I would like to provide an occasion for our colleagues to consider the past, present and future of radiation biology. As the field of radiation research covers a wide range of sciences, radiation biology also treat with many fields such as therapy, protection/sensitization, ecology and others. Every man has a unique way of thinking. That comes from his/her nature and environments as well. I have been working for 35 years in NIRS that is very unique in the world, in the field of biology for radiotherapy. I will start today's talk with history of radiation biology in radiotherapy. The most important findings in past are, I believe, the following 5 items. Namely, (1) colony formation, (2) sub-lethal- and potentially lethal damage and repair, (3) tumor hypoxia, (4) repopulation and 4R in radiotherapy and (5) apoptosis. Second, most important at present are (6) findings/proposals of linear-quadratic relation in dose-response and (7) proton therapy. The finally, future is difficult. Probably true is {“Never make forecasts, especially about the future”}, an aphorism made by Samuel Goldwyn, a Hollywood movie director. I dare to say here, however, the following two items would be most important in future; (8) predictive assay based on genomic information and (9) molecular imaging of intra- and/or extra-cellular information. Kinetics or sequential information might be important in the future items. Twenty years later, you will see whether the prediction be correct. P.S.: Other than therapy, important in the history is (10) thymic tumor induction by fractionated irradiation.

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In the history of more than 100 years using x-rays, radiotherapy has now established its firm place in cancer therapy. Recently, through development of 3-D conformal radiotherapy that permits high dose delivery to the tumor with sparing the surrounding normal tissues, improvement of treatment results and expansion of indications have been achieved. Within two months after the discovery of x-rays, their potential for treatment of disease was realized and tested, and 3.5 years thereafter the first patient was successfully cured of cancer by radiation alone. Since then, x-rays had been extensively used in cancer treatment but tumor control was not so good with severe skin complications due to low energy of x-rays available and lack of knowledge about “optimal” fractionation.
In the 1950's when the megavoltage radiotherapy became available using the modern linear accelerator and ⁶⁰Co unit, the potential ability of radiotherapy was remarkably expanded. In an effort to improve dose distributions using these machines, various techniques to improve dose concentration had been developed, and in early 1960's Takahashi developed cam-controlled multileaf collimators for conformational therapy with moving beams. Despite all these efforts, however, these analogue systems were never particularly successful and it soon became apparent that computerization was essential.
The introduction of CT scans developed in 1972, coupled with implementation of technologically advanced accelerators and sophisticated planning systems, have led us into a new era of high-tec radiotherapy. Charged particle with protons and carbon ions has also became available in more than 20 facilities worldwide. Advanced knowledge of radiobiology has also provided the impact on improvement of radiotherapy. However, it is important to realize not all developments have been without problem. The indications of various modalities have not been entirely known and costs are still expensive in many modalities.
As such, the major efforts of radiotherapy have been focused on technical developments to permit “more intensive and less toxic” treatment. In order to achieve this aim within 21^st century, further developments in the field of radiation biology may also be needed. This will be also discussed in my talk.

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The bystander effect for chromosomal aberrations and SCEs was examined in Chinese Hamster cell lines deficient in either non-homologous end joining or homologous recombination.
Dose-response curve for induction of chromosomal aberrations was curvilinear for all cell lines, with a great effect occurring at low fluences owing to aberrations arising in bystander cells. These aberrations were predominately of the chromatid type.
We are presently examining the HR repair defective cell lines in different Rad 51 paralogs, as well as a cell line deficient in BRCA-2. The frequencies of SCEs measured in three non-irradiated wild type cell lines were approximately 0.3 SCE per chromosome, whereas only 0.16 SCE per chromosome was observed in two Rad 51C -/- cell lines. SCE frequencies significantly higher than the baseline line level were induced in wild type cells. On the other hand, induction of SCE was minimal or absent after α-particle irradiation in all of the mutant cell lines. These results suggest that Rad 51 paralogs may contribute to DNA damage repair processes involved in the induction of SCEs in bystander cells by very low fluences of α-Particles.

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Space radiation presents significant health risks to astronauts on the International Space Station and for future missions to the Earth's moon or Mars. Risks of concern are carcinogenesis, degenerative tissue effects including early and late responses to the central nervous system, heart disease and cataracts, and acute risks. Methods used to project risks on Earth need to be modified because of the large uncertainties in projection models for space radiation risks, and thus impact safety factors and mission costs. We describe NASA's unique approach to radiation safety that applies uncertainty based criteria within the occupational health program for astronauts. The terrestrial criteria of the “point estimate”of the maximum acceptable level of risk is supplemented by a requirement that protects against risk projection uncertainties using the upper 95% confidence level (CL) in the radiation cancer projection model. NASA's 95% CL criteria links space flight safety to a vibrant ground based radiobiology program investigating the radiobiology of high-energy protons and heavy ions using the NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Lab. The near-term goal of this research is new knowledge leading to the reduction of uncertainties in projection models. Long-term goals include the development of biomarkers and countermeasures of space radiation risk. Risk projections involve a product of many biological and physical factors, each of which has a differential range of uncertainty due to lack of data and knowledge. The current model for projecting space radiation cancer risk relies on the three assumptions of linearity, additivity, and scaling along with the use of population averages. We describe mechanistic research that will reduce uncertainty estimates for this model by testing these underlying assumptions.

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Space radiation has been long acknowledged as a potential showstopper for long duration manned interplanetary missions. Our knowledge of biological effects of cosmic radiation in deep space is almost exclusively derived from ground-based accelerator experiments with heavy ions in animal or in vitro models. In an effort to gain more information on space radiation risk and to develop countermeasures, NASA started several years ago a Space Radiation Health Program, which is currently supporting biological experiments performed at the Brookhaven National Laboratory (Upton, NY). Accelerator-based radiobiology research in the field of space radiation research is also under way in Russia and Japan.
Space radiation research in Europe has been mostly driven by flight experiments, and remarkable results were gathered in the field of space radiation dosimetry in low-Earth orbit. The European Space Agency (ESA) has recently established an ambitious exploration program (AURORA), and within this program it has been decided to start a ground-based space radiation biology program. Europe has a wide tradition in radiobiology research at accelerators, generally focussing on charged-particle cancer therapy. This expertise can be adapted to address the issue of space radiation risk.
To support research in this field in Europe, ESA issued in 2005 a call for tender for a preliminary study of investigations on biological effects of space radiation (IBER). This study has been completed in 2006, and the study group has recommended ESA to support a research program on biological effects of heavy ions using GSI in Darmstadt (Germany) as main facility and GANIL in Caen (France) as secondary facility. The new accelerator currently under construction at GSI, FAIR, will be able to provide beams at very high energy in the future, thus covering an energy range (2-20 GeV/n) of great importance in space but poorly explored so far. New biology research topics identified as possible targets for large integrated projects were noncancer later effects, acute effects by large solar particle events, and interaction of space radiation with other space environment stressors.

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According to my view, the introduction of radiation research to Japan started after the accidental exposure of radioactive debris to the seamen on the fishing boat, the 5^th dragon in 1949 near the Bikini atoll. Immediately after the war in 1945, scientific research on A-bomb victims were carried out by Japanese scientists but it was classified by the occupation army. When the radioactively contaminated fishing boat came back to Japan, the contamination and its health effects were reported sensationally. The Japanese government immediately provided the research funds on radiation research and in the next year enacted the Fundamentals for Nuclear Development. Thus nuclear age started in Japan in 1955.
I was in an university hospital as a physician in Mie prefecture and interested in X-ray physics for diagnosis. Since I have read an introductory review on 1950 ICRP recommendation by R.S.Storn published in Radiology in 1952, I realized that our knowledge about radiation effects were quite limited. I moved to a new department of induced mutation in National Institute of Genetics in Mishima in 1955. When the Japan Radiation Research Society was established in 1959, I was regarded as a specialist of genetic effects of radiation, but what I have done was an introduction of published papers.
Fortunately new institutes and department were established one by one since 1956 in Japan, we were able to start new research activities on radiation research to catch up the activity to the international level. According to my personal impression, there seemed to be some similarities between our efforts and those of the scientists in the Meiji-era at the end of 19^th century in Japan, i.e., wakon-yohsai in Japanese, European technology with Japanese mind.

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Radiation research in Japan started after atomic bomb attack to Hiroshima and Nagasaki in summer 1945 as far as environmental study is concerned.
The next important event was the radioactive contamination of tuna-fishing boat Fukuryumaru no.5 in March 1954. This was caused by fall out ofsubstantial amount of radioactive particles produced by a large scale hydrogen bomb explosion test carried out at Bikini atoll.
Radioactive contamination of fishing boat and tuna, and exposure of fishermen to radiation gave great fear to the public of Japan. In addition to marine contamination it was found that terrestrial environment including agricultural crops and others were also contaminated by radioactive fallout.
Therefore, research scientists of various universities and research institutes set up many investigation teams of different specialities such as agriculture, fisheries, meteorology, veterinary science and others.National Institute of Radiological Sciences was establishes in 1957 and Japan radiation Research Society started in 1958.
During these period, investigation on the distribution and concentration of various fission products(¹³¹I, ⁹⁰Sr,¹³⁷Cs etc.)and neutron-induced nuclides(⁶⁰Co,⁶⁵Zn,⁵⁴Mn etc.) in environment including food materials and humans.
By the information is obtained from these investigations, useful knowledge on nuclides transfer mechanisms (Sr-Ca discrimination, relation between fall out rate and accumulated deposit in plants, and chemical states of nuclides etc.) and transfer coefficients of radionuclide were given to our research fields. Transfer coefficient of radionuclides such as air to plant, soil to plant, water to aquatic organisms were utilized for estimation of future radionuclide concentration in ecosystems and radiation dose to man.As nuclear power production started in Japan, the environmental radiation safety became so important.
Informations obtained by the past radiological studies in the environment were very useful for this purpose.
Furthermore, pilot plant for reprocessing of spent nuclear fuel in Tokai needed the same kind of transfer coefficient of many different radionuclides. For that purpose, a great effort to get such informations was made by field studies, experimental studies considering different chemical states of nuclides and also stable nuclide concentration studies. Difference of radionuclide metabolism among different age groups in man and experimental animals was studied with emphasis on very young stage of man an animals.
Natural radiation studies were also carried out including terrestrial radiation, cosmic radiation and natural nuclides in food and radon with its progeny.

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Radiation doses for the atomic bomb survivors have been reevaluated repeatedly. The first systematic dosimetry was done in 1957 (T57D). In 1965, this dose was evaluated by the collaboration between US and Japan and obtained as Tentative 1965 Doses (T65D)). The T65D was named with Tentative. However this was obtained according to the actual experiments with nuclear explosions etc. for 10 years of study, of which doses were thought to be the final evaluation. From the T65D radiation risks was obtained using the epidemiology study in Radiation Effects Research Foundation (RERF). After the T65D, US scientists found some problems comparing with the obtained dose results by using a super computer in 1970s. In 1981 reevaluation of this T65D was begun. In 1987, the new Dosimetry Study 1986 (DS86)) was obtained. The major change of DS86 was the decrease of neutron dose in Hiroshima, of which change was from 1/5 to 1/9 of T65D. In T65D neutron doses in Hiroshima was large and Nagasaki's was negligible and successfully explained the difference of leukemia incidence by assuming a neutron RBE. However, by the DS86, neutron doses in Hiroshima and Nagasaki became negligible; therefore neutron RBE data could not be estimated after DS86. Again in this DS86 there found some discrepancy between calculation and measurements in the Hiroshima's neutron doses. US-Japan joint study was begun and new Dosimetry Study 2002 (DS02)) was established. The difference between DS02 and DS86 was the increase of gamma rays about 10% for both in Hiroshima and Nagasaki. US group calculated organ doses and they will calculate radiation risks. These risks will be discussed at the International Congress of Radiation Protection (ICRP) and will be introduced in each country's law of radiation protection, which limits radiation exposures for radiation workers and general people.

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The epidemiological studies on health effects of A-bomb radiation in Hiroshima and Nagasaki continue to provide an important data source for the framework of radiation risk evaluation and radiological protection. The A-bomb radiation is a mixture of neutrons and γ-rays, and hence the health effects should be expressed against the equivalent dose, in which neutron dose be converted by weighting with relative biological effectiveness (RBE). However, it is generally reckoned that the direct derivation of any meaningful estimate of neutron RBE is impossible from the epidemiological data in survivors of Hiroshima and Nagasaki. To overcome this problem, as an alternative approach, we attempted at an experimental derivation of neutron RBE. The RBE of fission neutrons was first established for the induction of chromosome aberrations in human lymphocytes irradiated in vitro with fission neutrons with wide variety of energy spectra, and compared with that for tumor induction in the experimental animals by reviewing observations in literature. The validity of the established RBE system was then tested in the epidemiological data of A-bomb survivors. The RBE of fission neutrons was dependent on neutron dose but not on the energy spectra. The chromosomally derived RBE was essentially the same as that for the induction of solid tumors in mice and rats, and satisfactorily applied to the chromosome aberration frequencies and cancer incidence in A-bomb survivors as it was supreme in eliminating the city difference of the effects. The use of new RBE system applied to the DS02 dose system reduces the cancer risk by a factor of about 0.7 as compared with the current estimates using DS86 doses with neutrons weighted by a constant RBE of 10.

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Radon (²²²Rn) has been recognized as one of the most important contributors to exposure from natural radiation sources over the past 30 years. Recent studies also revealed a positive relationship between indoor radon concentration and lung cancer risk even at a low exposure level below 200 Bq m^-3. In the WHO International Radon Project launched in January, 2005, radon is regarded as a global cause of disease and the second leading cause of lung cancer next to tobacco smoking. Many countries world wide are about to take measures against radon. In general, residential radon is regulated by the action level at 200-600 Bqm^-3 based on the ICRP recommendation. However, WHO is planning to recommend a new guideline for radon exposure. The action level will be set at a lower level (100-400 Bq m^-3) than current one. The new guideline would necessitate the survey of indoor radon level; the measurement data have to be sufficiently assured from the viewpoint of their reliability.For radon measurements, there are many measuring devices: alpha track detectors, charcoal canisters, electrets and so on. In particular, alpha track detectors and electrets are suitable for large-scale and long-term surveys so as to obtain annual radon concentrations. Those detectors are also often used in epidemiological studies. They are generally calibrated in a well-controlled environment such as a radon chamber. However, Tokonami (2005) has pointed out that some of them are sensitive to thoron (²²⁰Rn). This finding implies that radon readings will be overestimated and consequently may lead to incorrect estimates of lung cancer risk. The present study describes thoron interferences on radon measurements in some of typical detectors, overviews of thoron concentrations in Japan, China, Korea and Hungary, and some related issues from the epidemiological viewpoint.

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The term “radon dose”is used most often to indicate the doses of inhaled radon decay products. Gaseous Rn-222 itself makes a contribution less than one percent to the total “radon dose”. The removal of inhaled airborne particles from the tidal airflow through the respiratory airways is governed by the state of local airflow condition and the physical properties of the aerosol particles. The former is related to the airway morphometry and respiratory physiology of the subject and the latter includes the size and density of the particles. The particles deposited in the respiratory tract are cleared mainly by absorption into the blood and by mucociliary movement to the pharynx. The location at which the radionuclide decays depends on the rates of these clearance mechanisms as well as its initial deposition site. Basal cells in bronchial epithelium are long considered to be cells at risk. In addition, secretory cell layers are also assigned to risk-relevant target tissues in the ICRP human respiratory tract model. The depth of them in bronchial and bronchiolar epithelium is important for calculation of dose to man. A method to derive dose coefficient by modeling the processes from particle deposition to energy absorption by target tissues is often called “The Dosimetric Approach”. On the other hand, ICRP has derived a value of dose conversion convention by another method often called “The Epidemiological Approach”, in which the detriment per unit radon exposure was assessed from the data on lung cancer induction in miners and was directly compared with the detriment associated with a unit effective dose, which was based mainly on the epidemiological studies on atomic bomb survivors. This paper will provide outlines of both approaches with emphasis on factors influencing dose and risk estimation.

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Radionuclides such as plutonium (Pu) and uranium (U) induce toxic effects by the synergism of radiation and chemical actions. The major cause for “uncertainty” in the assessment of effects is the toxicity of the chemical action induced by radionuclides. First are the changes of chemical forms of Pu and U after intake in the body; ionic Pu and U change to various forms such as hydroxide or phosphate, or are combined with protein. These cause differences in the characteristics and degrees of damage by the initial attack of radionuclide from those received thereafter. Second, the chemical changes of radionuclide are unequally distributed in organs, resulting in differences between the affinity for organs and the site where the damage is induced. In the case of U, acute and severe damage is induced in the kidneys, which retain only 15% of U, compared to the damage to the bone, where 85% of U is deposited. Third, the degrees of the chemical toxicity and radiation toxicity vary by doses of intake. In fact, with an increase in the dose of Pu, the acute death and shortening of life span that are apparently due to chemical toxicity appear before the onset of cancer. Fourth, the chemical action causes long-term retention and complex behavior of the retained radionuclides, which continue to attack the organs. Fifth, the damages by the chemical action tend to be irreversible and progressive compared to those by radiation exposure.
Thus, the chemical action and the toxicity induced by it might be said to cause “uncertainty” in the assessment of the biological effects that have been carried out only by radiation dose. Hereafter, a consideration of the contribution of chemical toxicity and chemical action as causes of uncertainty may be required in the assessment of toxic effects by radionuclides.

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Clinical use of intensity modulation radiation therapy (IMRT/IMXT/IMPT) is increasing year by year. In U.S.A., 60% of the cancer patients take radiation therapy, and more than 70% radiation therapy are by IMRT. In Japan, the number of patient taking radiation therapy is around 20% of all the cancer patient, and only 30 hospitals are ready to support IMRT (2004 investigation). IMRT is to modulate an intensity of radiation field, which is generally uniform intensity, depending on the geometrical positions of target and the organ at risk. Thus IMRT allows dose to be concentrated in the tumor volume while sparing OARs.
As well as U.S.A., that IMRT becomes the one of the mainstream of the future radiation therapy in this country is expected.
A whole body radiation exposure by the leakage radiation with the irradiation MU value increase and a normal tissue exposure with lower radiation dose near the target is often considered as a problem in IMRT and it may relate to possibility of the secondary malignancy incidence increase more. IMRT is potentially used for a curative case with radiation therapy and many patients treated by IMRT will get longer surviving period without serious acute toxicities. IMRT also represents a special case for children, who are more sensitive to radiation-induced malignancy and have so small body to receive scattered radiation. The late incidence of the secondary malignancy could not be ignored if a patient will survive longer.

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PET/CT has now been an indispensable imaging in the management of patients with malignancy. Hardware and its application in imaging procedures and diagnoses are evolving so fast. PET/CT is a non-invasive and highly useful imaging modality which enables whole body image acquisition. PET/CT is useful in cancer screening as well as evaluation of extent of disease, that is, staging of cancer. So far, it has been reported that PET/CT provides changes in treatment strategy in 20-30% of patients. Unsealed radionuclide therapy in some of cancers has recently been established in the treatment of malignant lymphoma and in the palliation of painful bone metastasis. New radionuclide therapy agents and good medical practices will bring nuclear medicine to an era of great evolution.

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[Purpose] To clarify the entrance skin dose (ESD) to both patients and operators during neurointerventional procedures.
[Materials and Methods] Institutional review board approved and oral informed consent was obtained from each patient. The ESD was measured for 32 patients (11 men and 21 women) between 15 and 76 years of age (median age, 61.5 years) using photoluminescence glass dosimeters (PLD). The ESDs for operators were measured simultaneously. Angiographic parameters including x-ray tube voltage, total exposure time, total fluoroscopic time, the number of digital subtraction angiography (DSA) studies and frames, and the dose-area product (DAP) were recorded during the procedure. The Pearson correlation test was used to determine the relationship between the maximum ESD and each of the angiographic parameters.
[Results] For 28 therapeutic IVR procedures, the maximum ESD was 1788 ± 1259 mGy (mean ± SD), and the ESD to the right temporal region, which showed the maximum dose point in average, was 1124 ± 1349 mGy (mean ± SD). The correlations between a patient's maximum ESD and the total exposure time, the DAP, the total number of DSA studies, and the total number of DSA frames were r² = 0.3622, P < 0.001; r² = 0.6422, P < 0.001; r² = 0.3955, P < 0.001; and r² = 0.7729, P < 0.001, respectively. The ESDs for operators were observed to be less than the threshold for deterministic effects.
[Conclusion] We demonstrated that the local ESD can be measured by the PLD method and that values of ESD with precise geometric information could be obtained. These results are valuable in that they may contribute to reduce x-ray dose accumulation in both patients and operators during neurointerventional procedures.

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To protect normal tissues from radiation-induced damages, usage of radioprotector is one possible way. The properties of radioprotectors required should be different depending on what occasion they are used. When they are used on the occasion of radiation exposure from nuclear accidents and radiological terrorism, which could result in large-scale casualties, the properties required are, for example, 1) effectiveness of post-irradiation administration, 2) easiness for administration, 3) stable and inexpensive. On the occasion of clinical use of radiation, the protectors can be administered before irradiation. When protectors are used for radiation therapy to reduce normal tissue damages, they should not protect tumors. When protectors are used for radiation diagnosis, they should be free of side effect, because they are used for a large number of normal subjects. In the present symposium, the present status of development of various kinds of radioprotectors including the results of our group will be shown.

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Recent crystal structure and cryo-electron microscopy studies have revealed that proteins called translation factors mimic the shape of tRNA. One of them, a polypeptide release factor, encodes a tripeptide that serves as an ‘anticodon’ to decipher stop codons in mRNA. These findings established the novel concept of macromolecular mimicry between protein and RNA. We aimed to prove this concept by creating novel RNA molecules that mimic proteins of interest. The systematic evolution of ligands by exponential enrichment (SELEX) method is based on in vitro selection of oligo-nucleotide ligands from large random-sequence libraries by repeated reactions of DNA transcription, RNA selection and RT-PCR amplification. The selected oligonucleotide ligands, having both high affinity and specificity to target molecules, are called ‘aptamers’. We have initiated SELEX experiments using several mammalian proteins. Selected RNA aptamers against target proteins acquired several properties equivalent to, or more importantly, superior to antibodies. One of these aptamers had a Kd on the picomolar scale, an affinity which is a thousand-times stronger than normal antibody. Structural and biochemical analysis revealed that RNA aptamers could achieve specific high affinity to target protein by capturing its global conformation even if it does not bear RNA recognition motif or strong affinity to RNA. This is completely different from the pinpoint (i.e., epitope <10 amino acids) recognition of target protein by antibodies. For this reason, the RNA aptamer has promising potential to substitute for or complement the antibody as a new diagnostic or therapeutic tool that we refer to as “RNA super-antibody ”.

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X-inactivation is initiated during early development of female mammals by the association of Xist RNA with the X chromosome, from which it is monoallelically transcribed. Targeted disruption of Xist clearly demonstrated that Xist is essential for X-inactivation to occur in cis. Although the discovery of Xist has accelerated the molecular study of X-inactivation, detailed mechanisms are still in mystery. This is partly due to the difficulty obtaining mutations in the mechanisms of X-inactivation as those females suffering from such mutations would most probably die during early embryogenesis. My team, therefore, has made a great effort to produce a series of mouse mutants, in which the Xist locus is genetically manipulated by gene targeting, and analyzes the effects of respective alterations on X-inactivation occurring in developing embryos. I will talk about some of these approaches.

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RNA interference (RNAi) has rapidly become a powerful tool for drug target discovery and validation in an in vitro culture system and, consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Cancer is an obvious application for RNAi therapeutics because abnormal gene expression is thought to contribute to the pathogenesis and maintenance of the malignant phenotype of cancer and thereby many oncogenes and cell-signaling molecules present enticing drug target possibilities. RNAi, potent and specific, can silence tumor-related genes and would appear to be a rational approach to inhibit tumor growth. In this presentation, we provide an overview of the emerging in vivo application of non-viral delivery systems for siRNA and miRNA, which in turn will provide a foundation for further development of RNAi therapeutics against cancer.

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Several spaceflight data suggest that alteration of gravity value induces a change in expression of many genes in cells. For some genes their transcription is up-regulated, while for other genes down-regulated. Such changes in gene expression are presumably attributable to alteration of cytoskeleton conditions. I will review the published data which support the idea, and discuss what changes may be expected for radiation-induced DNA damages in space.

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It is suggested that organic compounds necessary for the first life was formed in primitive atmospheres and/or interstellar environments. Complex organic compounds have been found in carbonaceous chondrites and comets. It is suggested that these organic compounds were originally formed in interstellar dust particles in molecular clouds. Our laboratory simulation experiments showed that large molecular weight complex organic compounds can be formed in molecular clouds environments by the action of cosmic rays. The products gave a wide variety of amino acids after acid hydrolysis. Here I discuss the relationship between organic compounds in interstellar dust particles, carbonaceous chondrites and comets and origins of life on the Earth

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Energetic heavy ions pose a great health risk to astronauts in extended ISS and future Lunar and Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Over the years, we have studied chromosomal damage in human fibroblast, epithelial, and lymphocyte cells exposed in vitro to energetic charged particles generated at several international accelerator facilities. We have also studied chromosome aberrations in astronaut's peripheral blood lymphocytes before and after space flight. Various fluorescence in situ hybridization techniques have been used to identify chromosome regions ranging from the telomere region to whole chromosome painting of all chromosomes simultaneously in one cell. We will summarize the results of the investigations, and discuss the unique radiation signatures and biomarkers for space radiation exposure.

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In recent, NASA has changed space program at several points in space science. One is space travel to Moon, Mars and beyond. Another is the stopping of large centrifugator in International Space Station (ISS). Therefore, we have to reconstruct the space experiments about biological effects of space radiations and human health from them. Since human being expands to space in near future, the effects of space radiation on human health will be analyzed from an aspect of molecular levels by the application of advanced techniques of molecular biology. In addition, the biological effects of space radiations are also important from cellular, organ and whole body levels. As experimental projects on the earth, there are many approaches such as the biological effects of radiations with low dose and low dose rate, high LET radiations, microbeams and bystander effects. The physical studies about characteristics of space radiations are also important from dosimetry, species and energy of space radiations, forecast of sun activity and protection from them.
Today, we will introduce the recent schedules of space science about space radiations in Japanese government. In addition, we show the project of our space experiments designed in next year.

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A nature of inverse physical depth-dose profile of heavy charged particles is biologically enhanced in carbon ions. The characteristic is considered to be beneficial when treating deep-seated tumour. Radiotherapy with those heavy charged particles was first initiated at Lawrence Berkeley Laboratory (LBL) in the United States, then at present followed with carbon ions at National Institute of Radiological Sciences (NIRS) and Hyogo Ion Beam Medical Center in Japan, and Gesellschaft für Schwerionenforschung mbH (GSI) in Germany. Biological or clinical effects of conventional X-rays are regarded as solely proportional to absorbed dose while those of carbon ions are known to be affected not only with dose but also radiation quality. Then, in order to achieve uniform tumour control in actual therapy, a model is required which converts an absorbed dose distribution of carbon ions into a distribution of corresponding clinical effect. Here RBE, relative biological effectiveness, is practically used for that purpose and clinical dose is given in terms of GyE derived by multiplying absorbed dose with the RBE. The enormous complexity of the RBE determination hinders itself from being understood even at this moment. In Japan, RBE is estimated from a combination of cell-survival response of human salivary gland tumour cell line (HSG) against carbon ions and clinical experiences in fast neutron therapy at NIRS. GSI, on the other hand, calculates RBE from a combination of tumour response against X-rays and microscopic dose distribution formed by individual ion in the beam. The difference in model or endpoint may result in different quantity in clinical dose between two facilities though apparently expressed with the same unit, GyE. In this talk, the difference in model and resultant clinical dose distribution is shown through an intercomparison on actual treatment planning. Clinical outcome (tumour control probability, TCP) is also analyzed by a TCP model in order to verify the RBE model as well as to clarify unique characteristics of carbon ion radiotherapy.

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Nuclear reaction, ¹⁰B (n,⁴He) ⁷Li is used for Boron Neutron Capture Therapy (BNCT). As a result of this, very high LET radiations of ⁴He and ⁷Li are emerged. LET values of these are similar, and about 160 keV/μm. 10B-compound (BPA and BSH) is injected to a patient's body through a vein. When the compound as these is concentrated in a tumor higher than in normal tissues, the tumor is selectively destroyed. The faculty of BNCT as a modality of cancer therapy has been made clear by co-operative endeavor of Kyoto University Research Reactor Institute (KURRI)-BNCT community. At the end of 2001, first trial in the world of BNCT to Head & Neck cancer (recurrent mucoepidermoid carcinoma) was done by the use of epithermal neutrons from the Kyoto university reactor (KUR). Clinical result of this treatment was very excellent, and the malignant tumor of this case is controlled for four years. KUR-BNCT group stimulated by this hoped to statistically explain on the basis of the abundant case data that BNCT is one of the best modality to control a cancer. Moreover, we hope to develop an accelerator- based BNCT to spread this modality for more and more patients.
In this report, I will talk about a summary of BNCT managed KUR-BNCT group, the existing state of an accelerator dedicated for BNCT and so on.

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In Proton Medical Research Center, University of Tsukuba, 1,046 patients have been treated from the time of its foundation in September 2001 to March 2007. Our facility focuses mainly on cancers commonly found in Japanese people, such as liver cancer, lung cancer, prostate cancer, esophageal cancer and brain or skull base tumors. Combined with the patients treated in the old facility, approximately 1800 patients have been treated so far in Tsukuba.
In this presentation, I would like to center on our recent trial for malignant gliomas. Glioblastoma, a most malignant type of glioma, is highly radioresistant, harboring mutated p53 in more than half of all cases. In addition, the kinase activity of DNA-PK, a non-homologous end-rejoining enzyme, is found to correlate with their radioresistance. These molecular profiles can partially explain its high radioresistance resulting in very poor outcome of the patients.
We have been treating glioblastoma patients by high dose hyper-fractionated concomitant boost radiotherapy combining x-rays and 200MeV proton beams. In this protocol, 96.4GyE is delivered to the gross tumor volume. So far, 17 cases have been enrolled and their treatments completed.The median survival time of these 17 cases is 21.4 months at present, which is approximately 5 months longer than that of patients treated with 64 Gy of x-ray irradiation in the same institute.
Immunohistochemical analyses of the tissue surgically taken after this treatment were performed in 7 cases. The details of these data will be presented at the meeting.
Our present protocol study is yielding even longer survival in patients with glioblastoma. However, it is mandatory to control peripherally invading tumors using other treatment modalities.

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High liner energy transfer (LET) particle therapy has various advantages in terms of radiobiological effects as well as dose distribution, and had been expected to offer a therapeutic gain over conventional photon therapy. The biologic advantages of high LET radiation including neutrons and heavy charged particles, compared with low LET radiation like photons, are summarized as a decreased oxygen enhancement ratio, a diminished capacity for sublethal and potentially lethal damage repairs, and diminished cell cycle dependent radiosensitivity. However, no proof of these effects has been provided by clinical trials and related clinical researches. Hence, the radiation biological aspect of the high LET carbon beam therapy of various cancers were investigated by analyzing clinical results of carbon beam therapy of various cancers at NIRS. At least radiation resistant tumors as adenocarcinoma, adenoid cystic cancer, malignant melanoma and osteosarcoma were well locally controlled by carbon beams. The similar local control rates between the oxygenated and hypoxic cervical cancers indicated that high LET carbon beam irradiation may reduce radiation resistant nature originated from tumor hypoxia. In addition, hypo-fractionated irradiation of carbon beams showed successful local control in lung cancers. These results elucidates radio-biological advantages of the high LET particle therapy for cancer treatment.

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In vitro transformation of X-irradiated Syrian/golden hamster embryo (SHE) cells has been examined so far. It was reported that the initial process involves two steps, one is immortalization, and the other is morphological transformation. Recently, it becomes possible to visualize DNA double strand breaks using radiation-induced foci of DNA damage checkpoint factors. We examined early passage of SHE cells and found that they have 3.6 foci of phosphorylated ATM in average. This indicates that endogenous DNA double strand breaks caused by so called "culture stress" might be a cause to inactivate p53 function whose process is indispensable for immortalization of rodent cells. It should be noted that the process involves genetic alterations, but is independent of radiation exposure. Dose-dependent changes were observed in morphology of the colonies formed by cells surviving X-rays. They commonly lost dependency of cell growth on plastic surface, and all the morphologically transformed clones demonstrated loss of a protein whose molecular weight is 240 kDa. This protein was equivalent to a human extracellular matrix protein, tenasin. Thus, it was suggested that suppression of gene transcription could be involved in a down-regulation of tenasin. Such epigenetic mechanism was confirmed by the introduction of the reporter gene into rodent cells. These results indicate that both genetic and epigenetic mechanisms are involved in neoplastic transformation of X-irradiated SHE cells, however, radiation only plays a role in the latter step. As radiation-induced deletions result in a large scale of chromatin remodeling, it is likely a cause of epigenetic suppression of gene functions.

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Chromosome instability is hallmark of most human cancers. DNA damaging agents increase the probability that whole chromosome or large fraction of chromosome are gained or lost during cell division. The consequence of chromosome instability is an imbalance in the number of chromosome per cell (aneuploidy) and an enhanced rate of loss of heterozygosity. Recently, we have demonstrated that phenyl hydroquinone (PHQ), a hepatic metabolite of Ames-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in yeast. We further found that PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell cycle at G1 and G2/M. We argued that PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy. In an effort to get precise insight of the action of PHQ, we observed that 1) Irregular bud formation by PHQ triggered yeast morphogenesis checkpoint, which arrested cell cycle at G2/M, 2) PHQ stabilized Swe1 protein (human Wee1 homolog), keeping Cdc2/CycB complex inactive thus cell cycle at G2/M transition, 3) swe1 mutation abolished the PHQ-induced aneuploidy, and 4) PHQ induced Hog1 (human p38 MAPK homolog) phospholyration, by which Swe1 can be stabilized. When human HCT116 culture cells are treated with PHQ, phospholyration of human p38, ATM/ATR dependent stabilization of p53, and increase of aneuploidy were observed. Thus, PHQ can activate MAPK and p53 pathway, arrest cell cycle at G2/M boundary, and as a consequence, lead aneuploidy both in yeast and human cells.

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Non-target influence of radiation is important as an object when we think about genetic effect of radiation. Because influence of radiation does not appear as direct event of radiation, we have to leave from the conventional paradigm that a target of genetic effect of radiation is DNA. It appears away from initial hit both in terms of time and space. This phenomenon is called generally as "genetic instability". However, induction mechanism is not made clear. This study planned to clarify how a p53 gene function contributed to genetic instability derivation. In this study, we used a primary culture cells derived from p53 gene normal (+/+) and knockout (-/-) C57B mouse. 10⁶ cells were inoculated into T75 culture flask with the Eagle's MEM culture medium containing 10% FBS. Each culture was subcultured at every 5 day. As a result, growth rate of the p53 normal cells fell down to nearly 1 at passage 5-7. However, the cells restored proliferation potency again when passage reached at 10. On the other hand, p53 knockout cells continued multiplying lively without passing through temporary breeding degradation. Moreover, we analyzed number of chromosome and aneuploid cells were observed in p53 normal and knockout cells. In addition, tetraploid cells were the main in p53 normal cells, and triploid cells were in p53 knockout cells. The p53 knockout cells also had the tumorigenesity. Furthermore, we analyzed the change of chromosomal karyotype. The structural aberrations were seen in p53 knockout cells. We will consider how the difference of p53 function, ploidy and chromosomal stability are took part in acquisition of tumorigenesis.

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Ionizing radiation induces malignant transformation in the mouse-embryo-derived cell line C3H10T1/2 cells. When cells cloned from these transformed foci are inoculated into mice, they are found to be tumorigenic. Thus, the transformation of cells is thought as the initial step of radiation carcinogenesis. However, the biological mechanisms of generation of transformation have not been clarified yet. In this study, firstly, we investigated the chromosome aberrations of X-ray-irradiated C3H10T1/2 cells until transformed cells were generated. The result showed that the frequency of the cell with an unstable number of chromosomes increased at 1 day after irradiation. Furthermore, we found that the progeny of irradiated cells also had an unstable number of chromosomes. The generation of an unstable number of chromosomes might be related to the generation process of radiation-induced transformation. Secondly, we examined the number of centrosome in X-ray-irradiated cells to clarify the mechanism of generation of an unstable number of chromosomes. The result showed that the frequency of the cell with an unstable number of centrosomes increased at immediately after irradiation. An unstable number of centrosomes were also observed in the progeny of irradiated cells. Based on these results, we made a hypothesis about the biological mechanism of generation process of radiation-induced transformation. An unstable number of centrosomes might be caused in cells at the early steps in the generation process of radiation-induced transformation. Since the unstable number of centrosomes divides the different number of chromosomes to the progeny cells, the chromosomes with oncogenes may be increased. When the mutation occurred at the oncogene sites in nature, the cells might be transformed.

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It is well known that the first target of cell transformation by radiation is DNA. DNA lesion produces mutation and causes carcinogenesis (multistage mutation theory of carcinogenesis). However, detail mechanism is not proved yet. Therefore, we examined mechanism of carcinogenesis by radiation using Syrian hamster cell and we found that there is a carcinogenesis process not based on mutation. We searched for an intracellular target related to carcinogenesis with a SHE cell to examine this hypothesis. As a result, we found the intracellular oxidation degree was elevated by both high density culture and X-irradiation. Elevated intracellular oxidation level produced centrosome and telomere aberration. Those cells did not cause chromosomal structural aberration, but did cause aneuploid. Cell transformation was induced with frequency more than 1,000 times of independent somatic mutation frequency. Radiation exposure enhanced membrane function of mitochondria, and elevated intracellular oxidative stress level to five or six times higher level. After then, a mitochondrial membrane function and an intracellular oxidative stress level in transformed cell was decreased remarkably. These results strongly show that a main target of carcinogenesis by radiation is not DNA. A target may be centrosome or its related proteins playing an important role in chromosomal distribution mechanism. Mitochondrial functional disturbance by radiation enhances level of intracellular oxidation radical that attack an important mitotic apparatus. Cell transformation via non-DNA target is produced with high frequency by normal cellular physiology action. Radiation only contributes to slightly raising the frequency. It is strongly suggested that we have to know naturally cancer-causing mechanism to reduce carcinogenic risk.

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It is well known that radiation caused disturbance of intracellular oxidation control mechanism. It was suggested that elevated intracellular oxidative stress became cause by changing of various prolonged heredity recently. We examined dynamics of the intracellular oxidative stress and analyzed how it acted on centrosome. We used a Syrian hamster embryo (SHE) cell for this study. Level of intracellular oxidation was analyzed with DCFH reagent using flow cytometer as intracellular H2O2 level. The intracellular oxidation degree increased in X-irradiated SHE cell as transient peak at 3 days after irradiation. The elevation of intracellular oxidation degree was the same as the stage that centrosome aberration was observed. During culture, the cell was distinguished in two groups, such as a group to stop cell growth and a group to continue cell growth, at 22 population doubling level. In the former, gradual elevation of intracellular oxidation degree was seen, and increase of number of centrosome was observed. And cell growth completely stopped when a value of an oxidative degree greeted a peak. On the other hand, in the latter, elevation of the temporary intracellular oxidation degree was observed. An oxidative degree returned to a level of a young cell group, and proliferation potency was maintained afterwards. Then the cell finally got unlimited life span. Aberration of a centric number by increase of quantity of oxidative stress was concerned with immortalization closely. This is accelerated by radiation exposure and is caused mitotic catastrophe. And this may be a cause of chromosomal aneuploid.

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Oxidative stress is thought as a difference between oxidative damage of reactive oxygen species (ROS) and anti-oxidative ability in cells. Essentially, ROS are produced in energy production, immune response and cell signaling, and are indispensable for living cells. Whereas excessive oxidative stress can oxidize lipid, protein, and DNA and it is thought to be involved in the critical cause of life-style related disease, aging and cancer. Quantity of oxygen (quantity of energy metabolism) consumed throughout the life does not depend on creature species. This suggests that there are differences in ability of oxygen metabolism control between in human cells and in rodent cells.
Therefore this study was carried out to compare the difference of response to oxidative stress with normal human cells (HE23), mouse cells (RRI1) and Syrian-hamster cells (SHE). We cultured human cells and rodent cells under atmospheric oxygen (20%) and hypoxic (2% and 0.5%) conditions, and measured cell growth, intracellular oxidation stress, quantity and function of mitochondria and anti-oxygen ability of each.
As a result, by culture in hypoxic conditions, human cells extended the replicative lifespan, but never made immortal under all the oxygen pressure. On the other hand, in hypoxically-cultured rodent cells, growth rate became small, but they immortalized under all the oxygen pressure. Intracellular oxidation stress of human, mouse and hamster cells rose through their culture, and that of hypoxically-cultured cells were enhanced in mouse cells. Mitochondrial functions of hypoxically-cultured mouse cells were enhanced. We will present the difference of oxidative stress control in human cells and rodent cells.

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In wildtype (p53+/+) mice, p53-dependent and -independent DNA repair mechanisms restore damaged DNA. Irreparably damaged cells are then effectively removed by p53-dependent apoptosis after low dose rate radiation (LDR). Therefore, the teratogenic rate is the same as that of non-irradiated controls. In contrast, only the p53-independent DNA repair mechanism works in knockout (p53-/-) mice; p53-dependent DNA repair and apoptosis do not work. So the teratogenic rate does not decrease to a control level in p53+/+ mice even at low dose rate irradiation. DNA damage after LDR is thought to be caused by mechanisms similar to those involved in the processes of carcinogenesis and teratogenesis. This study examined whether p53 can remove damage not only via a DNA repair mechanism but also by apoptosis during a radiation-induced carcinogenic process.
The backs of seven-week-old mice (p53+/+, p53+/-, and p53-/-) were irradiated with beta-rays three times a week until a tumor appeared or throughout the life of the mice. Group I received 2.5 Gy/day, while Group II received 5 Gy/day. Group III received 7.5 Gy/day, but was only for p53-/- mice. The p53 gene from each tumor was analyzed for mutations and loss of heterozygosity (LOH).
No tumors appeared in any of the p53-/- animals. It seems that life of this mouse will be too short to observe radiation carcinogenesis. However, tumors did occur in the p53+/- mice, with an incidence of 8/21 in Group I and 25/46 in Group II. Tumors were also found in the p53+/+ mice; 8/28 in Group I and 6/33 in Group II appeared about 150 days later than those of the p53+/- mice. Of 23 heterozygote tumors examined, 14 exhibited LOH of the p53 gene but no mutations. In p53+/+ mice, 7/9 of tumors had mutations and 3/9 had LOH.
The status of a p53 gene obviously affects the incidence and time of tumor formation. One of the reasons may be that the types of radiation induced abnormality of p53 gene vary with the status of a p53 gene.

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It is supposed that p53 gene function, as a gene guardian, controls cell cycle proceeding or induction of apoptosis, and suppresses the process of malignant transformation when DNA is damaged. However, we don't know exactly whether p53 gene function relates to malignant transformation or not. We studied to be clear whether p53 function related to cell immortalization or malignant transformation.In our study, we established four cell lines from C57BL embryo mouse, which included two normal p53 gene function cell lines (p53^+/+) and two p53 gene knock-out cell lines (p53^-/-). By the normal cell culture way, all of cells acquired the immortalization whether p53 function was positive or negative and whether the cells were irradiated or not. If p53, which responses to DNA damage stress, relates to cell malignant transformation, we expected that cell malignant transformation of p53^-/- cells promote by X-ray irradiation. So we studied the frequency of the chromosome aberration in X-ray irradiated cells. As a result, p53^+/+ cells became tetraploid cells both with or without irradiation. At 40-41 passages, 30-60% of p53^+/+ cells became tetraploid cells. On the other hand, p53^-/- cells became triploid (40-50%) rather than tetraploid both with or without X-rradiation. We observed that non-irradiated p53^-/- cells, at passage 38 or 90, initiated tumors upon transplantation of the cells under back skin of nude mouse, but non-irradiated p53^+/+ cells did not. Interestingly, both the cells before transplantation and the tumor cells were triploid cells mainly. This suggested that triploidy was related to cell malignant transformation closely.

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Transgenic mice with the added mutant p53 cDNA containing 9 bp deletion in exon 6 cloned from a radiation-induced tumor and ligated to the promoter of expression vector were generated. These mice showed a tumor incidence 1.7 fold higher than in wild-type mice, i. e., 42% excess, after subcutaneous injection of 0.02 mg 3-methylcholanthrene. Tumors produced in the mice were further treated with a siRNA#220 targeting to the promoter in the expression vector of the mutant p53 transgene using a delivery system with atelocollagen. This treatment resulted in suppression of 30% of 44 tumors, including 4/23 cures of autochthonous tumors. siRNA#220 was ineffective to wild-type tumors. The cure effect in the siRNA#220-senstive transplanted tumors involved induction of apoptosis as observed by TUNEL assay.

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Oxygen radicals are produced through normal cellular metabolism, and the formation of such radicals is further enhanced by ionizing radiation and by various chemical agents. The oxygen radicals attack DNA and its precursor nucleotides, and consequently induce various oxidized forms of bases in DNA within normally growing cells. The mismatch repair (MMR) system is implicated not only in the correction of replication errors but also in the response to DNA damage to maintain genomic stability. Increasing evidence suggests that MMR is involved in the process of avoiding mutagenesis caused by oxidative DNA damages in mammalian cells. We have recently established an experimental system to study oxidative DNA damage-induced mutagenesis and tumorigenesis in the gastrointestinal tracts of mice. To elucidate the role of MMR in the avoidance of oxidative stress-induced tumorigenesis, we performed KBrO₃-induced tumorigenesis experiments using Msh2-deficient mice. Chronic exposure to KBrO₃ resulted in multiple tumor formation in the small intestines of Msh2-deficient mice, indicating that MMR is involved in the suppression of oxidative stress-induced intestinal tumorigenesis in mice. We also present data obtained from mutation analysis of the tumor-associated genes such as ctnnb1 (b-catenin), k-ras, Trp53 genes in the intestinal tumors of Msh2-deficient mice treated with KBrO₃.

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The molecular mechanisms of heat-induced cellular responses are still unknown. For example, actual critical event in heat-induced cell killing has not yet been determined. Recently, we proposed that heat-induced cell killing might be associated with double strand breaks (DSBs) formation via protein denaturation for following results. (1) Using the neutral comet assay, we found that heat treatment induced DSBs in cellular DNA; (2) Heat treatment induced the formation of γH2AX foci, which is known to be quite sensitive and a specific indicator for the presence of DSBs; (3) An inflection point at 42.5^oC, and the thermal activation energies above and below the inflection point were almost the same between cell killing and γH2AX foci formation according by Arrhenius plot analysis; (4) The cell-cycle-dependent pattern of heat-induced cell killing was the same as the cell cycle pattern of γH2AX foci formation; (5) Heat-induced gamma H2AX foci formation was suppressed in thermotolerance development; (6) DNA damage recognition proteins such as ATM phospho-serine 1981, DNA-PKcs phospho-threonine 2609, NBS1 phospho-serine 343, CHK2 phospho-threonine 68 and SMC1 phospho-serine 966 all co-localized with γH2AX after heat treatment.
In this workshop, we will discuss the possible mechanisms of heat-induced DSB formation. We expect that these approaches will constitute a breakthrough in hyperthermic biology and oncology.

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Histone H2X is phosphorylated by ATM with Mre11/Rad50/Nbs1 complex at DNA double strand breaks (DSB). The phosphorylated H2AX (gamma H2AX) foci are thought to be a marker of DSB caused by radiation. However, recent research has revealed that nonDSB-causing chemicals and UV also make gamma H2AX foci, suggesting the presence of multi-pathways of H2AX phosphorylation. A purpose of our study is to elucidate what environmental chemicals induce gamma H2AX foci and to classify the chemicals by H2AX phosphorylation pathways. Among the atmospheric polycyclic aromatic hydrocarbons, benzo(a)pyrene, 1,8-dinitropyrene and 3-methylchoranthrene efficiently induce gamma H2AX foci but 1-nitropyrene and 3-nitrobenzanthrone do not. Well correlation among chemical structure, cytotoxicity and gamma H2AX inducibility was not observed so far. In this presentation, I will also show the correlation among DNA damage type, cell cycle and H2AX inducibility.

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DNA interstrand-crosslinks (ICLs) prevent the separation of DNA duplex strands, a step which is essential for replication, recombination, and transcription. Thus, unrepaired ICLs pose a considerable threat to cell viability and genome stability. However, most of the ICLs formed in mammalian cells are removed, although the mechanisms involved in this repair process remain to be determined. To elucidate details in ICL repair pathways, a highly sensitive PPB dot blot assay was developed which is capable of providing sensitive quantitative measurements of ICLs during the repair and removal process in genomic DNA. An analysis using this assay demonstrated a decreased rate of removal of ICLs in cells derived from the Fanconi anemia core-complex group, such as groups A and G, although cells with a defective recombination protein BRCA2 (the FA-D1 group) have normal repair kinetics. The presence of a normal ICL removal rate in recombination-deficient cells was confirmed by using XRCC3-defective Chinese hamster cells. Moreover, Xeroderma pigmentosum (XP) group F cells, devoid of incision ability, showed a decrease in the rate of removal of ICLs, whereas XP group A is normal in this respect. From these results, together with some data by others, we will discuss about the role of formation during ICL repair.

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Histone H2AX is rapidly phosphorylated by ATM and DNA-PKcs in response to DNA double-strand breaks induced by ionizing radiation and various cellular processes including V(D)J recombination, meiotic recombination and apoptosis. In addition, the H2AX phosphorylation takes place in S-phase cells subjected to UV irradiation or hydroxyurea treatment and seems to be mediated by ATR, which is recruited to single-stranded DNA regions (ssDNA) generated by replication arrest, with the aid of its partner ATRIP and ssDNA-binding protein RPA. We have recently found that UV-induced H2AX phosphorylation is also observed in G0-arrested human cells and that this phosphorylation depends on nucleotide excision repair (NER) as well as ATR. We proposed a model that NER process can be perturbed at a gap-filling step due to low levels of repair replication factors and the resultant ssDNA gap intermediates initiate the phosphorylation of histone H2AX (Matsumoto et al., J. Cell Sci. 120, 1104-1112, 2007). Here we show that the NER-dependent H2AX phosphorylation at G0 phase also occurs following treatment of chemical agents such as N-acetoxy-2-acetylaminofluorene and cisplatin, which generate DNA adducts reparable by NER. Furthermore, non-cycling peripheral T-lymphocytes isolated from wild-type mice exhibit H2AX phosphorylation following UV exposure, whereas those from NER-deficient mice do not, indicating this phosphorylation is again NER-dependent. In this workshop, we will further present our ongoing work and discuss on NER-dependent H2AX phosphorylation in vivo.

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Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooporation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18-/-) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild type HCT116 cells, RAD18-/- cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to camptothecin observed with RAD18-/- cells was limited to only the S phase due to the absence of the RAD18 S-phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18-/- cells was also demonstrated by alkaline comet assay.

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Malignant transformation depends on the accumulation of gene mutation that is important for cell growth, tumor suppression and cell cycle regulation. However, it takes decades to accumulate these mutations in adult cancer. In the cancer prone genetic disorders, cell cycle regulatory genes such as p53, BRCA1 and ATM are mutated. Recent clinical observations have revealed that leukemogenesis in childhood leukemia occurs already in intrauterine period. So called "infant leukemia" which occurs in less than one year-old children has MLL(Mixed Lineage Leukemia/Myeloid Lymphoid Leukemia)gene rearrangement frequently. This rearrangement also observed in secondary leukemia and break point is clustered in 8.3kbp of 90kbp MLL gene in both cases. In this breakpoint cluster region (BCR), topoisomeraseII(TopoII) binding sites are known to be clustered. TopoII is essential for structural maintenance of genomic DNA in cell metabolism, and forms a covalent linkage to both strands of DNA helix, makes a transient double-strand break (DSB) in the helix, and re-ligates the cleaved DNA. The agents with TopoII inhibitory activity such as etoposide, induce DNA DSBs by stabilizing covalent TopoII-DNA complexes and thereby exert their function as anticancer drug by inducing apoptosis. Exposure to bioflavonoid with TopoII inhibitory activity, which is contained in food also induces DSBs in BCR in MLL gene in vitro. We have recently reported that early G2/M checkpoint failure is a risk factor for etoposide-induced chromosomal aberrations in vitro. Furthermore, we have shown reduction of p53ser15 phosphorylation in normal peripheral lymphocytes after ionizing radiation in two out of seven infant leukemia patients. In one case, we have found heterozygote ATM mutation that has dominant negative effect in vitro. These observation indicate that exposure to agents with TopoII inhibitory activity and G2/M checkpoint failure could be a molecular mechanism for leukemogenesis.

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Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ATM and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATR kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53Ser18 phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53Ser18 in Ku-deficient cells at 12h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response.

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Reactive oxygen species such as superoxide radical, hydrogen peroxide and hydroxyl radical are generated in cells exposed to ionizing radiation. The reactive oxygen species oxidize nucleic acids, proteins and lipids. The oxidation of DNA produces single- and double-stranded breaks, apurinic/apyrimidinic sites and oxidative damage to base and sugar moieties. It was important to clarify whether or not altered levels of anti-oxidation enzymes such as catalase and superoxide dismutases can modify the sensitivity to the lethal effect of ionizing radiation in human cells. Hence, in this report, we constructed several clones of HeLa S3 cells with plasmids overproducing human SOD and other defensive enzymes. The lethal effects of gamma-rays were determined in HeLa with or without the plasmids.

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It is generally accepted that p53 phosphorylation by ATM is important for radiation-induced G1 arrest, however, relationship between spatiotemporal dynamics of activated ATM and p53 phosphorylation in individual cells remains unknown. Previously, we demonstrated that residual foci of activated ATM (Ser1981-phosphorylated ATM) grow in size time-dependently. Therefore, in the present study, we examined the relationship between the growth of phosphorylated ATM foci and p53 phosphorylation. G0-synchronized normal human diploid cells were treated with Nutlin-3, which stabilizes p53 by inhibition of p53-MDM2 interaction, to make p53 levels constant in all cells. Then, cells were irradiated with 1 Gy of X-rays, and were fixed at 2, 4, 8, 24 h after irradiation, followed by immunofluorescence staining for Ser1981-phosphorylated ATM and Ser15-phosphorylated p53. We measured fluorescence intensity of the both proteins in the nucleus, and found that intensity of both proteins decreased time-dependently on the whole. Interestingly, some of the cells persisted strong fluorescence intensity of the both proteins until 24 h after 1 Gy, like cells at 2 h after 1 Gy, though most of the initial foci disappeared by this time. All of such cells had the large foci (≥1.6 μm), therefore, we investigated p53-phosphorylating ability of a single phosphorylated ATM focus at 24 h after irradiation. We observed clear correlation between diameter of a single phosphorylated ATM focus and fluorescence intensity of phosphorylated p53. These results indicate that p53 phosphorylation is maintained by growth of residual phosphorylated ATM foci long after irradiation.

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Ionizing radiation induces DNA double strand breaks, which result in activation of ATM-dependent DNA damage checkpoint pathway. We have reported that a major mode of cell death induced by radiation is not apoptosis but senecsence-like growth arrest (SLGA). SLGA is an irreversible cell cycle arrest which resembles to the process induced in senescent cells. Because SLGA is dependent upon p53 function, it has been hypothesized that most human cancer cells, which abolish p53 function, may not be able to induce SLGA. As a result, human cancer cells proceed their cell cycle even with DNA double strand breaks, which lead to mitotic catastrophe. In the present study, we determine the process of cell death via mitotic catastrophe. Six human cancer cell lines were used. Every 24 hours, cells receiving 6 Gy of X-rays were fixed with 4% formaldehyde and stained with anti-phosphorylated ATM antibody together with DAPI. We found that mitotic catastrophe was observed in approximately 50% of cells, but all of them were phosphorylated ATM negative. Since 100% of cells were positive for phosphorylated ATM if they were exposed to 40J/m2 UVC, it was clear that apoptosis was not executed in cells induced mitotic catastrophe. Moreover, the cells were still phosphorylated ATM negative 48 hours after irradiation, however, we also found that parts of micronuclei were apoptosis positive, indicating that a mechanism degrading fragmented DNA is exist in cells. From these results it is indicated that mitotic catastrophe is a process of cell death which is independent of apoptosis. Although the cells progress cell cycle without successful cell division, they subsequently died by inducing irreversible SLGA.

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Ionizing radiation cause DNA double strand breaks (DSBs). H2AX that is one of a chromatin protein is phosphorylated by activated ATM and forms foci. Then DSBs are rejoined and most of the initial foci disappear. A few foci are growing to large foci. However, biological significance of remaining large foci is not clear. Therefore, we examined correlation between residual large foci and cellular radiosensitivity after ionizing radiation. Human normal diploid fibroblast cells (HE49) and HeLa cells irradiated X-rays then examined alterations of number and size of phosphorylated H2AX foci. We used replicating protein A (RPA) and methylated histone H3 as markers of S-G2 and G1 phase, respectively. As a result, unirradiated cells had few large foci. And also cells, which stopped their cell division after irradiation, had few large foci. These results indicate that remained large phosphorylated H2AX foci show loss of proliferating potential after irradiation. This suggests that we can use large phosphorylated H2AX foci to estimate therapy effect of radiotherapy treatment of cancer.

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