In order to prevent health hazards caused by long space flight, we have to establish necessary countermeasures for radiation protection and implement them to the operational rules. A primarily important task for this purpose is to quantify the health effects of long-term radiation exposure in space. According to the present system on radiological protection, it is predicted that astronauts will receive about 0.1Sv during a half-year stay in the ISS and about 1Sv in a future mission to Mars. JAXA has set a restricting exposure level as the dose increasing the cancer risk by 3 percent; astronauts can exceed this level in case of a long mission for years. However, these considerations are based on the information obtained on ground and the dosimetry system may be changed by coming medical data on astronauts' health. Particularly, biological effects of high-LET particles including the dose-rate effects and synergistic effects of other modifying factors are quite uncertain and to be studied. Keeping in mind living in space, we will be able to fundamentally discuss the definition of 'dose' (e.g. effective dose) with the advance of radiation research.

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The recent performance of manned space missions has raised the problem of astronaut exposure to various types of radiation, such as charged particles of solar origin (protons and electrons) and of galactic radiations (protons 85%, particles 14%, and heavy ions 1%). In spite of the low percentage of heavy ions, they contribute great effect to the total dose of cosmic rays delivered because they have very high LET. It is therefore important to assess the biological effects of heavy ions in order to estimate the radiation risk for human beings in future space missions of long duration and establish standards for radioprotection in space. Cytogenetic analysis of the lymphocytes of astronauts provides a direct measurement of space radiation damage in vivo, which takes into account individual radiosensitivity and considers the influence of microgravity and other stress conditions. We report chromosome aberrations in the lymphocytes of astronauts using fluorescence in situ hybridization (FISH) with chromosome painting probes.

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The research efforts in radiation biology in space has recently been accumulated in Japan since the first space radiation biology experiments was conducted by Dr. Sohei Kondo in the early human space flight research at United States. In Japan, frequent opportunities for the space radiation experiments have been offered by NASDA (currently JAXA) after the participation to the first International Microgravity laboratory (IML-1) mission at 1992. A series of Spacelab missions, Spacelab-J, IML-2, Space Shuttle/Mir, were dedicated internationally for the multidisciplinary research. The space radiation biology experiments in early phase conducted in Japan employed stable specimen during the mission such as plant seeds, bacterial spores, and Altemia eggs to survey the biological effects during the space flights. In the later phase, more sophisticated experiments were planned and conducted using fruit fly, rat, plasmid DNA and its repair enzymes. It was also recognized the importance of the physical dosimetry inside the space vehicle at the lower Earth orbit to quantitize the radiation environment. During the early space radiation biology experiments, the plastic nuclear track detector such as CR-39 was only available to obtain the LET spectra of the composite particle radiation field. A real-time radiation monitoring device (RRMD) was simultaneously developed by NASDA and introduced to the space radiation biology experiments to obtain more precise LET spectra as well as spatial and tome resolutions. This report summarizes the recent efforts from the past radiation biology experiments in space.

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The radiation environment in space consists of high energy protons and high energy heavy ions (HZE). The principal sources of HZE ions in nature are galactic cosmic rays (GCR) and the solar events (SPE), which consist mostly of protons with small components of heavy ions. The high energy heavy ion is the most biologically effective to humans. The NASA Space Radiation Health Program initiated the experimental assessment using a heavy particle accelerator in the dedicated NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory (BNL). Heavy Ion Medical Accelerator in Chiba (HIMAC) can accelerate heavy particles such as proton, helium, carbon, neon, silicone, argon and iron ions up to the maximum energy at 500 MeV/u. The experiments for research of HZE biological effects using HIMAC has been performed since 1994. Many participants were from foreign countries and all parts of Japan for collaborative experiments and research. In this symposium, I will review and summarize what has been happening in the space radiation research on the ground at HIMAC and other places in the world.

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Cosmos is another name which expresses the space. Although there are so many difficulties in considering the space exploitation, efforts and motivation to open the door is very strong. However, is it only exploitation we are forced to do? Name pf cosmoc includes many factors ranging from technical to artistic treasures. If one is not interested in things other than physical and technical matters, we must say that he lacks sense of essential charm points. Why have people been looking up stars considering their fortune? We will have various solutions.

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Japan's first commercial nuclear fuel reprocessing plant is now being constructed in Rokkasho, Japan. Since small amount of radionuclides will be released from the plant to the environment in normal operation, it is important to investigate the background levels of the radionuclides before full operation in order to evaluate them released in future. Distributions of ^239+240Pu and ¹³⁷Cs in soil samples collected from un-cultivated, agricultural upland and paddy fields in Rokkasho were examined. Inventories of ^239+240Pu in those fields were 111±52 Bq m^-2, 81±36 Bq m^-2 and 81±22 Bq m^-2, respectively, and those of ¹³⁷Cs were 3.2±1.5 kBq m^-2, 2.4±0.9 kBq m^-2 and 2.3±0.8 kBq m^-2, respectively. Variation ranges of their inventories in the agricultural fields were larger than the range in the un-cultivated field, because surface soil of the agricultural fields was removed or added with soil from other places in many cases. Concentrations of both nuclides in 25cm below were under detection limits in the un-cultivated fields. On the other hands, the nuclides were detectable under 1m depth for some cases in the agricultural upland and paddy fields. Atomic ratios of ²⁴⁰Pu/²³⁹Pu were 0.179±0.038, 0.164±0.023 and 0.175±0.014 in the upland, the paddy field and the un-cultivated field, respectively. These values agreed with the ratio of global fallout Pu within the range of one standard deviation.

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A small simulated ecosystem was used to get informations on accumulation of radionuclides by freshwater organisms. The simulated ecosystem was made up of an aquatic plant(Vallisneria sp.), a shrimp(Paratya compressa), clams(Unio douglasiae, Anodonta woodiana and Corbicula japonica), a snail(Cipangopaludina japonica) and fishes(Carassius auratus and Cyprinus carpio) in a glass aquaria containing freshwater and sediment. During the experiment, the aquaria was maintained with illumination for half a day, slow circulation of water and control of water temperature at 20 degrees centigrade and without aeration, filtration of water and feeding to the organisms. Radionuclides used in the experiment were ⁵⁴Mn, ⁵⁷Co, ⁸⁵Sr, ¹⁰³Ru, ¹²⁵I, ¹³⁷Cs and ¹⁴¹Ce. In several days after addition of the radionuclides into the aquaria, most part of the radionuclides was accumulated by sediment. Consequently activity level of the radionuclides in water became low. In spite of low radioactivity level in water, organisms showed relatively high accumulation of the nuclides. This suggested the participation of the radioactive sediment on accumulation of radionuclides by organisms in a freshwater ecosystem.

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Over the past several years, increasing attention has been paid on the protection of the environment from ionizing radiation. The ICRP is proposing in their radiation protection recommendations to select reference animals and plants to develop a common approach for the radiological protection of humans and nom-human species. The earthworm (worm) is nominated as one of the reference animals. Earthworms play important roles on ecosystems, and might be a good indicator of soil contamination and its effect on the ecosystem. Elemental composition of earthworm gives useful information on background level and possible accumulation of the metals as well as related radionuclides. In addition, change of the elemental composition itself might be a possible indicator of the effect on the earthworm and/or ecosystem. However, data for the elemental composition of earthworms were limited except for some specific heavy metals such as Cd, Zn, Pb and Cu. In this study, the earthworms and their growth media were analyzed for 37 elements, including radionuclide related elements such as Cs, Sr, Th and U, in order to obtain the basic information on the transfer parameters of the elements. The concentrations and transfer factors of the elements were determined both for laboratory and natural conditions.

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In the present work, the biological effect of tritium on tissue and erythrocyte DNA of fresh water fishes was estimated by the comet assay during long term exposure of tritiated water (HTO). Medaka or zebra fishes were exposed to HTO (146-1042Bq/ml) added to an aquarium for 10-26 days and the uptake, retention and release of tritium in fish body were investigated. Total tritium per g fresh weight of medaka equilibrated rapidly, around 40min after the start of exposure, to about 50% of the aquarium water concentration while organically bound tritium reached only 1% of the aquarium water concentration after 10 days exposure. The biological half time of tritium in the fishes transferred to fresh water after 26days exposure was 14 min. The effect of long term exposure to low dose tritium on fresh water fish was examined using medaka fed in HTO (2Bq/ml) containing aquarium for 232 days. No significant effect was observed in the liver cell.

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We investigate the effect by radiation and the response to radiation using microorganisms which is useful to detect the effects at the level of population or community. Experiments using a microbial model ecosystem (microcosm) elucidated that the effects observed as an index of changes in the population density of microbes were detectable at the radiation dose of more than 50 Gy. The result was considered to be owing to the character of generally high radio-resistant microbes, but low dose of radiation is known to stimulate the growth rate of some kinds of microbes. When the effects of radiation are evaluated by the change in the population density of organisms, the stimulating effects will affect the evaluation, especially for the dose-response relationships at the dose range of threshold. It is therefore very important for determination of the threshold value to analyze the response to very low dose of radiation. From this point of view, we started the preliminary experiments to analyze the microbial response to very low-level dose rate of radiation, especially to elucidate the realities of stimulating effects. We report the results of experiments using Euglena and Paramecium exposed to gamma-rays at the dose rate of 0.5 mGy/day and hope to discuss the significance of this kinds of study for environmental radiation protection.

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Population dynamics of heterotroph protozoa as predator and saprotroph bacteria as prey in the aquatic microbial ecosystem were analyzed by a particle-based computer simulation. Mathematical model based on the Lotka-Bolterra equation was adopted for describing their predator-prey relationship. When radiation impact was introduced chronically on the growth of bacteria, the reduction of carrying capacity of protozoa might be induced as an indirect response of the two species ecosystem to the chronic radiation exposure.

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The remarkable progress of research in genome science and regenerative medicine will bring new treatments for radiation casualties. This program try to reach to the goal of the establishment of the world center of excellence (COE) in 21st century for Radiation Casualty Medical Research. We also aim to nurture the next generation of young researchers and physicians in this field. Radiation exposure can cause genome damage and induce acute disorders such as multi-organ failure and late disorders such as cancers. In our research program, we are attempting to develop a diagnosis and prevention method for damaged genomes, new treatments for radiation-induced cancers, and methods of tissue-regeneration using pluripotent stem cells. Studies of molecular mechanisms of repair and cellular response to radiation-induced genome damage are also our major research projects. We are analyzing genes associated with leukemia and other cancers and establishing a monitoring system based on the information from genome alterations. From this, we can develop a new system of prevention and treatment of radiation exposed patients. Our ultimate goal is the achievement of a comprehensive medical system that is capable of dealing with all types of radiation injury based on the radiation dose estimated from the information of damaged genome and physical dosimetry.

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Nonhomologous end joining (NHEJ) is one of the major pathways that mediate the repair of DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK) complex plays important roles in this pathway, since mammalian cell lines that lack components of the complex are radiation sensitive and are defective in rejoining DSBs. DNA-PKcs is a serine/threonine protein kinase that is activated by DNA ends in vitro and that consists of the DNA-binding and regulatory subunit, Ku, and the catalytic subunit, DNA-PKcs. DNA-PK kinase activity is required for the repair of DNA DSBs in vivo; however, the molecular basis of this requirement is unknown. DNA-PKcs (DNA-PK catalytic subunit) is phosphorylated at various sites in vivo in response to ionizing radiation and the phosphophrylated DNA-PKcs is localized at the DNA double-strand breaks. Furthermore, mutations prevent DNA-PKcs phosphorylation lead to radiation sensitivity and impaired DSB rejoining, suggesting that phosphorylation of DNA-PKcs is required for the repair of DSBs by NHEJ pathway. Interestingly, IR-induced DNA-PKcs phosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs result in robust DNA-PKcs phosphorylation and localization to DNA damage sites. These results indicate for the first time that, while IR-induced DNA-PKcs phosphorylation is attenuated in S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis. The possible role of DNA-PK and NHEJ in repair of replication-associated DNA double-strand breaks in S phase will be discussed.

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Mutagenesis is a cellular response to the DNA damage. This biological function is essential for the maintenance of chromosomal integrity as well as the DNA repair function. It has been suggested that functions of the REV1, REV3, REV7 and POL32 genes are required for error-prone post-replication repair, essential for induction of mutations and prevention of cell death caused by ionizing radiation. Rev1 protein is the deoxycytidyl transferase and a member of the Y-family DNA polymease. The activity is capable of extending a primer terminus by insertion of dCMP opposite a variety of damaged bases. REV3 and REV7 encode another error-prone DNA polymerase, polζ. Pol32 is one of the subunits of a replicative DNA polymerase, polδ, and has a function interacting with PCNA. These genetic data suggest that those proteins form specialized machinery for translesion DNA synthesis. To elucidate molecular mechanisms of the induced mutagenesis in humans, the human proteins have been purified from overproduced Escherichia coli cells. We have demonstrated that the human REV1 protein is the deoxycytidyl transferase and forms a stable heterodimer with REV7. Recently, it has been found that the REV1 interacts with all of the Y-family DNA polymerases, polη, polι and polκ, suggesting the central role of REV1 protein in the translesion DNA synthesis. In this symposium, we present the biochemical property of the REV1 protein and discuss the molecular role of this protein in the translesion DNA replication.

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Mutations in the CSA and CSB genes cause devastating genetic disorder Cockayne Syndrome. Both complementation factors belong to the Transcription Couple Repair (TCR) pathway of the Nucleotide Excision DNA Repair (NER) but the relationship between CSA and CSB proteins is not well understood. We have shown recently that two NER factors CSA and DDB2 form similar Cul4A containing E3 ubiquitin ligase complex that also contain cul4A, Roc1 and DDB1. The substrates and role of different components of this ligase are not known. Here we demonstrate that CSB is a substrate of CSA ligase. Under certain conditions, CSB co-purifies with the CSA ligase, and these interactions are stabilized by proteasome inhibitor MG132. CSB is ubiquitinylated by in vitro reconstituted CSA/DDB1/cul4/Roc1 complex, but not by DDB2/DDB1/cul4A/Roc1 ligase. CSB is degraded in cells within 4 hours after UV-irradiation but is stabilized by proteasome inhibitor or in CSA-deficient cells. Thus, our work establishes the relationship between two complementation group of the Cockayne syndrome and demonstrates an essential role of ubiquitin-dependent proteolysis at recovery stage following transcription-coupled repair.

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Goal of the Hiroshima University COE program is the establishment of new age care protocol for radiation casualties; i.e., treatment for acute radiation syndrome and long-term follow up protocol for delayed radiation effects such as malignancies, based on the accurate assessment of individual radiation dose. With minimum treatment, 50% lethal dose (LD50) for young healthy adults has been estimated approximately 3.5-4 Gy. However, with appropriate supportive care and antibiotics, the LD50 would increase to 6-7 Gy. We are trying to further increase LD50. For assessment of individual radiation dose, we are establishing a rapid and easy method, by detecting the intranuclear accumulation of DNA repair proteins such as NBS1 and Rad52b, instead of time-consuming conventional chromosomal analysis. Multiple organ failure is the major target of acute radiation syndrome and would be only treated by stem cell transplantation. We are expanding our efforts in the field of stem cell biology to the clinical application. Long-term follow up for radiation casualties becomes more and more important because many patients who received relatively high dose radiation would be expected to overcome acute radiation syndrome. Early detection of leukemia cells using molecular tools followed by 'prophylactic' anti-leukemia therapy will be discussed.

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Mouse thymic lymphomas are one of the classic models of radiation-induced malignancies. We previously demonstrated the inactivation of a novel gene, Rit1/Bcl11b, in those radiogenic lymphomas. To confirm whether Rit1 functions as a tumor suppressor gene during lymphomagenesis, Rit1 knock-out mice were produced and analyzed. Thymic lymphomas were induced in Rit1 heterozygotes at a higher incidence, showing frequent Rit1 allelic loss, but did not develop without irradiation. However, Rit1 and p53 double heterozygotes spontaneously developed thymic lymphomas at a high frequency in 12 months after birth. Interestingly, most of those lymphomas did not show allelic loss at Rit1, suggesting haploinsufficiency of Rit1/Bcl11b function. Jurkat cells are one of human T cell lines expressing Rit1 proteins. To investigate Rit1 functions, we established three new cell lines of Jurkat cells that decreased Rit1 expressions by introducing an siRNA-producing recombinant plasmid. The cell lines all exhibited apoptosis and low expression levels of anti-apoptotic protein, BclxL, consistent with apoptosis found in the thymus of Rit1-knockout mice. Those cells also showed cell cycle arrest at the G1/S stage and reduced expression of a cell cycle inhibitor, p27. We will show results of Rit1 functions obtained from analysis of the Jurkat cell lines.

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Our COE program "International Consortium for Medical Care of Hibakusha and Radiation Life Science" was established in October 2002. This consortium is aiming at combining medical care based on epidemiological studies on these radiation victims with basic radiation biology. To understand radiation-induced "human" carcinogenesis either by low-dose or high-dose, we need a molecular epidemiology of those cancers with firm evidence of clinical epidemiology. Among the Atomic Bomb survicors, we are looking some important new findings that solid cancers seem to occur multiple in a single survivor, and recent observation suggests an increased incidence of myelodysplastic syndromes (MDS). MDS is a clonal disorder closely related to leukemia that appeared very early after the Bombings. Amazingly, cancer incidences for many different organs such as lung, stomach, colon, thyroid, bone marrow (MDS) and others, seem to be persisting over a half century after the Bombing. Multiple cancers in individual survivors are apparently increased in proportion to the distance from the hypocenter. MDS also seems to be almost two-fold higher in crude incidence among the proximately exposed group than the distally exposed. These cancer tissue and bone marrow samples are being intensively studied by using molecular technology to understand what kind of genetic abnormalities are actually induced by the radiation. Whether these genetic abnormalities can be explained by the findings of genetic instability induced by radiation is extremely important. Such basic researches are usually based on mouse experiments. We need to use human cells to fully elucidate true mechanism of radiation-induced human cancers including leukemia. To establish preventive medicine and better medical care for radiation-exposed victims our COE program will continue these molecular epidemiology and basic research on human cells.

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Ionizing radiation induces genomic instability, which is transmitted through many generations after irradiation in the progeny of surviving cells. We have hypothesized that radiation-induced large deletion causes potentially unstable chromosome regions, which are involved in delayed induction of radiation-induced genomic instability. Using phosphorylation-specific antibodies against ATM and histone H2AX, whose phosphorylation is induced by DNA double strand breaks, we detected delayed induction of phosphorylated ATM and H2AX foci in the progeny of X-ray-surviving cells, which indicated delayed induction of DNA double strand breaks. Furthermore, we found delayed chromosomal instability in X chromosomes in clones which contain large deletion involving the HPRT loci. It is suggested that large deletion involving ~Mb region causes unstable chromatin structure, and it results in delayed rearrangement of chromosomes involved. These findings provide the possibility that manifestation of radiation-induced genomic instability results from delayed DNA breaks, i.e., the breaks lead to delayed chromosome rearrangements, delayed cell death etc., many generations after irradiation.

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Previous studies from this laboratory have identified a number of causally linked genes including the novel tumor suppressor Betaig-h3 that were differentially expressed in radiation induced tumorigenic BEP2D cells that are virally immortalized. To extend these studies using a genomically more stable bronchial cell line, we show here that ectopic expression of the catalytic subunit of telomerase (hTERT) in primary human small airway epithelial (SAE) cells resulted in the generation of several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal. Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings. The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice. These cells show no alteration in the p53 gene but a decrease in p16 expression. Exponentially growing SAEh cells were exposed to graded doses of alpha particles or 1 GeV/nucleon of 56Fe ions. Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation. Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium. These findings indicate that hTERT-immortalized cells, being diploid and chromosomal stable, should be a useful model in assessing mechanism of radiation carcinogenesis.

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The current challenge of radiation biology research is to carefully define the response of cells and tissues to low dose exposure. With the observation of non-targeted responses, where cells do not respond according to the classical model of direct DNA damage, the response at low dose may not follow the predictions of the linear no threshold model. Our own approach has been to develop both charged particle and X-ray microbeams to accurately probe the spatial and low dose dependencies of cellular and tissue response. Our emphasis has been on the bystander response where cells, which have not been directly irradiated respond to their neighbours being exposed. These have been observed in a range of cell types under conditions where even exposure of only a single cell can lead to triggering of a bystander response. A common feature is that the responses predominate at low doses and saturate at higher exposure levels. Many factors have been shown to play a role in the downstream response. From our own studies we suggest that the release of the signal is a fundamental process which may not be related to the phenotype of the cell being targeted. However the ability of a population to respond to the released bystander signal is highly cell phenotype dependent, with cell type, cell cycle phase and DNA repair capacity all playing a modulating role.

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Cellular exposure to DNA damaging agents like ionizing radiation can result in mutations, gene amplifications, chromosomal rearrangements, carcinogenesis, and even cell death. The paradigm for understanding how induced damage results in these cellular endpoints dictates that cellular responses to the induced damage, e.g., DNA repair, and cell cycle arrest fix the damage and thereby seal the fate of the irradiated cell. This presentation will focus on delayed genetic effects occurring in the progeny of cells after exposure to ionizing radiation, including delayed chromosomal rearrangements, and recombination events as determined by a plasmid based assay system for homologous recombination. We will present new data on how changes in gene expression as measured by differential display and DNA microarray analysis provides a mechanism by which cells display a memory of irradiation, and introduce candidate genes that may play a role in initiating and perpetuation the unstable phenotype. In addition, we will describe how the cellular micro-environment can perpetuate instability in clonally expanded populations of cells surviving irradiation. These results will be discussed in terms of non-targeted bystander like effects where by cells that themselves were not irradiated exhibit many of the same detrimental effects as irradiated cells and what implication these effects may have for radiation carcinogenesis.

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The incidence of papillary thyroid cancer (PTC) in those areas exposed to fallout from the Chernobyl accident has increased dramatically, particularly in those under 19 at exposure. To determine whether there is a radiation signature, we studied the molecular pathology of post Chernobyl thyroid cancer using a variety of techniques on material supplied by the Chernobyl Tissue Bank (www.chernobyltissuebank.com). Ret rearrangement was studied by RT-PCR in 100 papillary cancers from patients aged under 15 at the time of the Chernobyl accident; 44% of these harboured ret rearrangements; the frequency in PTCs from children in England and Wales is 48%. There was no correlation with either age at exposure or latency. We found that the majority of PTCs (72%) harboured positive cells using FISH for ret rearrangement, and the maximum number of positive nuclei was 55%. Only 41% of cases were positive for ret on RT-PCR. Clustering of FISH positive nuclei was found in 31% of cases, suggesting subclonal growth. One case showed ret rearrangement as well as a chromosomal insertion ins (12;7)(q13;q11q22). The distribution of rearranged genes within the tumour studied by FISH suggests a distinct tumour heterogeneity in this case. Sampling of different areas of the tumour is therefore likely to account for differences between FISH and RT-PCR results. Expression analysis of 2400 genes was also performed 12 cases of PTC using Micromax cDNA array and compared with 9 PTCs from Belgium and France. Hierarchical clustering analysis on the 50% most expressed genes showed no differences between the 2 groups of tumours. Permutation analyses revealed differences in the expression of 10 genes; 5 of these related to lymphoid infiltration in the tumours, the other 5 showed small differences in expression, and are therefore likely to be of no biological significance. We have also compared the frequency of the common B-RAF mutation (T1796A) in a series of Ukrainian PTCs. 32 patients were aged over 30 and 35 cases aged under 16 at the time of diagnosis. 18 (58%) of the adult cases, but only 1 of the 35 cases aged under 16 harboured a B-RAF T1796A mutation. In the young cohort the overall frequency of RET rearrangement was 45.7%. Eight of the younger group of patients were born after 1st December 1986, and were therefore not exposed to radioiodine in fallout from Chernobyl, either in utero or as young children. None of the PTCs from these 8 patients were positive for B-RAF mutation. The frequency of RET rearrangement was 44% in the 27 cases exposed to radiation (median age 11.9, range 7.9-15.1) and 50% in the 8 not exposed (median age 13.8, range 7.9-15.7). Taken together, these results suggest that the different molecular biological profiles observed post Chernobyl are associated with the age of the patient at diagnosis with PTC, rather than being associated with a radiation aetiology.

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Since 1989, the incidence of thyroid cancer in children from areas afflicted by the Chernobyl NPP reactor accident increased considerably. Here the results of a treatment project that has been jointly started in April 1993 by partners from Belarus and Germany are presented. Up to now, 242 children (144 girls and 89 boys, mean-age 12.7 +/- 2.5 years) with differentiated thyroid cancer (240 papillary and 2 follicular) had been treated. In 152 cases (63%) tumor stage pT4 had to be diagnosed; 235 (97%) presented with lymph node metastases (pN1) and in 104 cases (43%) distant spread to the lungs was revealed by I-131 wholebody-scintigraphy. After surgery, which had been performed in Minsk, the children were treated in Germany with totally 935 courses of radioiodine therapy. The intervals between treatment courses amounted to 4-6 months. In 180 (86%) out of 210 children, who at least had received 2 courses of radioiodine therapy, complete remissions (CR) or stable partial remissions (SPR) were achieved (CR: wholebody scan negative, thyroglobulin not measurable; SPR: wholebody scan negative, thyroglobulin in hypothyroidism measurable, but below 10ng/ml). In the subgroup of children with pulmonary metastases, the rates of CR and SPR amounted to 68%. In 7 children clinical signs of pulmonary fibrosis were diagnosed: in two cases after radioiodine treatment only, in five cases after radioiodine- and pre-treatment with bleomycin. No single case of tumor progression has been seen up to now; in one case a recurrence was diagnosed after 10 years and follow-up. Radioiodine treatment of radiation induced thyroid carcinoma can induce complete or stable partial remissions even in cases with advanced tumor stages.

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It has long been assumed that DNA damage induced by radiation is directly involved in carcinogenesis. In this direct hypothesis, DNA damage is fixed as carcinogenic mutation in a irradiated cells within a short period of time after the exposure. However, it is known that some cancer has a long latency which could suggest that carcinogenic mutation is induced not at the time but a long time after exposure to radiation. In addition, we still do not have a direct proof that irradiated cells are the actual target of radiation carcinogenesis. Recent advances of radiation biology have excavated a series of biological responses which may have an important implication in radiation carcinogenesis. Genomic instability induces mutation not in the directly irradiated cells, but in their descendant through the indirect mechanism. Bystander effect induces mutation not in the irradiated cells but in the surrounding cells. In addition to these cellular radioresponses, the tissue level regulations in a body form yet another dimension when one consider the mechanism of radiation carcinogenesis. For example, the current radiation protection system assumes that radiation doses given at a low dose rate can be summed up to give cumulative dose on which radiation risk is evaluated. However, cells in a tissue are constantly turning over and for most of the cells, the summation of the doses is possible as long as the target cells are not discarded from the body. The above mentioned cellular and tissue mechanisms complement the epidemiological data on radiogenic cancer in human to better understand the mechanism of radiation carcinogenesis. For example, the linear increase of the solid tumors among atomic bomb survivors is in contradiction with the multistep mechanism of carcinogenesis if radiation functions as a direct carcinogen. However, the linearity can be explained if carcinogenic mutation is induced by genomic instability. Another example is the temporal pattern of the relative risk decline among atomic bomb survivors which might better be explained by the loss of the initiated target cells in the tissue. In the past, the two disciplines of the radiation carcinogenesis field, molecular/cellular/tissue radiation biology and epidemiology have worked independently. However, new discoveries made in the recent years on both sides are rich enough to complement each other. Thus, the time is ripe to have this symposium in which the two come together to discuss the important issue to better understand the biological mechanism and the risk of radiation carcinogenesis.

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The mitochondrial (MT) death pathway that involves cytochrome c (Cytc) release under regulation by Bcl-2 family proteins plays a crucial role in p53-dependent IR apoptosis. The Cytc-conducting pore remains controversial and unidentified in cells, despite several in vitro models presented. We aimed to delineate the role for Bax (Bak)/VDAC1 hybrids in Cytc release in vivo. For that purpose, we used the p53/Bax-expressing Black93 cells and the Bcl2/Bak-expressing, p53/Bax-deficient Reh cells. The p53-Bax-dependent MT death pathway determined Cytc release and apoptosis in 5 Gy-exposed Black93 cells, but Reh cells lacking p53/Bax despite Bak expression showed neither Cytc release nor apoptosis. The protein cross-linking and 2-step IP studies for isolated MT indicated that IR induced mono- and dimeric Bax/VDAC1 hybrids in Black93 MT, but only monomeric Bak/VDAC1 hybrid in Reh MT. The Bax and Bak oligomers were invariable with time post-IR. In Black93 cells, clotorimazol or a synthetic N-terminal hexokinase peptide recruited Bax to VDAC1, enhanced IR-induced Cytc release and apoptosis, but a VDAC inhibitor DIDS suppressed all of such events. On the other hand, a chemical BH3 peptide-like apoptosis agonist HA14-1 that binds to the Bcl2 pocket induced the Bak/VDAC1 hybrid dimer in Bak/Bcl2-expressing Reh cells, which, otherwise not, rendered the cells highly susceptible to Cytc release and apoptosis. These results demonstrate a new evidence for the important in vivo role of Bax (Bak)/VDAC1 hybrid dimer in apoptosis

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JNK is a subfamily of the mitogen-activated protein kinase(MAPKs). JNK is thought to be involved in the regulation of apoptosis, prolifiration, and differentiation, in response to stress. Resently, JIP was isolated as JNK interacting protein by yeast two hybrid methods. JIP is thought to tether different MAPK kinase kinase (MEKKs), MAPK kinase (MKKs), and MAPKs into close proximity so that the successive phosphorylation events can occur efficiently, thus conferring specificity to a particular combination of kinases for activation. However, the regulation of the interaction between JIP and MAPK family is still remained. Here, we isolated a novel JIP interacting protein that is a protien kinase, identified the interacting domain of the kinase, and studied the effect of JNK signaling.

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Mouse lymph node cell (M10) which is highly sensitive to X-ray irradiation is also sensitive against UV-ray irradiation. Growth of this cell depended on the UV-rays(310 nm, band B) irradiation, and the cell growth was suppressed in proportion to the UV irradiation time. UV irradiation doses (10, 20, 30 and 60sec) showed the induction of cell apoptosis and necrosis. Cell apoptosis and necrosis were assayed by FACS Calibur (Beckton Dickinson) using Annexin-V-FLUOS staining kit (Rochu Diagnostics). Cell sorting analysis distinguished the difference of the apoptosis and the necrosis of M10 cells. Apoptotic cells increased in proportion to UV-irradiation. We analyzed the gene expression in the UV-irradiated cells using DNA array technology. Dynamic changes of 597 genes after the UV-B (310 nm) irradiation of three doses (10, 20 and 30sec) were investigated on DNA array membranes. Radioactivities obtained from imaging analyzer were analyzed using software of ArrayGauge. The expression of mRNA was further analyzed by EX-ARRAY program. Time course of the gene expression was investigated at 2h and 5h after the irradiation. The expression of almost genes reduced in proportion to the doses. The comparison of the gene expression with the parental cells (L5178Y) demonstrated the remarkable activation of homeobox gene (Hox-2.4) and serine (or cysteine) proteinase inhibitor, clade B member 6 (Serpinb6).

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We investigated the mechanism of sodium orthovandate (vanadate)-mediated inhibition of the caspase cleavage of p42/SETβ to p41 in ionizing radiation-induced apoptotic cell death of MOLT-4. We found that vanadate suppressed caspase activation and the subsequent cell death, but it did not suppress caspase activity. We further investigated the effects of vanadate, compared with that of a caspase inhibitor or overexpressing Bcl-2. Consequently, like Bcl-2, the effects are attributed to suppressions of the release of proapoptotic molecules from mitochondria, loss of mitochondrial membrane potential, and Bax conformational change, while the latter two apoptotic events are not suppressed by a caspase inhibitor. Our findings indicate that vanadate acts on upstream event(s) of caspase activation and mitochondrial permeabilization by different way from a caspase inhibitor.

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Apollon, an anti-apoptosis molecule containing IAP repeat, was first cloned by Naito et al. Immunohistochemical staining of Apollon was performed on 43 cases of oral squamous cell carcinoma. The cases were divided into 4 groups according to treatments such as radiotherapy, chemotherapy, chemoradiotherapy and without therapy between biopsy and surgical operations. Compared with biopsy, the number of cancer cells positive for Apollon at surgery was higher only in radiotherapy group. Using a human oral cancer cell line SAS, real-time PCR revealed overexpression of the Apollon gene after the exposure to X-rays and the induction was canceled by simultaneous treatments with CDDP. Tumor tissues formed after inoculating SAS cells into nude mice subcutaneously showed the same tendency as in vitro cultures. These suggest that Apollon mediates radioresistance of oral squamous cell cancer and simultaneous chemotherapeutic treatments reduce the resistance partly through the suppression of Apollon gene expressions.

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To reduce the side effects of radiotherapy, radioprotective effects of lycopene on villi and crypts in the small intestine of abdominally radiated mice (15Gy) were examined with administration pre-, continuous and post-radiation. In the lycopene group, the ratio of the villus length to the crypt was significantly increased in comparison with the radiation only group at 2 days after radiation. At 7 days after radiation, the ratio of necrotic cells in crypt/total was significantly decreased and the ratio of necrotic cells in villus/total was significantly increased by lycopene administration, which indicated an acceleration of the recovery from the radiation injury with lycopene. Each lycopene administered group showed a significant radio protective effect, with the pre-radiation administration inducing a smaller effect than that of continuous and post-radiation administration. It is concluded that pre-, continuous and post-radiation administration of lycopene protects against radiation injury of the small intestine and accelerate the recovery. For side effects of gastrointestinal injury (diarrhea, vomiting and abdominal pain) after radiation therapy, clinical application of tomato and tomato juice including licopene is expected. Also, antioxidation nutrients of tomato and tomato juice would be effective as protection of radiation exposure of a pilot and an astronaut and space foods.

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α-amylase (EC.3.2.1.1) catalyzes the specific cleavage of α-1.4 glucan bonds in polysaccharides such as starch, and is produced by the pancreas (P-amylase) and the salivary gland (S-amylase). Stresses including radiation and major surgery are known to increase the activities of α-amylase in serum. However, the mechanism is not fully understood. Studies using Blue Starch as the substrate showed that X-ray irradiation of C3H/He mice increased the α-amylase activity in plasma at dose-dependent manner. A maximal activity of α-amylase was observed at 12 h after irradiation (about 3-fold). Non-denatured polyacrylamide gel fractionation of the plasma followed by starch-iodine staining showed that the S-amylase was dominant in mice and the activity was increased by irradiation, whereas that of P-amylase was not affected. On the other hand, irradiation failed to increase the activity or the protein level of α-amylase in human salivary gland cell lines HSG and HSY that were differentiated in Matrigel. Irradiation caused a decline in the pilocarpine-induced the flow rate of saliva and reduced the activity of α-amylase in saliva. Our results suggest that irradiation may not increase the synthesis but liberate α-amylase into blood via an increased permeability of cell membrane. Further studies are in progress.

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INTRODUCTION: Recently, human exposure to an extremely low frequency magnetic field (ELFMF) from various electrical appliances has increased significantly. Thus, the possible health effects of exposure to ELFMF have become a considerable public concern. On the other hand, occurrence of diabetes mellitus has increased progressively in recent years. Insufficient pancreatic islet function is the basis of various forms of diabetes. These two factors motivated us to investigate the effects of exposure to ELFMF on the function of insulin secreting cells. METHODS: We stimulated RIN-m, an islet-derived insulinoma cell line, to secrete insulin under exposure to an ELFMF (60 Hz and 5 mT) or under sham-exposure conditions. RESULTS: In the presence of a depolarizing concentration of potassium (45 mM KCl), exposure to the ELFMF significantly attenuated insulin release from RIN-m cells, compared to sham-exposed cells. The results of flowcytometric analysis indicated that ELFMF exposure prevented cell membrane depolarization. CONCLUSION: In this study, we detected the inhibitory effects of ELFMF on insulin secreting cells.

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DNA double strand breaks (DSBs) induced by ionizing radiation, are repaired by several pathways, such as non-homologus end joining (NHEJ), single-strand annealing (SSA), and homologous recombination (HR). Recently, it has been shown that ionizing radiation induces genomic instability, which is transmitted over many generations after irradiation through the progeny of surviving cells. This indicates that abnormal rejoining of DSB causes lesion, which persists as a radiation signature. Here we applied phosphorylation of histone H2AX induced by DSB as a marker of DNA lesion, and tried to establish a bio-dosimetry system, by which radiation signature on chromosome in irradiated nomal human diploid cells can be detected. We found that a number of phosphorylated H2AX foci per chromosome increased in a dose-dependent manner, and the foci colocalized at the site of chromosome aberrations. Interestingly, phosphorylated H2AX foci was also present even on chromosome which aberration was not left. Number of foci increased with dose dependent manner and it was about 11 times more than the number of chromosome aberrations. From these results, we concluded that phosphorylated H2AX foci could be a suitable marker for detection of radiation signature.

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X-ray-induced senescence-like growth arrest (SLGA) gives some similar phenotypes to senescent cells, which are flattened morphology, expression of senescence-associated-&beta-galactosidase (SA-&beta-gal) and p53-dependent irreversible G1-arrest. Although it has been thought that residual DNA double strand breaks induce SLGA, the causal relation is not clear. In this report, we investigated localization of X-rays induced phosphorylated histone H2AX in mitotic cells to examine if residual DNA double strand breaks are involved in SLGA induction.
In response to 4 Gy of X-rays, 40 % of normal human diploid cells became SA-&beta-gal positive 4 days after irradiation, and approximately 80 % of cells showed phosphorylated H2AX foci (within 5 foci per nucleus). We found that all foci colocalized with phosphorylated ATM foci. In metaphase cells, phosphorylated H2AX foci were observed on chromosome fragment, but in addition, they were detected on chromosomes without any detectable aberration. Furthermore, phosphorylated H2AX foci were located on 80 % of observed chromosomal bridge, which results from illegitimated DNA-rejoining. These results indicate that not only DNA double strand breaks but also altered chromatin structure are associated with SLGA induction.

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Phosphorylation of H2AX (γH2AX) is induced by various genotoxic stresses and associated with the recruitment of damage-signaling factors to damaged DNA. Deletion of H2AX causes hypersensitivity to ionizing radiation (IR), a G2/M checkpoint defect and genomic instability in mice. However, the precise role for γH2AX in DNA repair is less understood. To investigate the role for gH2AX in DNA repair, we generated a mutant line incapable of H2AX phosphorylation by introducing a H2AX mutant allele in which serine 139 is replaced by alanine (S139A) into H2AX^+/- cells. In the mutant, phosphorylation of H2AX is abolished after IR. The mutant was sensitive to camptothecin (CMP), which efficiently induces replication blocks, and ionizing radiation (IR). Interestingly, the mutant showed reduced gene targeting frequencies and an impaired Rad51 focus formation, indicating a requirement of γH2AX for homologous recombination (HR). In addition, in a XRCC3^-/-/H2AX^-/S139A double mutant, spontaneous and CMP-induced chromosomal aberrations, probably caused by defective HR, synergistically increased in number. These results suggest that γH2AX participates in HR-mediated repair during replication, thus maintaining the genomic stability in vertebrates.

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The Escherichia coli MutT protein hydrolyzes 8-hydroxy-dGTP (8-OH-dGTP) in vitro, and mutT gene deficiencies cause increased spontaneous A:T to C:G mutations. However, no direct evidence exists for the enhanced mutagenicity of 8-OH-dGTP in mutT cells. In this study, 8-OH-dGTP was introduced into wild type and mutT E. coli cells, and mutations on a chromosomal gene were monitored. 8-OH-dGTP induced mutations in the rpoB gene, and the degree of the mutation induction in the mutT strain was ~6-fold higher than that in the wild type strain. On the other hand, 2-hydroxy-dATP, which is not a substrate of the MutT protein, increased mutations to similar degrees in both strains. These results provide the first evidence showing that the MutT protein suppresses mutations by 8-OH-dGTP in vivo.

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We have analyzed the chromatin structure of the promoters of the c-FOS and U6 genes of mitotic HeLa cells, using dimethyl sulfate, ultraviolet light, and 4,5',8-trimethylpsoralen, as in vivo footprinting agents. The distribution of the damage induced by these agents was visualized by ligation-mediated PCR or terminal transferase-dependent PCR. We observed that transcription factors were displaced from their recognition sequences in these promoters during mitosis, and we found that the nucleosome positioning, which seems to potentiate transcription in these promoters during interphase, disappeared during mitosis. Thus, the complex nucleoprotein structures of these promoters were dissociated during mitosis, and this seems to contribute to the suppression of transcription in this phase of the cell cycle.

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Detection of DNA strand breaks by radiation was carried out using real-time PCR amplification. The PCR method is able to target to given sequences. Therefore, differences of the radiation sensitivity might be detected. Gamma irradiation to pBR322 plasmid DNA solutions was done by 137Cs, gammacell 40, as dose from 0 to 250Gy. 1000~125bp fragments were amplified by combined primer sets for rDNA, retrotransposon R1Bm and R2Bm to irradiated Bombyx mori genomic DNA solution including 1~5mM Tris-HCl as radical scavengers. Two types DNA solutions were prepared. Genomic DNA extracted has the size variations from 20kb to 40kb and DNA digested with EcoRI has the sizes from 10kb to 17kb for the target rDNA cluster region. EcoRI site is only one in the each region of three different units and the location is out of the region between those primers. The results were shown that radiation sensitivity was depending on the scavenger concentration. Comparisons between digested and non-digested genomic DNA showed the different sensitivity patterns. These differences might be derived from structures of those DNAs in the solution. We want to discuss the reasons occurred the differences including the feasibility for the simulation analysis.

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We studied radiation-induced mutations in a transgenic mouse, HITEC, which carries the rpsL gene as a reporter for mutations. We also performed a partial hepatectomy to induce cell proliferation and thereby increase the detection of mutations. HITEC mice were irradiated with 5 Gy of gamma rays before hepatectomy, and the remnant-regenerating liver was excised after hepatectomy. The mutation frequencies for both the resected liver at the partial hepatectomy and the remnant-regenerating liver were analyzed. We previously reported that in mice in which hepatectomy was performed 2 days after irradiation and the remnant liver was excised 3 days after hepatectomy, the mutation frequency was increased approximately twofold. In mice in which hepatectomy was performed 10 days after irradiation and the remnant liver was excised 3 or 10 days after hepatectomy, the mutation frequency was two to three times greater. These results indicated that partial hepatectomy increases the frequency of radiation-induced mutations, and that the mutations remained in the liver cells for at least 10 days after irradiation. In this study, the periods between irradiation and hepatectomy were set to 30 and 60 days and the remnant liver was excised 3 days after hepatectomy. We did not see an increased mutation frequency in either group. This result shows that the mutation repair system is obviously working even in terminally differentiated cells.

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Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. The life span of fibroblasts derived from WS patients is much shorter than that of normal fibroblasts. WS cells also show genomic instability and sensitivity to certain genotoxic agents. The responsible gene for WS, WRN belongs to RecQ helicase family and exhibits 3' to 5' helicase and 3' to 5' exonuclease activities, and interacts with Ku, DNA-PKcs, RPA and NBS1. DNA repair-related genes such as NBS1, BRCA1 and MDC1 are recruited to DNA damage sites for DNA repair. AS WRN might function for DNA repair, WRN could be recruited to DNA damage sites. Therefore, we investigated the foci formation of WRN in response to DNA damage.
The formation of WRN foci was detected in response to DSBs induced by X-ray, bleomycin or etposide in HeLa cells. AT and NBS cells showed these foci formation. Camptothecin, or hydroxyurea, which stall the DNA replication and induce DSBs, also induced WRN foci formation. Moreover, S phase cells showed WRN foci in response to DNA damage, but G1 phase cells didn't. Therefore, WRN is recruited to DNA damage site only in S phase and function for DNA repair.

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Following whole-body exposure of p53(-/-) and p53(+/+) mice to fast neutrons from a 1-W reactor UTR-KINKI, the frequency of micronuclei (MNs) was measured in reticulocytes at multiple time points after exposure. The resulting time-course curve of MN frequency showed a clear peak 36 h after exposure, irrespective of the dose used. We calculated the area beneath the curve (A) as an index of total MN response. The A values for p53(-/-) and p53(+/+) mice did not differ significantly, suggesting near normal sensitivity of p53(-/-) mice to fast neutrons for MN induction. Since p53(-/-) mice are hypersensitive to X-ray-induction of MNs in reticulocytes, this finding implies that p53-gene function is not involved as a defense of somatic tissues from DNA damage caused by high-LET radiation. This hypothesis was tested by examining the frequency of apoptotic lymphocytes in the thymus and spleen of p53(-/-) mice 4 h after exposure to fast neutrons. In both organs, the frequency of apoptotic cells increased significantly over the control level. For example, the frequency in mice exposed to a neutron dose of 0.2 Gy relative to the control was 7.2±2.4 in thymus and 7.2±1.9 in spleen. These results support the conclusion that apoptosis induced by high-LET radiation is controlled by gene(s) other than p53.

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The genome as a carrier of genetic information in living cells is vulnerable to DNA-damaging agents of both endogenous and environmental origins. To remove DNA lesions, cells have two major repair pathways; nucleotide excision repair (NER) operating on bulky helix-distorting damage caused by environmental mutagens and base excision repair for non-bulky and non-helix-distorting DNA modifications caused by endogenous and some chemical carcinogen-induced damage. When RNA polymerase II in the elongation phase encounters DNA damage that blocks transcription, transcription-coupled repair (TCR), a specialized pathway that efficiently removes lesions on the transcribed strand, operates to counteract the immediate and cytotoxic response of the interference. The transcribed strand is repaired much faster by TCR than is the non-transcribed strand or inactive regions by global genome repair (GGR). The focus of this project is to investigate how the DNA lesion causes the stop of pol II during transcription elongation which is thought to be a trigger step of TCR. To analyze effect of DNA lesions on transcription elongation, purified pol II and oligo dC tailed template containing a DNA lesion were employed for transcription elongation assays. The results showed that pol II stalled at DNA lesions by incorporation of nucleotides and formed pol II/lesions/transcrips complexes. We discuss these observations together with the other properties of pol II and indicate that stalled-pol II itself has function as damage sensor.

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Mutation in NBS1 protein causes Nijimegen breakage syndrome (NBS) which cells exhibit hyper-radiosensitivity, abnormal S phase checkpoint and chromosomal instability. NBS1 forms a complex with Mre11/Rad50 to play critical roles in DNA double strands break (DSB) repair. We have reported that the Mre11 complex with functional NBS1 is essential for homologous recombination (HR) repair pathway. This suggest that NBS1 regulates the complex by controlling their subnuclear localization and enzymatic activities. To analyze functional domains of NBS1 in HR repair pathway, a SCneo reporter was introduced into NBS fibroblast cells. Full length or mutant NBS1 was expressed in the cells, and then DSB was induced by transient expression of I-SceI endonuclease. We found that Mre11-binding domain of NBS1 is the most critical domain for HR, and that ATM-phosphorylation sites of NBS1 is not essential for normal HR function whereas slight decrease in efficiency was observed.

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Eukaryotic cells have at least two major pathways to repair DSBs, i.e., non-homologous end joining (NHEJ) and homologous recombination (HR). In the present study, we examined the role of each repair pathway in the formation of chromosome aberration by using knockout DT40 cell lines.
We constructed Ku70^-/- and Rad54^-/- cell lines and chromosome aberration that induced in G₁ or G₂ phase by X-irradiation was examined.
The results in G₂ phase irradiation showed that higher frequency of chromatid type aberration was observed in Rad54^-/- cells than that in other cell lines. This suggests that HR in which Rad54 plays an important role is important for the relief of formation of chromatid type aberration. When cells were irradiated in G₁ phase, higher yield of chromosome type aberration was observed in Ku70^-/- cells. Interestingly, dicentric was suppressed in Ku70^-/- cells in G₁ irradiation. These results suggest that chromosome type aberration is suppressed by NHEJ in which Ku70 plays an important role, although Ku70 is important for formation of dicentric.

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We have reported that DNA-dependent protein kinase (DNA-PK) activity correlates with radiation sensitivity using esophageal cancer cell lines. It has been reported that suppression of DNA-PK activity by PI3-kinase inhibitor wortmannin, antisense Ku70/DNA-PKcs or small inhibitory RNA for DNA-PKcs sensitize cells to ionizing radiation. These results indicate that levels of DNA-PK activity have relevance to cellular sensitivity to ionizing radiation. In colorectal cancer, DNA-PK activity and protein/mRNA levels of Ku70, Ku80, DNA-PKcs and Sp1 were significantly higher in the tumor tissues compared with the normal tissues. Ku70, Ku80 and DNA-PKcs have consensus Sp1 recognition elements in their promoter region, which suggest their transcription is Sp1-dependent. In esophageal cancer, DNA-PK activity and protein/mRNA levels of Ku70, Ku80, DNA-PKcs and Sp1 were significantly higher in the tumor tissues compared with the normal tissues. In immunohistochemical analysis, DNA-PKcs, Ku80 and Ku70 were expressed almost exclusively in the nuclei. In normal squamous epithelium, the cells in basal cell layer highly expressed them and the differentiated cells express them at low intensity. Their expression varies in the same cancer tissue, which makes the estimation of radiation sensitivity by biopsy difficult. For radio-sensitization of tumor tissue, we found that phosphorothioate oligonucleotides and suramin inhibit DNA-PK activity. Suramin is supposed to be applicable to clinical use.

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To clarify the roles of two major DNA double-strand break (DSB) repair pathways, i.e., non-homologous end-joining (NHEJ) and homologous recombination (HR), in hyperthermic radiosensitization, we examined chicken B lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR. Hyperthermic radiosensitization could be seen in all of the mutants, including those lacking both NHEJ and HR. Therefore, hyperthermic radiosensitization cannot be explained simply by the inhibitory effects, if any, on typical NHEJ and/or HR alone. However, in NHEJ-defective cells, consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered cells in G1 and early S, was not sensitized: substantial sensitization was seen only in the radioresistant subpopulation, which is considered cells in late S and G2, capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G1 and early S phases and also suggested some inhibitory effects of hyperthermia on HR. Thus, partial contribution of NHEJ and HR in hyperthermic radiosensitization, especially that depending on the cell cycle stages, remains to be considered.

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In co-culture with osteoblastic cell line MC3T3-E1 and mouse bone marrow cell, the number of osteoclasts with in carbon ion irradiation group were fewer than that of gamma ray group or non-irradiated group. The outlines of an osteoclast in the carbon line irradiation group were irregular and small of a size. On the other hand, osteoclasts in the gamma ray irradiation group were large and stained for TRAP activity. Carbon ion irradiation had a more marked effect on osteoclast suppressed their maturation to a greater extent than did gamma irradiation. These observations suggest that carbon ion irradiation induces differential modulation of osteoclasts growth factor expression.

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In order to contribute to the establishment of sophisticated radiotherapy, a research project for developing simulation systems utilizing advanced computational technology is in progress. In this project there are two subjects: 1) development of a dose calculation system for remotely supporting X-ray therapy, 2) development of a simulation system for demonstrating the realization of therapy using laser-drive protons. In the first subject, a dose calculation system for providing accurate dose distribution in a patient body is under developing. In this system, a voxel human model, a precise accelerator head model, and a Monte Carlo calculation are utilized to perform realistic simulation. The dose distribution is calculated by this system at a dose calculation center, and the related data are transferred through a network. The system is intended to support the current therapy widely, and also to apply to the advanced therapy of IMRT (Intensity Modulated Radiation Therapy) and CTRTx (CT radiotherapy). In the second subject, the simulation system for proton production process by laser, for proton transport and for dose calculation are under developing. The feasibility of laser-driven proton therapy will be investigated using the simulation system, which will be expected to lead to the less expensive and small-scaled proton treatment equipments.

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Stress-induced senescence may play an important role as an acute, drug- or ionizing radiation(IR)-induced program along with interphase apoptosis and mitotic catastrophe. The aim of the study was to evaluate whether IR can induce senescence-like phenotype (SLP) associated with terminal growth arrest in the thyroid cancer cells, and if so, to evaluate impact of terminal growth arrest associated with SLP in intrinsic radiosensitivity of various thyroid carcinomas. The induction of SLP in thyroid cells were identified by: 1) SA- β -Gal staining method, 2) Dual flow cytometric analysis of cell proliferation and side light scatter using vital staining with PKH-2 dye, 3) Double labeling for 5-BrdUrd and SA- β -Gal. IR induced SLP associated with terminal growth arrest in ARO, FRO, KTC-2, NPA and TPC-1 thyroid cancer cells lines in time- and dose dependent manner. Analysis of relationship between induction of SLP and radiosensitivity revealed a trend in which more radioresistant cell lines strongly tended to show lower specific SLP yields (r=-0.93, p=0.068). We find out that SA- β -Gal staining is detectable in irradiated ARO xenotransplants, but not in control tumors. We, therefore, conclude that induction of SLP with terminal growth arrest contribute to the elimination of clonogenic populations after IR.

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