Ionizing irradiation directly causes DNA damages as well as indirectly influences genomic integrity via modifying some cellular functions. The mechanism of the indirect effects has not been well studied, however, because it is difficult to separately investigate the direct and indirect effects. We recently developed human cell lines to generate DNA double strand breaks (DSB) in the genome using meganuclease I-SceI (Honma et al., EMM 42, 288, 2003). They are suitable models to study the fate of DSB directly cause by irradiation. We now investigated the effect of low-dose irradiation on the repair of DSB using these cell models. Human ymphoblastoid cell lines TSCE5 and TSCER2 are heterozygous (+/-) or compound heterozygous (-/-), respectively, for the thymidine kinase gene (TK), and were introduced an I-SceI endnuclease site into the gene. NHEJ for a DSB at the I-SceI site results in TK-deficient mutants in TSCE5 cells, while HR between the alleles produces TK-proficient revertants in TSCER2 cells. Because the expression of I-SceI enzyme produced a DSB at the TK gene 10,000-times more efficiently than ionizing irradiation, we could ignore the irradiation-inducing direct DNA damages. We examined the pretreatment effect of 30 mGy gamma ray for 24h on the repair of I-SceI-inducing DSB. While NHEJ efficiency was never changed, the frequency of HR increased approximately 2-times comparing non-irradiated cells. This indicates that low-dose irradiation influences something on the HR mechanism. We now investigated the manner of HR modified by irradiation.

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To compare gene expression with time in murine NIH/PG13Luc cells exposed to low dose-rate radiation (LDR) and middle dose-rate radiation (MDR), total 10 genes including p53-dependent genes (CyclinG1, p21, Ephx1), extra-cellular-matrix related-genes (Tnc, Col1a2, Fbln5) and cell-cycle related-genes (Mapk9, Rbl1, Mcm6, Cdc2A) were selected based on the result of the previous microarray analyses. p53-dependent genes (CyclinG1, p21, Ephx1) were up-regulated at more than 15 mGy/hr. The early and late responses of p53-dependent genes were confirmed by using a high dose-rate radiation (HDR) (1.1 Gy/min). On the other hand, extra-cellular matrix related-genes (Tnc, Col1a2, Fbln5) were up-regulated at a dose rate higher than 1 mGy/hr. The expressions of extra-cellular matrix related-genes were not up-regulated on early response, but were up-regulated on late responses by exposure to HDR. Expressions of Tnc and Col1a2 increased by only adding human rTGF-β1. And Tgfb1 increased by irradiation. Expressions of cell cycle related-genes (Rbl1, Mcm6, Cdc2A) were down-regulated at 15 and 60 mGy/hr, but not at 1.1 Gy/min. These results indicate that the up- and down- regulation pathways for p53-independent genes are different from those for p53-dependent genes induced by LDR and MDR. (This study was supported by a grant from Aomori prefecture, Japan)

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Genetical technique in Drosophila melanogaster can hold unevenness of spontaneous mutation frequency (about 0.3%) in check small, because of the leveling of genetical background. This technical advantage made clear mutation frequency reduction at the low dose, low dose rate irradiation than spontaneous mutation frequency. Irradiation to the Drosophila immature sperm using gamma ray of 0.5 mGy in one minute from the ⁶⁰Co reduced the frequency of the sex-linked recessive mutation up to 0.1% (p<0.05). To explain this effect, the transcript profiles were evaluated using oligonucleotide arrays from the same stage embryos which have an immature sperm. We report here, a set of 160 genes, including hsp 70A gene, which is known as stress response protein gene, with transcripts that were affected by this level. However, genetic expression was not able to observe the significant change of expression of the gene group, (e.g. mei-41, p53) which participated in repair, cell cycle or apoptosis and were affected by high dose, high dose rate irradiation. We suggested that the expression of the gene group as mei-41 and p53, have minimum dose to response.

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In the past decade, it has been reported that the non-irradiated cells which are close to the irradiated cells respond indirectly to radiation. This phenomenon is so called radiation-induced bystander effect. We would report new findings about high- or low-LET radiation adaptive response induced through radiation-induced bystander effects mediated by nitric oxide (NO).
1. Induction of adaptive response by low-dose X-irradiation: At 3 to 12 hr after low-dose irradiation at 0.02 Gy, adaptive response was obviously observed in p53-deficient human non-small lung cancer H1299 cells transfected with wild-type p53 gene. This response was partially diminished by the addition of carboxy-PTIO.
2. Induction of adaptive response by low-dose Carbon-beam (290 MeV/u, 70 keV/μm) irradiation: At 3 to 24 hr after low-dose irradiation at 0.02 Gy, adaptive response was obviously observed. This response was partially diminished by the addition of carboxy-PTIO.
We found that radiation adaptive response was induced by not only low-LET but also high-LET radiation and that these responses were induced through NO production. It is suggested that radiation-induced bystander effects mediated NO may contributes to the induction of these responses.

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Apurinic/apyrimidinic (AP) site, an intermediate in base excision repair, is the most common DNA lesion that is lethal and premutagenic. We have developed a method for rapid detection and quantitation of AP sites in DNA with the Aldehyde Reactive Probe (ARP). In this study, we have developed a novel method for in situ analysis of intracellular AP sites using fluorescein-conjugated ARP, FARP-1 (5-[N'-(2-Aminooxyacetamidoethyl) thioureido] fluorescein). It is conceivable that the long patch sub-pathway is relatively dominant in S phase. Using synchronized HeLa cells, we examined the number of apparent MMS-induced AP sites in G1 and S phase cells by ARP method. The numbers of AP sites were not different significantly in both phases. By the FARP-1 method, we successfully analyzed AP sites in MMS-treated and propydium iodide-stained cell nuclei using a flow cytometer. The reaction of FARP-1 was specific, since the signal was suppressed by the pre-treatment of cell nuclei with methoxyamine. More AP sites were detected in S phase after MMS treatment, suggesting that both G1 and S phase cells efficiently repair the methylated bases by BER, since more DNA is included in a single S phase cell than in a G1 cell.

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Apurinic/apyrimidinic endonuclease plays an essential role in DNA base excision repair (BER). APE1 (aka APEX1, or Ref1), the mammalian AP endonuclease, is also known to have gene regulation functions as a redox-enhancing facotr or as the acetylation-mediated co-repressor. APE1 null embryos die just after blastocyst formation, and there has been no report of isolation of its mouse embryonic fibroblast (MEF) cells. To understand APE1's in vivo role more precisely, a conditional ko MEFs were isolated in which APE1 could be removed by excising the APE1 gene with the Cre recombinase, and the fate of the cells without APE1 was analyzed. The cre gene was introduced by a direct nuclear microinjection. The MEF cells became apoptotic within 12 h after the cre injection, and almost all the cells were in a late apoptotic stage in 24 h. The MEF cells were normal when the cre as well as the wild-type APE1 cDNA were co-injected, indicating that APE1 is essential even in cultured cells. A "complementation test" was carried out where the cre gene was co-injected with missensed APE1 cDNA, each of which lacks a particular function of APE1. Based on this experiment, we concluded that the AP endonuclease activity as well as the aceytlation-mediated co-repressor functions are absolutely required for the cells.

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Oxidative stress constantly threatens the integrity of cellular DNA under physiological conditions. Flap endonuclease 1 (Fen1) is believed to play a critical role in the repair of oxidized DNA lesions by serving as an integral part of the long-patch (LP)-base excision repair (BER) pathway. The degree to which LP-BER is involved in BER in higher eukaryotes, however, is not clearly understood. Using Fen1-disrupted DT40 cells, we investigated the impact of Fen1 on BER and on DNA replication and cell death under oxidative stress. We found that nuclear extracts from cells deficient in Fen1 had a significant reduction in their LP-BER capacity, and supplementing the extracts with Fen1 later restored LP-BER. Exposure of Fen1-deficient cells to hydrogen peroxide (H2O2) resulted in caspase-dependent cell death within 3 hours. In addition, Fen1-deficient cells had an increase in the amount of stalled replication forks after exposure to? H2O2 as detected by visualization of replication units in combed DNA fibers before and after treatment. Overall, these results strongly suggest that Fen1 has a critical role in the LP-BER process in living higher eukaryotes and imply that Fen1 works as an anti-apoptotic factor during DNA replication possibly by avoiding replication stalled under oxidative stress.

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Ionizing radiation produces a unique form of DNA damage called clustered damage, which contains two or more lesions such as single-strand breaks and oxidative base damage induced within the one or two helical turns of DNA. Clustered damage would be less readily repaired than isolated lesions. Therefore clustered damage might be biologically significant. In this report we show that E. coli mutM nth nei triple mutant was less sensitive to the lethal effect of sparsely ionizing radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of MutM, Nth and Nei increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Increased sensitivity to gamma-rays also occurred in E. coli overproducing human hOgg1. Treatment of gamma-irradiated plasmid DNA with purified MutM converted the covalently closed circular to the linear form of the DNA. Furthermore, the present study revealed that HeLaS3 cells transfected by Ogg1 type1a or type2a plasmid were more sensitive to gamma-rays than HeLaS3 cells without plasmid. Ogg1 protein localized in nuclei and Ogg1 type2a in mitochondria. Overexpression of these Ogg1 proteins in HeLa cells was confirmed by RT-PCR procedures. Excessive excision by DNA glycosylases converts nearly opposite base damage in clustered DNA damage to double-strand breaks, which are potentially lethal. We are currently investigating the biological effects and repair mechanisms of clustered DNA damages in human cells.

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Ionizing radiation and reactive oxygen species induce various oxidative base lesions in DNA. Oxidative DNA base lesions are generally repaired by the base excision repair (BER) pathway initiated by DNA glycoxylases. This pathway is conserved from bacteria to humans. In the BER pathway of E. coli, oxidative pyrimidine lesions are removed by Endo III and Endo VIII that exhibit similar substrate specificities. The redundant repair roles of Endo III and Endo VIII in cells have also been demonstrated. The human Endo III (hNTH1) and Endo VIII homologues (hNEIL1 and hNEIL2) remove oxidative base lesions, but their in vivo repair roles have yet to be determined. In this study, we have compared the damage specificities of hNTH1, hNEIL1, and hNEIL2. hNTH1 and hNEIL1 exhibited distinct preferences for the stereoisomers of thymine glycol (Tg). The activity ratio of hNTH1 for 5R-Tg:5S-Tg was 13:1, whereas that of hNEIL1 was 1.5:1. Similar to hNTH1, HeLa cell extracts preferentially excised 5R-Tg (5R-Tg:5S-Tg = 13:1), indicating that hNTH1 is a dominant activity for both Tg isomers in human cells. hNTH1 and hNEIL1 excised other oxidative base lesions, but their preferences for these lesions were different. hNEIL2 exhibited a high activity for AP sites, but the activity for oxidized base lesions was marginal. The present results indicate that, while displaying distinct preferences, hNTH1 and hNEIL1 have redundant substrate specificities. Thus, like Endo III and Endo VIII, hNTH1 and hNEIL1 may have redundant repair roles in human cells.

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Reactive oxygen species and ionizing radiation induce a wide variety of oxidative modifications to purines and pyrimidine bases in DNA. Bacteria, yeast and eukaryotes have evolved base excision repair mechanisms for oxidative base damage in DNA. Base excision repair is initiated by DNA glycosylase through the removal of modified bases from the DNA. E. coli has three kinds of DNA glycosylase, MutM, Nth and Nei, that recognize and remove oxidatively damaged bases from DNA. These DNA glycosylases are able to recognize and remove 5-formyluracil and 5-hydroxymethyluracil from DNA. Moreover, several evidence showed that there are residual activities to repair 5-foU-containing duplex DNA in crude extract from E. coli mutM nth nei mutant, suggesting possible roles of another DNA glycosylase(s). We identified and purified the novel protein to recognize and repair the duplex oligonucleotides containing 5-foU. We are currently investigating the structure and functions of the protein. Furthermore, we found some novel proteins in C. elegans that rescue the spontaneous mutations in E. coli mutM nth nei mutant.

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It is widely accepted that cancer results from the accumulation of mutations in the genes that directly control cell birth or cell death. But the mechanisms through which these mutations are generated are the subject of longstanding debate primarily between two models; one holds that cancer specific phenotypes are the result of multiple, specific gene mutations where mutation either generate dominant oncogenes or inactivates recessive tumor suppressor genes, and the other holds that cancer specific phenotypes are the result of the thousands of normal genes whose dosage is altered by aneuploidy. The proponents of mutation hypothesis postulate that among 6 to 10 mutations are necessary for carcinogenesis. Assuming mutation rate is 10-6/cell/generation, and 6 mutations are involved in cancer formation, we can calculate that only 1 in 10³⁶ human cells would ever become cancer cells. Since human consist of 10¹² cells, only 1 in 10²⁴ humans would get cancer. In other words, cancer would hardly exist. The mutation hypothesis also predicts that carcinogens can mutate cellular genes. On the other hand, about 50% of carcinogens are categorized as non-mutagens. Thus, the mutagenic reactions of genotoxic carcinogens may be coincidental rather than causal factors for tumorigenesis. In this workshop, we presented the experimental results which support the hypothesis that aneuploidy is the cause rather than a consequence of tumorigenesis.

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Chromosome aberrations as well as point mutations are genomic alterations that play important roles in carcinogenesis. In general, it is thought that radiation efficiently induces chromosome aberrations via DNA strand breaks, and chemical carcinogens and ultraviolet light (UV) irradiation induce point mutations via modification of bases in DNA. To dissect the genotoxic effects of various environmental carcinogenic factors in vivo, we have developed gpt delta transgenic mice, which allow analysis of deletions (chromosome aberrations) and point mutations, and examined the genomic alterations induced by radiation, UV and chemical carcinogens at the molecular level. Heavy-ion irradiation induced large deletions in size more than 1 kb in the liver but no point mutations. In contrast, gamma-ray and X-ray irradiation induced both deletions and point mutations. UV irradiation induced not only base substitutions (G:C to A:T) in the skin but also induced large deletions. Carcinogen PhIP contained in the burnt food induced base substitutions (G:C to T:A) and 1 bp deletions in the colon but no large deletions. Mitomycin C efficiently induced both base substitutions (tandem base substitutions) and large deletions in the bone marrow. These results suggest (1) low LET radiation such as gamma-ray and X-ray induces base substitutions due to modification of bases in DNA along with induction of DNA strand breaks (2) UV irradiation and chemical carcinogens induce deletions via DNA strand breaks by replication stall along with induction of base substitutions via modifications of bases in DNA.

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Reactive oxygen species (ROS) are produced through normal cellular metabolism, and formation of such radicals is further enhanced by ionizing radiation and by various chemicals. ROS attack DNA, and the resulting oxidative DNA damage is considered to attribute to carcinogenesis, aging, and neurological degeneration. Among various classes of oxidative DNA damage, 8-oxo-7, 8-dihydroguanine (8-oxoguanine, 8-oxoG) is the most abundant, and plays significant roles in mutagenesis because of the ability to form a mispairing with adenine. Enzymatic activities that may be responsible for preventing 8-oxoG-evoked mutations were identified in mammalian cells. We have focused on the following three enzymes; MTH1, OGG1, and MUTYH. MTH1 is a mammalian ortholog of E. coli MutT that hydrolyzes 8-oxo-dGTP to its monophosphate form in the nucleotide pool, thereby preventing the incorporation of the mutagenic substrate into DNA. OGG1, a functional counterpart of E. coli MutM, has an 8-oxoG DNA glycosylase activity. MUTYH, a mammalian ortholog of E. coli MutY, excises an adenine paired with 8-oxoG. These three enzymes have been considered to prevent mutagenesis caused by 8-oxoG in mammal. To analyze the function of the mammalian MTH1 (Mth1), OGG1 (Ogg1) and MUTYH (Mutyh) in vivo, we established gene-knockout mice for these three enzymes by gene targeting, and investigated spontaneous tumorigenesis as well as mutagenesis. I will discuss our recent progress on mutagenesis and carcinogenesis with these mutant mice lines.

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The finding of cancer specific chromosome abnormalities has contributed to the recognition and isolation of oncogenes and tumor suppressor genes. Chromosome changes have been used for the analyses of genetic changes involved in the progression, such as invasion and metastasis, of cancer cells. Renal cell carcinoma (RCC) is the most commonly observed malignancy in the human adult kidney. The RCC is histopathologically classified into two types; non-papillary and papillary types. In non-papillary RCC, a frequently observed deleted region on the short arm of chromosome 3 suggested that tumor suppressor genes which are crucial for the RCC development may be localized at this region. With the analyses of primary event in RCC carcinogenesis, some candidate genes were isolated as a tumor suppressor gene involved in the genesis of sporadic RCC as well as the genetic disorder, VHL syndrome. In the present study, we have analyzed the chromosome change in sporadic RCC and considered the possible mechanism of RCC carcinogenesis and progression based on the chromosome abnormality. As above mentioned, loss of a short arm of chromosome 3 was most frequently observed in non-papillary RCC and the deleted region was spreading over the short arm of chromosome 3. On the other hand, numerical changes were frequently observed in the papillary RCC. Thus, the deletion in the wide range of chromosome arm and gain of whole chromosome may have the effects on the genome balance in the cell and such genome imbalance may also have some possible roles in the RCC development and progression.

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It has been believed that the first target of radiation carcinogenesis is DNA.However, this expectation is not proved directly yet.Therefore, we investigated mechanism of radiation carcinogenesis by using primary syrian hamster (SHE) cell.As a result, I presented that transformation frequency is 500-1,000 folds higher than that for somatic mutation frequency.-This contradicts "the multistage mutation theory" which carcinogenesis produces in accumulation of 3-5 independent mutations. Therefore, We searched for an intracellular target related to carcinogenesis with a SHE cell to solve this contradiction. As a result, it was recognized that the intracellular oxidation degree was elevated by high density culture and radiation exposure. And oxidative radicals attack centromere or centrosome and destroy their structure. In those cell culture, structural aberration of chromosome does not occur, but aneuploid is seen in high frequency. These results suggest a possibility that a main target of radiation carcinogenesis is not DNA, but is centromere and centrosome which are the proteins to constitute chromosomal homeostasis maintenance mechanism.

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We already developed an X-ray microbeam irradiation system using monochromatic SR X-rays. Energy of X-rays is restricted to 5.35 keV, since the beam is deflected right angle upward using diffraction of Si(311), in order to irradiate the samples through the bottom of the dish. Presently we are usually using a microbeam cut out by a slit system, and beam shape is 5 micron square(minimum) or larger. Dose rate is about 40 R/s, which corresponds to about 10⁴ photons/s/100 micron². In region scan mode we can obtain survival data with statistical significance. We have another microscope, into which information of the coordinate of the cells irradiated by the irradiation system can be transferred. Recently, we have installed a confocal laser microscope into this system, which enables highly sensitive detection of fluorescent image of the irradiated cells.
One of the advantages of using SR as light source is that we can choose any energy of monochromatic X-rays with practical intensity. In order to study the signal induction process from energetic viewpoint, we are planning to build an energy-tunable X-ray microbeam system, by which we can irradiate with monochromatic X-ray of inner-shell absorption edges of certain elements. We have to use the horizontal beam as emitted from the electron storage ring, hence we need to overcome some difficulties such as vertical positioning and sample chamber containing cells. This system will be tested this autumn.

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Bystander Effect is one of the interesting researches in the field of low dose radiation effect. It occurs due to some unknown response between the directly irradiated cell and normal ones. For the study of Bystander Effect, a microbeam cell irradiation system becomes a strong tool. SPICE (Single Particle Irradiation system for Cell) of NIRS was constructed and we start its operation in 2003. On the SPICE, some newly developed systems are mounted. One is a microscope system which is consists of OLYMPUS fluorescent microscope parts mounted on a granitic level block, and high performance voice coil motor system. And the other is single ion hit system consisted from single ion counter, signal control PC board, high-voltage pulse generator, and electrostatic beam deflector. In this conference, we'll make a presentation about these developments, and test examination of cell. Additionally, we'll show the development of new type cell dish use for accurate targeting of beam.

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Research and development of ion irradiation of living micro-volume materials, such as cells and cell organelles, has been in progress at RIKEN. Using a tapered glass capillary with an inner diameter of about 1 um at the outlet, we have already succeeded to focus ion beams down to the size of its outlet inner diameter. Recently, we have also developed a thin glass end-window for this type of tapered glass capillary. Inserting this capillary into a liquid target, focused ion irradiation of the target while keeping the beam line under high vacuum has been realized. The volume of the energy deposition region is controllable in three dimensions with about 1 um resolution by adjusting the beam energy and the thickness of the end-window. In order to verify the size of this energy deposition region, the capillary was inserted into a liquid scintillator and scintillation was observed with a microscope. For example, in the case of 3 MeV alpha rays entering a capillary with a end-window thickness of 7 um and outlet inner diameter of 1.5 um, the observed scintillation volume was 6 um in length and 3 um in diameter, consistent with calculations performed by SRIM-2003. Therefore, using a tapered capillary with a thin end-window, we have succeeded in controlling the size of the energy deposition region down to the same order as a typical biological cell.

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We have been studying the biological effects of low-dose radiation using a synchrotron X-ray microbeam irradiation apparatus developed in KEK-PF. This system is able to provide an X-ray microbeam of arbitrary size larger than 5 microns square. In this research, by using this advantage, we have compared the survival fractions of V79 cells irradiated with X-ray microbeams of different sizes. We have already reported that low-dose hypersensitivity was observed in nucleus-irradiation with a 10 microns square X-ray beam more clearly than in broad-field irradiation. In order to elucidate the contribution from irradiated cytoplasm, we irradiated the whole cell with a 50 microns square X-ray beam and compared the survival fractions with those obtained in irradiation only to nucleus. The survival curve obtained in 50 microns square X-ray beam irradiation is almost the same as that obtained in broad-field irradiation. At the low-dose region, the low-dose hypersensitivity, which was clearly observed when only cell nucleus was irradiated with X-ray beam, became less observable. Considering the situation that in 50 microns square X-ray beam irradiation, the entire cytoplasm, as well as the nucleus, was irradiated uniformly with X-rays, while the cytoplasm was not irradiated in 10 microns square X-ray beam irradiation, these results suggest that the energy deposition in cytoplasm might induce some kind of intracellular signaling and enhance the repair activity of the cell, and hence the low-dose hypersensitivity might be suppressed in whole cell irradiation.

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The system in PF BL27 to irradiate culture cells with microbeam soft X rays is useful to observe the phenomena which are occurring in situ in the cells with irradiation and to clarify the important fundamental problems in radiation biology. The problem why the cells in M phase has high radiosensitivity has been interested in radiation biology, however, the exact mechanism of the radiosensitivity remains unclear. In this study, abnormal cell-cycle progression in the M phase was investigated using the microbeam irradiation system in PF BL-27B, when a single M phase cell in each mitotic stage was irradiated with a 5.35 keV X-ray microbeam focused on the cell nucleus. HeLa cells, genetically modified and expressing EGFP-tagged aurora kinase B, were used as irradiated samples, in order to recognize the stage of each cell in the M phase, such as prophase, prometaphase, metaphase anaphase and telophase. Each single cell at each stage was irradiated with 10 micro-m x 10 micro-m monochromatic microbeam focusing on the nucleus, and each irradiated cell was pursuited on line to watch mitotic cell division to the end. Each cell at each stage was irradiated with 10 Gy at the dose rate of 20 Gy/min and the average times to take in each stage were measured. From the results, it is clearly observed that cell-cycle progression in the M phase has mitotic arrest due to a delay of the metaphase/anaphase transition. The dose dependence of the elongation of the M phase was also examined and one of checkpoint proteins was observed by immunostaining assay.

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DNA double-strand breaks (DSBs) are the most lethal damage in the radiation-induced DNA damage and are subject to misrepair. In higher vertebrate cells, there are at least two major DSB repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR). It has been identified many DSB repair proteins, which are engaged in NHEJ and/or HR. Recently, imaging analysis of foci formation of DSB repair proteins, such as phosphorylated histone H2AX, is one of the available methods to elucidate the mechanisms of DSB repair. However, it is difficult to observe the initial response of DSB repair proteins on the site of DSBs induced by X-rays, because conventional X-ray machines cannot perform target irradiation. In this study, we irradiated the targeted part of cell nuclei using synchrotron X-ray microbeam system and observed the localization and phosphorylation of DSB repair proteins by immunofluorescence. We first identified the induction of DSBs in the irradiated site using the phosphorylated histone H2AX specific antibody. The localization of DSB repair proteins was observed in normal human fibroblast MRC-5 cells irradiated with X-ray microbeam and then fixed 30 min after irradiation. Localization of NBS1 and phosphorylated DNA-PKcs and ATM on the site of target irradiation could be observed predictably, but we identified a few proteins, which have been reported nuclear foci formation after conventional X-rays irradiation, were not localized. The localization of cell cycle checkpoint proteins will be presented.

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Silkworm is an experimental insect good to investigate developmental biology or cell differentiation. About 400 silkworm strains are collected and maintained, especially larval pigmentation mutations or morphotypes are mainly stored. One of them, termed Knobbed (K) mutant is a quite unique and important model of cell differentiation, in that cells in the knob region consist of abnormally proliferated and stratified cells.
In this study, the new application of irradiation with heavy ion microbeam for the first instar silkworm larvae was developed to clarify that when and where the knob mutant would form by suppressed proliferation of epidermal cells. The holed aluminum plates were specially designed to fix the first instar larvae of silkworm during irradiation. After irradiation with 180-μm-diameter microbeam of 220 MeV ¹²C ions, larvae were reared to evaluate the accuracy of irradiation. The deletion of knob was observed in over 70% of the larvae at fifth instar (LET=127.9 MeV/μm ions, where the absorbed dose of epidermal cells are equivalent to 500 Gy). The epidermal cells stayed as it was a monolayer at irradiated region. These results indicate that heavy ion beam irradiation can control the abnormal cell division of epidermis in the knob mutant.

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There has recently been an upsurge of interest in ionizing radiation-induced bystander effects. However, its dependence upon linear energy transfer (LET) remains to be elucidated. Utilizing our systems of collimated heavy-ion microbeams available at Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) of JAEA, the current investigation here aims to characterize bystander killing by three types of heavy ions with differing LET in confluent density-inhibited normal human fibroblast AG01522 cells under conditions that intercellular interactions are facilitated. Clonogenic survival analyses revealed that targeted irradiation of a few cells within the whole population of around million cells resulted in nearly 10% loss of clonogenicity, indicating the induction of bystander responses. Besides, apoptosis in the bystander population was detected with TdT-mediated dUTP nick end labeling assay, of which induction peaked at 24 hours post-irradiation. Meanwhile, to an extent similar to bystander apoptosis frequency, apoptotic frequency increased at least up to 72 hours following the random irradiation with 10% survival doses of corresponding broad beams of heavy ions. Our present findings suggest the modal and temporal distinction of cell death between irradiated cells and non-irradiated bystanders. Moreover, very little difference in these bystander responses was found among number of heavy ions targeted (1, 5, 10 ions), and between LET values (103, 294, 375 keV/μm) as well.

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