[in Japanese]

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Despite expectations, no evidence of germ cell mutation, seen as genetic disease and adverse pregnancy outcomes (APO), has been documented in children of survivors from atomic bombs in Japan nor in offspring of survivors of childhood and adolescent cancer. Continuing basic research and animal model assesses the risk of genetic disease in 23,889 children born to 14,519 survivors of cancer diagnosed up to the age of 35 years in Denmark and Finland through population-based record linkage. Comparison children include those born prior to cancer diagnosis (n=15,740) and 98,465 children of 45,037 siblings of cancer survivors. Genetic disease and APOs are being evaluated further with dose-response analyses over categories of radiation dose and administered chemotherapy. Together with the two US studies, these findings in population-based studies in two countries are reassuring that the children of cancer survivors are not at high risk of genetic disease apart from the known genetics or familial predispositions.

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[in Japanese]

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The RERF has worked for 60 years studying the effects of radiation. These studies have been used the world over for treatment and the setting of standards. In the long run, such studies will be vital to maintaining human health. In the short run, we have to stop nuclear weapons.
RERF research makes it obvious that nuclear weapons are cruel, inhumane and cannot be limited to the battlefield in either time or space. As such, they are illegal weapons. A majority of scientists who worked on the Manhattan Project believed the atomic bombs should never have been used. They should have been banned and eliminated or at least put under international control. Today, the vast majority of nations and people on this planet wish to be liberated from the threat of these weapons, and yet, we continue to allow a tiny group of greedy, competitive, dominance-seeking men to threaten all life on Earth with annihilation.
Next May, the human family will decide whether to eliminate nuclear weapons or let everyone have one. If we choose the latter, we will be choosing to solve our myriad global problems through a radical, violent reduction in human population. We will close out for decades, centuries or, perhaps, permanently any hope of graduating to a culture of peace. If the international community fails at the next NPT Review Conference (May 2010, New York) to take a convincing step toward complete disarmament, the number of nuclear-armed states could double or triple before the following review conference in 2015.
What we need to do is outlined in the Hiroshima-Nagasaki Protocol. We need to start negotiations immediately, and we need to be working toward a deadline. We have no more time for playing games. Disarmament has become a matter of do or die, literally.

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The Journal of Radiation Research (JRR) comes to issue Vol.50 in this year. The journal has been bearing the key role on transmitting academic informations to the world from society member's since it established. Papers from the nonmember were accepted after 1998. JRR has been opened on internet to the public as Open Access Journal in 2000. All the articles from the first issue (1960) are open now on Web. It is possible to inquire in the document search engines PubMed, CrossRef, ChemPort, JDream, Google Scholar and so on. The journal got an Impact Factor of 1.5 in average after 2000. In 2010, Japanese Society for Therapeutic Radiology and Oncology will join us to publish JRR, and oncology related fields will be added. The circulation of the journal will be increased to exceed 3000 copies from about 1000 copies at present. At this occasion, JRR must be enhanced further more, and to publish a fairer thesis, this seminar was planned.

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The purpose of this luncheon seminar is to encourage discussion on publication ethics, especially on definition of authorship, gift authorship, conflict of interest, and peer review system in scientific journals for preventing scientific misconduct. As Frank Davidoff, past editor of the Annals of Internal Medicine, points out “Science does not exist until it is published”, the issues on publication ethics and good publication practice become more important one recently. I also discuss on the development of the Uniform Requirements for Manuscripts Submitted to Biomedical Journals (URM) that was founded in Vancouver, Canada, in 1978. The URM have been changing from style manual to ethical guideline for writing medical papers. Now, the URM have gained widespread acceptance by journal editors, but is not well known either to researchers and faculty members with active research careers who publish frequently. Focusing on publication ethics as an essential aspect of responsible scientific conduct is now requested.

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In recent, we can find many advanced reports about signal transduction pathways of cell survival, DNA repair, cell cycle and angiogenesis of cancer cells. These pathways were induced after the cancer cells were treated with cancer therapies. To aim much high efficacy in cancer therapies, It is important to control these pathways. In this symposium, we aim to introduce new advanced experimental results targeting these pathways from basic research and combine to cancer therapies for patients. For example, we schedule to presentation and discussion about the control for cell survival signal and cell cycle signal such as mTOR, the depression for DNA repair and the sensitization among radiation, hyperthermia and anti-cancer drugs.

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Despite that much effort has been made for the development of radiosensitizers, useful drugs are still unavailable in usual clinics of radiotherapy. Antiangiogenic agents are thought to be promising in cancer therapy and many drugs possessing such properties have been developed. Recently, a consensus is being made that the combination of such drugs with radiotherapy is quite effective. Sulfoquinovosylacylglycerol (SQAG) is sulfoglycolipids, which we originally extracted from natural products. We also succeeded in chemical synthesis of the agent. In this study, we show that SQAG could be a unique antiangiogenic radiosensitizer. The agent by itself had an antiangiogenic activity at high doses, however, it synergistically inhibited angiogenesis at low doses when combined with ionizing radiation using different in vitro methods. Combined treatment with SQAG and radiation appears to promote the adoption of a senescence-like phenotype rather than apoptosis by vascular endothelial cells in vitro. The agent remarkably enhanced the radioresponse of several human tumors transplanted into nude mice, accompanied by a significant reduction in the vascularity of the tumors as evaluated by immunohistochemical staining of CD31. Collagen IV, which constitutes the basal membrane of the vessels, was significantly reduced by the combined treatment. The effect of the combined treatment on vascular normalization as evaluated by the time course of α-Sma expression, a marker of pericytes, and hypoxic fractions as determined by pimonidazole staining will also be discussed. We conclude that SQAG could be a potent antiangiogenic radiosensitizer.

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Transcriptional regulation of DNA double-strand break repair genes by Sp1
DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand breaks (DSBs) repair, and it consists of Ku70, Ku80 and DNA-PKcs. It has been shown that the promoter regions of these three genes have Sp1 binding sites, and the expression levels are correlated with that of Sp1. The purpose of this study is to clarify the contribution of Sp1 to radiation sensitivity of cells through the transcriptional regulation of DSBs-repair genes. We investigated whether Sp1 affects the protein and mRNA levels of Ku70, Ku80, DNA-PKcs, XRCC4, NBS1, MRE11 and MDC1. In addition, we examined the DSBs-repair, DNA-PK activity, cell cycle, and radiation sensitivity in Sp1-dawn-regulated cells. A human transformed kidney cell line 293T was transfected with siRNA vector targeting Sp1 (Sp1-siRNA) or control vector. The vector-transfected cells were selected with G418. After 7 days selection, protein and mRNA levels were evaluated by Western blotting and RT-PCR, respectively. The protein and mRNA levels of Sp1, Ku70, Ku80, DNA-PKcs, XRCC4, NBS1, MRE11 and MDC1 were down regulated by the Sp1-siRNA treatment. The DSBs-repair after 80 Gy irradiation and DNA-PK activity were suppressed by the Sp1-siRNA treatment. The surviving fraction after irradiation was also suppressed by the Sp1-siRNA treatment. However, cell cycle was not affected by the Sp1-siRNA treatment. These results suggest that Sp1 regulates the radiation sensitivity by the transcriptional regulation of DSBs-repair genes.

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Radiotherapy has been the most effective nonsurgical treatment for cancer. However, the recurrence is frequently occurred by the acquisition of radioresistance of tumors and resulting failures of clinical outcome. Therefore, the determination of underlying mechanism of the radioresistance of tumor cells may provide novel therapeutic approaches to eradicate radioresistant cells.
This study is the first to demonstrate that long-term fractionated radiation (FR) for more than 31 days conferred radioresistance of tumor cells with cyclinD1 overexpression. The time course analyses revealed that the level of cyclinD1 was unchanged until day 14, but was significantly elevated on day 31. The radioresistance was stably maintained in the tumor cells even on 31 days after the cessation of irradiation.
CyclinD1 overexpression was brought about by a feedback loop mediated by DNA-PK/AKT/GSK3b-mediated down-regulation of cyclinD1 proteorysis. CyclinD1 overexpression produced DSBs in S-phase which again activated DNA-PK, compleating the loop. Inhibition of the AKT/GSK-3b/cyclinD1 pathway by treating HepG2 and HeLa cells either with an AKT inhibitor or siRNA targeting cyclinD1 suppressed the radioresistance of long-term FR cells.
Present observations give a mechanistic insight for acquired radioresistance of tumor cells through long-term FR exposures, and provide novel therapeutic targets to radiosensitize recurrent radioresistant cells.

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Roles of intracellular oxidative stress in apoptosis induced by radiation and hyperthermia were studied. Various endpoints of apoptosis, DNA fragmentation, morphological changes, and phosphatidylserine externalization, etc. were examined. Enhancement of radiation-induced apoptosis in human lymphoma U937 cells by sanazole was found. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential, and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca²⁺ concentration ([Ca²⁺]i) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis. Here roles of intracellular oxidative stress in radiation as well as hyperthermia-induced apoptosis will be discussed.

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It is well known to enhance the cell proliferation by the activation of survival signal and to suppress the apoptosis and non-apoptosis such as autophagy, mitotic catastrophe and necrosis by the inactivation of death signal in cancer cells. In recent years, cancer therapy research has focused on molecular targets on survival and death signal transduction pathways. For efficient cancer therapy, potential targets of interest are selectively activated signal transduction factors which can inhibit survival signal or induce death signal. The signal transduction pathways affected by radiations (X-rays and heavy particles), anti-cancer agents or hyperthermia include p53 mediated pathways, JNK (Jun N-terminal kinase) mediated pathways, Akt (protein kinase B)/mTOR (mammalian target of rapamycin) mediated pathways, NBS1 (Nijimegen breakage syndrome 1) mediated pathways, classic MAP (mitogen activated protein) kinase mediated pathways, and p38 MAP kinase mediated pathways. Events such as cell death, cell survival, cell proliferation, and/or cell cycle arrest can be affected by these pathways. We have reported that targeting of signal transduction of life and death can enhance cellular sensitivity via radiation, anti-cancer agents and heat. This presentation reviews our strategy for cancer therapy.

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At first, to identify a set of genes related to progression and metastasis of cervical carcinoma after radiotherapy (RT) and to establish a predictive method, genome-wide data were investigated using a cDNA microarray. A total of 28 patients with stage III-IV cervical cancer who underwent definitive RT were included in this study. The expression profiles of 14 tumors with local failure and multiple distant metastasis and 14 tumors with no evidence of disease (NED) obtained by punch biopsy before treatment. The most accurate prediction was achieved at 63 genes (sensitivity: 78.8%, specificity: 38.1%). One of these genes was already known to be associated with metastasis via chromosomal instability (TTK). A “Predictive Score” system was developed that could predict the probability for development of metastases.
Next, to identify the most important gene related to prognosis after RT, we have validated some genes by mRNA expression from blinded 60 test patients using a real-time PCR. We compared mRNA expression of 29 with NED and 31 with cancer-caused death (CD) tumors after RT obtained by punch biopsy before treatment using a real-time PCR. Taqman probe was designed according to the sequence of BAX, TEGT (BAX-inhibitor), XRCC5, PLAU, HIF1A, CD44, and TTK cloned genes, with normalization to housekeeping genes. The endpoint of this study was the correlation between mRNA expression and the overall survival rate of these patients according to Cox’s proportional-hazard model. HIF1A and TTK mRNA expression values in CD group were higher than in NED group, however, not significantly (p = 0.079, p = 0.092, respectively). On univariate and multivariate analysis, TTK was a statistically significant independent poor prognostic factor for overall survival rate after RT (HR, 1.06; 95% CI, 1.0-1.12, p=0.044, and HR, 1.35; 95% CI, 1.02-1.78, p=0.035). In conclusion, the relatively high incidence of TTK may explain, at least in part, the poor prognosis of advanced cervical carcinomas after RT.

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The UNSCEAR (United Nations Scientific Committee on the Effects of Atomic Radiation) reports summarize a variety of findings on radiation effects from molecular/cellular-level studies to epidemiological studies. These reports are utilized as a scientific basis in establishing recommendations and safety standards for radiation protection by international bodies such as ICRP and IAEA. The UNSCEAR has recently published the volume 1 and volume 2 of the 2006 report, and is now preparing the 2008 report. Many outcomes from Japanese studies are cited in the UNSCEAR reports, which show how much Japanese scientists contribute internationally in the field of radiation research. In this symposium, recent activities of the UNSCEAR will be introduced by several members of Japanese delegations to the UNSCEAR, and then significance of the dissemination of the information derived from Japanese studies through the UNSCEAR will be discussed.

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An outline of risk evaluation on cancer and non-cancer disease made in the UNSCEAR 2006 report had been discussed at the time of the 50th meeting of the Japanese Radiation Research Society. Therefore, the contribution of Japanese epidemiological studies in the UNSCEAR 2006 report will be discussed this time.
Radiation and Cancer Risk:
The study which is widely utilized in the report is the Life Span Study (LSS) of Radiation Effects Research Foundation (RERF), as has been in the past.
In site-specific risk evaluation, the results of LSS have been cited in almost all cancer sites. In particular, risk estimation by gender and age at the time of exposure can be made only by LSS. The dose response observed in the LSS is utilized as one of the bases of risk estimation at lower doses. Results of follow up of in-utero exposed population are also utilized.
In addition, as studies on occupational exposure, nuclear workers study being conducted at Radiation Effects Association (REA) and radiologic technologists study conducted at National Institute of Radiological Sciences (NIRS) are cited in the report. Follow-up of Thorotrast patient is cited as study on medical exposure.
In addition to the above, although not conducted in Japan, studies in high background radiation area in China, and studies on residents in Marshall islands are cited, in which Japanese epidemiologists are involved.
The LSS will continue to provide useful information for risk estimation of cancer, however, it can not directly provide information on the effects of protracted low-dose exposure. It is, therefore, necessary to develop more studies in Japan in order to produce useful in formation on effects of low dose exposure.
Radiation and Non-cancer Disease (Cardiovascular Disease) Risk:
The epidemiological study that is most widely utilized in the report is the LSS of RERF. The RERF Adult Health Study is also cited which provides evidence to complement weakness of mortality data.
In addition, as studies on occupational exposure, nuclear workers study being conducted at REA and radiologic technologists study conducted at NIRS are cited in the report.
Studies of non-cancer disease are insufficiently conducted because most of epidemiological studies put main emphasis on cancer risk.
It is essential to expand studies of this area in order to make more contribution in the future.

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UNSCEAR 2006 REPORT is composed of two volumes and the second volume deals with non-targeted radiation effects, effects on immune system, and radon. In this symposium I will focus on the trend of research from UNSCEAR 2006 REPORT volume 2 and their future prospects. Today, the biological effect of radiation is believed to be based on energy deposits on nucleus in target cells by radiation. Non-targeted radiation effect is a phenomenon where energy deposits on cellular components other than nucleus or even on neighboring cells can cause mutation or apoptosis. If such a non-targeted radiation effect occurs effectively in vivo, it may induce a paradigm shift in estimating radiation risk at low dose or low dose rate, especially by high LET radiation. As to radiation effect on immune system, new concepts and technologies in basic immunology have been introduced into radiation research. As to radon, systematic review on indoor radon-lung cancer case control studies and pooled analyses will be highlighted.

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Further to its previous reports in 2000 and 2001, United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) has published the latest volumes of its report “UNSCEAR 2006 Report –EFFECTS OF IONIZING RADIATION—“ (Vol.1 and 2) . And moreover, the 2008 Report had been approved in 2007 and 2008 meetings and is expected to be published in the upcoming years. The UNSCEAR secretariat has now started to dissscuss the scientific subjects and the strategic plan of next following term (2008-2015).
On UNSCEAR 2007 meeting, almost 40 scientific programmes proposed by the various scientific groups in different countries, and we can probably say that some of these will become the Scientific Annexes of the future volume of the UNSCEAR report, were provided to the committee members. These proposed programmes were then examined by its scientific importance and societal need, and merged together. Finally, 12 candidates (e.g., uncertainties in radiation risk estimation, attributability of health effects due to radiation exposure, etc) were considered and will be subjected to the further discussion in the working group. The results will be further discussed at the forthcoming UNSCEAR meeting in 2009.

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The health effects of radiation is well established. However, as Upton pointed out (Upton, Cancer Invest 1989), biological effects of low-dose and dose-rate radiation exposure have a lot of uncertainties in their quality and quantity. They appear to be different from those of high-dose radiaton exposure not only in their magnitude but also in their nature. In this paper, discussions will be made as to solid cancer risk of low-level external exposure, mainly on the basis of results obtained from epidemiological studies. The epidemiological studies that are important for evaluating the health effects of low-level radaition are the studies of atomic bomb survivors, nuclear workers, and the residents of high-background radiation areas (HBRA) in India and China. Although much attention had not been paid to HBRA studies until recently, the studies in India are particularly important. In the HBRA in Kerala, India, collaborative studies with IAEA, IARC, France, USA are already initiated in addition to the studies led by Dr Sugahara, the former chariman of the Health Reasearch Foundation (Chairman: Dr Torizuka).

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Evaluation of lifelong health risks from ionizing radiation, especially from low-dose radiation, needs comprehensive understanding of radiation response at multi-cellular levels. Recent studies have shown both direct and indirect interactions between individual exposed and unexposed cells, which might affect manifestation of various late effects caused by low-dose ionizing radiation. Integration of such multi-factorial interactions can not be accomplished by currently available methods but requires new approaches such as “Systems Radiation Biology”. In this symposium, present status of systems radiation biology research is presented by three investigators, and its possible future will be discussed.

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Ionizing radiation has been a primordial part of the planet earth and it might be the main reason why live could florish here. All living organisms, like humans, are being exposed to many natural, civilisatoric and technical irradiations from various sources and to various degrees. Often individuals, groups, administrations and government are very much concerned about at most very small individual radiation risks, i.e. about possible health effects of low dose rates of ionizing radiation (below, say, 10 mSv/yr). Radiation research over more than 50 years in vain has tried to improve our quantitative knowledge on such radiation health effects.
A main reason for this failure of answering such important questions is the principally wrong research strategy of concentrating main efforts on radiation effect investigations of single molecules (e.g. DNA) or of single cells (mainly in vitro). However, most health effects of living, adaptive organisms most likely actually result from rather indirect, systemic, emergent, responses at different levels of tissue organisation which, in principle, can never reductionarily be studied with single objects in isolation. In this contribution, a more promising research strategy, namely that of Systems Radiation Biology (SRB), will be outlined and justified. In this quantitative, more top-down, SRB - approach quantitative hypotheses on relevant action pathways of organismic homoestasis and its disturbances by external agents are being formulated and tested in close co-operations of theorists and experimentalists educated in different relevant disciplines (mathematics, physics, chemistry, biology, medicine, epidemiology, etc.).

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Annexin A2 was recently identified as a paracrine factor that mediates, in part, the anchorage-independent growth (AIG) response to low dose radiation (Weber et al., Rad. Res, in press, 2009). Paracrine-dependent AIG did not fully account for the low dose radiation AIG response and we undertook a genomics approach to identify additional sensitive markers of AIG responses. Our experimental design exploited irreversible regulation of AIG by 12-O-tetradecanoyl phorbol-13-acetate (TPA), relative to reversible regulation of AIG by basic fibroblast growth factor (bFGF). 142 differentially expressed genes were common to colonies arising from bFGF- and TPA-treated JB6 cells. The majority of genes exhibited comparable patterns of regulation in terms of increased or decreased expression, while 30 genes exhibited reciprocal regulation patterns. Hepatic leukemia factor (HLF) and D-site albumin promoter-binding protein (DBP) expression were increased in both bFGF- and TPA-induced colonies. Ectopic expression of human DBP and HLF increased low dose X-ray radiation (10 cGy)-, TPA- and bFGF-induced AIG responses. HLF and DBP expression were increased in human basal cell carcinoma tumor tissue, relative to paired uninvolved tissue from the same donor. HLF and DBP mRNA expression were also increased in a normal human skin equivalent model system (MatTek). Collectively, our approach has identified sensitive candidate biomarkers of the AIG response that are expected to enable detailed investigations of possible risk factors for radiation carcinogenesis.

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Systems biology predicts that some properties result from altered relationships between components. Systems radiation biology asks not only how radiation affects specific components (i.e. cells) but also how these alterations affect interactions that maintain tissue integrity. While radiation can alter genomic sequence as a result of DNA damage, it can also induce signals that alter multicellular interactions and phenotypes that underpin carcinogenesis. Our previous studies characterized the composition of irradiated mouse tissues, identified transforming growth factor β1 (TGFβ) as a key cytokine activated by radiation, and developed novel models of radiation effects in both mice and cultured human epithelial cells. This presentation will focus on how TGFβ regulates the intrinsic DNA damage response, tissue composition and the carcinogenic effects of low dose radiation. Rather than being accessory or secondary to genetic damage, we propose that radiation induced signaling via TGFβ creates the critical context for cancer development.
Research funded by US DOE Low Dose Radiation Program
and NASA Specialized Center of Research

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More than 60 years have passed since the atomic bombings of Hiroshima and Nagasaki. Epidemiological studies of atomic bomb survivors and of high background radiation area residents have provided information on radiation-related cancer risks. However, basic studies are still needed regarding some important issues such as the risk modification by the age at exposure, the low dose rate radiation effect, and the target cells of radiation carcinogenesis. In this workshop, we will learn from young and energetic radiation biologists about the latest studies of radiation-induced carcinogenesis in animal models and discuss whether these results are extrapolated to the knowledge from epidemiological studies. The goal of this workshop is to search for future direction of the radiation carcinogenesis studies.

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Little is known about carcinogenesis following low-dose-rate (LDR) irradiation. Cancer is originated from cancer stem cell with self-renewal and multipontency. We tried to elucidate at which cell differentiation stage cells were transformed into leukemic stem cells (LSCs) by LDR irradiation. Three groups of C3H/He mice were irradiated with 8, 4 and 3 Gy at dose rates of 20 mGy/day, 400 mGy/day and 1.0 Gy/min, respectively. Radiation-induced and spontaneous leukemias were compared regarding chromosome aberration detected by array CGH analysis, CD-antigen expression profiles by FACS and identification of LSC by transplantation. Array CGH showed that approximately 50% of leukemias in all groups commonly had 30Mb hemizygous deletion nearby centromere of chromosome 7. In contrast, hemizygous deletion of PU.1, a leukemia-related gene, on chromosome 2 was found in a dose-rate-dependent manner in 6% of spontaneous leukemias in non-irradiated mice, and 30%, 56%, and 90% of leukemias from mice irradiated respectively at 20 mGy/day, 400 mGy/day and 1.0 Gy/min. The leukemias with hemizygous PU.1 deletion (PU.1^del-leukemias) frequently had mutation on the remaining allele of PU.1. PU.1^del-leukemias showed an increase in common myeloid progenitors (CMPs), while leukemias without PU.1 mutation (PU.1^wt-leukemias) showed an increase in common lymphoid progenitors (CLPs). Furthermore, transplantation assay by injection of each cell populations showed that LSCs were present in subpopulations of hematopoietic stem cells (HSCs) and CMPs in PU.1^del leukemias, while LSCs were in subpopulations of CLPs and granulocytes in PU.1^wt-leukemias. Our results indicate differences in the origins of LSCs and gene mutations responsible for radiation leukemogenesis with different dose-rates. This study was performed under contract with the Aomori Prefectural Government, Japan.

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Ionizing radiation is the only firmly established human breast carcinogen. Rodent mammary cancer is a useful model of human breast cancer and has been the central player in experimental breast cancer research. The dependence of cancer risk upon the age at carcinogenic exposure is an important issue.
Chemical carcinogenesis studies have clarified that there is a narrow window of susceptible period in the peripubertal stage, during which extensive mammary gland growth occurs. This window of susceptibility may be explained by either differentiation state of the mammary gland, maturation of the potential for metabolic activation of carcinogens, or transition of the DNA repair capacity.
We have examined the effect of the age at exposure on radiation carcinogenesis. In contrast to chemical carcinogenesis, the susceptibility is relatively low during the juvenile period. Our data indicate that, although the immature gland may be susceptible to carcinogens, juvenile radiation exposure leads to relatively low incidence of mammary cancer through premature ovarian dysfunction and subsequent insufficient tumor promotion, suppressing hormone-dependent cancer development. Molecular mechanisms of radiation-induced carcinogenesis will also be discussed.

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Thymic lymphoma is very common in mouse malignancies and provides a model to delineate aberrant cells at the early precancerous stage. The existence of prelymphoma cells or lymphoma-initiating cells in γ-ray induced mouse atrophic thymus has been noted by the finding that transplantation of thymocytes in atrophic thymus can produce thymic lymphomas. Importance of leukemia/lymphoma-initiating cells is underlined in the analysis of relapsed acute lymphoblastic leukemia (CML) in humans, which showed that the cells responsible for relapse are ancestral to the primary leukemia cells. Here we study phenotypic and genetic changes of the probable prelymphoma cells in atrophic thymus. Also, we investigate changes in DNA damage checkpoint because a hallmark of precancerous cells in major human cancer types is aberrant stimulation of cell proliferation and the subsequent activation of DNA damage checkpoint. For atrophic thymuses at 40 and 80 days after fractionated whole-body γ-irradiation to mice, we examined clonality by assaying D-J rearrangement patterns at the TCRβ locus. Clonally expanded thymocytes (designated as C type) were found in approximately 40% (43/111 in 40 day thymus and 21/45 in 80 day thymus), while others exhibited similar D-J rearrangement patterns to normal thymus (T type). The C type thymocytes mostly consisted of CD4⁺CD8⁺ double positive (DP) cells despitess clonal expansion, suggesting that the thymocytes are aberrant DP cells having passed b-selection. C type thymocytes exhibited pausing at an early G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as γHA2X, Chk1/2, or p53. This is unexpected because the checkpoint activation is assumed to function as an inducible barrier against g-ray induced genomic instability. Of interest, is that 17 of the 52 Ttype thymuses at 40days after showed allelic loss at Bcl11b tumor suppressor locus. This suggest that allelic loss contributes to clonal expansion of the thymocytes that still possess the capacity to differentiate. These results suggest that formation of prelymphoma cells probably requires changes in two distinct steps, cell proliferation and differentioation arrest, as described in human CML and lymphoma malignancies.

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At the time of the JCO criticality accident 10 years ago in 1999, several methods of the retrospective dosimetry were applied for dose estimation of irradiated people. The methods include clinical, biological and radiochemical methods. By starting from a review of the JCO accident, the workshop will summarize recent applications of the retrospective dosimetry on the detection of low-level contamination and on the dose reconstruction for atomic bombs in Nagasaki and Hiroshima, Semipalatinsk nuclear test site, and Chernobyl contaminated site. Since development and progress of analytical techniques strongly contribute to the retrospective dosimetry, available new techniques such as accelerator mass spectrometry will be introduced in the talks.

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Exposure to radiation rarely causes immediate signs or symptoms after exposure. However, prodromal symptoms could appear soon when a whole body is exposed to high dose radiation for a short duration. A criticality accident, which occurred at Takaimura of Ibaraki prefecture, Japan in 1999, caused exposure of three people to γ-rays and neutrons. They developed prodromal symptoms soon after exposure. In this accident, thus, three victims were heavily exposed. Dose assessment is performed by the biological/medical method and the countermeasures based on radiation physics and calculation. However, most of the data, such as the dose rate of radiation, its distribution, and the quality needed to evaluate the average dose, are not available when the decision for treatment should be made. In the accident, therefore, the temporary dose estimation of these victims was performed from the onset time and severity of prodromal symptoms. More detailed doses were assessed later by serial changes of lymphocyte numbers, chromosomal analysis, ²⁴Na, ⁴⁵Ca, or ³²P activity, and theoretical calculation. The results obtained from these methods were fairly consistent. One of the problems in dose assessment for the exposure to γ-rays and neutrons is how to evaluate the effect of neutrons on victims and the contribution of neutrons in the exposure. Considering the relative biological effectiveness (RBE) of the neutrons to be 1.7 from our own results and the ratios of neutrons to γ-rays 1.4-1.9, we estimated doses of 3 victims as 16-25, 6-9, and 2-3 grays equivalent as biological doses (GyEq), respectively. The other problem is that the accidental exposure is always inhomogeneous. Therefore, the estimated average dose of whole body is not necessarily in parallel to symptoms or signs previously reported. From the accident we have learned that there is no identical accident to other and that dose assessment from various methods is required for evaluation of damages to the body is required.

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Dosimetry system 2002 (DS02) has been established according to the collaboration among many institutions and universities in US, Japan and Germany. DS02 calculates organ doses of atomic bomb survivors. At first emitted neutron and gamma-ray energy spectra from the Hiroshima and Nagasaki atomic bombs were calculated and were used to calculate air transport, absorption and transmission through Japanese houses. Finally organ doses of each atomic bomb survivor were obtained. On the other hand bricks and roof tiles were collected and gamma-ray doses were measured. Similarly iron, granite and copper samples were collected and neutron induced radioactivities such as Co-60, Eu-152, Cl-36 and Ni-63 were measured. The measured data were compared with DS02 and its accuracy was verified. These doses will be combined with the epidemiological studies of the Radiation Effects Research Foundation and the radiation risks were obtained. The risks will be used to limit exposure for radiation workers and also for general people. The major part of the radiation of DS02 is gamma rays, which were increased about 10% both in Hiroshima and Nagasaki comparing with the former DS86. In this paper obtained results of DS02 and the background of the construction of DS02 are shown. Among those data only Co-60 data were not agreed with DS02 at the longer distances. Here we introduce new Co-60 measurements, of which data show good agreement with DS02 at longer distances.

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With recent developments of analytical instruments and measurement techniques able to detect ultralow amount of ²³⁶U (T_1/2=2.342×10⁷ y, α-decay) in the environment. U-236 is being recognized as a potential tracer not only for monitoring of uranium contamination release from nuclear cycle, but also for geochemical dynamics studies.
For accurate determination of ²³⁶U in the environment at low level, the ²³⁶U input from global fallout has to be considered. However, information on ²³⁶U background has not been clearly defined. In current study, ²³⁶U along with ^239+240Pu and ¹³⁷Cs, were measured to estimate their isotope ratio, concentration and inventory in soil samples from forest in Ishikawa, Japan. This area has not been affected by local or regional ²³⁶U releases from nuclear facilities or the Chernobyl accident. The activity of ¹³⁷Cs, ^239+240Pu, ²³⁸U and ²³⁶U/²³⁸U atom ratio in the soil (0-10, 0-20 and 0-30 cm) were measured with γ-ray and α- spectrometries and accelerator mass spectrometry (AMS).
The ²³⁶U/²³⁸U isotopic ratio and ²³⁶U concentration ranged from 1.85×10^-8 to 1.09×10^-7 and 8.92×10⁸ to 3.76×10⁹ atoms/g dry soil, respectively. The average ²³⁶U/^239+240Pu ratio (atoms/Bq) was (1.56±0.10)×10¹¹. That translates into the ²³⁶U/²³⁹Pu atom ratio of 0.235 using the global fallout ratio for ²⁴⁰Pu/²³⁹Pu of 0.176. This is within the range of 0.05 - 0.5 reported for the locations with high ²³⁹Pu and low ²³⁸U soil concentrations. No significant correlation was found between ²³⁶U and ¹³⁷Cs concentrations in measured soils as opposite to ^239+240Pu.
Our results indicate that surface soil has been contaminated with ²³⁶U from globalfallout of 10⁸ - 10⁹ atoms/g (10¹³ atoms/m² in soil from this area). The total surface soil deposition of ²³⁶U due to global fallout was estimated as ca. 900 kg.
The study was undertaken to characterize the Black Rain area of Hiroshima atomic bomb. The distributions of ²³⁶U, ^239+240Pu and ¹³⁷Cs isotopes were measured in more than 60 surface soil samples from Hiroshima. The results of this study will be presented and discussed in the presentation.

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A large amount of radioiodine isotopes (mainly ¹³¹I, T_1/2=8days) were released from the accident at Chernobyl Nuclear Power Plant (CNPP) in April-May 1986. An increase in childhood-thyroid cancer in the contaminated area was assumed to be the cause of radioiodine released at the time of the accident. However, there is a lack of relevant data on the ¹³¹I levels in the local environment (e.g. air, plant, soil), and therefore, it is difficult to assess radiation doses received by the patients from this nuclide following the accident. However, a long-lived iodine isotope, ¹²⁹I (T_1/2=15.7 million years), which was also released with a certain ratio to ¹³¹I from CNPP, could be used for estimating the ¹³¹I levels in the environment. In this presentation, we will discuss analytical results of the ¹²⁹I concentrations and ¹²⁹I/¹²⁷I atom ratio in soil samples collected from CNPP exclusion zone (30-km zone) with the aim of assessing current contamination levels and distribution patterns. For the analysis of iodine fraction in soil samples, pyrohydrolysis method was utilized for the separation of ¹²⁷I and ¹²⁹I nuclides and subsequently determined using inductively coupled plasma mass spectrometry (ICP-MS) and accelerator mass spectrometry (AMS), respectively. The concentration of ¹²⁹I and ¹²⁹I/¹²⁷I atom ratio in surface soil samples in the 30km-zone of CNPP ranged from 4.6 to 170mBq/kg and from 1.4 x10^-6 to 13 x10^-6, respectively. These values are significantly higher than the global fallout values of ¹²⁹I, indicating the most of ¹²⁹I was attributed to the fallout of the accident. Stable iodine concentrations in this area were found to be very low (below 1ppm) for most of the samples, suggesting the environmental iodine levels in this area are potentially low. The ¹²⁹I /¹³⁷Cs activity ratio in surface and sub-surface soils was not so constant, i.e. in the range 7.3 - 20.2 x 10^-7. This might be due to the different behavior of deposition and/or migration of these nuclides. The results suggest that the obtained data of ¹²⁹I should be useful reconstructing the ¹³¹I deposition in the contaminated areas.

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For the purpose to evaluate external radiation exposure of inhabitants who were living in settlements around the Semipalatinsk Nuclear Test Site, Kazakhstan, current levels of Pu-239,240 and Cs-137 contamination have been measured in soil samples taken around the test site. The trail of the radioactive plume could be clearly observed in the radioactivity distribution around several settlements. For example, the shape of the radioactive trail created by the first USSR atomic bomb test in 1949 was confirmed near Dolon village located 110 km from the ground zero. As a result of a Gaussian fitting of the radioactivity distribution, the center-line of the radioactive plume was supposed to have passed over about 2 km north of Dolon village. The initial deposition of Cs-137 due to the 1949 test was evaluated by subtracting the contribution of the global fallout from the measured values, and estimated to be 15 kBq m^-2 at the center-line of the plume, while it was 7 ± 2 kBq m^-2 in the Dolon village area. The deposition levels of fission products other than Cs-137 were estimated by considering their relative ratios of fission yield to Cs-137 as well as the fractionation effect during the transport-deposition process. Assuming a refractory/volatile fractionation factor of 5, external gamma-ray exposure was calculated at 1 m above ground from deposited radionuclides. A cumulative exposure of 350 ± 100 mGy was obtained for Dolon village, 80 % of which was delivered within one month after the deposition. Our cumulative exposure seems to be consistent with values of 440 - 480 mGy by other investigators based on TL measurement using brick samples in Dolon. Thus, local fallout contamination more than 50 years ago could be effectively reconstructed using the recent contamination data in soil.

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The nucleus is a place where genetic information is maintained by correct replication and segregation of chromosomal DNA and expressed by transcriptional machinery. To exert such fundamental reactions that are required to maintain biological functions efficiently, inner spaces in the nucleus are functionally separated to a high degree without membrane, displaying dynamic organization. To gain further insight into the mechanisms of systemic disorders resulting from radiation exposure, in addition to each fundamental cellular function, dynamic organization of the nucleus should be explored. In this workshop, we will discuss novel findings of nuclear organization, mainly repair pathways that respond to DNA damage, in association with diseases and try to understand the mechanisms underlying radiation-induced disorders, which are hardly understood by conventional researches.

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Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. On the other hand, increased expression of a DNA recombination protein RAD51 has been shown to induce chromosome translocations. However, how these DNA repair related proteins are implicated in chromosome translocations is still unclear.
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. Here we show that ATM deficiency results in the excessive binding of RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. 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.

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Chromosomal DNA in eukaryotes is wrapped around a histone octamer to form nucleosomes, the fundamental units of chromatin. Post-translational histone modification plays critical roles in regulating genome integrity, chromosome segregation, and gene expression. For example, DNA double strand breaks induce the modifications of histone H2AX, including acetylation, phosphorylation, and ubiquitylation. Phosphorylation of histone H3 is associated with chromosome condensation and segregation. The dynamics of such histone modifications have been analyzed using fixed or biochemically prepared samples so far. We now developed methods to visualize the dynamics of those modifications in living and permeabilized cells by using monoclonal antibodies. I will present here (1) visualizing the dynamics of histone H3 phosphorylation and chromosome missegregation in living cells using phospho-specific antibodies, and (2) detection of histone H2AX phosphorylation in permeabilized cells. These methods that allow the detection of protein modifications in situ will be useful for future studies on the radiation and chromosome biology.

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Epigenetic regulation has been recently shown to play a central role in cardiovascular pathologies. Chromatin remodeling factors including histone modifiers and enzymes have been shown to regulate developmental processes to pathologies such as cardiac hypertrophy and even aging at the molecular, cellular and animal levels. This has brought upon a paradigm shift from the classical thinking weighed on signaling pathways to that of emphasis on nuclear regulation and processes. Research in this field is still in early stages, however.
Data from our lab showing involvement of epigenetic regulators in cardiovascular pathologies will be discussed. Notably, involvement of chromatin remodeling molecules and DNA damage/repair molecules in cardiovascular pathologies at both the cellular and animal levels will be discussed.

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DNA double-strand breaks induced by ionizing radiation and chemotherapeutic agents activate damage response pathways initiated by ATM or ATR, resulting in the recruitment of DNA repair proteins at the damaged sites. Dynamics of RAD51, a central player at early stages of homologous recombination, in the nucleus have been well analyzed with being an excellent model in this field. Since homologous recombination consists of complex processes, it is regulated by a variety of proteins. However, dynamics of these proteins are only analyzed from the viewpoint of their association with RAD51. Identification of their dynamics in the nucleus contributes to exploration of the network of nuclear functions as well as a better understanding of their molecular functions. We have therefore examined the dynamics of DNA repair proteins that function in the proximity of RAD51 using human cells as models. It is well established that RAD52 plays a critical role in homologous recombination in yeasts, whereas its role in higher eukaryotes is unclear because its mutation does not exhibit drastic changes. When dynamics of RAD52 were examined using GFP-tagged RAD52, its focus formation reached the peak later than that of RAD51. Double immunofluorescence revealed that some foci colocalized with each other but others did not. These observations indicate that RAD51-independent role of RAD52, in addition to its RAD51-dependent role, is important in DNA repair. Such examination of dynamics in the nucleus proposes that DNA double-strand break repair may be more complicated than current models.

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It has been considered that environmental biota (living organisms) could be protected, when human beings are properly protected from ionizing radiation. However, radiation protection on non-human biota is recently becoming a matter of concern. Are there any common mechanisms in the radiation effect in a variety of living organisms?
This workshop is planned by Dr. Y. Kubota and Dr. S. Fuma, Environmental Radiation Effects Research Group, NIRS, where many studies are carried out on the environmental radiation effects. A diversity and similarity will be discussed in the radiation effects of rice crop, woody plants, microbial ecosystem, and soil arthropods. Interesting discussion will be anticipated from the views of biology as well as radiation protection.

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Conservation of biodiversity requires understandings of the effects of chemicals and ionizing radiation on diverse organisms not only at population levels but also at molecular levels. We identified ionizing radiation-inducible genes in the collembolan Folsomia candida. Transcripts induced by 26 Gy of acute gamma-ray irradiation were screened using high-coverage expression profiling. Those included the gene encoding proteins involved in detoxification of oxidative stress, DNA repair, and molting. Some of them were also significantly induced by 200 mGy of acute X-ray irradiation.

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We have been investigating molecular changes in rice (Oryza sativa -crop/grass/genome model) leaves against various environmental stimuli. Our examination was extended to stress responses of rice to ultra low-dose ionizing radiation (IR) first using radioactively contaminated Chernobyl soil (CCS) from exclusion zone around Chernobyl reactor. For this purpose we established a two-week-old rice seedling in vitro model system. Rice leaves were irradiated 72 and 96 h above CCS (contained mainly Cs-137 as gamma-ray emitter), giving 5.34 microGy/day. First results revealed induction of stress-related marker genes and secondary metabolites in irradiated leaf segments over appropriate control. Secondly, employing the same in vitro model system, we replicated the first experiment using in-house fabricated Cs-137 sources with various gamma-ray intensities (2 microGy/day–100 microGy/day) and selected genes by RT-PCR. Results imply that ultra low-dose radiation elicits a defense/stress response in rice, a novel finding, suggesting rice plant as a simple and good model for investigating IR responses. Our colleague, Kimura S, will present details of experimental method at the poster session.
Reference. Rakwal, R. et al., 2009. Int. J. Mol. Sci. 10:1215-1225.

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Effects of ionizing radiation on trees (woody plants) have been demonstrated in the studies on the effects of atomic bomb radiation, the irradiation experiments in gamma-fields, the biological investigations after the Chernobyl accident and so on, resulting in growth inhibition, morphological changes, mutation and death of trees. Woody plants are subjected to abiotic and biotic stresses throughout their long lives, thus it is assumed that they are developing defense mechanisms against various environmental stresses. However, the defense mechanism against ionizing radiation in woody plants remains unclear. In herbaceous plants such as Arabidopsis thaliana and rice, several genes including DNA repair genes were characterized, whose expressions were increased by γ-irradiation. Therefore, woody plants are expected to have the similar or unique mechanisms for protection to ionizing radiation.
We study on the effects of γ-irradiation on Lombardy poplar (Populus nigra var. italica) and the defense mechanisms against γ-radiation. Gamma-irradiation on the poplar caused growth inhibition, death of plantlets, abnormal leaf shape and color, shortening of internodes, cell death in roots and nuclear DNA damage. To elucidate the molecular defense mechanism against γ-radiation, we performed DNA microarray analyses. The up-regulated genes by γ-irradiation in the poplar shoots contained the DNA repair genes such as DNA ligase IV, XRCC4 and RAD17 and the oxidative stress-responsive genes such as peroxidase, cytochrome P-450 and glutathione metabolism enzymes. These results suggest that the poplar plants have the defense mechanisms which suppress the direct and indirect effects of γ-radiation.

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The public concern about radiation protection of non-human species and environment is increasing. The development of an international framework for environmental radiation protection is encouraged by international organizations such as ICRP, UNSCEAR and IAEA. The purpose of environmental radiation protection is to conserve plants and animals, to maintain the diversity of species, the health and status of natural habitats. To achieve the purpose, the ICRP task group is developing a reference-animals-and-plants approach. This approach is useful for the estimation of the dose-response relationships at the individual level, but it may be difficult to estimate the effects of radiation on community and ecosystem levels by that approach because ecosystems consist of biotic and abiotic factors, where these factors can be organized into species, populations and communities. Ecosystems are really complicated systems. In addition to the reference-animals-and-plants approach, a holistic approach to the ecosystem is also necessary to ensure environmental radiation protection.
We have tried to assess the influence of ionizing radiation on ecosystem by adopting top-down approach. In the presentation, we describe the impact of ionizing radiation on microbial community in a paddy field and on the release of elements and ions from the paddy soil as an example. Although it is thought that bacteria are not sensitive to ionizing radiation exposure, the results of our study revealed that bacterial community structure, that is bacterial species compositions and the relative amount of each bacterial species, were influenced by a dose of about 5 Gy. In addition to the changes in the bacterial community structure, concentrations of sulfate and iron in the paddy water significantly increased and decreased, respectively. These results suggest that the functioning of the microbial ecosystem in the paddy field is influenced by radiation exposure.

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Pyrimidine dimers are the major DNA damage induced by solar UV light, and are repaired by nucleotide excision repair (NER) in human cells. Defects in NER are associated with 8 types of autosomal recessive diseases including xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, which have diverse clinical features in addition to skin hypersensitivity to sun exposure. One of other UV-induced DNA damage is DNA single strand break (SSB). Aprataxin which plays an important role in SSB repair is a protein causative for autosomal recessive ataxia (EAOH/AOA1). Interestingly, impaired nuclear import of aprataxin was discovered in both triple A syndrome and cerebellar ataxia type 14. This WS focuses on understanding the molecular pathogenesis of DNA repair diseases with diverse clinical symptoms.

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To get a clue to understand how mutations in the XPD gene result in different skin cancer susceptibilities in patients with xeroderma pigmentosum (XP) or trichothiodystrophy (TTD), a thorough understanding of their nucleotide excision repair (NER) defects is essential. Here, we extensively characterize the possible causes of NER defects in XP-D and in TTD fibroblasts. The 3 XP-D cell strains examined were similarly deficient in repairing UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) from genomic DNA. The severity of NER defects correlated with their UV sensitivities. Possible alterations of TFIIH (which consists of 10 subunits including XPD) were then examined. All XP-D cell strains were normal in their concentrations of TFIIH, and displayed normal abilities to recruit TFIIH to sites of UV-induced DNA damage. However, replication protein A (RPA; single-stranded DNA binding protein) accumulation at DNA damage sites, which probably reflects the in vivo XPD helicase activity of TFIIH, is similarly impaired in all XP-D cell strains. Meanwhile, all 3 TTD cell strains had ∼50% decreases in cellular TFIIH content. Importantly, 2 of the 3 TTD cell strains, which carry the major XPD mutations found in TTD patients, showed defective recruitment of TFIIH to DNA damage sites. Moreover, RPA accumulation at damage sites was impaired in all TTD cell strains to different degrees, which correlated with the severity of their NER defects. These results demonstrate that XP-D and TTD cells are both deficient in the repair of CPDs and 6-4PPs, but TTD cells have more multiple causes for their NER defects than do XP-D cells. Since TFIIH is a repair/transcription factor, TTD-specific alterations of TFIIH possibly result in transcriptional defects, which might be implication for the lack of increased incidence of skin cancers in TTD patients.

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The biological importance of nucleotide excision repair (NER) in humans has been indicated by studies of autosomal recessive human genetic disorders: xeroderma pigmentosum (XP) and Cockayne syndrome (CS), in which NER activity is impaired. XP patients are hypersensitive to Ultraviolet (UV) in sunlight and show an increased incidence of UV-induced skin cancers. Although CS patients are sensitive to UV, they have no predisposition to sunlight-induced skin cancer, but instead show severe developmental and neurological abnormalities as well as premature aging. Seven NER-deficient complementation groups have been identified in XP (XP-A to XP-G), and two in CS (CS-A and CS-B). In addition, XP-B patients and certain patients with XPD or XPG gene show features of CS in addition to symptoms of XP (XP-B/CS, XP-D/CS and XP-G/CS). The clinical features of CS in XP-G/CS patients are difficult to explain on the basis of a defect in NER.
We found that XPG forms a stable complex with TFIIH, which is active in transcription and NER. Mutations in XPG found inXP-G/CS patient cells that prevent the association with TFIIH, also resulted in the dissociation of CAK and XPD from the core TFIIH. These results provide an insight into the role of XPG in the stabilization of TFIIH and the regulation of gene expression, and provide an explanation of some of the clinical features of XP-G/CS.

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Mutations in CSB gene give rise to two kinds of disorders. One is Cockayne syndrome (CS) group B showing photosensitivity, abnormalities in physical and neurological development, and another is UV-sensitive syndrome (UVsS) characterized by only mild photosensitivity without any abnormalities in physical and neurological development. One of the UVsS patients, UVs1KO, has no CSB protein due to homozygous null mutation in CSB gene, while various types of mutant CSB proteins are produced in CS-B cells. We have hypothesized that mutant CSB proteins produced in CS-B cells may have an inhibitory function, leading to CS-features. It is known that transposable element PGBD3 is integrated in the intron 5 of CSB gene. We found that the CSB-PGBD3 fusion mRNA is not targeted to nonsense-mediated mRNA decay even when 5'-terminal part of CSB has a nonsense mutation, resulting in the stable production of truncated CSB protein in CS-B cells. We also found that the wild type and truncated CSB proteins interacted with topoisomerase I (Top1), and the truncated CSB proteins inhibited catalytic cycle of Top1, implicating that the CS features are caused at least by an inhibition of catalytic cycle of Top1.

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Aprataxin is a protein causative for autosomal recessive ataxia that occurs most commonly in Japan. We previously found that aprataxin has an enzymatic activity and that mutants lose their activities. Further experiments using aprataxin knock-down cells, patient cells, and patient cerebellum demonstrate that this protein is involved in vivo in repair of oxidative stress-induced DNA single strand break (SSB). Other researchers demonstrated that aprataxin catalyzes removal of 5’-AMP and 3’-phosphate at SSB, suggesting that this protein plays an important role in SSB repair.
We then identified impaired nuclear import of aprataxin in an autosomal recessive disease, triple A syndrome. This disease shares some neurologic features with amyotrophic lateral sclerosis, but infrequently shows ataxia. Other symptoms include esophageal achalasia, alacrima, and adrenal insufficiency. The causative protein is ALADIN, a component of nuclear pore complex. In triple A syndrome oxidative stress increased DNA damage and cell death along with decreased nuclear aprataxin.
Further research demonstrates that nuclear import of aprataxin is regulated by its phosphorylation. Protein kinase C gamma, a protein causative for autosomal dominant cerebellar ataxia type 14 (SCA14), phosphorylates aprataxin at the residue near nuclear localization signal, and disturbed the nuclear import. A PKC inhibitor increased nuclear aprataxin, DNA repair capacity, and cell survival.
In conclusion, these findings demonstrate that qualitative and quantitative alterations in the DNA repair protein aprataxin are associated with neurological diseases and that oxidative stress causes accumulation of DNA damage leading to neurodegeneration.

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Radiobiological effect is a complex multistep-event containing the physical, chemical and biological sub-steps. Among the sub-steps, DNA damaging (between chemical and biological steps) is the most important one leading the final consequences such as cell death and transformation, because it is the initial step with bio-molecular changes directly ad initium. Thus, to study DNA damage has always occupied an important place in radiation biology. Recently, DNA damage in radiation biology is thought to be stating of obvious matter, and reports for DNA damage are decreasing in the late meetings. However, there have been the most studies for only simple structural damage such as DNA double strand break and oxidative base lesions. We have less knowledge about more complex structural damage like clustered DNA damage and cross-link damage, and about DNA damage induced by heavy ion beams. Additionally, we often tend to conventional analyzing method without necessary deliberation. In this workshop, we provide three progress reports about such important themes for undefined problems including clustered DNA damage and cross-link damage, and also present an issue about analyzing method for radiation DNA damage. Finally, we offer the special featured histrical review for this theme by Dr. Osamu Yamamoto, a great emeritus professor for radiation DNA damage.

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Spectra of DNA damage (strand breakage, base lesion, damage distribution, etc.) induced by ionizing radiation are considered to be different among radiation qualities. Although investigators are obtaining information about the difference, it is difficult to conclude overall nature of DNA damage by means of only a method for detecting a DNA damage. So, it is expected that we can have new knowledge by comparing data obtained by two or more individual methodologies.
So far, we have obtained some DNA damage information of ⁶⁰Co gamma-rays, etc., by comparing data from analytical methods. For example, in yields for single strand break on dry DNA sample induced by ⁶⁰Co gamma-rays, we found that the yield obtained by using phosphodiesterase I [1], by which calculating an absolute strand break yield, is 1.3 - fold larger than that obtained by super coiled plasmid method (utilizing its conformational changes by a strand breakage). This result implies that it is necessary for us to conclude nature of the damage under sufficient discussion about nature of a methodology. Particularly, it is quite important for us to review and reconsider the methodology, particularly when investigating, e.g., chemical and spatial existence of clustered damage.
In this presentation, we will discuss an approach to obtain beneficial and ungarbled DNA damage information for contributing connection of radiation chemistry with radiation biology.
[1] Akamatsu, K., Anal. Biochem. 362 (2007) 229-235.

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The higher biological effect of the ionizing radiation suggests some specific character of damage induced by them. When ionizing radiation passes cell as a beam, it might produce multiple lesions gathering around the passing beam on the target DNA. This unique form of DNA damage induced by ionizing radiation is termed “clustered DNA damage”, which occurs rarely in events of other damaging agents. There have been several studies showing that clustered DNA damage more strongly inhibits DNA replication and resists DNA repair than isolated DNA damage. Thus clustered DNA damage is thought to be a major cause for the serious biological effects induced ionizing radiation. High-LET radiations such as heavy ion beams usually conduce to more severe victims than low-LET radiations like X-rays and γ-rays. This severity with high-LET radiations might be derived form its higher condensation of ionizations than low-LET ones. Also it suggests some differences of the yields and/or characters of induced clustered DNA damage between high and low LET radiations. In this study, we compared the yield and characters of clustered DNA damage induced by γ-rays, carbon ion beams and iron ion beams. The LETs were 0.2, 13, and 200keV/μm, respectively. The yield of clustered DNA damages in both purified target DNA molecules and the chromosomal DNA in irradiated CHO cells showed decreasing trends with increase of LET [γ > C > Fe]. We show the quantitative analysis of the intracellular clustered DNA damage with pulse field gel electrophoresis. We also characterize the clustered DNA damage induced by these radiations with oligonucleotide target molecules. Our results suggest that the most accountable cause of the biological effect with heavy ion beams is the quality of induced clustered DNA damage rather than its quantity.

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