at T2609 clusters further modulating the HR pathway of DSB repair.

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It is demonstrated that ataxia telangiectasia-mutated (ATM) protein is activated not only by DNA double-strand breaks (DSBs), but also by alteration of higher-order chromatin structure in the absence of DSBs. In the present study, we hypothesized that ATM activation might persist long after completion of DSB rejoining, if the DSB-disrupted structure of chromatin flanking rejoined DSB is not properly recovered, and that such case might be more frequent when DSB rejoining occurs illegitimately between different chromosomes. We found that ionizing radiation (IR)-induced foci of Ser139-phosphorylated histone H2AX, whose phosphorylation is mainly performed by ATM, continued to form interstitially on already-rejoined chromosomes, including dicentric/tricentric chromosomes (kinds of chromosome translocation), long (≥15-19 hr) after DSB rejoining. Treatment of ATM inhibitor dramatically elevated the frequency of chromosome translocation appearing in mitosis-progressing cells which had been irradiated in G0/G1. Treatment of p53 siRNA also increased chromosome translocation similarly, indicating that ATM suppresses propagation of chromosome translocation through p53-dependent checkpoint. Furthermore, delayed restoration of ATM activity in ATM-inactivated cells harboring chromosome translocation conferred growth disadvantage in such cells. These results indicate that ATM-dependent checkpoint signaling persists in response to misrejoined chromosomes long after completion of DSB rejoining, which minimizes propagation of chromosome translocation.

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Genome DNA maintains gene integrity with its higher structure. However, once DNA damage is generated into genome DNA, cells activate cell cycle checkpoint and repair damaged DNA via chromatin modification, including histone modification and chromatin remodeling. Phosphorylation of H2AX is known as one of major DNA damage-related histone modification. Previously, we reported that H2AX is important for the activation of ATM-dependent cell cycle checkpoint and that acetylation of H2AX/H2A contributes to homologous recombination (HR) repair. Therefore, we attempted to identify the components of H2AX-related complex by proteomics analysis.
One of identified components, nucleolin is confirmed to be recruited and accumulated at DNA damage sites by immunoprecipitation, chromatin immunoprecipitation and laser micro-irradiation analyses. nucleolin-knockdowned cells showed decreases in activation of ATM-dependent checkpoint, HR activity and repair efficiency of DNA double-strand breaks (DSBs) following irradiation. Knock-downed cells also revealed that reduction of RPA and Rad51 accumulation into chromation, which are important for HR repair. Recently, nucleolin was reported to facilitate a transcription through its eviction activity to H2A/H2B. Therefore, nucleolin could be important for chromatin modification in DSB-dependent DNA damage response.

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One of the most striking features of the cellular response to DNA double strand breaks (DSBs) is the accumulation of proteins into large and microscopically discernible structures in the vicinity of the lesions- the so-called ionizing radiation induced foci (IRIF). This accumulation process is orchestrated by post-translational histone modifications, which serve as affinity platforms for DNA damage response (DDR) proteins. The most proximal of these modifications seems to be the phosphorylation of the histone variant H2AX by the ATM kinase (gamma-H2AX), which leads to the recruitment of Mdc1 and its associated protein partners. One of these is the ubiquitin ligase RNF8, which mediates K63-linked poly-ubiquitylation of histones, which in turn is required to target downstream DDR factors such as 53BP1 and BRCA1 to sites of DNA damage. Since our initial discovery of RNF8-mediated histone ubiquitylation, we and others have uncovered the identity and functions of additional components of this pathway, highlighting the exquisite complexity of this reaction. While the relatively inactive RNF8 ligase is critically required to prime the ubiquitylation reaction, a much more active ligase, RNF168, plays a crucial role in amplifying the response. We recently identified Herc2 as a novel factor that is required for DDR-associated histone ubiquitylation. Herc2 is a giant 500 kDa protein that plays a key role in the DDR by orchestrating the assembly of an active RNF8 ubiquitin ligase complex, and directing this activity towards histones H2A and H2AX. In my talk, I will review how DDR-induced histone ubiquitylation is orchestrated, and present our latest findings on the functions of Herc2. I will also present how DNA damage induced SUMOylation of several of the above components regulates the response.

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Ionizing irradiation induces various types of DNA damages including DNA double strand breaks. Such DNA damages activate the DNA damage response system including DNA repair and signal transduction to keep genomic integrity. Therefore, failure of DNA damage response system to exclude cells with inaccurately repaired DNA, such as point mutations and chromosome abnormalities, leads to human diseases like cancer, neurodegenerative diseases and progeria by the altered genetic information. Recent studies have revealed that reorganization of damaged chromatin, such as posttranslational modification and/or exchange of histones, plays an important role in the regulation of DNA damage response.,
In this symposium, we will introduce recent progresses in the field of DNA damage response. We would like to discuss the mechanism of inappropriate DNA damage response leading to human diseases.

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Homologous recombination (HR) is essential for cells to maintain genome stability through the precise repair of DNA double-strand breaks and other lesions that are produced by a variety of exogenous and endogenous agents. Defects in HR due to mutations or epigenetic silencing of the HR genes are associated with cancer predisposition. We have recently found that meiosis-specific synaptonemal complex proteins are aberrantly expressed in various cancer cells, whereas their roles in mitotic cells are poorly understood. In this symposium, we will show that HR can be inactivated by aberrant expression of the meiosis-specific synaptonemal complex protein SYCP3 in mitotic cells, raising a novel mechanism for genome instability and cancer development. We generated epithelial cells expressing SYCP3, and found that these cells demonstrated phenotypes reflecting impaired HR, such as increased DNA double-strand breaks, hypersensitivity to DNA damage, reductions in radiation-induced RAD51 foci formation and sister chromatid exchanges, and aneuploidy. The molecular mechanism underlying the inhibition of HR by SYCP3 will be discussed based on our recent data on dynamics of SYCP3 and key molecules involved in HR.

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Double Strand Breaks (DSBs) are highly toxic lesions, which can be repair by homologous recombination or non-homologous end-joining (NHEJ). Recently has emerged the concept of alternative NHEJ (A-NHEJ) pathway(s).
We used intrachromosomal substrates to monitor NHEJ of DSBs targeted by the meganuclease I-SceI, in living mammalian cells. This substrates allow to characterize A-NHEJ in a chromosomal context; it is highly efficient but extremely inaccurate in the absence of KU. Using this substrate, we have shown that the accuracy of the canonical NHEJ (KU/XRCC4-dependent) pathway is directed by the structure of the extremities rather than the enzymatic machinery itself. The consequences of KU versus XRCC4 depletion, on the choice of the NHEJ pathway, will be discussed.
The above results suggest that the initiation of A-NHEJ is promoted by a nuclease or a helicase to expose single strand DNA allowing annealing of the strands and microhomologies. The involvement of MRE11 (and more generally of the MRN complex) and of CtIP, at the initiation of A-NHEJ will be also discussed here: we conclude that MRE11 is implicated in the two NHEJ pathways but the nuclease activity of MRE11 is only implicated in the A-NHEJ. Other partners are under investigations for the initiation of A-NHEJ.

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DNA double-strand breaks (DSBs) are a potent threat to genome integrity. DSBs elicit a signaling cascade that modifies the chromatin surrounding DSBs first by ATM-dependent phosphorylation and then by regulatory ubiquitylation. The early steps of DSB-induced ubiquitylation are catalyzed by the RNF8 and RNF168 E3 ubiquitin ligases, and the E2 ubiquitin conjugating enzyme, UBC13 (UBE2N). Here we report that OTUB1, a deubiquitylating enzyme, is a negative regulator of DSB-induced chromatin ubiquitylation. We made the surprising observation that OTUB1 suppresses RNF168-dependent poly-ubiquitylation independently of its catalytic activity. OTUB1 is able to do so by directly binding to UBC13, the cognate E2 enzyme for RNF168. OTUB1 inhibits the ability of UBC13 to catalyze the attachment of ubiquitin to another ubiquitin moiety but it does not interfere with the formation of the ubiquitin-UBC13 thioester intermediate or with the discharge of ubiquitin from UBC13. Chromatin ubiquitylation in response to DSBs is therefore kept in check by OTUB1 via a non-canonical mode of action by a deubiquitylating enzyme. Moreover, depletion of OTUB1 restores the accumulation of 53BP1 on chromatin and OTUB1 depletion mitigates the DSB repair defect associated with defective ATM signaling.

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Chromatin reorganization involving histone modifications, histone variant exchange, histone eviction and nucleosome remodeling play an important role in DNA repair, apoptosis and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetylase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response to DNA damage is largely unknown. To better understand the mechanism of TIP60 complex in DNA repair, TIP60 complex was purified from chromatin soluble fraction after DNA damage. We found that the TIP60 complex is associated with histone H2AX, a variant form of H2A, after DNA damage. Furthermore, DSBs facilitate the association of TIP60 with the ubiquitin-conjugating enzyme UBC13. The TIP60-UBC13 complex regulates the acetylation and ubiquitination of H2AX following the formation of DSBs. The DSB-induced acetylation of H2AX is required for this ubiquitination and occurs independently of the phosphorylation of H2AX. We also show that damage-induced acetylation and ubiquitination provoke the eviction of H2AX from chromatin immediately after induction of DSBs. Interestingly, the eviction of H2AX depends not on phosphorylation, but rather on acetylation and ubiquitination by TIP60. We conclude that sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promotes enhanced histone H2AX dynamics, which in turn stimulates a DNA damage response. We will discuss about the role of the eviction of H2AX upon DNA damage in chromatin remodeling and checkpoint activation.

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Chromosome translocations are one of the most common genetic alteration identified in cancer and leukemic cells. Fusion of two loci on different chromosomes by translocations causes the disruption of appropriate gene expression or formation of chimeric gene products, resulting in the malignant transformation. In lymphoma and lympoid leukemia, system for the physiological recombination of the immunoglobulin or T cell receptor genes by inducing DNA double strand breaks and rejoining the two DNA ends is suggested to play a role in chromosome translocations. On the other hand, DNA damaging agents, such as ionizing radiation and certain chemotherapies, induces secondary malignancy with recurrent chromosome translocations. However, the mechanism of chromosome translocations in secondary malignancy is largely unknown. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. We found that deficiency of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, results in the excessive binding of the DNA recombination related proteins RAD51 and RPA at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots. Mechanism of chromosome translocations in secondary malignancy will be discussed.

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Many anti-cancer strategies are based on the induction of DNA double strand break (DSB). The effectiveness of these strategies can be counteracted by the cell's ability to repair DSBs through homologous recombination (HR). Therefore, we want to understand the mechanism through which HR operates. We are analyzing the effect of mutations in the ATPase of Rad54, which is essential for HR. Rad54 possess a variety of biochemical activities that can affect Rad51 function in the critical steps of HR. Our in vivo study shows that Rad54's ATPase activity is not only important for HR but also affects its cellular behavior. Specifically, we show that it is not necessary for recruitment of the protein into DNA damaged induced structures at sites of DNA damage, known as foci, but rather that it influences Rad54 dissociation from foci. Furthermore, our data show that Rad54 but not its ATPase activity is required downstream of DNA break resection. In addition, our data reveal Rad54's ATPase activity differentially affects the behavior of the pool of Rad54 in a focus that is bound to DNA versus the pool that is not bound to DNA; the DNA-bound fraction can no longer dissociate, while the unbound fraction that is not actively engaged in DNA repair can still reversible interact with the focus. Finally, we show that the ATPase activity of Rad54 affects the locations of foci, because after preferential movement to the nuclear periphery, they remain there in the mutant cells.

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It is well known that "black rain"containing FP radioactivity fell down in the Koi-Takasu area located 3 km west from the hypocenter in Hiroshima. Meanwhile, the radiological situation is yet unknown about black rain that fell down in other areas around Hiroshima, including the mountainous suburbs. In this symposium, the followings are reported: previous studies including black rain in Nagasaki, analysis of questionnaires on black rain experience, recent uranium isotope measurements with TIMS and AMS techniques, radioactivity measurement of soil samples under houses built during the period between A-bomb and global fallout. Then, a trial is presented to reconstruct radiation exposure from deposited radionuclides as well as a proposal of meteorological simulation for the black rain. The general discussion will be at the end of the symposium about future tasks to be tackled with.

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Many scientists have been studied about "black rain" problems from the atomic bombings. Our group also have been studied about black rain problems from 1980. However, we could not find clear results related to the activities or radiation doses from the "black rain". Main reason of its difficulties is many additional fallouts from the nuclear experiments performed after the atomic bombings. Their levels are more than 10 times of the Hiroshima atomic bomb fallout, therefore we could not separate such as Cs-137 from the Hiroshima atomic bomb. Recently, from long continued study of the radiation for the residents living near Semipalatinsk nuclear test site, we find some ways to estimate radiation doses. These will be applicable. Also we could collect soil samples from old Japanese houses built immediately after the black rain and also before the global fallout. We show our studies in this symposium. About the area of the black rain, we are categorizing into 3 places. (1) 3km area relatively near area of the direct exposure, and (2) within 30km area, where we usually call "black rain" area. This area is sometimes called as "Uda are" or "Masuda area". (3) over 30km area there is almost no study until now. Our study is mainly in the area of (2). Recently from the joint study of Hiroshima city hall and our group, new Ohtaki area was obtained which is more wider than we thought before. Our final purpose is to estimate radiation doses for the people living in this area. Studies of radiation effects such as Hiroshima and Nagasaki, Semipalatinsk and so on, is to estimate radiation risks for the people combining with epidemiological study. However such study was not exists here so from the estimated radiation doses we will consider radiation effects in this area.

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Scientific records of Nagasaki "black rain" are limited. According to the records, the black rain began to fall about 20 minutes after the explosion in Nishiyama area located about 3 km east of the hypocenter. Wind at the time of explosion was reported about 3 m/s from south-west. But significant ^239,240Pu and ¹³⁷Cs contaminations were found mainly in the area due east of the hypocenter. ^239,240Pu concentration and external dose rate of Nishiyama area was very much higher than the "Black rain" area of Hiroshima. Rainfalls were reported at many places near around the hypocenter. Rainfall in the sunshine was reported from about 10 minutes after the explosion at Omura Navy Hospital about 19 km north-east of the hypocenter. But the rainfall was not black.
Falls of dry "black ash" and light fine dust were reported in the area due east of the hypocenter and they reached over 10 km from the hypocenter. It was reported that farms turned dimly white with the dust and characters could be written by a finger on leaves of taro.
People who were not officially acknowledged as atomic bomb victims have been accusing Japanese government to request acknowledgement by asserting that there was very strong radioactivity of the fallout immediately after the explosion in a large area including Nishiyama area. The maximum accumulated external dose for a person from 1 hour after the explosion in Nishiyama area was estimated about 0.4 Gy. And no signs of high dose internal exposure were found from whole body counting and chromosome study. However, the direct measurements of the external dose started from 48 days after the explosion and the whole body counting and chromosome study started after 1969. Many of inhabitants of Nishiyama district at the time most likely ate vegetables growing in the district and drank water of Nishiyama water reservoir. A resident of those days who drank water of Nishiyama water reservoir every day says that his throat began to swell and pain from one year after the explosion. Estimation of internal exposure of early days is important.

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In 2008, Hiroshima city conducted a self-writing questionnaire survey for 31598 inhabitants of Hiroshima city and its suburb who might encounter with "Black Rain", and investigated rainy intensity, color, start time, end time and the location. The subjects were restricted to those who met "Black Rain" on 6 August 1945 with having exact information on the exposed location, and then 1565 subjects were available for statistical analyses. The individual's locations were classified to representative spots such as the investigation local government office and school. For each spot, the answer results were summarized with a ratio or a mean value. Using nonparametric smoothing method based on local linear regression model, we estimated spatial-time distributions of rain area, rainy color and rain intensity. It was revealed that "Black Rain" began to rain at around western suburb of Hiroshima city at about 9 am then became heaviest at about 10 am with spreading toward north-west direction, and disappeared at about 3pm on 6 August 1945. As a result, the circumference of the domain where rain time after a linear adjustment was estimated to be less than one hour was wider than the Uda rainy area. It was suggested that the rain area contains almost whole area of current Hiroshima city except the East and northeast side.

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There was "black rain" after the Hiroshima atomic bomb (A-bomb) exploded over the Hiroshima city on 6th August 1945. In the Hiroshima black rain area, previous attempts failed to detect fission products such as ¹³⁷Cs and ⁹⁰Sr due to global-scale fallout that occurred mainly in 1960s because the global fallout of nuclear test in the atmosphere seemed to contribute larger than the local fallout in 1945. According to unofficial reports, about 51 kg of ²³⁵U was loaded in the Hiroshima bomb, of which 912 g was consumed by the 16-kt explosion. Considering that the precise determination of uranium isotopic composition will give some information about radioactive equilibrium, enrichment of ²³⁵U or presence of ²³⁶U, thermal ionization mass spectrometry (TIMS) and accelerator mass spectrometry (AMS) have been employed to detect uranium isotopes from the Hiroshima A-bomb in soil samples previously collected in 1976 from the black rain area around the Hiroshima city. The results of uranium isotope measurements as well as inter comparison between TIMS and AMS will be presented. The ²³⁶U measurement can provide valuable information about the local fallout contamination by the Hiroshima A-bomb.

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Recently, in relation with enlargement of social compensation for A-bomb survivors, concern on radiation exposure due to close-in fallout has been raised among the people who experienced the black-rain. Radioactive characteristics as well as spatial distribution of the close-in fallout by the Hiroshima A-bomb have not been specified even 60 years after the A-bombing. In this work, measurement of Cs-137 in soil samples under houses built after 1945 has been attempted to evaluate the close-in fallout deposition at the time of Hiroshima atomic explosion. As a result, we detected the low-level Cs-137 in soils under houses. However, problem is how we evaluate the levels of detected 137Cs.

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It is well known that "black rain" containing FP radioactivity fell down in the Koi-Takasu located about 3 km west from the hypocenter in Hiroshima. The radiological situation of the black rain that fell down in the mountainous areas around Hiroshima is not yet clear because no radiation survey was carried out in these areas just after the bombing. In the present study, the amount of FP deposition with black rain in the mountainous area was estimated based on the Cs-137 data in soil samples taken in 1976 as well as recent Cs-137 data in soil under houses built soon after the bombing. By excluding the contribution from the global fallout, the initial Cs-137 deposition of 0.5 - 2.0 kBq/m² was estimated with the black rain. The deposition of other FP nuclides than Cs-137 was obtained from their radioactivity ratios to Cs-137 that were calculated based on JNDC FP Library, taking into consideration the effect of volatile/refractory fractionation. As a result, a cumulative radiation exposure of 10 - 60 mGy at 1 m above ground was estimated for two weeks after deposition on an assumption that the deposited radionuclides remained at the same place during this period.

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To study of local fallout of Hiroshima A-bomb at present, direct evidences could be obtained by measurements of soil samples which is not affected by global fallout. We also could do model simulations. The basic concept of the simulation is to simulate behavior of fission products and induced radionuclides, especially to simulate wet and dry deposition of them. To do model simulations, we are going to prepare datasets of cloud height, distribution of fission products, distribution of Cs-137 originated Hiroshima A-bomb and area of rainfall etc. as initial and boundary conditions.

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It has been reported that radiation effects are manifested not only in irradiated cells but also in unirradiated cells that are close to irradiated cells (called as radiation-induced bystander response). Recent studies revealed that reactive oxygen species, nitric oxide, cytokine and nucleotide transferred from irradiated cells to unirradiated cells through gap junction and/or culture medium induce bystander responses including reductive cell death and chromosomal aberration. However, the details of molecular mechanism and biological significances are still unclear. Microbeam system is often utilized as a powerful tool for the studies of radiation-induced bystander responses since it can precisely discriminate irradiate cells and others in cell population. In this workshop, we will learn from the specialists about their latest studies and discuss on the future direction of radiation-induced bystander effects.

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The bystander effect is the phenomenon whereby ionizing radiation induces biological effects in non-irradiated bystander cells having received signals from directly irradiated cells. Bystander cells manifest genetic changes (e.g., chromosome aberrations, mutagenesis and micronucleation), epigenetic changes, alterations in gene expression, activation of signaling pathways, and delayed effects in their progeny. Proposed mechanisms mediating the bystander effect involve gap-junctional intercellular communication, reactive oxygen/nitrogen species, secreted soluble factors, lipid rafts, and calcium fluxes. Here we briefly overview the current knowledge of the bystander effect, particularly focusing on the observed phenomenon and its proposed mechanisms.

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We have investigated radiation-quality dependent bystander cellular effects in normal human fibroblasts induced by low fluences of either broadbeam or microbeam with different radiation types. The cells were irradiated with low-fluence broadbeams of ¹³⁷Cs gamma rays, ²⁴¹Am-Be neutrons, helium, carbon and iron ions or microbeams of monochromatic X rays at High Energy Accelerator Research Organization, protons at National Institute of Radiological Sciences and heavy ions (carbon, neon and argon) at Japan Atomic Energy Agency. In broadbeam experiments, no difference was observed in cell killing irradiated with X-ray acute dose in cells pre-treated with low-fluence irradiations of different radiation types. For mutation at hprt locus detected as 6-thioguanine resistant clones, the X-ray acute dose induced mutation frequencies were 4.0 times higher in carbon- and 1.9 times higher in helium-ion pre-treated cells than those in un-pretreated cells. The mutation frequency pre-treated with neutrons was reduced to 15%, comparing to the control. In the microbeam experiment, the percent cell survival in carbon-ion microbeam irradiated dishes was ranging from 83% to 94%, while the survivals in X-ray, proton, neon-ion and argon-ion microbeam irradiated dishes were around 100%. The mutation frequencies in cells irradiated with proton, carbon-ion and neon-ion microbeams were 4.0 times, 5.8 times and 2.7 times higher than those of non-irradiated control cells. Because in our microbeam-irradiation method, we estimated that only 0.2% of total cells on the dish was irradiated with microbeams, these results suggest that bystander cellular effects depend upon radiation quality and biological endpoints.

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(Purpose) In order to know the mechanisms of bystander effects, we examined the involvement of slow releasing long-lived radicals (SRLLRs) induced by the conditioned medium from the irradiated cells (IR-conditioned medium). (Method) Bystander effects were detected by the medium transfer method. Inductions of mutations at HPRT locus by bystander effects were examined in CHO cells. The levels of SRLLRs induced by conditioned medium in bystander cells were determined by ESR method. Ascorbic acid and N-acetyl cysteine (NAC) were used as radical scavengers for SRLLRs, and the suppressive effects of radical levels and mutation inductions were examined. (Results) The results showed that the levels of SRLLRs were increased in the cells treated with IR-conditioned medium, suggesting that the IR-conditioned medium can induce SRLLRs in bystander cells. The irradiation to only medium could not induce the level of SRLLRs in cells. Also, the induced levels of SRLLRs by IR-conditioned medium were suppressed by the treatment of ascorbic acid, but not by the treatment of NAC. Moreover, the mutation frequency at HPRT locus in cells treated with IR-conditioned medium were higher than those in control cells with non-IR-conditioned medium, and the induced mutation levels by IR-conditioned medium were completely suppressed by the treatment of ascorbic acid in bystander cells, but not by the treatment of NAC. The analysis by multiplex PCR method on HPRT locus showed that these induced mutations were point mutation. The analysis of mitochondria specific O₂^- marker, MitosoxRed showed that IR-conditioned medium induced the O₂^- in mitochondria, suggesting that signal through the mitochondria are involved in the production of SRLLRs. (Conclusion) These results suggest that an indirect induction of SRLLR through the IR-conditioned medium play an important role for the inductions of bystander mutagenesis response in non-irradiated cells.

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