T4 endonuclease V is an enzyme that repairs DNA by cleaving the 5'-side of the thymine base in thymine photo-dimers in a DNA duplex as a pyrimidine dimer glycosylase. The mode of recognition of unusual base pairing between a thymine photo-dimer and adenine base by the enzyme was studied by X-ray analysis.¹⁾
Mutagenesis and carcinogenesis of damaged bases are well known. 7,8-dihydro-8-oxoguanine (8-oxoguanine).²⁾ and pyrimidine photo-dimers³⁾ in c-Ha-ras genes, were investigated by using synthetic genes containing modified nucleotides.
The genes for monoclonal antibodies specific for cyclobutane pyrimidine dimmers, the (6-4) DNA photoproduct and the Dewar isomer, have been coned and the amino acid sequences of the variable domains were determined. Site directed mutagenesis of the scFv's was used to investigate the origin of the binding affinity of the antibody to the DNA lesion.⁴⁾
1) Vassylyev, D.G. et al., Cell 83, 773-782 (1995).
2) Kamiya, H. et al., Cancer Res 52, 3483-3485 (1992b)
3) Kamiya, H. et al., Nucleic Acids Res 21, 2355-2361 (1993).
4) Kobayasi, H. et al., Biochemistry 38,532-539 (1998)

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Purpose: 2-Nitroimidazole nucleoside analog, RP-170 was developed as a potential radiosensitizer by POLA Corporation. We succeeded in synthesizing [18F] labeled fluoroRP-170 ([18F]FRP-170). A purpose of this study is to investigate the potential of this new drug as a new hypoxia marker. Materials and Methods: WHT/Ht albino mice bearing the squamous cell carcinoma (sqcc) or fibrosarcoma in the both axillas were used. Biodistribution and the tumor-to-blood(T/B)ratios of [18F]FRP-170 were investigated 120 hours after iv injection. Double-tracer autoradiography(DARG) for tumors was also performed with [14C]IAP and [18F]FRP-170. [14C]IAP was used as a tracer of blood flow. Patients: 16 patients with lung cancer and recurrent esophageal cancer were injected 185 MBq of [18F]FRP-170, and PET images (Siemens ECAT EXACT HR+) were obtained 120 min after injection. Results: The uptake rates of various tissues except for liver and kidney were less than that of tumors. The T/B ratios were 1.97(1.36-2.9) for sqcc and 2.50 (1.72-3.60) for fibrosarcoma. DARG with [18F]FRP-170 and [14C]IAP revealed that the distribution of [18F]FRP-170 was just reversed to the distribution of [14C]IAP. Obvious uptakes of [18F]FRP-170 were found in the most of 16 patients with lung cancer and recurrent esophageal cancers. Their tumor/muscle ratio were approximately 1.25 to 2.14. Conclusions: [18F]FRP170 surely showed tumor image in anywhere except for liver and kidney. We conclude that [18F]FRP-170 is a promising new hypoxia marker in cancer patients.

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Boron neutron capture therapy (BNCT) had a big subject into which high-concentration boron must be made to take in the cancer cell. If there is such an effective boron compound, a nuclear reaction occurs with epithermal neutrons, and it can become an effective cure. It turned out that boronophenylalanine (BPA) carries out high concentration accumulation in cancer cells. Then, since accumulation of boron can be evaluated in a living body by PET with quantitative nature, fluoroboronophenylalanine (FBPA) was a candidate and resembled BPA as a PET tracer. As a result, the active region and boron concentration of a tumor can be evaluated and it is recognized by medical treatment as an indispensable method on BNCT treatment. To avoid over-irradiation, it should be more prudent possible also by BNCT to come to utilize PET and to treat safely.
It became clear for adaptation expansion of clinical use on BNCT in the head and neck, lungs, etc. by FBPA-PET study. There is especially much air fraction in lungs and a neutron is in advantageous environment in adaptation of BNCT. Moreover, exact boron concentration can be predicted now also by the special medication, such as arterial injection methods. At multiple metastatic lung cancer in the limited area, if an exposure field is made large, it will be thought possible to treat simultaneously. Then, accelerator-based BNCT is also positively taken up by research projects in recent years, and has shown reality. If a practical use stage is reached, it is expected that it will contribute to the medical treatment of cancer greatly.

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Many solid tumors exhibit hypoxic regions as well as possess high interstitial fluid pressure (IFP). While hypoxia in tumors limits the efficacy of radiation treatment, high IFP limits the efficacy of chemotherapy by inhibiting the accumulation of effective levels of chemotherapeutic drugs. Additionally, hypoxic cells in tumors are more reducing and may offer opportunities for bioreductive drugs or selective normal tissue radioprotectors which do not modify the tumor radioresponse and improve treatment outcome.
We have evaluated a variety of non-invasive techniques to monitor tumor oxygen status, tumor IFP and the tumor redox status in experimental animals bearing murine as well as human tumors. Using Electron Paramagnetic Resonance Imaging (EPRI), we have been able to non-invasively map tumor pO2 in three-dimensions with high spatial, temporal resolution with sensitivity to discriminate oxygen status differences of +/- 2 mm Hg. We have also used Overhauser enhanced Magnetic Resonance Imaging (OMRI) to map anatomically co-registered pO2 maps as well as tumor IFP. Using conventional MRI with nitroxyl radicals as T1-sensitive contrast agents which are metabolized selectively to diamagnetic form in tumors, the tumor redox-status was examined.
In my presentation, I will present results on some recent studies using these techniques to non-invasively examine tumor physiology.

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Magnetic Resonance Imaging (MRI) has provided significant clinical utility in the diagnosis of diseases and will become a powerful tool to assess phenotypic changes in genetically engineered animals. Several studies have demonstrated non-invasive assessment of free radical reactions and redox state in small animals using low frequency electron spin resonance (ESR) spectroscopy and nitroxyl radicals. In vivo ESR signal intensities of nitroxyl radicals decrease with time after injection; and the decreases are enhanced by ROS, generated in oxidative disease models in a site-specific manner. Overhauser enhanced MRI (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity via the Overhauser Effect. In this paper, we show in vivo ESR analysis of free radical reactions in various disease models and images of dual labeled nitroxyl probes by changing the external magnetic field for ESR irradiation in field-cycled OMRI, which should offer significant applicability to nanometer scale molecular imaging and simultaneous assessment of redox state in gene-modified animals. These techniques may become powerful tools to clarify mechanisms of disease and to monitor pharmaceutical therapy.

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Health effects of low-dose-rate or low-dose radiation should be evaluated in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. Female gpt delta transgenic mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. The NNK treatments enhanced the frequency of base substitutions significantly, but no obvious combined effects of gamma irradiation were observed. In contrast, the NNK treatments appeared to suppress radiation-induced large deletions. The frequency of large deletions more than 1 kb in size increased in a dose-dependent manner of gamma irradiation. When NNK treatments were combined, however, the dose-response curve became bell-shaped where the frequency at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks in DNA. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed.

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To determine whether mutations continually induced by low-level radiation persist and accumulate in somatic stem cells in vivo, mice heterozygous at the Dlb-1 locus (Dlb-1^b/Dlb-1^a) were irradiated at a dose rate of 0.04, 0.86 or 15.56mGy/day for 483 days from 8 to 78weeks of age to attain total doses of 0.02, 0.42 or 8.0Gy, respectively. Small intestines were sampled from control and irradiated mice 2weeks after the completion of irradiation and subjected to the Dlb-1 mutation assay in which evidence of mutations of Dlb-1^b gene in the stem cells was detected as mutant clones on the surface of villi. About 10000 villi were observed for each of mice. Frequency of mutant clones(F) was defined as the number of clones detected per 10000 villi observed. F values in the control and the low, middle and high dose groups were 25.60±0.8, 25.62±0.8, 26.1±0.8, 35.2±1.0, respectively (N=10 for each group). Induced rate of mutant clones by unit dose of 1Gy is 1.38±0.33. Within experimental errors, this value is equal to induced rate of 1.20±0.08 that has been estimated based on F values determined for young heterozygous mice at ages of 8 to 13weeks after gamma irradiation with doses up to 8Gy in a dose-rate range of 0.3 to 3mGy/min. These results support the conclusions that (1) mutations continually induced by low-level radiation accumulate in somatic stem cells and persist for prolonged time; (2) susceptibility of stem cells to radiation mutagenesis does not change with age.

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Generations of students in radiation biology have been taught that heritable biological effects require direct damage to DNA. Radiation-induced bystander effect represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the biological consequences of exposure to low doses of radiation. Although radiation induced bystander effects have been well documented in a variety of biological systems, including 3D human tissue samples, the mechanism is not known. There is recent evidence that the cyclooxygenase-2 (COX-2) signaling cascade plays an essential role in the bystander process. The observations that heritable DNA alterations can be propagated to cells many generations after radiation exposure and that bystander cells exhibit genomic instability in ways similar to directly hit cells indicate that the low dose radiation response is a complex interplay of various modulating factors. A better mechanistic understanding of cellular and tissue responses to low dose / low dose rate radiation will provide important insights into how radiation induces its effects.

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Exposure of cell populations to ionizing radiation results in communication of signaling molecules between irradiated cells and between irradiated and non-irradiated cells in the population. This phenomenon, termed the 'bystander response', has been shown to occur both in vitro and in vivo. It affects the overall response of exposed cells and results in induction of significant biological effects in unirradiated bystander cells and their progeny. Genetic alterations, changes in gene expression and lethality have been shown to occur in bystander cells that neighbor directly irradiated cells. The roles and mechanisms of oxidative metabolism and gap-junction intercellular communication in modulating bystander responses in human cells are under intense investigation. Molecular and biochemical aspects of these processes in determining overall responses of cell populations exposed to high and low linear energy transfer radiations will be discussed.

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Transcriptome analysis permits to explore the field of low doses of radiation and potentially it can provide a global view of radiation responsive pathways. We have exposed a human lymphoblastoid cell line to either 0.02 or 2 Gy of ionizing radiation that yielded relatively little or faint cytotoxicity and little or no apoptotic DNA fragmentation. We used cDNA microarrays to examine the modulation of gene expression at various time points within 72 hours following gamma radiation exposure. We observed that 1) a lower number of genes are deregulated after 0.02 compared to 2 Gy, 2) some genes are specifically deregulated according to the dose while others are similarly deregulated whatever the dose, 3) all responsive genes after both doses and those specifically deregulated after 2 Gy are mainly involved in signal transduction, cytoskeleton, protein metabolism and catabolism, intracellular trafficking and transcription factors whereas genes specifically deregulated after 0.02 Gy are mainly related to signal transduction, cytoskeleton, stress response, ionic transport and channel 4) after both doses, responsive genes related to cell survival and death are in good agreement with data obtained on cell survival and death and 5) overall results support the hypothesis that low doses of ionizing radiation lead to a typical stress-induced translation inhibition and RNA processing alteration. This work underlines the need of further efforts to explore the effects of low doses of radiation.

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Low doses in vitro and in vivo induce an adaptive response that reduces both radiation-induced and spontaneous risks. A single low dose of low LET radiation increased the latency (with no change in frequency) of radiation-induced or spontaneous cancer in both normal and cancer prone (Trp53 heterozygous) mice. A prior low dose given during the time of fetal organ development lowered the risk of radiation-induced birth defects, and a low dose prior to a high dose protected the offspring of male mice from heritable mutations produced by a subsequent large dose. Chronic exposures protected against age related ulcerative dermatitis in Trp53 normal (but not Trp53 heterozygous) mice. These observations challenge the Linear No Threshold Hypotheses and other principles and practices used for radiation protection. Dose thresholds for increased risk of cancer are apparent. Below those dose thresholds overall risk is reduced below that of the unexposed controls, indicating that dose rate reduction factors (DDREF) approach infinity. Different tissues have different thresholds for detriment, indicating that individual tissue weighting factors (WT) are also not constant. Because risk from low LET radiation is not constant with dose, and dose responses from high LET are non-linear due to detrimental bystander effects, radiation-weighting factors (WR) for high LET radiation cannot be constant at low dose.

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The breast and ovarian tumor suppressor BRCA1 acts as a hub protein that coordinates many cellular pathways to maintain genomic stability. Its major biological functions includes DNA double-strand break repair by homologous recombination and regulation of cell cycle check points, transcription, apoptosis, and centrosome duplication. As a clue to elucidate the mechanisms underlying the BRCA's multiple biological functions we previously discovered that BRCA1 forms a heterodimeric RING-type ubiquitin ligase with BARD1. BRCA1-BARD1 catalyzes untraditional Lys-6-linked polyubiquitin chains that could be a signal for a process other than proteolysis. We reported that Nucleophosmin/B23 (NPM1) is one of substrates of BRCA1 and the NPM1 ubiquitination by BRCA1-BARD1 was abolished by CDK2-Cyclin A or CDK2-Cyclin E. This could be important for the maintenance of genomic stability through regulation of centrosome duplication. In addition we recently identified RPB8, a common subunit of RNA polymerases, as another substrate of BRCA1-BARD1 by a proteomics screen. Interestingly, HeLa cells stably expressing a ubiquitin-resistant form of RPB8 exhibited UV hypersensitivity, a phenotype ascribed to BRCA1 deficiency. Revealing the roles of BRCA1's enzyme activity, including its specific substrates, could translate into the clinic by providing a means to predict the sensitivity of breast cancers to DNA damaging anti-cancer agents or to predict a molecular target for therapeutic strategy.

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Fanconi anemia (FA) is a rare hereditary disorder characterized by bone marrow failure, compromised genome stability, and increased incidence of cancer. FA is caused by abnormalities that occur in components of the FA core complex, a key factor FancD2, and other related factors. In DNA damage responses, FancD2 is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. This monoubiquitination is critical for regulating nuclear dynamics of FancD2 as well as DNA repair through homologous recombination. In this talk, we will show previously hidden functions of the FA core complex.

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DNA double-strand break (DSB) formation is essential in several normal cellular processes, but accidental DSB formation may lead to cancer and death. A universal cellular response to a DSB is the phosphorylation of histone protein H2AX to form γ-H2AX in the chromatin flanking the break site. Immunostaining with anti-γ-H2AX reveals each nuclear DSB as a focus of γ-H2AX (γ- focus). Normal human cells exhibit increasing numbers of γ-foci as they senescence---from ~0.4 per early passage cell to >2 per late passage cell---and similar increases in the incidence of γ-foci occur in both the somatic and germline tissues of mice as they age. When nascent DSBs are generated in hu-man cell cultures with ionizing radiation, the numbers of nascent γ-foci are similar in cells at different stages of se-nescence but the rates of dimensional focal growth and focal accumulation of DSB-repair proteins are substantially slower in late passage cells and even slower in cells taken from Werner Syndrome patients, who exhibit premature aging and genome instability. Young fibroblast cultures induced to prematurely senesce by exposure to DNA damaging agents exhibit increases in DSB incidence similar to those found in replicative senescence, indicating that accumulating unrepairable DSB-containing lesions may be a casual factor in aging. The results demonstrate that mammalian cells aging in vitro and in vivo accumulate DSB-containing lesions that may play a causal role in aging.

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Medical management of the acute radiation injury might be an important area to which procedure of regeneration medicine and stem cell therapy could contribute, since organ stem cells might be targets of radiation injury. Furthermore, the recovery of blood flow is an important factor in the restoration of many tissues, therapeutic angiogenesis is assumed to become essential treatment in this field.
We have started a project of therapeutic angiogenesis by implantation of autologous bone marrow mononuclear cells (BMMNCs) since 2003 in conformity to the protocol of TACT (Therapeutic Angiogenesis Using Cell Transplantation) trial, which treated patients with severe lower limb ischemia.
Two cases of Buerger's disease were treated and their symptoms were improved within two to four weeks after the transplantation. Angiography indicated that there emerged new blood vessels in patient's treated limb. Augmentation of blood flow was suggested by thermography in one case. In both cases, any serious adverse symptoms due to this treatment were not observed.
In the present trial, we could recognize effectiveness and safety of therapeutic angiogenesis. We need to improve the present procedure with introduction of evolving knowledge regarding mechanisms of physiological, pathological and utilizable therapeutic angiogenesis, and should develop the best strategy of tissue regeneration medicine for victims of radiation exposure.

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Radiation-induced thyroid cancer studies have been one of the focuses of the International Medical Cooperation and Epidemiology project conducted in frame of Nagasaki University’s 21^st Century COE program.
Papillary thyroid carcinoma (PTC) is a prototypic human malignancy known to develop with an increased rate in the individuals exposed to external or internal radiation, especially if exposure takes place at young age. Since the recognition of the phenomenon of radiation-induced thyroid cancer, extensive efforts have been made to elucidate its distinctive molecular features and to determine a “radiation signature”.
Analysis of mutational frequencies of PTC-specific oncogenes (ret/PTC, TRK, BRAF, RAS and Gsa) or TP53 tumor suppressor demonstrated patients’ age-associated trend for some of them but no correlation with radiogenic nature of PTCs was found. To learn more about individual genetic characteristics of PTC patients, a comparative pilot study was performed to profile SNPs in several DNA damage response genes (ATM, MDM2 and TP53). Initial results demonstrated that some polymorphisms display a weak association with certain age groups of radiation-related or spontaneous PTCs.
Results of molecular investigations as well as those of molecular epidemiology studies seeking to identify genes that modify susceptibility to radiation-induced thyroid carcinogenesis in humans will be discussed.

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Despite considerable research effort, the mechanisms underlying the unequivocal association between accidental, occupational or therapeutic exposures to ionizing radiation and the development of leukaemia remain unknown. Conventionally, the responsible genetic lesions have been attributed to DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes. However, many reports of, so called, non-(DNA)targeted effects of ionizing radiation in the unirradiated descendants of irradiated cells (radiation-induced genomic instability) or in cells that have communicated with irradiated cells (radiation-induced bystander effects) challenge this conventional paradigm. In the context of the haemopoietic system, the two phenomena are inter-linked and an instability phenotype need not necessarily be a reflection of genomically unstable cells but a reflection of responses to ongoing production of damaging bystander signals in the tissue microenvironment. Both the production of and the response to such signals are influenced by genetic factors and the cell interactions have properties in common with inflammatory mechanisms. Thus, superimposed on damage induced directly in target stem cells, cell interactions make important contributions to determining overall outcome after radiation exposure and have significant implications for the potential health consequences.

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Conventional radiation is supposed to cause stochastic effects such as the development of cancer or genetic effects in proportion to the amount of radiation to which the subject is exposed. On the cellular level, it is believed that even small doses can damage DNA and the cell membrane. Olivieri and Wolff et al. discovered in 1984 that lymph cells of humans receiving a weak [3H]thymidine treatment show a radio-adaptive response. The elucidation of this radio-adaptive response drew attention in terms of understanding of radiation hormesis phenomena and evaluation of risk associated with radiation. In the 1990s, phenomenological studies of the cell and on an individual animal level were conducted and the conditions that induce a radio-adaptive response began to be clarified. However, though elucidation of conditions that induce a response indicates the involvement of cell membrane function and signal transduction system, the details were still unknown. In particular, how the radio-adaptive response of cells relates to the radio-adaptive response of individual animals was a mystery. The elucidation of this mystery will be a formidable challenge in future.

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In radiolysis of water, hydrated electrons and OH radicals are produced as the transient reactive species. They react quickly with solute molecules and corresponding products are formed. In biological system organic polymers and DNA molecules are damaged. Therefore, the understanding of water radiolysis is inevitably important to evaluate the radiation effects in living system. It is also significantly dependent on LET of radiation and, thus, related to RBE and OER. In the present lecture, (1) time and spatial dependence of water radiolysis, (2) behaviors of radiation induced transient species such as OH radical and hydrated electrons, (3) LET dependence of the radiolysis, and (4) direct effects and indirect effects would be presented together with recent experimental and theoretical results.

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The survival rate of mice after acute high dose irradiation is markedly improved by low dose whole-body preirradiation. The number of hematopoietic stem cells (HSCs) is related to the kinetics of the improvement in survival rate. One of the possible mechanisms underlying the adaptive response of mice is the acquisition of radioresistance by HSCs. Conventionally, preirradiation is carried out at a high dose rate (HDR). Therefore, the adaptive response induced by low dose-rate chronic irradiation is virtually unknown. We have been investigating the induction of such adaptive response in vivo in terms of the radioresistance of HSCs under very low dose-rate (around 1 mGy/hr) irradiation for different periods. As results, the survival rate of HSCs after acute challenge irradiation was enhanced by the low dose-rate irradiation but the acquisition showed specific intervals. We are interested in how HSCs acquire radioresistance with low dose-rate chronic irradiation to establish methods of evaluating adaptive response without performing challenge irradiation. Thus far, we have observed colony forming units in spleen (CFU-S), which was originally established for HSCs. Alternatively, we have evaluated HSC populations that indicate a more primitive stage than CFU-S populations without performing challenge irradiation. Further analysis of the effect of low dose-rate irradiation on such primitive stem cells will provide new directions of future research on the adaptive response of mice.

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Risk estimation for low dose of radiation is based on extrapolations from existing high dose data. This idea assumes that cellular responses, including DNA repair, play an equally efficient role at low and high radiation doses. However, the validity of this assumption in the recent year is challenged by some radiobiological phenomena, such as the bystander effect and radio-adaptive response. These phenomena have a significant impact on assessment of risk of exposure to low doses of ionizing radiation. In this study, to investigate the biological effect of low dose radiation, we analyzed the amount of initial phosphorylated ATM forms discrete foci in non-dividing primary normal human fibroblasts (MRC-5 cells) that irradiated of X-rays with doses ranging for 1.2 mGy to 1000 mGy and showed that phosphorylated ATM forms discrete foci are capable of detecting the radiation-induced DNA damage after radiation doses as low as 1 mGy. Also the induction of foci showed sharp linear dose-response relationship with doses ranging for 1.2 mGy to 5 mGy. This result indicated that very low dose of x-ray might occur bystander effect. In addition we investigated the time course analysis of radiation-induced DNA damage based on phosphorylated ATM forms discrete foci in MRC-5 cells. Here we present evidence that foci of phosphorylated ATM focus induced in culture of non-dividing MRC-5 cells by very low dose of X-rays remain unrepaired for a long time. This result suggested that the threshold in the repair of DNA damage exists.

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Biological effect of neutrons is generally larger than that of low-LET radiation. For the risk assessment of neutron exposure, it is important to evaluate RBE. ICRP determines radiation-weighted factor (w_R) for neutron by referring to the RBE of chromosome aberration, mouse life shortening and rat tumor induction (ICRP Publication 92).
Many animal studies on neutron carcinogenesis have been conducted to determine RBE, but its value varies among the species and strain of animals used and the tissue examined. The data regarding energy dependency is still lacking. To extrapolate the animal data to human, the precise dose estimation and energy distribution in target tissues are also needed. Taken together, both experimental and theoretical evaluation of physico-biological effects of neutrons on target tissues is important for the estimation for w_R.
In this workshop, current topics of neutron RBE and neutron sources in NIRS will be discussed.

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HeLa and CHO-K1 cells were irradiated with ions near the Bragg peak to determine how many ion traversals through a cell nucleus are necessary to induce cell inactivation. The ion traversals through a cell nucleus were visualized by immunostaining the phosphorylated histone H2AX (gamma-H2AX), as an indicator of DNA double strand breaks (DSBs), to confirm that DSBs are actually induced along every ion traversal through the nucleus. The survival curves after irradiation with ions decreased exponentially with the ion fluence without a shoulder. The inactivation cross sections were calculated from the slope of the survival curves, and the cross-sections for Fe ion, which was the highest LET, and the standard errors were 96.9 +/-1.8 and 57.9 +/-5.4 um2 for HeLa and CHO-K1 cells, respectively. This corresponding to 0.442 and 0.456 of the mean value of each cell nucleus area. Taking the distribution of the cell nucleus area into consideration with an equation proposed by Goodhead et al. (1980), which calculates the average number of lesions per single ion track through the average area of a sensitive organelle (mainly nucleus), these two ratios were converted to 0.705 and 0.659 for HeLa and CHO-K1 cells, respectively. These ratios were less than one, suggesting that the average numbers of lethal hits per cell produced by a single ion traversal were less than one.

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In the history of radiation biology, until relatively recently, major attention had been paid to the effects of radiation of high doses. This was the case not only for the basic biology, but also for the application of biological radiation effects in medicine, agriculture and other areas; cell killing effects or mutagenic effects of high dose radiation has been utilized. Recent research on biology at low dose range revealed a number of phenomena that cannot be explained by the effects observed at high doses, including radiation adaptive responses, bystander effects, and genomic instability. Certain types of radiation responses are thought to be involved in such phenomena. They are intriguing subjects as a basic science, and they also have an impact on the utilization of radiation and the framework of risk estimation.Here in this session, biological effects of low dose/dose rate radiation are reviewed and their significance will be discussed.

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Clinical treatment of high-dose radiation exposure depends on radiation dosage. It is very difficult to cure the victims exposed to more than 10 Gy even by high-level medicine currently available. Under the circumstances, we only expect the development of generative medicine against DNA damage. In the case of less than 10 Gy exposure, we can expect the curability that depends on the quality of medicine. We have to discuss how to improve the quality of radiation casualty medicine for less than 10 Gy exposure. The problem of medicine for high-dose exposure is that it rarely happens. However, we can learn many things from hematopoietic stem cell transplantation. In the case of total body irradiation, a total of 12 Gy divided irradiation and lethal doses of DNA-damaging agents are administered. It is very important to evaluate complication, particularly cardiac and pulmonary functions as well as organs that may be damaged by infection. It is therefore needed to pay attention to such problems to improve the outcome of acute radiation casualty medicine. We have to insist that prevention of diseases associated with life-style and treatment of infectious diseases are also important for radiation casualty medicine.

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Recently, significant interest has developed on the effects of exposure to electromagnetic fields on the human body, especially the issue whether or not the electromagnetic fields have carcinogenic potential. In our living environment, there are many devices and environments that generate electromagnetic fields, such as MRI machines used in medical care, the alternating magnetic fields near transformer stations and beneath power lines, electric household appliances, and linear motor cars that will be realized in the near future. In addition, exposure to high-frequency electromagnetic fields from the recent increase in use of mobile phones has jumped, with more than 90 million mobile phones in use in Japan today. Therefore, we are exposed to electromagnetic fields much more frequently than in the previous natural environments. Here, I review the present status of investigations on the effects of extremely low frequency (ELF) and radio frequency (RF) electromagnetic fields on living organisms, especially on the response of cells and genes to these electromagnetic fields, including studies conducted by ourselves. Animals and epidemiological studies on the effects of ELF electromagnetic fields are also reviewed. On the other hand, I introduce the evaluation for carcinogenic effects of electromagnetic fields by the International Agency for Research on Cancer (IARC), in which I had participated in the evaluation meeting at IARC as a member of working group.

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As early as 1950's in Japan, it was shown that electrical potential differences appear in dry bone subjected to mechanical stress. Since this finding, there are many researches that the application of an externally applied voltage, in particular, of electromagnetic fields (EMF) might have an effect on bone development. Although there are many application of EMF in the therapeutic use from the middle of 1970's, the concern has been raised over the effects of the exposure to 50/60Hz EMF (ELF-EMF) on human health after the publication of Wertheimer in 1979. Since then, many research efforts began to investigate possible association between ELF-EMF exposure and human health risk. The topics focused on leukemia and brain tumor in children and adult, breast cancer in adult. Experimental areas have included nervous system, physiology, reproduction, immunology, endocrinology. behavior etc in human and animals. Although IARC concluded that ELF magnetic field is possibly carcinogenic to humans (Group 2B), it remains that there are other explanations for the observed association between the exposure to ELF magnetic field and childhood leukemia. This presentation will shortly discuss the biological and health effects of ELF-EMF exposures from the points of human and animal studies, along with the discussion on the therapeutic application of electromagnetic phenomena.

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Biological effects of ultraound are classified as 1) thermal effects, 2)non-thermal cavitational effects, and 3)non-thermal and non-cavitational effects. First, the chemical effects of ultrasound in aqueous solutions are due to acoustic cavitation which refers to the formation, growth and collapse of small gas bubbles in liquid. The very high temperatures and pressures of collapsing gas bubbles lead to the thermal dissociation of water vapor into OH radicals and H atoms which are the same as observed in radiation chemistry. There is evidence for free radical formation in aqueous solutions and biological fluids, produced in vitro by exposure to continuous waves, tone-burst, and microsecond pulses of ultrasound at acoustic pressures similar to those which may be encountered during medical applications. Although there is evidence that free radicals produced by ultrasound are associated with cell killing in vitro, the extent of their role has not been established. Acoustic cavitation induced by ultrasound is of interest because of the wide use of diagnostic imaging and therapeutic application of safety considerations for posible deleterious effects due to physical and chemical effects of cavitation. Here, the role of cavitation in the induction of apoptosis and the change of gene expression is discussed.

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Therapeutic ultrasound has mainly been applied for its thermal or mechanical effects. Medical applications of high-energy ultrasound to ablate cancers are now under investigation. Recently, there have been numerous reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles and nanobubbles as a carrier/enhancer of drugs has added a whole new dimension in therapeutic ultrasound. Progress in the past decade from the pharmaceutical side has further added much excitement in applying this technology especially in the field of molecular biology, gene therapy and regenerative medicine. Alternatively from the device side, therapeutic ultrasound catheters and extracorporeal ultrasound probes are under development specifically for this purpose, some already in clinical trials. Non-invasive focused ultrasound in conjunction with anti-cancer drugs may help to reduce tumor size, lessen recurrence as well as reduce severe drug side effects. Chemical activation of drugs by ultrasound energy for treatment of atherosclerosis and tumors is another new field recently termed as "Sonodynamic Therapy". Lastly, advance in molecular imaging has also initiated great expectations in applying ultrasound for both diagnosis and therapy at the same time. Microbubbles or nanobubbles targeted at the molecular level will permit medical doctors to make a final diagnosis of a disease by ultrasound and immediately proceed to therapeutic ultrasound.

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High LET ionizing radiations show higher RBE than low LET ones. It is thought that clustered DNA damage specifically induced by high LET radiation is involved in higher RBE. Since recent study of DNA repair leads to utilization of various DNA repair enzymes, it is able to study more complex and underlying clustered DNA damage including base lesions (cluster base damage). To evaluate involvement of cluster base damage in the biological consequences of high LET radiation, we estimated amount of cluster base damage in DNA irradiated with heavy ion particles. Two species of DNA molecule, pDEL19 (~4.6 kbp) and lambda phage DNA (~46 kbp) were used as target substrates. These DNA were irradiated with gamma-rays (0.2 keV/μm), carbon (13 keV/μm) and iron ions (200 keV/μm) in 10 mM Tris-HCl (pH7.5). For detection of base lesions, the samples were digested by Escherichia coli endonuclease III for oxidative pyrimidines and E. coli Fpg or human OGG1 for oxidative purines, and subjected in gel electrophoresis and aldehyde reactive probe method. The result showed that amount of base lesions localized sparsely and in cluster decreased with increase of LET (gamma-rays > carbon > iron), like single and double strand breaks. The result indicates that not quantity but quality of cluster (base) damage is more important to elicit the biological effect of high LET radiation. Alternatively, cellular process of cluster base damage and variety of the final products of such process among different LET radiation seem to be important in the biological consequence of high LET radiation.

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Clustered DNA damage, defined as two or more lesions within one to two helical turns of DNA by a single radiation track, is a unique feature of ionizing radiation. We are currently focusing our interest on the biological effects of non-dsb clustered damage in vivo, whose effect has remained largely unknown. We have chosen an experimental approach that utilizes synthetic DNA containing base damage as a model of radiation induced clustered damage. One of the advantages of this strategy is that the effect of a specific type of damage could be clarified in detail. Using a bacterial plasmid-based assay, we have investigated the mutagenic potential of bistranded clustered damage sites which consist of 8-oxo-7,8-dihydroguanine (8-oxoG) and dihydrothymine (DHT) at defined separations. We found a significantly higher mutation frequency for the clustered DHT + 8-oxoG lesions than that for either a single 8-oxoG or a single DHT in wild-type and in glycosylase-deficient strains of E. coli. A marked increase in mutation frequencies was observed in mutY mutant compared to wild type. Even higher mutation frequencies were found in fpg mutY double mutant, reaching values around 35% of the rescued plasmids. These results suggest that (1) Fpg activity is compromised for removal of lesions within clustered damage sites (2) the loss of the DHT strand contributes, at least partly, for the enhancement of mutation frequency by the cluster, and (3) MutY is the most important glycosylase for reducing the mutagenic potential of the cluster.

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The oxygen effects that are prominent for low LET radiations become small with increase of the LET. Radio-chemical reactions are generally believed as the fundamental mechanisms underlying oxygen effects. In this research, we studied here significance of cell survival, initial DNA-DSB and unrejoined DNA-DSB in CHO cells after low- and high-LET radiation. The CHO cells were exposed to 200 kVp X-ray or HIMAC carbon ions (80 keV/μm) under oxic or hypoxic conditions. Amounts of DNA-DSB in the cells were analyzed by a static field gel electrophoresis. The oxygen enhancement ratio (OER) of cell killing after irradiation of carbon ion was 1.9, and that after X-ray was 2.8. The OER of DNA-DSB by carbon ion was 2.1, and was not affected by rejoining treatment. On the other hand, the OERs of initial DNA-DSB (3.7) and unrejoined DNA-DSB (5.8) after X-ray were affected by rejoining treatment. The quantity of initial DNA-DSB after carbon ions under oxic condition was larger than hypoxic condition. However, oxygen did not affect on the quality of DNA-DSB.

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It is well known that high-LET heavy ion is more effective for lethal effect than low LET radiation. It is considered that this high effectiveness is caused by heavy ion-induced clustered DNA damages, which are thought to be non-repairable or difficult to be repaired. However, much less is known about difficultly of clustered DNA damages repair in DNA repair process. The major DNA repair pathway in mammalian cells is non-homologous end joining (NHEJ). In this study we investigated NHEJ pathway by analyzing responses of Ku to DNA damage induced by high-LET heavy ions. pGFP and pGFP-Ku80 were transfected into Ku80-defective xrs-5 cells. Cells were irradiated with 7.2 MeV /u Ar¹³⁺beams (LET=1610 keV/μm) at TIARA JAEA-Takasaki. Sensitivity of Xrs5-GFP-Ku80 cells and xrs5-GFP cells to Ar¹³⁺was comparable. This result indicates that DNA damage induced by Ar¹³⁺is difficult to be repaired. To evaluate the response of Ku80 to DNA damage induced by heavy ions, GFP and γH2AX foci in the nuclei were observed. These foci were co-localized at 10 min after irradiation, while GFP foci were not clear and co-localized signal were not observed at 20 min. it was inferred that Ku proteins recognized DNA damage induced by Ar ions and dissociate from DNA ends without repair.

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DNA-PK is composed of a catalytic subunit (DNA-PKcs) and DNA end-binding Ku70 and Ku86, and is essential to the repair of DNA double-strand breaks (DSBs) via by non-homologous end joining (NHEJ). Accelerated heavy ions with high LET can induce complex clustered DNA damage involving two or more DSBs. The complexity of clustered DNA damage shows a strong LET dependence, which makes it a good candidate prime determinant of the higher RBE of high-LET heavy ions than that of low-LET X-rays. Here we show the cell killing effect of heavy ions and the response of DNA-PK to heavy-ion-induced DSBs. We determined that the surviving fractions using human DNA-PKcs-defective M059J and control M059K cell lines irradiated with X-rays and heavy ions. The peak RBE value of M059J cells was significantly lower than that of M059K cells. Similar results were obtained using HeLa cells treated with an inhibitor of DNA-PK. Next we observed that phosphorylated DNA-PKcs was recruited to the vicinity of DSBs induced along the track of heavy ions. In addition, phosphorylation level of XRCC4 after heavy-ion irradiation was higher than that after X-ray irradiation, according to LETs. The time course of XRCC4 phosphorylation, however, showed no difference between X-rays and heavy ions. These results suggest that DNA-PK can efficiently recognize DSBs induced by high-LET heavy ions and is activated, but NHEJ cannot precisely repair because of the complexity of DNA damage. The relationship of phosphorylation of DNA-PKcs and XRCC4 will be presented.

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DNA double strand break (DSB) is thought to be the most critical damage induced by ionizing radiation (IR), and non-homologous end joining (NHEJ) pathway is regarded as the most prevalent mechanism to repair DSBs in mammalian cells. Although several key proteins such as DNA-PK and ligase IV/XRCC4 have been identified for NHEJ, it is not clear if another critical protein, ATM is directly involved in this pathway. Some researchers suggested the involvement of ATM in repair of complex (or dirty) type DSBs. We observed kinetics of gammaH2AX foci in normal human cells irradiated with X-rays, carbon (290 MeV/n, 70 keV/um) and iron ions (500MeV/n, 200 keV/um). These heavy ions are a good source to induce complex DSBs. The appearance of gammaH2AX foci is similar for all three radiation sources, but the disappearance is significantly delayed in carbon and iron irradiation when compared that with X-rays. In contrast, when AT heterozygote cells were used, significant delays were observed in gammaH2AX foci appearance in carbon and iron irradiated cells. Moreover, we were not able to observe the phosphorylation of DNA-PKcs in AT cells irradiated with heavy ions, while X-irradiation induced DNA-PKcs phosphorylation in AT cells. In addition, we observed a significant delay in ATM phosphorylation in normal cells irradiated with 200keV/um iron ions. These studies indicate that 1) high LET irradiation induces complex type DSBs which significantly affects NHEJ repair proteins, 2) ATM seems to play an active role in NHEJ process, and may be critical in repairing complex type DSBs.

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To identify the dynamics of DNA damage-recognizing proteins at high LET particles-induced track, we analyzed the focus formation and phosphorylation of these proteins after exposure to iron-ion beams (500 MeV/u, 200 KeV/microm) using immunocytochemistory and flowcytometry. Since the focus formation of phospho-H2AX (gamma H2AX) is well understood to be detected at radiation-induced double strand breaks (DSBs), we performed the visualization of spatial distribution of lesions from an aspect of dose dependency. The number of this track induced by iron-ion beams was well corresponded with the value of a calculation well. Although the phosphorylation of H2AX is increased with dose dependent manner after exposure to Fe-ion beams like X-rays, more gamma H2AX remain in iron-ion beams irradiated cells than in X- irradiated cells. This result suggests that the HZE particles-induced lesions are irreparable. In addition, we demonstrate that DNA damage-recognizing proteins such as phospho-serine 1981 of ATM, phospho-threonine 2609 of DNA-PKcs, phospho-serine 343 of NBS1 and phospho-threonine 68 of Chk2 co-localized with gamma H2AX at high LET-radiation-induced portion. These findings suggest that iron-ion beams were quite effective for detection of DNA damages of DSBs recognized with DNA repair enzymes used here after phosphorylation of them, because iron-ion beams can be used to generate extremely localized at DNA damages within restricted regions of the nuclei.

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Oxidized DNA precursors as well as oxidative DNA lesions seem to contribute to mutagenesis induced by ionizing radiation. Likewise, oxidation of RNA precursors could disturb transduction of correct genetic information encoded in DNA [1]. In this study, we focused on effects of oxidized RNA precursors on RNA produced in in vitro transcription.
We used T7 RNA polymerase as a model enzyme, and 8-hydroxy-GTP (8-OH-GTP) and 2-hydroxy-ATP (2-OH-ATP) as representative oxidized RNA precursors. We analyzed amount of full-length mRNA and nucleotide sequence of the RNA after reverse transcription. We found that (i) addition of 8-OH-GTP and 2-OH-ATP reduced amount of RNA and that (ii) 8-OH-GTP induced T-->G (U-->G) and T-->C (U-->C) mutations, and 2-OH-ATP caused T-->C (U-->C) mutations.
These results suggest that formation of oxidized RNA precursors affects transcription quantitatively and qualitatively, and disturbs expression of correct genetic information.
[1] Taddei et al. Science 278, 128 (1997)

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The recently identified Y-family DNA polymerases comprise proteins from different species including bacteria, eukarya and archaea. The most distinct feature of the polymerases is their ability to replicate DNA past damaged sites (translesion DNA synthesis). Some reactions they catalyze are error-free (correct bases are inserted opposite template lesions), while others are error-prone. Thus, the polymerases appear to play important roles in DNA damage tolerance and mutagenesis. However, damages are induced not only in DNA but also in its precursors, i.e., dNTP pool. Indeed, the incorporation of oxidized dNTPs into DNA is a major source of spontaneous mutagenesis and carcinogenesis. Here, we report that human DNA polymerase eta (pol η) incorporates oxidized dNTPs erroneously. The pol η preferentially incorporates 8-OH-dGTP opposite template A and incorporates 2-OH-dATP opposite template C, G or T. Pol η also efficiently elongates from primers having 8-OH-dG or 2-OH-dA as their 3'-termini. In addition, when the dinB gene encoding Pol IV and/or the umuDC genes encoding Pol V are disrupted in an sodAB fur strain of E. coli, the high spontaneous mutations were diminished by 80-90%. In the sodAB fur strain, superoxide stress and ion overload occur, which leads to high rates of spontaneous A:T to C:G and G:C-to-T:A mutations. The targets contributing to the mutator phenotypes are 8-OH-dGTP and 2-OH-dATP rather than DNA. Our results suggest that Y-family DNA polymerases may promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis.

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8-Hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), oxidatively damaged DNA precursor, is produced by reactive oxygen species formed by ionizing radiation, and is incorporated by DNA polymerase(s) opposite incorrect bases to form mispairs. It has been shown that 8-OH-dGTP is highly mutagenic in Escherichia coli and various in vitro replication systems. Here we studied the mutagenicity of 8-OH-dGTP in living mammalian cells by direct introduction using cationic lipids, Lipofectamine reagent. 8-OH-dGTP and plasmid DNA, including supF gene as a mutational target, were co-introduced into COS-7 cells. After 48 hr incubation, replicated plasmid DNA was recovered and were electroporated into indicator E. coli, and then mutant frequencies were determined. 8-OH-dGTP efficiently induced A:T to C:G transversion mutations, although total mutant frequency was not increased compared with dGTP control. This result is consistent with the previous observations that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro and that 8-OH-dGTP induces A:T to C:G mutations in E. coli. Thus, we showed the first direct evidence that 8-OH-dGTP was actually mutagenic in living mammalian cells.

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Damaged DNA precursors are formed by reactive oxygen species induced by ionizing radiation. After damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, we examined whether a nucleotide excision repair enzyme, UvrABC, could suppress mutations induced by oxidized nucleotides in vivo .
We introduced oxidized DNA precursors, 8-hydroxy-dGTP and 2-hydroxy-dATP, into uvrA and uvrB Escherichia coli strains, and analyzed mutations in the chromosomal rpoB gene, as a mutagenesis target. Oxidized DNA precursors induced mutations in the wild type, but not in the uvrA and uvrB strains. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with hydrogen peroxide (H₂O₂), and the rpoB mutant frequencies were calculated. Frequency of the H₂O₂-induced mutations was increased in the all strains tested, and the increase was two- to three-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, it is suggested that the UvrABC nuclease is involved in fixation, but not suppression, of the mutations induced by oxidized nucleotides. We hypothesized that incision of the complementary strand, not that of the strand containing oxidized base damage, by UvrABC might fix the mutation. Although duplex oligonucleotides containing 8-hydroxyguanine and 2-hydroxyadenine were treated with the purified Thermus thermophilus HB8 UvrABC proteins, the expected activity was not observed. Thus, the UvrABC proteins would fix the mutation by another mechanism.

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Endogenous DNA lesions are produced through the misincorporation of damaged nucleotides during DNA replication, in addition to direct acting of endogenous mutagens to DNA. Oxidation and deamination are the most established endogenous sources for nucleotide lesions. Thus to know the sanitizing mechanisms for the endogenous nucleotide lesions is important for understanding the mechanism of spontaneous mutagenesis and genomic instability. For this purpose we first isolated the sanitizing enzymes in Saccharomyces cerevisiae for oxidized nucleotide lesions, Ylr151c, and for deaminated nucleotide lesions, Ham1, and characterized them as pyrophosphatases for these nucleotide lesions. We next constructed the strains deficient in either YLR151c or HAM1 in both haploid and diploid cells, and examined spontaneous frequency of mutation in haploid cells and of loss of heterozygosity (LOH) in diploid cells. In haploid cells, the deficiency in YLR151c caused mild mutator phenotype, while that of HAM1 did not show any effects on spontaneous mutagenesis. However, in diploid cells, the deficiency in HAM1 caused 20-fold induction of LOH through hyper-induction of homologous recombination. These results indicate that endogenous nucleotide lesions cause not only mutagenesis in haploid cells but also genomic instability through hyper-recombination and LOH in diploid cells. Referense; Nucleic Acids Res. 32, 5339-5348 (2004), Biochem. Biophys. Res. Commun. 325, 928-933 (2004)

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DNA and its precursor nucleotides are constantly oxidized by reactive oxygen species produced through the exposure of ionizing radiation and various types of chemicals in the environment as well as through normal metabolic processes in the cells. The resulting oxidative lesions in DNA and nucleotides are considered to be a major source of spontaneous mutations in many organisms. 8-Oxo-dGTP, an oxidized form of dGTP, can be incorporated into a position opposite of adenine as well as of cytosine during DNA replication. Thus, 8-oxoguanine incorporated with adenine can form a pair with cytosine in the next round of DNA replication, leading to an A:T to C:G transversion; 8-oxoguanine incorporated with cytosine can form a pair with adenine during DNA replication, leading to a G:C to T:A transversion after two round of DNA replication. Organisms are equipped with mechanisms to avoid the mutagenesis caused by the oxidized nucleotides. Bacterial MutT protein hydrolyzes 8-oxo-dGTP and 8-oxo-dGDP to 8-oxo-dGMP, thus preventing the incorporation of the mutagenic substrate during DNA replication. In mammals, proteins belonging to the NUDIX hydrolase family have similar enzymatic activities: MTH1 (NUDT1) and NUDT15 hydrolyze 8-oxo-dGTP while NUDT5 hydrolyzes 8-oxo-dGDP. Thus, these enzymes are considered to function in the preventive mechanisms for mutatgenesis caused by 8-oxo-dGTP. Here we show the data obtained from the mutagenesis and tumorigenesis experiments using Mth1-deficient mice, and discuss mechanisms for preventing mutagenesis caused by oxidized nucleotides in mammals.

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The accumulation of modified or damaged bases in genomic DNA is a major threat for the alteration of genetic information as a result of mutagenesis or even for programmed cell death. It has been established that such damaged bases in genomic DNA arise from two independent pathways: one is a consequence of the direct modification of the normal bases in the DNA and the other is that of the incorporation of modified nucleotides generated in resident nucleotide pools. 8-oxo-dGTP is one of the major causes for spontaneous mutagenesis, because 8-oxo-dGTP is formed by the spontaneous oxidation of dGTP in the nucleotide pool, and it is incorporated into the nascent strand opposite adenine as well as cytosine in the template strand during DNA replication. We have demonstrated that 8-oxo-dGTP is hydrolyzed by MutT family proteins from prokaryotes to humans, and, as a result organisms maintained a low spontaneous mutation rate. Recently, genome projects revealed the existence of many MutT-like proteins with different substrate specificities, and a structure-based approach identified another novel enzyme, ITPase which hydrolyzes deaminated purine nucleoside triphosphates, such as (d)ITP and (d)XTP. In Escherichia coli, a mutant of rdgB gene coding ITPase protein is viable but it shows synthetic lethality with recA or recBC mutation. It is likely that dITP or dXTP accumulated in the nucleotide pools exhibits cytotoxicity in the absence of recombination repair. I will review the molecular pathophysiology caused by deficiency in human or mouse MutT homologs as well as ITPase homologs.

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Nucleotide excision repair (NER) is the most versatile DNA repair system of living organisms and the basic reaction is conserved from E. coli to humans. The NER reaction is composed of multi-step processes and can be achieved by ~30 polypeptides in a well-orchestrated manner. The biochemical studies including reconstitution experiments and immunofluorescent studies combined with an elegant technique of micropore UV irradiation have revealed the detailed molecular mechanism of the basic NER reaction. Current interests in this field is to uncover the regulatory mechanism of NER under the more complex environment in living cells and the link between NER and signaling cascades initiated by DNA damage. We have been working on DDB protein consisting of two subunits, DDB1 and DDB2, which exhibits a high affinity to various types of DNA damage, and demonstrated its stimulatory role in NER especially for CPD. DDB has been recently shown to have another function as a component of E3 ubiquitin ligase and to be implicated in the modification of NER factors as well as histones. On the other hand, we have recently found that in human quiescent cells the NER process can be perturbed at a gap-filling step and the resultant ssDNA gap intermediates initiate the phosphorylation of histone H2AX. These findings provide a new insight into the quality control of DNA repair process that can be perturbed halfway. In this symposium, we will present our recent work and discuss on the regulatory mechanism of NER in human cells.

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Nucleotide excision repair (NER) is a DNA repair pathway, which eliminates various helix-distorting DNA damage including some oxidative damage and UV-induced (6-4) photoproducts (6-4PPs). Here, to get clues to understand why patients with NER-defective disorders develop progressive neurological abnormalities, NER capabilities in neural cells were investigated. Primary neurons and astrocytes from rat embryonic brains were prepared in mixed-cell cultures, and fibroblasts from the same embryos were cultured for comparison. The original method for in situ 6-4PP determination using antibodies against the lesion was applied to determine NER capabilities in individual neural cells, which were identified by staining of specific cell markers. The results demonstrated that NER pathway is functional in both neurons and astrocytes, but their repair capabilities are significantly lower than that of fibroblasts. The degree of DNA repair deficiency was comparable to the case of NER-defective human fibroblasts (TTD2VI) that expressed only half levels of NER proteins (TFIIH) of normal cells. Consistent with these results, the levels of an NER protein (PCNA) recruited at localized UV damage sites were apparently lower in neurons and astrocytes than those in fibroblasts. Thus, the present study suggests that the neural cells constituting the central nervous system may generally have the attenuated NER capabilities because of reduced expression of NER proteins.

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UV light forms three types of DNA damage, cyclobutane pyrimidine dimers (CPD), pyrimidine(6-4)pyrimidone photoproducts (64PP) and Dewar valence isomers at dipyrimidine sites in DNA and induces specific types of mutations at those damaged sites in mammalian genome, finally leading to skin cancer. Triplet mutations, which are characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence including a dipyrimidine, have been identified recently as a new type of UV-specific mutation and found to occur preferably in nucleotide excision repair (NER)-deficient genetic backgrounds. However, the mechanism of formation of the triplet mutation is unknown. Here, we show that 64PP as well as CPD (and probably Dewar isomers) contribute to the triplet mutagenesis, depending on the types of the triplet mutations. We further propose several models of error-prone translesional DNA synthesis (TLS) on those lesions as underlying mechanisms for triplet mutagenesis. These models would promote the study of TLS mechanisms by providing a number of concrete molecular objects to analyze.

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UVB radiation can damage DNA by causing formation of cyclobutane pyrimidine dimer (CPD). Photoreactivation mediated by an enzyme, photolyase, is the major pathway for repairing CPD in plants. We previously reported that the different degrees of UVB sensitivity in rice are caused by mutations in the CPD photolyase gene, and CPD photolyase activity plays an important role in preventing UVB-caused growth inhibition. However, it is poorly understood the characteristics and functions of CPD photolyase in plant. We present two topics about the CPD photolyase in rice as mentioned below.
(1)Characterization of CPD photolyase from rice plant. Native CPD photolyase was purified from rice leaves by anion exchange and heparin affinity chromatography. At final step, enzyme was bound to CPD-containing DNA conjugated to magnetic beads and released by blue irradiation, with an overall increase in specific activity of about 8,000-fold. SDS-PAGE and western blotting analyses indicated that native rice photolyase contains of two 54- and 56-kDa isoforms. The purified native enzyme activity was much higher than enzyme expressed in E. coli.
(2)CPD photolyase encoded by a single-copy gene in nuclei functions in nuclei, chloroplast and mitochondria in rice plant. We found that UVB-induced CPD on not only nuclei but also chloroplast and mitochondrial genome was repaired dependently by exposure of blue irradiation in rice. Furthermore, the CPD photoreactivation in each organelle was not detected in antisense transgenic plant with little CPD photolyase activity.

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It has been generally accepted that LDRE results from the SLD repair. To study the molecular mechanism of LDRE, we analyzed the knock-out mutants KU70^-/-, RAD54^-/-, and KU70^-/-/RAD54^-/- of the chicken B-cell line, DT40. Survival enhancement by LDR irradiation was observed in parent DT40 and RAD54^-/- cells but not in NHEJ deficient KU70^-/- and KU70^-/-/RAD54^-/- cells. Under continuous LDR irradiation, dividing NHEJ-deficient cells will be irradiated and killed in G1 phase. In the LDRE, NHEJ pathway was more important than HR pathway. We studied further the effect of low dose-rate irradiation using the deficient KU70^-/- and KU70^-/-/53BP1^-/- cells since 53BP1 is reported to play a role in new NHEJ repair. KU70^-/- cells survived well under 0.5 Gy/day compared to 1.0 Gy/day dose rate irradiation. KU70^-/-/53BP1^-/- cells were sensitive than KU70^-/- cells under 0.5Gy/day dose rate irradiation. This suggests that 53BP1 dependent NHEJ is independent Ku/DNA-PK NHEJ and also that both NHEJ pathway play a role in the LDRE.

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We have investigated effects of low-dose-rate radiation (LDR) on human cells immortalized by the hTERT gene. GI-phase-arrested cells were irradiated by LDR (0.3mGy/min) continuously for a maximum of 2 weeks or high-dose-rate radiation (HDR; 2Gy/min). In normal human cells, the survival after LDR radiation was significantly high, while the induction of micronuclei was significantly low, when compared to those after HDR irradiation. In contrast, in AT cells significant difference between LDR and HDR were not observed. Only few γH2AX foci formation was observed in LDR irradiated normal cells, while in AT cells significant number of γH2AX foci were induced after LDR irradiation. We have suggested that ATM played an important role in repairing the DSBs induced by LDR radiation. However, it is still not clear ATM function in repair of DNA damage induced by LDR radiation. We are now investigating the effects of LDR radiation on DSB repair deficient cells (Artemis, DNA Ligase IV and NBS) arrested in the G0/G1 phase.

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Lethal effects of ionizing radiation are different in its extent depending on its dose-rate. At a cellular level, it is known that ATM gene is involved in this dose-rate effect. In order to study the dose-rate effect at an individual level, we irradiated third instar larvae of wild-type Drosophila and measured their viability. When they were irradiated with gamma-rays at dose-rate higher than 36Gy/h, LD50 was constant at 36.1±1.5 Gy and independent on the dose-rate. At dose-rate lower than 36Gy/h, LD50 became larger, according to an approximate equation LD50=35.7×log₆₀{irradiation period(min)}-4.5Gy. Although an irradiation with 55Gy caused 100% lethality when the irradiation period was less than 3h, a part of irradiated larvae survived when irradiation period was longer than 5h. When mei-41, a Drosophila homolog to human ATM was used instead of wild-type flies, the dose rate effect disappeared. In mei-9 which is homologous to XPF, on the other hand, the dose-rate effect was evident though radiation sensitivity was higher than wild-type. These results suggest that the double strand break repair function under control of mei-41 gene is involved in the expression of dose-rate effect.

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