Ionizing radiation generates various genomic lesions such as base damage, strand breaks, and DNA-protein crosslinks (DPCs). The formation of base danmage and DNA double-strand breaks (DSBs) is promoted by the presence of oxygen, whereas that of DPCs is inhibited, showing an opposite trend of oxygen dependence. Base damage is repaired by base excision repair, and DSBs are repaired by homologous recombination (HR) and nonhomologous end joining. However, the principal repair mechanism and biological effect of DPCs remain largely elusive. We irradiated transplanted mouse tumors under normoxic and hypoxic conditions and analyzed DSBs and DPCs. The yields of DSBs were greater under normoxic than hypoxic conditions, and those of DPCs were greater under hypoxic than normoxic conditions. It was also suggested that DPCs induced by ionizing radiation are stable relative to those induced by aldehydes. The roles of nucleotide excision repair (NER) and HR in the repair of DPCs were investigated using mammalian cells and their cell-free extracts. The results showed that the maximum size of DPCs amenable to NER was 8 kDa, eliminating its repair role for DPCs. It was also suggested that the replication fork stalled by remaining DPCs is reactivated by HR. In in vitro transcription reactions with T7 RNA polymerase, DPCs in the transcribed, but not nontranscribed, strand strongly inhibited transcription. DPCs significantly reduced the transcriptional fidelity.

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It had been accepted that non-cancers such as vascular disease and cataracts are the effects caused by high-dose exposure, thereby limitedly considered for radiation protection purposes. In January 2011, Task Group 63 of ICRP issued a draft report dealing with such non-cancer effects in a radiation protection context, followed by public consultation. In this draft report, vascular disease and cataracts were considered as important late effects, and proposed the reduced threshold dose of 0.5 Gy. Subsequently, the ICRP statement issued in April 2011 recommended a reduced equivalent dose limit for the lens of the eye. IAEA has already set out to include such reduced dose limits in its next Basic Safety Standards. In this contribution, we shall demonstrate the outline of such influential draft report and its potential issues.

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Exposure to radiation is thought to affect the immune system, but little is known about direct relationship between radiation-associated immunological alterations and disease development. Among A-bomb survivors, significant changes are still observed in the immune system in association with radiation dose, such as decreased naive T-cell populations, impaired IL-2 production, reduced TCR repertoire, and increased less competent memory T cells. Similar attrition of T-cell mediated immunity appears in the ordinary aging process. A link between alterations in T-cell immunity and inflammation among A-bomb survivors is suggested. Known to increase with age, plasma levels of inflammatory proteins, such as IL-6, TNF-α and CRP, increase dose-dependently among A-bomb survivors. Percentages of both Th1 and Th2 cells also increase with age and radiation dose, a phenomenon that may be involved in the enhanced inflammatory cytokine production in A-bomb survivors. Interestingly, there are inverse, significant associations between plasma levels of inflammatory cytokines and the relative number of naive CD4 T cells, suggesting that the elevated levels of inflammatory markers found in A-bomb survivors are in part ascribed to T-cell immunosenescence. Thus, it can be proposed that radiation exposure accelerates immunosenescence linked to enhanced inflammatory responses that may lead to development of inflammation-related diseases including cancers in A-bomb survivors.

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Recent accumulating evidence including epidemiologic study of atomic bomb survivors suggests that chronic incidence of non-cancer diseases such as cardiovascular diseases increases by the exposure of low dose radiation, which is going to be taken into account in radioprotection regulation. It is, however, controversial that risk estimation model based on data obtained by high dose/high dose rate radiation can be applied to risk estimation for low dose/low dose rate radiation as in the case of risk estimation for cancer. Inflammation is one of the biggest risk factors for non-cancer diseases. We have demonstrated that continuous exposure of low dose rate gamma-radiation ameliorates type II diabetes and rheumatoid arthritis in mouse models. Through meta-analysis, we detected significant reduction of gene expression such as IL-1alpha, CD80, and iNOS by continuous low dose rate irradiation for 17 days, in mice implanted with allogeneic tumor cells, while that of p53-downstream targets such as p21 and mdm2 was significantly reduced. Continuous low dose rate irradiation significantly reduced footpad swelling evoked by concanavalin A, and suppressed pulmonary metastasis of Lewis lung carcinoma that is know to depend on inflammatory response. These results suggest that biological responses to continuous low dose/low dose rate radiation are qualitatively different from those to high dose/high dose rate radiation. We propose that dose rate effect should be taken into account for the risk estimation of non-cancer diseases for low dose/low dose rate radiation.

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We previously reported a significant increase in body weights in B6C3F1 female mice continuously exposed to low dose-rate (20 mGy/ 22 h/ day) gamma-rays as compared to that of age-matched non-irradiated control mice. To clarify the underlying mechanisms of body weight gain observed after exposure to continuous low dose-rate irradiation, we examined adipose tissue weights, liver and serum lipid contents, and factors related to glucose and lipid metabolism (serum insulin and adipocytokines), as well as ovarian dysfunction in female B6C3F1 mice continuously irradiated with gamma-rays at 20 mGy/ 22 h/ day from 9 to 44 weeks of age. Significant body weight gains, related to tissue adiposity, were observed from 28 to 44 weeks of age (accumulated dose = 2.7–4.9 Gy) in the irradiated mice as compared to those of non-irradiated mice. Histopathological analyses of ovaries and vaginal smears showed depletion of viable oocytes leading to premature menopause occurs at the same period when increased body weight gain was observed in the irradiated mice. These results suggest a possibility that the radiation-induced premature menopause could trigger the induction of body weight gain due to adiposity in B6C3F1 female mice continuously irradiated with low dose-rate gamma-rays at 20 mGy/ 22 h/ day. This study was performed under contract with Aomori Prefectural Government, Japan.

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Apoptosis can trigger compensatory proliferation of surrounding cells to maintain tissue homeostasis. While oxidative stress is tightly associated with apoptosis and necrosis, it remains unknown whether oxidative stress contributes to compensatory proliferation. We show that interleukin (IL)-11, a member of the IL-6 family, is produced by cells in an oxidative stress-dependent manner. IL-11 production largely depends on oxidative stress-mediated ERK2 activation, which in turn induces phosphorylation and accumulation of the transcription factor Fra-1 by preventing its proteasome-dependent degradation. Fra-1 is subsequently recruited to the Il11 promoter and activates transcription. Upon acute liver injuries in mice, IL-11 is mainly produced by hepatocytes in response to reactive oxygen species that are presumably released from dying hepatocytes. IL-11 induces phosphorylation of STAT3 in adjacent live hepatocytes, resulting in compensatory proliferation. Furthermore, administration of an IL-11 agonist enhances proliferation of hepatocytes and ameliorates oxidative stress upon acetaminophen (APAP)-induced liver injury. Conversely, APAP-induced liver injury is exacerbated in Il11 receptor alpha 1^-/- mice. Collectively, these results suggest that IL-11 provides a functional link between oxidative stress and compensatory proliferation.

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Hematopoietic stem cells (HSCs) can self-renew and differentiate into all hematopoietic lineages. Since the hematopoietic system is a regenerative tissue with a high proliferative potential, HSCs are sensitive to extracellular oxidative stress such as radiation and chemotherapeutic agents. The human HSCs enriched by selection for CD34 antigen, which the highly glycosylated transmembrane protein, have widely been used in studies of in vitro hematopoiesis. However, CD34⁺ cells are heterogeneous populations that contain various functional cells such as lineage-committed progenitors, early progenitors. Although the relationship between the cell surface antigens expressed on HSCs and hematopoiesis has become clearer, little information has been reported on the relationship between the heterogeneity of different populations of HSCs and their radiosensitivities. Therefore, we focused on the expression of the early hematopoiesis-related antigens CD38, CD45RA, CD110 and Tie-2 (tyrosine kinase with immunoglobulin and the epidermal growth factor homology domains 2) antigens including CD34 in HSCs. Consequently, their radiosensitivities were dependent on the presence of immature HSCs expressed Tie-2 antigen. We evaluated the relationship between radiosensitivity and expression of NFE2-related factor 2 (Nrf2) target genes, such as heme oxygenase 1 (HO-1) and NAD(P)H dehydrogenase, quinone 1 (NQO1), in HSCs. Nrf2 is a key protein in the coordinated transcriptional induction of expression of various antioxidant genes. A statistically significant negative correlation was observed between the surviving fraction of HSCs and the intrinsic NQO1 mRNA expression, indicating that HSCs in which NQO1 mRNA levels are low may also be radioresistant. The results suggest that the antioxidant system associated with Nrf2 is involved in the radiosensitivity of HSCs.

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We examined two biological endpoints in mouse neurosphere cells for evaluating the characteristics of stem cells; the repair kinetics of X-ray-induced DNA double strand breaks (DSBs) and the pathway toward immortalization by successive subculture of the cells. Fibroblast cells and neurosphere cells were obtained from embryos of ICR mice at day 14.5 of gestation. First, we examined the repair kinetics of DSBs after 1 Gy of X-irradiation by scoring phosphorylated histone H2AX (gamma-H2AX) foci and chromosome breaks visualized by premature chromosome condensation (PCC). The results indicated that the significantly less gamma-H2AX foci and PCC breaks were evident in the neurosphere cells than in the fibroblast cells until 1 h after irradiation, suggesting that the repair ability of DSBs in the neurosphere cells is more efficient than that in the fibroblast cells. Second, to establish a stable neurosphere cell line, we examined three successive subculture protocols whereby the neurosphere cells were plated at a density of 2X10⁵ cells into a 25-cm² flask and subcultured every 3 days (3T2), 5 days (5T2) or 10 days (10T2), with two additional trials (3T2 and 10T2) for the fibroblast cells as controls. We found that the neurosphere cells subcultured by the 5T2 and 10T2 protocols and the fibroblast cells subcultured by the 10T2 protocol extremely extended the lifespan (>100 PDN) whereas both cell strains subcultured by the 3T2 protocol senesced. Chromosome analysis revealed that chromosome numbers in the 50% lifespan extended neurosphere cells showed diploid whereas those in the fibroblast cells showed triploid and tetraploid. These results suggest that the pathway toward immortalization is different between the neurosphere cells and the fibroblast cells.

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Tissue stem cells can produce cancer if they are exposed to carcinogens such as ionizing radiation and accumulate sufficient oncogenic mutations. In 1950s, existence of mammary gland stem cells was first inferred by an observation that transplantation of a small number of mammary epithelial cells can reconstitutes a complete gland. During 1970–1990s, Kelly Clifton and his coworkers, by their quantitative transplantation/colony formation experiments, suggested that some cells with a potential of producing progeny population (termed “clonogenic cells”) survive irradiation and contribute to forming mammary cancer. Later in 2000s, a prospective method of isolating mammary stem cells based on their surface antigens accelerated identification of a hierarchy of cells within the mammary gland, including a progenitor cell population (called “luminal progenitor cells”) which gives rise to a special subtype of breast cancer called basal-like cancer. When reinterpreting Clifton’s work, with such and other new evidence in mind, it becomes clearer that acute irradiation of luminal progenitor cells or their ancestral cells leads to mammary gland carcinogenesis. Direct evidence for this hypothesis is awaited.

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Radiation-induced cancer mortality is presumed to be proportional to the dose of ionizing radiation, but it remains unclear whether the radiation-induced DNA damage and gene mutations accumulate in the tissue stem cells, putative targets of carcinogenesis. To elucidate the mechanism of the stem cell turnover, we used the lineage tagging system of Lgr5-cre/Rosa-lacZ mice to determine the kinetics of stem cell competition after ionizing radiation exposure. In this study, we found that the efficacy of tamoxifen (4OHT) in lineage tagging induction is different between small intestine and colon, and by the age of induction. Furthermore, a frequency of tagged crypts gradually decreased after 4OHT treatment. These results demonstrate that the dynamics of tagged crypt is regulated by both of the recombination frequency and monoclonal conversion in each crypt. Using this experimental model, we exposed 4 Gy of X-rays to the mice after 4OHT treatment and found that the frequency of tagged-crypts immediately decreases after irradiation. These results indicate that ionizing radiation may accelerate the turnover rate of intestinal stem cells.

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Bcl11b encodes a transcription factor that is expressed in various types of cells including T cells and the crypt cells of intestine. Mutations or deletion of BCL11B gene were found in 16% of human T-cell acute lymphoblastic leukemias and in ganma-ray induced mouse thymic lymphomas, indicating a tumor-suppressive role for Bcl11b in lymphoma/leukemia. We previously showed that loss of one Bcl11b allele contributes to clonal expansion of premalignant thymocytes in Bcl11bKO/+ mice after ganma-irradiation. However, what cells in the thymus are radiation targets leading to malignancy and how Bcl11b heterozygosity affects the irradiated cells remain open. We thus addressed these questions by developing Lck-Cre;Bcl11bflox/+ mice, where cells at a specific developmental stage lose a Bcl11b allele. The first half of the talk will be on this issue, and the rest will be about radiation effect on mouse intestinal crypt cells that comprise Lgr5+ CBC cells, known as active stem cells. Attenuated Bcl11b activity in mice impaired intestinal homeostasis and partially abrogated radiation-induced suppression of CBC cell proliferation, together with reduced induction of p53 tumor suppressor. This suggests that attenuation of Bcl11b activity promotes the regeneration capacity of CBC cells by elevating radio-resistancy. I will discuss whehter or not CBC cells are radiation targets leading to malignancy.

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Nematode shows a marked resistance to radiation as follows:

1. Radiation resistance decreased slightly throughout the first, proliferative phase of embryogenesis. This might be due to the increase in target size, since most cells of nematode are autonomously determined. Eggs irradiated in the second half of embryogenesis were about 40-fold more resistance to the lethal effects of X-rays. This is probably due to the absence of cell divisions during this time. X-irradiation of the eggs resulted in significant life-shortening of the animals. The radiation resistance increased more with advancing larval stages and adult stages.
2. The time spent as a dauer larva does not affect adult life span in Caenorhabditis elegans, as if aging is suspended in this quiescent developmental stage. Modest doses X-irradiation of dauer larvae increased their post-dauer longevity. Post-irradiation incubation of young dauer larvae did not modify this beneficial effect of radiation. Conversely, holding dauer larvae prior to irradiation rendered them refractory to this X-radiation-induced response.
3. Pre-exposure of wild-type Caenorhabditis elegans to oxygen conferred a protective effect against the lethality imposed by subsequent X-irradiation. In contrast, two mutants (rad-1 and rad-2) that are UV and ionizing radiation hypersensitive but not oxygen sensitive, did not exhibit this adaptive response.

As X-ray-induced single-strand breaks in DNA can be rapidly and efficiently rejoined by a repair mechanism, it is suggested that nematode has strong repair system.

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Reactive oxygen species (ROS) are generated in living cells during normal cellular metabolism as well as by exogenous stimuli such as ionizing radiation and various chemical oxidants. DNA carrying vital genetic information of cells constantly suffers from spontaneous oxidation by ROS. ROS produce a wide variety of oxidative damage to DNA, including various types of base modifications. Oxidatively damaged bases in DNA cause deleterious effects such as mutation, cell death and aging. Recently, relationship between oxidative damage to DNA and aging has been suggested, but the molecular mechanisms remain unsolved. Base excision repair (BER) is the main pathway to repair oxidative base damage. DNA glycosylase and AP endonuclease are essential to maintain genome stability by excising various types of DNA lesions. It is necessary to examine whether the mutation in the BER enzymes affects the life span of cells and organisms. The nematode C. elegans is a useful model organism for studying aging because its lifespan can be determined easily and quickly. However, there are little reports on BER enzymes of C. elegans. In this study, we identified and analyzed DNA glycosylases and AP endonucleases of C. elegans. CeNTH- and Ung-1-deficient C. elegans showed no significant difference in lifespan compared to the wild type worm N2. The results suggested the existence of other DNA glycosylase(s) that functions to repair oxidized pyrimidines and uracil in DNA. That is, the deletion of one DNA glycosylase could be complemented by other DNA glycosylases that are able to excise the same types of damaged bases. In addition, we are studying roles of the Exo-3 and Apn-1proteins in relation to aging and development in C. elegans.

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Oxidative DNA damage occurs directly in DNA and in nucleotides (dNTPs) in the nucleotide pool. Oxidized nucleotides such as 8-oxo-dGTP or 2-OH-dATP could be incorporated into DNA during replication, which could result in mutagenic consequences. Many organisms have enzymes for eliminating the abnormal nucleotides in the nucleotide pool as well as repair enzymes for damage in DNA. In this study, we have identified enzymes involved in sanitization of the nucleotide pool in C. elegans and analyzed the RNAi phenotypes of C. elegans under environmental stress. Together with our recent observations, we will introduce the mechanism of elimination of the oxidized nucleotides in C. elegans.

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The hormetic effect, which extends lifespan by various stressors, has been confirmed in Caenorhabditis elegans (C. elegans). We have previously reported that oxidative stress resistance in a long-lived mutant age-1 is associated with the hormesis. In an age-1 allele that activates an intracellular insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling pathway, the superoxide dismutase (SOD) and catalase activities increased during normal aging. We now demonstrate the changes of mitochondrial superoxide radical (^.O₂^-) levels in age-related strains under a hormetic condition. The ^.O₂^- levels in age-1 strain significantly decreased after intermittent hyperoxia exposure. On the other hand, this phenomenon was not observed in a daf-16 null mutant. This hormesis-dependent reduction of the ^.O₂- levels was observed even if the mitochondrial Mn-SOD was experimentally reduced. Therefore, it is indicated that the hormesis is mediated by any event suppressing the mitochondrial ^.O₂^- production. Moreover, we describe change in the oxygen consumption levels of the age-1 mutant in the oxygen radical-induced hormesis. The oxygen consumption levels in age-1 animal significantly decreased after intermittent hyperoxia exposure. These data suggest that oxidative stress-inducible hormesis is associated with reduction of mitochondrial ^.O₂^- production by activation of an antioxidant system via Ins/IGF-1 signaling pathway.

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Humans mount an immediate innate immune response to pathogen infection and also a slower but more specific adaptive immune response. While adaptive immunity is only shown in vertebrates, many aspects of innate immunity are highly conserved in metazoans. The nematode C. elegans is susceptible to several pathogens, and they colonize the digestive tract and finally kill the nematode. It has been reported a series of genes coded for antimicrobial compounds and defense proteins specifically induced by the bacterial infection. Many of the host genes involved in pathogen defense in mammals also function in host defense of C. elegans. We here found that the certain genes including lysozyme and mucin-like genes were also induced by exposure to ionizing radiation (IR). A mucin-like gene F49F1.6 codes a secreted surface protein with a part of similarity to mammalian Mucin-2. It seems that the induction occurs in the intestine from the result of irradiation with different depth. The F49F1.6 RNAi animals caused much severer growth retardation and slower recovery following IR exposure than those of mock RNAi control. These results suggest that the gene functions as a repair and barrier protein secreted of intestine in response to the bacterial infection and IR damage. We also clarify the mechanism of transcriptional regulation by GATA- and FOXO-transcriptional factors, and p38 MAPK signal transduction pathway. Altogether, these suggest that there is a cross-talk response between IR irradiation and innate immune response.

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To better understand the effects of radiation inherent to human activities such as manned space flight, diagnostic imaging, and radiation therapy, it is important to focus on not only the risks of carcinogenesis but also the potential for effects on critical functions such as learning, memory and/or motor control. The nematode Caenorhabditis elegans is a good in vivo model for the examination of radiobiological effects. Using this model, we recently found that whole body irradiation reduced both the locomotion using body-wall muscles and pumping motion (chewing and swallowing) using pharyngeal muscles, and that both movements were restored within several hours. However, there was an obvious difference in the aspect of the reduction between the locomotion and pumping. Though it is well-known that heavy ions charging particles with a high linear energy transfer (LET) induce higher relative biological effectiveness than low-LET radiation such as gamma-rays, effects of heavy ion beams on C. elegans's motility were same as those of gamma-rays. In this presentation, we will give an outline of the radiation effects on motor functions in C. elegans and discuss about further studies to understand the detailed mechanism underlying the irradiation-induced reduction and restoration of movements.

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The presence or absence of molecular oxygen dramatically influences the biological effect of low LET radiations. To produce oxygen effect, molecular oxygen must be present during the radiation exposure or at least during the lifetime of the free radicals generated by the radiation. Little study has been done to actually investigate the influence of oxygen after the radiation exposure. The present study was undertaken in order to explore the rejoining activity of DNA-DSB induced by anaerobic X-ray or carbon ion (∼80 keV/µm) irradiations under oxic and hypoxic holdings (37ºC). DNA-DSB in CHO cells were analyzed by a static-field gel electrophoresis. The kinetics of the rejoining could be described by a sum of fast and slow components. The slow component of DNA-DSB induced by X-ray under oxic incubation was faster than that under hypoxic incubation. Furthermore, the percentages of non-reparable DNA damage were 5% and 20% under oxic and hypoxic incubation conditions, respectively. However, no difference between oxic and hypoxic incubation conditions was found for carbon ion irradiation. There results indicate that molecular oxygen influences the rejoining of DNA-DSB after low LET radiation exposure.

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Tissue hypoxia is now recognized to contribute to the emergence of therapeutic resistance, including radiation-therapy. It has been known that hypoxic stress down-regulates variety of DNA repair gene expressions, but details of DNA-damage responses under hypoxic conditions remains unclear. Here, we tried to clarify details of the molecular mechanism of down-regulations and effects on DNA-damage responses in hypoxic cancer cells. Hypoxic down-regulations of MLH1, MSH2, MBD4, MRE11A, RAD51, and BRCA1 genes in an oral squamous cell carcinoma cell line, HSC-2, were confirmed by real-time RT-PCR. Their promoter analyses demonstrated that DEC transcriptionally repressed those promoters of the 6 genes via their HDAC-dependent transcriptional activities or physical competitions. Interestingly, hypoxic pre-culture of HSC-2 might delay sensing to DNA damages induced by irradiation and their repair monitored with gH2AX. Importantly, those altered responses to DNA damage in hypoxia-pretreated cells affected radiation sensitivity that was recovered by knock down of DEC2. These results suggest that the HIF-1-DEC system may play an important role in DNA damage responses, relating to anticancer therapies and protections against radiation exporsure, via transcriptional regulations in hypoxic cells.

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Tumor recurrence frequently occurs after radiotherapy, but the characteristics, intratumoral localization, and post-irradiation behavior of radioresistant cancer cells, remain largely unknown. Here we developed a sophisticated strategy to track the post-irradiation fate of cells which existed in perinecrotic regions at the time of radiation, and obtained direct evidence in vivo that this population predominantly causes tumor recurrence. Although the perinecrotic tumor cells were originally hypoxia-inducible factor 1 (HIF-1)-negative, they obtained HIF-1 activity after surviving radiation, which triggered their translocation toward tumor blood vessels and subsequent tumor recurrence. A HIF-1 inhibitor suppressed the translocation and decreased the incidence of post-irradiation tumor recurrence. For the first time, our data unveil the HIF-1-dependent cellular dynamics behind post-irradiation tumor recurrence and provide a rational basis for targeting HIF-1 after radiation therapy.

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Purpose: To evaluate the effect manipulating intratumor hypoxia on local tumor response and lung metastatic potential in radiotherapy including boron neutron capture therapy (BNCT). Methods and Materials: B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumors received γ-ray irradiation or reactor thermal neutron beam irradiation following the administration of a ¹⁰B-carrier (L-para-boronophenylalanine-¹⁰B (BPA) or sodium mercaptoundecahydrododecaborate-¹⁰B (BSH)) in combination with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH). Immediately after the irradiation, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of the quiescent (Q) and total (= P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumor-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Results: In the total cells, a more marked enhancement in sensitivity was observed with nicotinamide than MTH. In Q cells, MTH combination induced a more marked enhancement than nicotinamide. BPA-BNCT increased the sensitivity of the total tumor cell population more than BSH-BNCT. However, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. With or without irradiation, nicotinamide treatment decreased the number of lung metastases. With irradiation, BPA-BNCT, especially in combination with nicotinamide treatment, showed the potential to reduce the number more than BSH-BNCT. Conclusion: Acute hypoxia-releasing nicotinamide may be promising for reducing the number of lung metastases. BSH-BNCT in combination with MTH improves local tumor control, while BPA-BNCT in combination with nicotinamide may reduce the number of lung metastases.

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Hypoxia has been known to be a feature associated with tumor radioresistance. So far, clinical strategies to overcome chronic hypoxia due to the limitation of the oxygen diffusion have been designed. However, acute or intermittent/cycling hypoxia, which frequency can range from a few cycles per minutes to per hours, is receiving increased attention because this type of hypoxia has been reported to have an influence on tumor malignancy. Therefore, a priori information on fluctuating hypoxia may be important in clinical treatment planning, but complicated dynamics makes it difficult to elucidate biological significance of intermittent hypoxia.
Here, we introduce a newly-developed oxygen imaging method, pulsed electron paramagnetic resonance imaging (EPRI). The monitoring of EPR spectrum of paramagnetic radical tracer administered to mice, which line width is dependent on molecular oxygen, enables us to estimate quantitative oxygen concentration in tumor. We established rapid scanning and imaging by minutes, providing the existence of intermittent hypoxia and its frequency. We also report the magnitude of intermittent hypoxia was associated with the maturation of tumor vascular.
To clarify the biological effect of intermittent hypoxia, we focused the influence on radiosensitivity of cancer cells. We used rat glioma C6 cell line, which was revealed to develop intermittent hypoxia in its intracranial tumor by immunohistochemistry for two distinct hypoxic markers. When C6 cells were exposed to intermittent hypoxia with a few cycles of 1-hour hypoxia interrupted by 30-minutes reoxygenation, the radioresistance of cells was significantly enhanced as compared to the persistent hypoxia. Here, we reported new findings including the mechanism of radioresistance induced by intermittent hypoxia.

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This is the first workshop on carrier path in the radiation research field in Japan, which is organized by the Future Planning Committee of the JRRS. Through this workshop, we would like to offer constructing a network for education, research, and job opportunity within the Japanese Radiation Research Society.

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To survey the carrier path in radiation research field, we collected questionnaire from JRRS members, who hold full-time posts (including tenured).

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Young scientists have found it difficult to attain a permanent academic position in the radiation research field, and a system for carrier paths should be hence established. After obtaining a Ph.D degree, I was in term-limit positions at four different laboratories for a total of six years, and then gained an age-limit position. I shall give a few words from such experience.

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In our country, in spite of the excess number of Ph. D.s, the number of permanent academic positions has decreased, and as results, the limited-term researchers have increased. The limited-term researchers are certainly contributing for activation of research activities in our country, but they are feeling some kind of uneasiness because their future career paths seem not very clear. I also have kept my research career somehow, by getting limited-term postdoctoral positions so far. In the last year of my term, I have to look for the next job. For each job hunting, to make a decision, I referred to my research interests and direction at the time, the workplace and salary of the job, and the opinions of my family members and my teachers. I do not know whether my choice in each crossroads was right. However, I did my best in each situation, and have grown up through various experiences. In this workshop, I will talk about my research career path, and I am hoping you could make it reference for finding your career path.

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Exploring your science career in Japan

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Human tumors and cultured cells contain elevated levels of endogenous DNA damage resulting from telomere dysfunction, replication and transcription errors, reactive oxygen species, and genome instability. However, the contribution of telomere-associated versus telomere-independent endogenous DNA lesions to this damage has never been examined. In this study, we characterized the relative amounts of these two types of DNA damage in five tumor cell lines by noting whether γ-H2AX foci, generally considered to mark DNA double-strand breaks (DSBs), were on chromosome arms or at chromosome ends. We found that while the numbers of non-telomeric DSBs were remarkably similar in these cultures, considerable variation was detected in the level of telomeric damage. The distinct heterogeneity in the numbers of γ-H2AX foci in these tumor cell lines was found to be due to foci associated with uncapped telomeres, and the amount of total telomeric damage also appeared to inversely correlate with the telomerase activity present in these cells. These results indicate that damaged telomeres are the major factor accounting for the variability in the amount of DNA DSB damage in tumor cells. This characterization of DNA damage in tumor cells helps clarify the contribution of non-telomeric DSBs and damaged telomeres to major genomic alterations.

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DNA mismatch repair (MMR) is an important genome caretaker system, and is highly conserved from bacteria to humans. MMR corrects base-base mismatches and small insertion / deletion mispairs generated during DNA replication, recombination and repair. In addition, MMR can also repair oxidized base lesions. MMR consists of four steps: (1) recognization of mismatches in DNA and binding to mismatch sites (2) creation of a strand break as a starting point for the excision of the mismatched base (3) excision of the nicked strand from the nicked site up to and slightly past the mismatch (4) repair resynthesis and ligation. In Escherichia coli, MutS, MutL and MutH have been identified as MMR proteins. In human, MSH2, MSH3, MSH6, MLH1, MLH3, PMS1 and PMS2 have been identified. It is well-known that defects of these genes lead to an increased mutation rate. Several MMR genes, MSH2, MSH6, MLH1 and PMS2 have been identified in C. elegans. C. elegans is an animal model suitable for studies of aging and lifespan. Our purpose is to evaluate contribution of MMR for growth or lifespan. We measured phenotype and resistance to various mutagens of each mutants. In addition, we examined various activities of MMR proteins by using purified MMR proteins.

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Three members of phosphoinositide-3-kinase-like protein kinase (PIKK) family, ATM, ATR and DNA-PKcs, play distinct but sometimes redundant roles in regulating DNA damage response. Histone H2AX is one of the well-known substrates and is phosphorylated at Ser139 in response to double strand break (DSB) as well as stalled DNA replication forks or their collapse. We previously reported that histone H2AX is also phosphorylated in serum-starved quiescent cells exposed to UV in a nucleotide excision repair (NER)-dependent manner. Recently, we found that this phosphorylation is mediated not only by ATR in response to ssDNA gap formation but also by ATM possibly activated by DSB formation.
In this study, we focused on detecting DSB in G0-arrested cells following UV irradiation and analyzing a biological role of ATM signaling under this condition. A neutral comet assay revealed that longer comet tails were detected in G0-arrested normal cells but not in XP cells following UV irradiation, demonstrating NER-dependent DSB formation. The time course of DSB formation was correlated well with that of ATM signaling activation. Moreover, under G0-arrested condition, AT cells were found to be more sensitive to UV than normal cells, indicating the significant contribution of ATM signaling pathway to UV response in G0-arrested cells. We will also discuss more detailed mechanism of NER-dependent DSB formation.

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Ciona intestinalis (Chordata, Ascidiacea) is probably the most studied and cosmopolitan species of ascidians. The Ciona genome contains ~16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. Although the whole pathway of Ciona DNA damage repair is to be illustrated, Ciona has similar DNA repair proteins as those of human. We have already characterized 8-oxoguanine DNA glycosylase (ogg1) and endonuclease _III_ (nth) homologues. It is suggested thatCiona DNA damage repair pathway is similar to that of human. Apurinic/apyrimidinic (AP) site is a typical DNA damage that occurs spontaneously or as an intermediate in base excision repair pathway. AP site causes impaired DNA replication and transcription. AP endonuclease incises DNA adjacent to abasic sites to initiate DNA repair and counteract the cytotoxic and mutagenic effects of AP sites. We study on the Ciona intestinalis AP endonuclease homologue (CiAPE). First, we cloned the CiAPE gene and purified CiAPE protein expressed as a GST-fusion protein in E. coli. Second, we used an E. coli mutant that is deficient in AP endonucleases and therefore hypersensitive to DNA-damaging agents that produce AP sites. The E. coli mutant was partially rescued by expression of CiAPE. CiAPE protein exhibited cleavage activity toward THF (tetrahydrofuran) AP site. In addition, CiAPE localization in Ciona embryo is under investigation by expressing venus protein from 3 kbp upstream region of CiAPE.

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To study the induction of bipyrimidine DNA photoproducts by natural sunlight, ice-cold aqueous DNA solution in dishes was exposed to sunlight on fair days from June to October in 2010 on the roof of Nara Medical University's building. UV-B irradiated DNA was also prepared as a reference. DNA photoproducts (CPDs, 6-4PPs or DewarPPs) were then measured by ELISA with lesion-specific monoclonal antibodies (TDM-2, 64M-2 or DEM-1). Both sunlight and UV-B induced CPDs in a dose-dependent manner, and almost same numbers of CPDs were formed between 100 J/m2 UV-B and 1-2 hrs sunlight exposure. UV-B produced 6-4PPs efficiently, while sunlight poorly induced 6-4PPs with the peak at 3-4 hrs after exposure. In contrast, UV-B produced only low levels of DewarPPs, while sunlight induced DewarPPs efficiently. In a comparison between the sum of each 1 hr-exposure damage by sunlight and corresponding consecutive-exposure damage, the latter showed much less 6-4PPs formation, but there was no difference in the induction of DewarPPs. These results indicate that the conversion rate of 6-4PPs into DewarPPs increases with increasing sunlight exposure compared with the constant induction rate of 6-4PPs, and thus DewarPPs become the second frequent bipyrimidine photoproducs following CPDs.

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The oxidative stress caused by low LET radiation is considered as a risk factor to induce alteration of genetic information. In DNA, among four bases, guanine is most susceptible to oxidation, and its simple oxidized form is 8-oxoguanine (8-oxoG). 8-OxoG is a potent pre-mutagenic lesion because it can pair with adenine as well as with cytosine during DNA replication. To investigate the oxidative DNA damage caused by reactive oxygen species as an indirect effect of low LET radiation on various cell type, we examined the intestines of wild-type mice received a whole-body irradiation. Using immuno-histochemical method, we detected 8-oxoG in both nuclear DNA and mitochondrial DNA. At 1 hour post irradiation (X-ray, 4 and 10 Gy, 1 Gy/min), we observed only slightly increased 8-oxoG in nuclear DNA of intestinal epithelial cells in both villi (non-proliferative cells) and crypt regions (proliferative cell rich). In contrast to the nuclear signal, a significant increase of mitochondrial 8-oxoG in villous epithelial cells (but not in cryptic cells) was observed. To clarify the oxygen effect, we analyzed cellular oxygen consumption of intestinal epithelial cells by Hypoxiprobe-1. In the intestine of non-irradiated mice, the villous cells were more hypoxic compared to the cryptic cells, however after radiation, oxygen consumption in villous cells increased. These results suggest that X-ray radiation increases the level of 8-oxoG predominantly in mitochondrial DNA and may lead prolonged oxidative stress caused by mitochondrial disfunction.

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Heavy ion irradiation is known to cause serious biological damage due to the complex DNA damage it induces. However, the recovery/repair mechanism associated with this kind of damage is not well-understood. Recently it was found that the choice of DSB (double strand breaks) repair pathways depends on the chromatin structure. Heterochromatic DSB are generally repaired more slowly than euchromatic DSB, and ATM signaling is required for DSB repair within heterochromatin (Noon et al., 2009 Nature Cell Biology). Therefore, we decided to study the relationship between DSB and chromatin structure using gamma-H2AX assay. We compared the characteristics of gH2AX foci in normal human fibroblasts (48BR), ATM defective (AT1BR) cells, and XLF defective cells exposed to X-rays, carbon ions (LET 70 keV/μm), and Fe ions (LET 200 keV/μm) using a high resolution microscope (Delta Vision). Gamma-H2AX foci and clusters of them were evaluated and characterized under the criteria we determined. Gamma-H2AX foci with heavy ions were larger than foci with x-rays, and distribute in cluster inhomogenously. Over 60% of foci induced by X-rays were classified as single focus which were repaired quickly (by 2hrs post irradiation) and the rest in clustered forms were repaired more slowly and completed by 24 h post-irradiation. Following Fe ions, 70% of foci were categorized as clusters; most of clustered foci did not disappear 24 h after irradiation. The clustered foci with carbon ions were repaired with the slow kinetics (2-24 h).

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Fanconi anemia (FA) is a rare hereditary disorder characterized by malignant tumors, chromosomal instability, and an extreme sensitivity to DNA cross-linkers. Altogether 15 FA genes constitute the FA pathway, which is thought to stabilize the stalled replication forks. DNA damage and replication stress trigger the monoubiquitination of the key FA proteins FANCD2 and FANCI, which is mediated by a multi-subunit E3 ubiquitin ligase (called the FA core complex). Although monoubiquitination of FANCD2 is critical for DNA crosslink repair, how FANCD2 functions in the DNA damage response is still poorly understood. In this study, we tried to identify FANCD2/FANCI-associated proteins. We have purified FANCD2 or FANCI complex by two-step immunoprecipitation from HeLa S3 cell stably expressing HA-FLAG-tagged FANCD2 or FANCI that was challenged with the replication inhibitor hydroxyurea, and associated proteins were identified by mass spectrometry. The FANCD2/FANCI complex contained recently reported FAN1 nuclease, the FA core complex components, and novel interacting proteins that may have an important role in the FA pathway. We will discuss functional implications of these findings.

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We estimated the rate of repair of UV-induced DNA lesions in HeLa cells arrested at mitosis. The removal of (6-4) photoproducts from the genome overall in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that DNA in condensed mitotic chromosomes is not necessarily inaccessible to DNA-transacting factors or metabolically inert. However, not all modes of DNA repair seem feasible during mitosis. The removal of cyclobutane pyrimidine dimers from certain genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression.

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Ionizing radiation induces base damage, DNA strand breaks, and DNA-protein crosslinks (DPCs). We have previously shown that DPCs are repaired by nucleotide excision repair and homologous recombination in bacterial cells, whereas they are repaired by homologous recombination in mammalian cells. Since DPCs are extremely large compared to conventional bulky lesions such as pyrimidine dimers and aromatic adducts, it is likely that they impede the progression of replication and transcription machineries. However, not much is known about how DPCs affect replication and transcription. In the present study, we have analyzed the effect of DPCs on transcription using an in vitro transcription system. Transcription reactions were performed with DPC-containing templates and T7 RNA polymerase (T7RNAP), and products were analyzed by denaturing PAGE. With templates containing DPCs in the transcribed strand, transcription was strongly arrested by DPCs, resulting in truncated transcripts. In addition, transcription passed through DPCs, resulting in weak bands of runoff transcripts. The amount of runoff transcripts decreased with increasing the size of corsslinked proteins. With templates containing DPCs in the nontranscribed strand, transcription passed through the DPC site, resulting in no truncated transcripts, but the amount of runoff transcripts decreased (about 50%) relative to the control template.

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DNA double strand break (DSB) induced by ionizing irradiation is one of the most serious damage, which causes such as cell death, mutation, and cellular malignancy. To maintain genomic stability, DSB is repaired by various repair pathways including homologous recombination (HR) and non-homologous end joining (NHEJ). In present study, we established Nbs1/Ku70 double knockout cell line using chicken DT40 cells and analyzed its phenotypes, especially their response to radiation. Although Nbs1/Ku70 cells were extremely sensitive to radiation, and they displayed significant slow DSB rejoining rate, about 50 % of DSB was rejoined within 30 minutes even in the double knockout cells. This suggests that there should be another dominant DSB repair pathway(s) substitute for typical HR or NHEJ. In addition, Nbs1/Ku70 double knockout cells were died by apoptosis similar rate in wild type cells, while induction of apoptosis following irradiation was significantly suppressed in Nbs1 deficient cells.

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Ionizing radiation efficiently induces DNA double strand breaks (DSBs). There are two major pathways to repair those DSBs. One is homologous recombination (HR) and the other is non-homologous end joining (NHEJ). HR is thought to occur during late-S and G2 phase of cell cycle because it requires sister chromatid as a repair template. NBS1, a component of MRN (MRE11/RAD50/NBS1) complex plays a crucial role in HR. We investigated whether the partial inhibition of NBS1 could affect HR efficiency or not. A mutant form of NBS1 was constructed and was introduced into HeLa or MRC5 cells. Expression of the mutant NBS1 resulted partial inhibition of radiation induced foci formation and significant reduction of HR frequency. Cells expressing the mutant NBS1 were slightly sensitive to X-rays compared to the cells ectopically expressing wild-type NBS1. This difference in radiosensitivity between wild-type and mutant NBS1-expressing cells was enhanced when the cells were irradiated with a split dose. These observations suggest that partial inhibition of NBS1 suppress HR and inhibits recovery from sublethal damage.

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The monoubiquitination of FANCD2 and FANCI proteins (ID complex) is central to Fanconi anemia (FA) pathway activation under replication stress. Our previous study indicated that damage-induced FANCI phosphorylation serves as a molecular switch to activate FANCD2 monoubiquitination. It was reported that knockdown or hypomorphic ATR reduces damage-induced FANCD2 monoubiquitination. To further evaluate the contribution of ATR-ATRIP kinase complex to FA pathway, we established ATRIP conditional knockout DT40 cell line. After excision of the exons encoding the ATR-interacting domain of ATRIP, ATRIP expression was abolished, leading to the loss of mitomycin C-induced FANCD2 monoubiquitination and FANCI phosphorylation. We also show that ATR phosphorylates recombinant FANCI protein in vitro, which is facilitated by the presence of FANCD2. In addition, we found that FANCD2 monoubiquitination was abrogated in ATRIP mutants lacking interaction site for RPA but not for TopBP1, both essential for Chk1 phosphorylation. These results establish a key role of ATR-ATRIP kinase complex for FA pathway activation in a manner that is independent of TopBP1.

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DNA-PK is well known as a kinase which phosphorylates many repair factors of DNA double-strand breaks. Previously we reported that the radiation-sensitive mutant RIC1 strain has defects in the foci formation of phosphorylated histone H2AX (gH2AX), G2/M checkpoint, Apoptosis induction and homologous recombination (HR) after g-irradiation. Here we examined the involvement of the ric1 gene in ATM and DNA-PK function. ATM inhibition resulted in reduced gH2AX intensity and HR efficiency after DSBs in wild-type cells, but no significant effect in RIC1 cells. These results suggest that the ric1 gene associates with DNA-PK and aberrant DNA-PK function or regulation causes various defects after DSBs in RIC1 strain. Furthermore we suggest that DNA-PK mainly phosphorylates H2AX in medaka. We will discuss ric1 gene function and DNA-PK function in medaka.

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ATM is known to be a key regulator in DNA damage response. Ataxia-Telagiectasia disorder, which mutated ATM, shows hyper radio-sensitivity, abnormal cell cycle checkpoint and genome instability. Recently, several AT-like disorder have been reported and their responsible genes might have a functional interaction with ATM in DNA damage response. Therefore, we investigated DNA damage responses in cells derived from one AT-like disorder, AOA3 (ataxia oculpmotor apraxia 3).
AOA3 cells showed sensitivity to DNA-damaging treatments, an increase in oxidant stress and abnormal cell cycle checkpoint. Furthermore, AOA3 cells showed reduction in ATM-dependent phosphorylation in response to exposure to γ-ray or H2O2. In vitro ATM kinase assay revealed that AOA3 cells have a defect in ATM activation. Interaction of ATM with NBS1, which is indispensable for ATM activation, also reduced in AOA3 cells, Moreover, pre-treatment of H2O2 in normal cells repressed activation of ATM-related responses. These results suggested that increases of oxidative stress could abrogate ATM-related damage response.

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Genomic stability in higher organisms is established by elaborate DNA repair systems and cell cycle checkpoints. We try to dissect the molecular pathology of human genomic instability diseases in order to understand the protective mechanism against various stresses such as ionizing radiation.
BUBR1 is a central molecule of the mitotic spindle assembly checkpoint. Germline mutations in the BUB1B gene encoding BUBR1 cause premature chromatid separation (mosaic variegated aneupoidy)[PCS(MVA)]syndrome, which is characterizes by constitutional aneuploidy and a high risk of childhood cancer. Patient with the syndrome often develop Dandy-Walker complex and polycystic kidneys; implying a critical role of BUBR1 in morphogenesis. However, little is known about the molecular function of BUBR1 other than mitotic control.
Here we report that BUBR1 is essential for the primary cilium; a microtubule-based organelle on the surface of most vertebrate cells in G0 phase, and that PCS(MVA) is thus a novel ciliopathy. Morpholino knockdown of bubr1 in medaka fish also caused ciliary dysfuncion characterized by defects in cerebellar development and perturbed left-right asymmetry of the embryo. Biochemical analyses demonstrated that BUBR1 is required for ubiquitin-mediated proteasomal degradation of CDC20 in the G0 phase and maintains anaphase-promoting complex/cyclosome (APC/C)-CDH1 activity that regulates the optimal level of dishevelled for ciliogenesis.

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The E3 ubiquitin ligase RNF20 regulates chromatin structure by monoubiquitinating histone H2B in transcription. Here, we show that RNF20 is localized to double-stranded DNA breaks (DSBs) independently of H2AX and is required for the DSB-induced H2B ubiquitination. In addition, RNF20 is required for the methylation of H3K4 at DSBs and the recruitment of the chromatin-remodeling factor SNF2h. Depletion of RNF20, depletion of SNF2h, or expression of the H2B mutant lacking the ubiquitination site (K120R) compromises resection of DNA ends and recruitment of RAD51 and BRCA1. Consequently, cells lacking RNF20 or SNF2h and cells expressing H2B K120R exhibit pronounced defects in homologous recombination repair (HRR) and enhanced sensitivity to radiation. Finally, the function of RNF20 in HRR can be partially bypassed by forced chromatin relaxation. Thus, the RNF20-mediated H2B ubiquitination at DSBs plays a critical role in HRR through chromatin remodeling.

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DNA double-strand breaks (DSB) repair requires the processing of the broken DNA ends to accomplish the ligation. Non-homologous end-joining (NHEJ) is a main mechanism of DSB repairs in mammalian cells. DNA polymerases mu (pol mu) and lambda (pol lambda) belong to mammalian pol X family members and form a specific complex with Ku and XRCC4-LigIV, which are essential for DNA end-processing in NHEJ. The live cell imaging has shown that these NHEJ factors rapidly recruit to the DSBs. However, it has not been clarified how pol mu recruits to the DSBs in vivo. We report here the detailed mechanism of pol mu's recruitment to the DSBs. We first observed pol mu at DSBs in living cells after laser micro-irradiation. pol mu accumulated at DSBs immediately after irradiation. By photo-bleaching and photo-conversion techniques, we found that pol mu exchanges between the DSBs and the nucleoplasm within a few seconds. Furthermore, pol mu accumulated at DSBs independently of Ku80. These observations are quite different from those of Ku80-interacting NHEJ core factors, i.e., Ku70, DNA-PKcs (DNA-dependent protein kinase), XRCC4, and XLF (XRCC4-like factor), which Ku80-dependently accumulate at DSBs. Whereas the N-terminal region of pol mu containing BRCT domain, which binds to Ku80, Ku80-dependently accumulated at DSBs, the C-terminal pol beta-like region of pol mu Ku80-independently accumulated at DSBs. The pol beta-like region of pol mu contains DNA-binding motif HhH (Helix-hairpin-Helix) and PCNA-interacting motif (PIM)-like region. Both of them retained the ability of accumulating at DSBs. Biochemical analyses showed that gamma-ray irradiation stimulates pol mu-PCNA interaction and the interaction is sensitive to ethidium bromide, suggesting that dsDNA affects on the pol mu-PCNA interaction. Our studies show that pol mu recruites to DSBs at the first step during the NHEJ process and three functional domains in pol mu individually have abilities to accumulate at DSBs after irradiation.

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NBS characterized by high sensitivity to IR and UV, chromosome instability, and predisposition to lymphoid cancer. NBS1 functions to the cell cycle check point and the HR repair after IR. However, the function in the UV damage response mechanism is unknown. We show that NBS1 is recruited to UV-induced damage and DSBs by distinct mechanisms. NBS cells were high sensitivity to UV and NBS1 accumulated in the stalled replication fork. Moreover NBS1-deficient cells showed reduced RAD18 and Pol eta focus formation, and severely reduced mono-ubiquitination of PCNA. NBS-deficient cells were high mutation frequency in similar Pol eta-deficient cells. We show that NBS1 binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Pol eta focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting motif of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting motif of RAD6. These motifs of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously, and are crucial for UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis.

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Radiation-induced bystander responses are biological effects observed in cells that receive signals from irradiated cells but are not directly exposed to ionizing radiations. Using a synchrotron X-ray microbeam irradiation system developed at the Photon Factory, KEK, we recently showed that nitric oxide (NO)-mediated bystander cell killing is parabolically enhanced in a dose-dependent manner. In this study, we irradiated 5 nuclei of V79 cells with 10 × 10 µm² 5.35 keV X-ray beams and then measured the mutation frequency in the bystander cells by using the HPRT mutation assay. The mutation frequency with the null dose was 2.6 × 10^-5 (background level) and decreased to 5.3 × 10^-6 with a dose of approximately 1 Gy (absorbed-dose in the nucleus); at higher doses, however, the frequency returned to the background level. Same type of biphasic dose-response was observed during the bystander cell killing, as shown in our previous study. Similarity of these behaviors in dose-response may suggest a correlation between the enhancement of bystander cell killing and the suppression of spontaneous mutagenesis in the bystander cells. Increase in oxidative damage in unstable cells which have lost an antioxidative ability is responsible for spontaneous mutagenesis. The genetically unstable cells in the population of bystander cells might be selectively excluded by the bystander cell killing as NO caused mitochondrial degeneration, and hence the suppression of mutagenesis was observed in the bystander cells.

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Recently, heavy-ion radiotherapy attracts attention as a new cancer therapy, but the response of cancer cells which are irradiated by heavy-ion beams has not yet been clarified. Particularly, bystander effects are important to upgrade heavy-ion radiotherapy, because they may bring some influences to the outside of the irradiated area. So, it is required to clarify the mechanisms of bystander effects and their influences in the therapy. The purpose of this study is to detect bystander cell-killing effects between normal fibroblasts and lung cancer cells with different p53 status and to elucidate its mechanisms. In the study, we used human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wtp53 line which produces normal p53 proteins in their DNA damage response. We irradiated cells with carbon-ion broad beams (LET = 108 keV/μm, Dose = 0.5 Gy) or gamma-rays (LET = 0.2 keV/μm, Dose = 0.5 Gy), then calculated survival rates of bystander cells after 6- or 24-hours co-culture of irradiated and non-irradiated cells using colony formation assay. When we co-cultured irradiated H1299/wtp53 cells with non-irradiated WI-38 cells, it was found that survival rates of WI-38 cells were not decreased at all. Consequently, it was suggested that bystander cell-killing factors were not released from irradiated H1299/wtp53 cells.

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There are many reports available concerning bystander cellular effects after exposure to alpha particles derived from ²³⁸Pu or He-ion microbeams. However, only limited sets of studies have examined bystander effects after exposure to different ion species heavier than helium. In this study we investigated bystander cell-killing effect in both normal human fibroblasts and human tumor cell lines irradiated with low energy (6MeV/n) carbon ions generated with the Medium Energy Beam Course at the HIMAC. Cells were inoculated onto the Mylar film in the dishes and then irradiated with carbon ions. Irradiation was carried out using 4 different protocols; (1) All cells attached on the Mylar film were irradiated; (2) Irradiated and un-irradiated cells were pooled in a 1:1 ratio and plated as a single culture; (3) Half of cells were irradiated using ion-beam stopper; and (4) Half of cells were irradiated with treating a specific inhibitor of gap-junction mediated cell-cell communication. The surviving fraction, which was assayed with a colony formation assay, in the cells with wild-type p53 gene was the same between the irradiation condition (1) and (3) and significantly lower than that of the condition (2). Also, the surviving fraction of the condition (4) was the almost the same with the condition (2). On the other hand, in the cells with mutated-type p53 gene it was the same level among the condition (2), (3) and (4) and significantly higher than that of the condition (1). The results showed that bystander lethal effect was observed in both normal and tumor cells harboring wild-type p53 gene, but not in p53-mutated tumor cells. Moreover, observed bystander effect was suppressed by treating with a specific inhibitor of gap-junction mediated cell-cell communication. The results suggest that both p53-mediated cellular response and gap-junction-related bystander effect are an important role of carbon-ion induced bystander lethal effect.

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[Background] Recent studies have suggested that ionizing radiation enhances metastasis and invasion of tumor cells. Although some factors such as MMP, which degrades extracellular matrix, and RhoA, which regulates cellular cytoskeleton, are thought to be involved in this phenomenon, its precise mechanism has not been clearly shown. Assuming that radiation-induced bystander effect influences on cellular invasive ability, we here studied whether irradiated cell condition media (ICCM) enhanced invasive ability of non-irradiated cells.
[Methods] Human lung cancer-derived A549 cells were irradiated with 2 or 4 Gy (2.55 Gy/min) of X-rays, and the media (ICCM) were collected for the experiments. In order to evaluate cellular invasive ability, ICCM containing 1% FBS were added to the lower chamber of Matrigel Invasion Chamber (BD) and non-irradiated cells suspended in fresh media were loaded into the upper chamber, and the number of invaded cells was counted. The effect of ICCM on ERK phosphorylation was examined by Western blotting.
[Results] ICCM enhanced cellular invasive ability in a dose-dependent manner, and induced ERK phosphorylation. These results suggested that ICCM enhanced cellular invasive ability via ERK.

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