Caffeine sensitizes a number of cells to ionizing radiation. The radiosensitizing effects of caffeine are believed to be associated with the disruption of DNA damage-responsive cell cycle checkpoints controlled by ATM. We investigated the effects of caffeine on non-growing G0 human fibroblast cells by determining cell survival and scoring chromosome aberrations. We found that G0 cells exposed to X-rays were sensitized by post treatment of caffeine from cell survival study. Analysis of chromosome aberrations using FISH (fluorescence in situ hybridization) revealed that high frequency of aberrant cells and misrejoining was induced by caffeine. There seems to be another mechanism of cytotoxic effects of caffeine on cell killing other than inhibition of cell cycle checkpoint control.

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Little is known about the mutagenic effects of clustered DNA damage. We have designed a plasmid-based assay to determine whether closely-opposed sites of base damage lead to higher mutation frequencies than single sites of damage. The bistranded clustered damage consists of 8-oxo-7,8-dihydroguanine (8-oxoG) and dihydrothymine (DHT), with 8-oxoG placed within a restriction site on one strand and DHT positioned at sites 1, 3, 5 bp away on the opposing strand. Damaged DNA was transfected into wild type or glycosylase deficient mutant cells (fpg, mutY and fpg mutY) of E. coli, and mutations were determined by inability to cut the restriction site. Mutation frequencies were found to be significantly higher for clustered lesions than for single lesions. Mutation frequencies were similar to wild type in the fpg mutant, while a marked increase was observed in mutY mutant compared to wild type. Even higher mutation frequencies were found in fpg mutY double mutant, reaching values around 35% of the rescued plasmids. We suggest that (1) at clustered damage sites, the action of Fpg is compromised, (2) MutY has an important role for reducing the formation of mutations by 8-oxoG within a cluster, and (3) an effective back-up mechanism for excision of 8-oxoG may exist in the cell.

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Benzo[a]pyrene (BaP) is an ubiquitous environmental pollutant with potential carcinogenicity. It has been shown that BaP, upon UVA irradiation, synergistically induced oxidative DNA damage, but other DNA damage was not confirmed. In this study, we examined whether coexposure to BaP plus UVA induces double strand breaks (DSBs) using xrs-5 cells, deficients in the repair of DSBs (Ku 80 mutant) and whether Ku translocates involving the formation of DSBs. BaP plus UVA had a significant cytotoxic effect on CHO-K1 cells and even more drastic effect on Ku80 deficient, xrs-5 cells, suggesting that the DSBs were generated by coexposure to BaP plus UVA. The DSBs were repaired in CHO-K1 cells within 30 min, but not in xrs-5 cells, indicating the involvement of a nonhomologous end joining, which needs Ku proteins. Furthermore, we succeeded in visualizing that Ku80 rapidly assembled to the exposed region, in which DSBs might be generated, and clarified that the presence of both Ku70 and Ku80 was important for their accumulation.

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The oxygen effects that are prominent for low LET radiations become small with increase of the LET. Radio-chemical reactions are generally believed as the fundamental mechanisms underlying oxygen effects. However, DNA repair in biological systems could also affect the oxygen effect because oxygen enhancement ratio (OER) values obtained for survivals of E. coli are different when the DNA repair systems are different. In this research, we studied and reported here significance of DNA-DSB repair in OER after low- and high-LET radiation. The CHO cells were exposed to 200 kVp X-ray or HIMAC carbon-ion beams under oxic or hypoxic conditions. Amounts of DNA-DSB in the cells were analyzed by a static field gel electrophoresis.
The OER of DNA-DSB after X-ray irradiation was 4.0 without repair, and increased to 7.5 when a repair time of 3 hours was introduced. On the other hand, the OER after carbon-ion beams were 2.2-2.3, and not affected by introducing repair time. We hypothesize that the oxygen-dependent DNA-DSB is different from the oxygen-independent DNA-DSB, and that the production ratio of these DNA-DSB is different between X-ray and carbon-ion irradiation.

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8-oxoguanine (8-oxoG) is an abundant and critical base modification. It is excised from the DNA by MutM in Escherichia coli and Ogg1 in budding yeast and mammalian cells. However, any structural and functional homologs of them are not yet identified in Shizosaccharomyces pombe. Nth1 protein, an ortholog of E. coli Nth, was cloned from the S. pombe. It removes oxidative pyrimidines from the DNA. E. coli Nth and Nei remove 8-oxoG from the 8-oxoG:G mispairs in DNA. We examined whether the Nth1 protein functions as a DNA glycosylase/AP lyase for 8-oxoG in DNA as E. coli Nth and Nei. The Nth1 protein was expressed in E. coli and partially purified using glutathione affinity column chromatography. The DNA glycosylase activity was assayed for 8-oxoG:A, 8-oxoG:G or 8-oxoG:C containing duplex oligonucleotides. The Nth1 protein efficiently removed 8-oxoG from 8-oxoG:A and 8-oxoG:G pairs from DNA. The activity to remove 8-oxoG from 8-oxoG:A and 8-oxoG:G mispairs was much higher than that from 8-oxoG:C. Furthermore, we report about the effects of Nth1 expression on some mutator strains deficient in DNA N-glycosylases.

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Purpose: To clarify the cellular processing and repair mechanisms for radiation-induced clustered DNA damage, we examined the correlation between the levels of DNA glycosylases and the sensitivity to ionizing radiation in Escherichia coli.
Materials and methods: The survival of gamma-rays, X-rays, alpha-particles and H₂O₂ were determined in E. coli with different levels of DNA glycosylases. DSB formation by post-irradiation treatment with DNA glycosylases was assayed with gamma-irradiated plasmid DNA in vitro.
Results/Conclusion: An E. coli mutM nth nei triple mutant was less sensitive to low-LET radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of E. coli MutM, Nth, Nei and human OGG1 increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Treatment of gamma-irradiated plasmid DNA with MutM protein converted the supercoiled to the linear form of the DNA. On the other hand, overproduction of MutM conferred resistance to H₂O₂ on the E. coli mutM nth nei mutant. The levels of DNA glycosylases affect the sensitivity of E. coli to low-LET radiation. Excision by DNA glycosylases converts nearly opposite base lesions in clustered damage to DSBs which are potentially lethal. Post-irradiation treatment of H₂O₂ increased the probability of clustered damage formation.

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It's well-known that ionizing radiation, such as X-rays, gamma-rays and alpha-rays, produces a unique form of DNA damage called "clustered damages", which contains two or more lesions induced within the one or two helical turns of the DNA. Many of the lesions induced by ionizing radiation are chemically indistinguishable from those induced as by-products of oxidative metabolism. However, it has been hypothesized that clustered damages induced by ionizing radiation would be less readily repaired than isolated lesions. So clustered damages might be biologically significant. In this report we showed HeLaS3 cells transfected by hOGG1 type1a or type2a plasmid was more sensitive to gamma-rays than HeLaS3 cells without plasmid. Clustered damages formed by ionizing radiation might be converted to lethal double-strand breaks during attempted base excision repair. We are now studying the biological effects of clustered damages in human cells.

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Ionizing radiation induces various DNA damage localized closely along its track. The resulted damaged site including two or more lesions within two helical turns of DNA is called clustered damage. Especially, bistranded clustered damage is thought to be unrepairable, and lead to cell death and mutations. Double-strand break, as bistranded strand break cluster, is processed by non-homologous end-joining. On the other hand, bistranded clustered base damage is supposed to be processed by base excision repair. However, enzymatic efficiency of each DNA-glycosylase for clustered base damage has been unclear. We examined the enzymatic activity of DNA-glycosylases for oxidative base lesions in clustered damage in this study. Oligonucleotide substrates containing two bistranded base lesions were prepared, such as thymine glycol, urea, and 8-oxoguanine. The distance between two lesions was set 1, 3 or 5 nucleotide away for upstream or downstream on each substrate. Activities of Escherichia coli endonuclease III, VIII, and Fpg, and these human homologues for the clustered substrates were assessed by nicking assay. As a result, activities of tested DNA-glycosylases were affected by the distance and the species of lesions.

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DNA double strand-breaks (DSBs) caused by ionizing radiation and some chemical agents can induce great damage to genetic material. All cells maintain repair systems for such breaks, however, these activities are fluctuated in a tissue specific and/or age dependent manners. We have studied the effects of radiation on meiosis and reproductive development using an experimental model organism, the nematode Caenorhabditis elegans. In the adult C. elegans hermaphrodite, germ line nuclei in the tip of the gonad arm divide mitotically and thereafter enter meiotic prophase I, progressing from the leptotene/zygotene stages to diakinesis stage in maturing oocyte. The linear array of the developmental phases of oogeneisis makes the adult hermaphrodite a convenient model system to study these effects.
We found that meiotic pachytene nuclei of C. elegans are hyper-resistant to X-rays, gamma rays and heavy ion particle irradiations, whereas early (post-fertilization) embryonic cells and diakinesis stage oocytes are not. The high level of expression of enzymes involved in meiotic homologous recombination accounts for these differences in resistance. In addition, we would like to discuss the radiation response of the pachytene checkpoint system including apoptosis and to introduce the construction of bystander effects in germ line cells using micro-beam system.

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The yields of DSBs in plasmid DNA in aqueous solution by monochromatic X-rays increase toward lower X-ray energies. This energy dependence is explained that DSBs induction correlates with the primary-event density which increase with the decrease in photon energy through higher LET of secondary electrons by lower photon energies. Since the X-ray energy dependence of DSB induction in cellular DNA, however, has not been studied, we investigated the yields of DSBs after irradiation of V79 cells by 5, 10, and 15 keV X-rays. DSBs in chromosomal DNA was measured using the pulse field gel electrophoresis (PFGE) after cells were irradiated and embed in agarose plugs at 4oC, and then, treated with protease K. The yields of DSBs were determined using gel images of ethidium bromide fluorescence and the random breakage model. The yields of DSB induction in chromosomal DNA by X-rays of 5, 10, 15 keV were 7.8410-3, 7.2110-3, 6.2010-3 DSB/Mbp/Gy, respectively, which indicated the similar tendency to those of plasmid DNA.

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The Effect of Track Structure on the Induction of DNA Damage Seiichi WADA, Tomoo FUNAYAMA, Tetsuya SAKASHITA, Yasuhiko KOBAYASHI, JAERI-TakasakiThe different particles indicate different biological effects even if their LET value is same. This difference in biological effects by different particles with same LET is considered to be caused by a difference in their pattern of energy deposition in target materials. Thus we investigated the biological effects of track structure by different particles with the same LET value using visualization of DNA damage induced by hit-ions. CHO-K1 cells attached on ion track detector CR-39 were irradiated with the same LET value (about 437 keV/µm) but different nuclide (¹²C and ²⁰Ne). DNA strand breaks were immunohistochemically detected with exonuclease and tdt for total breaks, and with γH2AX antibody for double strand breaks. The fluorescent signal observed in nuclei by immunochemistry was co-localized at the site of etched pit where the ion was traversed on the cell. To evaluate the spatial distribution of DNA damage, the diameter of the area of fluorescent signal was measured. The area of γH2AX induced by C ion and Ne ion were not significantly different, however, the area of DNA strand breaks induced by Ne ion was larger than that induced by C ion. This result indicates that the difference of ion track structure influences the pattern of induction of DNA damage.

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We investigated the difference in rejoining kinetics of chromosome breaks between normal human lymphocytes and normal fibroblasts irradiated with X-rays using the fusion-based G1-type PCC method. To detect the initial damage, ice-cold normal cells were exposed to 0, 1, 2Gy of X-rays and immediately processed for the PCC assay. The number of excess chromosome fragments in lymphocytes (without repair) was about 1.6 fold higher than that in fibroblasts. Furthermore, if a DSB repair inhibitor wortmannin was applied during the PCC incubation period, the initial dose response for lymphocytes was identical to that for fibroblasts. In addition, the chromosome rejoining of lymphocytes for the first two hours after irradiation was significantly delayed when compared to the repair of lymphocytes using the PCC method with wortmannin. These results indicate that the reduced repair, not the initial chromosome damage, of lymphocytes may be a main contributing factor for the known hyper-radiosensitivity of lymphocytes. Studies at the molecular level are underway to elucidate the mechanism underlying these interesting phenomena.

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To investigate whether the photoelectric effect on DNA constituent atoms is more effective than low-LET γ-irradiation at inducing DNA damage, hydrated plasmid DNA was irradiated with synchrotron ultrasoft X-rays (2.147 keV). The yields of strand breaks do not significantly depend on the level of hydration, as has already been shown in our previous γ-irradiation studies. The ratio of the yield (SSB/DSB) is about 16, which is slightly higher than those for γ-irradiation (8-12). The yields of base lesions determined by post-irradiation-treatment of the DNA with enzymatic probes (Fpg and EndoIII) which excise base damage significantly increase with increasing level of hydration. The maximum ratios of the yields of the base lesions to SSB are 1.9:1 (Fpg) and 2.2:1 (Nth), respectively. The yields of enzymatically induced DSB are about half of those of γ-irradiation, indicating that enzymatically revealed, clustered DNA damage sites are lower when induced by ultrasoft X-rays. These results indicate that photoabsorption event at C, N and O in DNA and ejected low-energy secondary electrons may produce these types of DNA damage with different efficiencies to those by γ-irradiation. The role of photoelectric effect including phosphorus K-absorption and low-energy electrons will be discussed in terms of their efficiency to induce DNA damage.

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Single tracks of ionizing radiation tend to create patterns of damage consisting of two or more damages within one or two helical turns of the DNA. These closely packed patterns are called Clustered-DNA-Damage or Locally-Multiply-Damaged-Sites. Although it is thought that DSB is the most critical damage, recent studies have shown that clustered-DNA-damage involving base damages may play an important role in biological consequences of radiation damage. The purpose of this study is to calculate the spectrum of initial damage consisting of strand breaks and base damages for ionizing radiations of different linear energy transfer (LET). Induction of strand breaks and base damages were simulated with direct as well as indirect effect using Monte Carlo track structure methods. The number and configuration of damages on the DNA segment of a few helical turns were estimated. The calculations show the contributions to the spectrum of damage involving more than 2 damages are about 10% for 100 keV electrons and 40% for 1 keV electrons, while the value of more than 60% has been calculated for 3.2 MeV alpha particles. In particular, the contributions involving more than 3 damages are 2%, 15% and 45% for 100 keV and 1 keV electrons and 3.2 MeV alpha particles, respectively. The detail of the positional distribution of damages which may be related to the inhibition of repair processing will be presented.

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We have analyzed delayed mutation as a part of cellular response to genomic damage and found that this occurs at the pink-eyed unstable locus in the somatic cells of F1 mice born to irradiated sperm. However, a whole body mouse system and tissue culture cells are too complex to elucidate the molecular mechanism of delayed mutation.
We have now examined delayed mutation in fission yeast. We have constructed three yeast strains which carry tandem duplications differing in length at the ura4 gene and with which reversion mutation trough homologous recombination (HR) is easily detectable. The frequency of spontaneous HR was linearly related to the length of the repeat. One of the strains carrying a 200 bp repeat was X-irradiated (25-500 Gy) and in the frequency of the reversion was found to be dose dependent. In addition, the increase mutation frequency persisted for 6-10 cell generations after irradiation where damage repair is thought to be completed.
These results suggest that in fission yeast untargeted mutation can occur and delayed mutation is induced after genomic damage.

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Ionizing radiation and reactive oxygen species induce various oxidative lesions in DNA. Oxidative base lesions are repaired by the base excision repair pathway that is conserved from bacteria to humans. In E. coli, oxidative pyrimidine lesions are removed from DNA by Endo III and Endo VIII that have redundant damage specificities. In the present study, we compared the damage specificity of human homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2) for oxidative base lesions and abasic (AP) sites. hNTH1 and hNEIL1 exhibit differential activities for stereoisomers of thymine glycol (5R-Tg and 5S-Tg). hNTH1 excised 5R-Tg preferentially over 5S-Tg, whereas hNEIL1 excised 5R-Tg and 5S-Tg comparably. HeLa cell extracts excised 5R-Tg preferentially, and this preference was similar to that of hNTH1. hNTH1 and hNEIL1 also exhibited the activity for other oxidative base lesions and AP sites, although they displayed distinct preferences. Therefore, hNEIL1 may have back-up function for hNTH1 in cells. hNEIL2 exhibited a high activity for AP sites, but the activity for the oxidized lesions was marginal.

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Reactive oxygen species are continuously generated in living cells. They interact with DNA, which produce a wide variety of base damage and cause mutations. Bacteria and eukaryotes have evolved DNA repair systems to maintain the genetic stability and prevent mutations. Many enzymes are involved in the base excision repair pathways in E. coli and eukaryotes. For instance, MutT dephosphorylates 8-oxo-dGTP and prevents the incorporation of 8-oxodGTP produced in nucleotide pool. 8-oxoG mispairs with A and causes from G:C to T:A transversions. E. coli Nth has glycosylase activity that recognizes and excises thymine glycol (Tg). Tg blocks DNA replication in vitro. The phenotypes of DNA repair defective mutant are well studied in E. coli and S. cerevisiae, while little in multicellular organisms. In humans, the defect of nucleotide excision repair results in serious hereditary diseases. However, there are little indications about nematode repair system, despite of their great convenience. C. elegans, a multicellular organism of approximately 1,000 cells, has short life span and small genome size. Recently, we found C. elegans has an uracil DNA glycosylase, indicating that repair systems are also conserved in nematodes. We are currently identifying other putative enzymes involved in C. elegans BER pathways.

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Base excision repair (BER) is one of thepathways to avoid mutations in living cells. Damaged bases are excised by DNA glycosylasesand then AP lyases nick DNA, followed by repair synthesis and rejoining by DNApolymerases and DNA ligases. Increased frequency of mutations in BER-defectivemutants in E. coli and S. cerevisiae indicates that BER plays animportant role in cells. However, the effects of BER deficiency in multicellularorganisms are yet unknown. In human, defect of nucleotide excision repairresults in serious hereditary diseases, while no desease has been found torelate to BER deficiency. Previously, ung knockout mice were generated. Indeed, they have deficit in immune system, but no obvious phenotype relevantto ung function in BER system.
So we investigated the influence of BERdeficiency in multicellular organsim using C. elegans. Recently, weidentified the uracil DNA glycosylase homolog gene (Ceung) in C. elegans,and clarified the glysocylase activity of purified recombinant CeUNG. The RNAimediated UNG mutant of C. elegans is viable and show apparently normalphenotype. Here we report further analysis of the phenotype.

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Damaged DNA bases are removed by a DNA glycosylase to generate AP sites. AP sites have two types of chemical structure, regular AP sites and reduced AP sites. DNA containing both regular and reduced AP sites are recognized and cleaved by AP endonuclease. After this step, there are two pathways of BER in mammalian cells. One is the short-patch BER pathway, in which DNA Polymerase beta inserts a single nucleotide into the repair gap and removes the dRP residue. The DNA ligase III-XRCC1 complex completes DNA repair by sealing the DNA strand. The other BER pathway is long-patch BER. It is involved in the repair of reduced AP sites. Different subsets of enzymes from the short patch pathway are involved in the long patch pathway. DNA Polymerase beta initiates repair synthesis by adding the first nucleotide. After that polymerase delta/epsilon, PCNA, FEN1 and DNA ligase I are used. DNA is repaired by gap filling of several (2~8) nucleotides. So far, the fidelity and mutational specificity of short-patch BER and long-patch BER pathways associated with mammalian BER have not been directly investigated. In this study we compared the fidelity and mutational specificity of short-patch and long-patch BER pathways.

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DNA double strand break (DSB) is the most critical damage that are induced by radiation. Many DSB repair proteins have been identified using several approaches such as positional cloning and phenotype analysis of knockout cells. Among these, Nbs1, the underlying gene for Nijmegen breakage syndrome, forms a complex with Mre11/Rad50 and is essential for homologous recombination (HR) and S-phase check point. To clarify the role of Nbs1 protein on DSB responding pathways, we analyzed apoptosis induction in Nbs1 deficient cells after irradiation. In sharp contrast to wild type cells, NBS1 deficient cells did not display apparent induction of apoptosis after exposure to X-rays. Because DT40 cells are not expressing p53, our results suggest that Nbs1 acts in a critical pathway for induction of apoptosis independent of p53. We report results from the analysis of Nbs1-dependent but p53-independent pathways for apoptosis induction.

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Three radiation sensitive mutant strains of Medaka which showed significant increase of malformation by irradiation at organogenesis stage were established. We reported that one of these strains, RIC1, showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages and has a defect in the repair of DNA double-strand breaks (DSBs). We examined the responses of early embryonic cells in the other mutants, RIC2 and RIC3. Genetical complementation analyses demonstrated that these three strains have mutation at three different genetic loci. After irradiation with 1Gy at the morula stage, although no significant increase of hatchability was observed in CAB strain embryos, lower hatchabilities than those of non-irradiated were shown in RIC2 and RIC3. Moreover, RIC2 was more sensitive than RIC1. The TUNEL assay showed higher frequency of apoptosis in RIC2 and RIC3 embryos immediately after gamma-irradiation at the pre-early gastrula stage. These results suggest that RIC2 and RIC3 show high radiation sensitivity even at early embryonic stages. However, the comet neutral assay showed that almost of all DSBs induced were rejoined within 1h after irradiation at the pre-early gastrula stage like CAB. This result suggests that the products of ric2 and ric3 gene are associated with DNA surveillance mechanisms except for DNA DSBs repair mechanism in which ric1 gene participates at early developmental stages.

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The purpose of this study is to reveal the surveillance mechanisms to DNA damage in male germ cells using Medaka, Oryzias latipes, because of the ease with which the differentiation stages of its spermatogenic cells can be identified. In our laboratory, the three radiation sensitive Medaka mutants (RIC; Radiation Induced Curly tailed malformation) were isolated by ENU mutagenesis screening in the Medaka. We reported that RIC1 mutant has a defect of DNA double strand breaks repair during early embryogenesis. We examined the effect of DNA double-strand breaks (DSBs) in the mutant testis. We investigated the testis by histological analysis in wild type (CAB strain) and RIC1 after 2.5Gy gamma-irradiation. At day 30 after irradiation, there were no spermatogonial stem cells (SSCs) and histological recovery like in the CAB was not observed in the RIC1 testis. RIC2 is sensitive at morula stage than RIC1. In the RIC2 testis, there were SSCs and partial recovery was observed at day 30 after 2.5Gy gamma-irradiation. These results suggest that SSCs in RIC1 testis are very sensitive compared to those in CAB, and that there are differences in the responses (e.g. DNA repair system and apoptosis) of SSCs to DSBs after gamma-irradiation between RIC1 and RIC2.

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Thymineglycol is an oxidative base damage in DNA and is a lethal damage because of the interference of a polimerase action. We have found a novel monofunctional DNA-glycosylase activity for thymineglycol in mouse organs. The nuclear fraction was prepared by homogenizing mouse frozen stomachs. We exclude the contaminating APendonuclease activity as well as mNTH activity from the nuclear fraction by high salt extraction method with 0.42 M NaCl buffer. Previous experiment revealed that the activity was retained in nuclear fraction after the salt tretment. The treated nuclear fraction was sonicated and centrifuged to obtain nuclear extract. Following polyethyleneimine precipitation to remove DNA, we performed ammonium-sulfate fractionation, hydrophobic column chromatography (HiTrap Phenyl FF, Amasham Biosciences), hydroxyapatite column chromatography (Bio-Scale Ceramic Hydroxyapatite Type1 Column, BIO-RAD), ion-exchange column chromatography (UNO-S1, BIO-RAD), then acquired purified protein fractions containing the target activity. After SDS-PAGE and silver stain (Silver Stain MS kit, Wako), we found several candidate protein bands; 4 major bands (61, 56, 46, 33 kDa) with 6 minor bands (70, 53, 41, 37, 27, 25 kDa). We analyzed them with MALDI-FTICR Mass Spectrometry. There were not mNTH1, mNEIL series nor heterogeneous nuclear ribonucleoprotein A2/B1, which had found during the last MALDI-FTICR Mass Spectrometry analysis.

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Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing DNA double-strand breaks (DSBs). Artemis is involved in V(D)J recombination and DNA repair. Artemis has a role in T and B lymphocyte immunodeficiency and in predisposition to lymphoma through the NHEJ pathway of DNA repair. Artemis mutations cause human T-B-severe combined immunodeficiency associated with increased cellular radiosensitivity (RS-SCID). To address the role Artemis in human cells, we used gene targeting in human somatic tissue culture, human HCT116 colon cancer cell, to functionally inactivate the Artemis locus. Human Artemis heterozygous cell lines displayed haploinsufficiency with reduction in cell proliferation. Second round of gene targeting generated homozygously null Artemis cell lines. These cells showed a growth defect.

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DNA ligase IV is one of the key proteins that are associated with DNA double strand break (DNA DSB) repair through the non-homologous end joining (NHEJ) pathway. We designed 21 bp small double stranded RNA (siRNA) on the basis of its specific DNA sequence, and transfected HFLIII, normal human primary fibroblasts, with the DNA ligase IV siRNA. The RNA and protein expression levels of DNA ligase IV and several key radiological characteristics were investigated in the siRNA-transfected and control cells. Compared to the cells with mock-transfection, the RNA and protein expressions were reduced in the siRNA-transfected cells. An increase in chromosomal fragmentation and a decrease in cell survival were observed in X-irradiated cells transfected with the siRNA. The transfection with the DNA ligase IV siRNA did not make a significant effect on cell growth and the XRCC4 protein expression. These results indicate that human cells could be radiosensitized by reducing the expression of DNA ligase IV using RNA interference technology.

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Non-homologous end-joining is the primary repair pathway for DNA double-strand breaks in human cells. The rejoining of DNA ends is catalyzed by the complex of DNA ligase IV and XRCC4. Using human XRCC4-knockout cells that have no detectable yield of Ligase IV, we analyzed the end-joining (EJ) activity in their extracts. As a result, the activity in XRCC4-knockout cells is lower than that in the parental cells with XRCC4 but is still high under our in vitro conditions. The XRCC4-independent EJ activity was partially purified through chromatographic procedures, leading into the separation from DNA-PK components, DNA-PKcs and Ku70/Ku86. Interestingly, the addition of DNA-PK components to the active fraction had no effect on its EJ activity, while they completely suppressed the activity of T4 phage-derived DNA ligase. Also, in combination with DNA-PK, the coexistence of a kinase inhibitor wortmannin greatly abolished the EJ activity. These results suggest that the XRCC4-independent EJ is associated with the function of DNA-PK complex, whose kinase activity is a regulatory factor for the EJ.

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To study the molecular mechanisms which determine the sensitivity to gamma-irradiation in vivo, we identified three types of mutants with high incidence of radiation induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established. Both in the early embryo or germ cells, RIC1 strain, is highly sensitive to gamma-ray than the wild-type strain.
In Medaka, polymorphisms among wild population enable us to map genes of interest. RIC1, which belongs to the southern Japanese population, was crossed with HNI inbred strain derived from the northern ones. After backcrossing their offspring with RIC1, acquired embryos were irradiated at the organogenesis stage. Then, radiation-induced curly tailed embryos were identified as the ric1 phenotype, and their DNA were extracted. Ric1 locus was located in LG9, and 2cM genomic region containing ric1 gene was determined. In this region, gene order seems to be conserved among medaka and two puffer fish species. Now we are examining genes related to DNA double-strand break repair from pufferfish genome database as candidate gene for ric1.

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Many types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to bypass altered DNA bases. To overcome this block, cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. The TLS polymerases are also characterized in terms of their low-fidelity synthesis on undamaged DNA, leading to the prediction that unregulated action of these polymerases give deleterious effects on cells. Thus, activity of these polymerases must be regulated strictly. In fact the amount of TLS polymerase is kept constant at protein level even if the level of RNA increased more than 30 times by ectopic expression in Drosophila. By deletion analysis of dRAD30B we have identified a region responsible for degradation of this protein. When the region was deleted, stability of he truncated protein increased drastically. The degradation is inhibited by treatment with MG132, suggesting an involvement of ubiquitin-proteasome pathway on the regulation of dRAD30B stability.

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We have recently constructed a novel Human Artificial Chromosome (HAC) vector called 21delta-qHAC, generated by deletion of a distal portion of the long arm of human chromosome 21 and insertion of a loxP sequence to facilitate insertion of any desired gene construct. We intend to use this HAC vector to rescue phenotypes associated with DNA repair gene deficiency in cells. To evaluate the usefulness of 21delta-qHAC vector for this purpose, we first attempted in vitro studies using CHO cells (V3) exhibiting radiosensitive phenotype due to a DNA-PKcs deficiency. In this experiment, we inserted a ~21 kb tetracycline-inducible DNA-PKcs expression cassette into 21delta-qHAC vector by Cre-mediated recombination. The resulting 21delta-qHAC/DNA-PKcs construct was then introduced into the V3 cells. RT-PCR results from cells containing the 21delta-qHAC/DNA-PKcs vector showed doxycycline (a tetracycline derivative)-dependent regulation of the DNA-PKcs gene. Furthermore, radiosensitivity assays showed phenotype rescue in these cells due to the production of functional DNA-PKcs protein. To our knowledge, our study is the first to report successful artificial control of gene expression by placing the multiple regulatory elements of the tetracycline system in a single expression cassette. Our results also show that 21delta-qHAC is a useful tool in the introduction of a therapeutic gene with multiple regulatory elements; and correction of a defective phenotype.

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It is known that deficiencies of the XPG gene cause a genetic disease of xeroderma pigmentosum (XP). In some cases with more destructive mutations of the gene, mixed symptoms of XP and a severe type of Cockayne syndrome (CS) were also found. Xpg-knockout mice show severe growth retardation and premature death as well as DNA repair defects, supporting the correlation between the severity of the gene mutation and severe-type CS association. However, the contribution of CS phenotypes is unclear in patients with less severe XPG mutations. We studied UVB-induced mutations in skin using lacZ-transgenic mice with a C-terminal truncation in both alleles of the Xpg gene which causes a mild DNA repair defect but not discernible growth deficiencies in affecting mice. We found a remarkable suppression of mutation induction in the skin epidermis from this mouse at higher UVB dose range.

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Patients with Cockayne syndrome are characterized by UV sensitivity, developmental abnormalities and premature aging. Cockayne syndrome has two complementary group proteins, CSA and CSB. Cockayne syndrome shows increased sensitivity to UV-induced DNA leasions. These lesions are repaired by NER (nucleotide excision repair), which has two pathways, GGR (global genome repair) and TCR (transcription-coupled repair). Cockayne syndrome defect selectively in TCR and RNA synthesis is not recovered after TCR. Also, cockayne syndrome cells induce apoptosis after UV irradiation in low dosage compared with normal cells. >From these observations, it seems that CSA and CSB proteins work in transcription, DNA repair, and signaling pathway of apoptosis after UV irradiation. In this study, for the purpose of revealing the signaling pathway that is concerned by CSB protein, we have analysed gene expression pattern after UV irradiation with CSB deficient human fiblobrast cells.

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In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause an early onset of Cockayne syndrome (CS) in some patients (XPG/CS) with characteristics such as growth retardation and a short life span. To identify the XPG region that causes the onset of CS, we have generated Xpg mutant mice with C-terminal truncations or a base substitution mutation, and examined their growth and life span. The CS-causing mutation XpgD811stop completely inhibited the NER activity of Xpg, but the non-CS causing mutations, XpgD811A or Xpgdelex15 (deletion of the last 183 amino acids), resulted in the retention of residual NER activity. To generate a completely defective NER condition in mice with the non-CS causing Xpg mutation we crossed these mutant mice with Xpa-null mutant mice. When the Xpa-null mutant allele was introduced, mice with the non-CS causing deletion mutation (Xpgdelex15) exhibited the CS phenotype (growth retardation and short life span), but the base substitution mutant (XpgD811A) mice did not. These results indicate that both the C-terminal truncation of Xpg and a complete loss of NER activity are required for the expression of the CS phenotype.

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The 78-kDa glucose-regulated protein (GRP78) is known as an ER stress-induced molecular chaperone. There has been little information about the roles of GRP78 in response to UVC in mammalian cells. In this annual meeting last year, we found that GRP78 is involved in the resistance of human cells to UVC using RSa cells with down-regulation of GRP78 expression by transfection of antisense cDNA for GRP78. In the present study, we investigated the mechanisms underlying the GRP78-involved UVC resistance. To estimate the DNA repair capacity of cells, the amounts of damaged DNAs in vivo were measured in the transfected cells after UVC irradiation with the specific antibodies against the damages. In addition, repair activities in whole cell extracts from the cells were measured by in vitro repair synthesis assay using UVC-irradiated plasmid as a substrate. We found that the cells with down-regulation of GRP78 expression showed lower ability to repair the damaged DNA in vivo and in vitro than the vector control cells. Furthermore, the antisense-cDNA transfected cells also showed higher sensitivity to cisplatin, by which the DNA damage induced is primarily repaired by nucleotide excision repair, like UVC-induced DNA damage. These results suggest that GRP78 has a protective role against UVC-induced cell death possibly via functioning in nucleotide excision repair, at least in the human RSa cells tested.

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UVB radiation sensitivity among rice cultivars varied widely. The possibility that UV-resistant rice cultivar might exhibit higher cyclobutane pyrimidine dimer (CPD) photorepair ability compared with UV-sensitive one, has been indicated. Here, we show that UVB sensitivity among rice cultivars is tightly linked with CPD photolyase activity by quantitative genetic approach using UV-resistant Sasanishiki (japonica rice cultivar) and UV-sensitive Surjamkhi (indica rice cultivar). Firstly, we confirmed that the CPD photolyase activity in Surjamkhi was significantly lower than that in Sasanishiki. There were two variations at codons 126 and 296 in the deduced amino acid sequence of CPD photolyase between both cultivars, and these mutations affected the CPD photolyase activity. Next, to test whether the alteration of the CPD photolyase causes the difference in UVB sensitivity in rice, QTL analysis for linkage between UVB sensitivity and CPD photolyase with DNA makers developed in flanking region of CPD photolyase gene, using the population derived from a Sasanishiki/Surjamkhi was carried out. There was a peak with LOD score 24.5 at the region located photolyase gene. Thus, these results suggest that the spontaneously occurring mutation in CPD photolyase gene could lead to difference in UVB sensitivity in rice.

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IL-12, one of the cytokines produced in human skin by UV-B exposure, has recently been reported to enhance UV-induced DNA repair. In this study human keratinocyte cell line (HaCaT) was irradiated with UV-B at physiological dose rate, and the repair kinetics of DNA photoproducts was examined by ELISA. The amount of cyclobutane pyrimidine dimer (CPD) removed in 6h following the irradiation was significantly increased by the presence of IL-12 in a dose-dependent manner. No such effect of IL-12 was observed on repair of (6-4)photoproduct. The number of survived cells after UV-B irradiation, as revealed by colony forming ability, was also increased by IL-12. These results suggested that IL-12 accelerated the repair of CPD in UV-B irradiated keratinocytes, resulting in prevention from cell death.

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PURPOSE: Heparin-binding EGF-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes. HB-EGF is synthesized as a membrane-anchored form (proHB-EGF) and proHB-EGF is cleaved on the cell surface to yield soluble HB-EGF by the mechanism called "ectodomain shedding". Ultraviolet is one of the stimuli inducing shedding of proHB-EGF. The aim of this study is to determine the intracellular signal pathway lead to shedding. MATERIALS AND METHODS: Vero-H cells were cultured for 15h in serum-free MEM-NEAA, then treated with inhibitors for 30 min. For UVB irradiation, Vero-H were exposed to 40 mJ/cm² UVB using a Spectrolinker. Following incubation for 30 min, the cells were collected and shedding was detected by western blot analysis. RESULT AND DISCUSSION: An inhibitor of p38 MAPK inhibited the stress-induced ectodomain shedding of proHB-EGF, but inhibitors of PKC, MEK and PI3K did not. On the other hand, anti oxgen reagent inhibited shedding of proHB-EGF induced by not only UVB, but also TPA and LPA. gamma ray did not induce ectodomain shedding of proHB-EGF. These results indicate that the UVB-induced proHB-EGF shedding is mediated by p38 MAPK and that reactive oxgen speacies is not involved in the signaling pathway induced by ultraviolet radiation.

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The cataract is promoted by UV irradiation. It is well known that the main causes of UV induced cataract are reactive oxygen species which are generated by UV irradiation. Metallothionein (MTs) regulates detoxification from heavy metals, and free radical scavenging in living tissues. In this study, we analyzed the inductions of metallothionein-I (MT-I) and metallothionein-II (MT-II) by ZnCl₂ treatment, and the protective effect of MTs against UV-A, UV-B and UV-C using mouse lens epithelial-derived cultured cells, alphaTN 4-1 cells. AlphaTN4-1 cells were treated by various concentration of ZnCl₂, and then the expressions of MT-I and MT-II mRNA were analyzed by the real-time RT-PCR. ZnCl₂ treated cells were irradiated by UV-A, UV-B and UV-C, and the cell viabilities were analyzed. The cell viabilities were measured by ³H-thymidine incorporation. This study revealed that MT-I and MT-II mRNA were induced concentration-dependently by ZnCl₂ treatment, and induced MTs by ZnCl₂ treatment significantly increased the cell viability against UV-A irradiation. This study indicates that a novel protection mechanism by MTs against UV-A may exist in the lens.

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We have found long-lived radicals(LLR) with long half-time (T1/2 is over 20 hr) in X-irradiated cells, previously. LLR is a cause of mutation, but it is not a cause of cell death. Therefore, LLR may contribute to cancer and senescence through mutation. Recently, we also found that LLR was induced in a cell irradiated with UV-B. In this experiment, we examined whether vitamin C could scavenge LLR induced by UV-B, and whether it could depress induction of mutation. Actually, the cells were treated with 5mM of vitamin C both for 2 hr before X-irradiation and for 2hr from 20min after X-irradiation. Vitamin C didn't affect the survival of cells exposed to UV-B, which indicates that vitamin C didn't scavenge active short life time radicals but LLR. However, both pre- and post-treatment with vitamin C reduced mutation frequency in the UV-irradiated cells. These results suggest that LLR are involved in the induction of mutation by UV-B light.

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Radiation generates reactive oxygen species that interact with cellular molecules. We investigate the effect of hydrogen peroxide compared to X-ray using repair proficient chicken DT40 cell and repair deficient DT40 cells (RAD54^-/-, KU70^-/-, RAD54^-/-/KU70^-/-). Repair proficient normal DT40 and KU70^-/- cells were resistant to low dose of hydrogen peroxide (under 5μM) but RAD54^-/- and RAD54^-/-/KU70^-/- cells were sensitive to. At higher dose of hydrogen peroxide (5~1000μM), both normal DT40 and KU70^-/- cells had a huge tail of survival curve. Homologous recombination repair of DSB is very important for biological effects of hydrogen peroxide, suggesting that hydrogen peroxide make only SSB but this become DSB in S phase. Effect of oxygen radical might be not similar to ionizing radiation.

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Reactive oxygen species attack DNA and its precursor nucleotides, and consequently induce various oxidized forms of bases in DNA within normally growing cells. Among such modified bases, 8-oxoguanine (8-oxoG) and 2-hydroxyadenine (2-OH-A) are highly mutagenic lesions, if not repaired, in DNA. MUTYH, a mammalian homolog of E. coli MutY protein, is a DNA glycosylase that excises adenine and 2-OH-A incorporated opposite guanine or 8-oxoG from DNA, thus expected to prevent G:C to T:A transversion mutation in mammalian cells. To investigate the role of the MUTYH gene in mutagenesis, we introduced the rpsL transgene to the MUTYH-null and wild-type mice by crossing. Using rpsL gene as a reporter, we compared the mutation frequencies and spectra between these two genotypes at the age of 24 weeks. The analysis of mutation spectra revealed that the frequency of G:C to T:A transversion was significantly increased in MUTYH-null mice compared to that of wild-type mice. This result suggests that MUTYH prevents occurrence of G:C to T:A transversion spontaneously induced by oxidative damage. Consistent with this notion, oral administration of KBrO₃, an oxidizing reagent, to the mice resulted in a significant increased frequency of G:C to T:A transversion in MUTYH-null mice. Our observations indicate that MUTYH prevents mutagenesis caused by oxidative damage in mammals.

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For the cancer imaging therapy the development of appropriate drugs has been carried out actively. Cu-PTSM is a kind of drug which was made in the development work. Cu-PTSM is not incorporated selectively only to cancer cells but it is known that the accumulation ratio in cancer cells is very high. In this study Cu-PTSM was incorporated to tumor cells, then the cells were irradiated with monochromatic X rays having energy around Cu-K edge at KEK-PF. The radiation energy of the monochromatic Cu-K X rays should be absorbed at Cu atom selectively and the special effect should be expected through the photo-electric effect in Cu atoms. In this study chromosomal aberrations, especially isochromatid breaks, were measured as radiation effect. The energy of the monochromatic X rays used for the experiments were 8.944 keV (Cu-K lower) and 9.034 keV (Cu-K higher). As a result, the induction of isochromatid breaks were clearly shown in the both cases of Cu-K lower and Cu-K higher irradiation depending linearly on the concentration of Cu-PTSM involving in U2OS cells. In the case of irradiation with Cu-K higher X rays at 500 nM Cu-PTSM concentration, the yield of isochromatid breaks was 1.8 times larger than that with Cu-K lower X rays.

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DNA double strand break (DSB) is well known as a huge DNA damage introduced by irradiation. On the other hand, in meiosis, scheduled DSBs formation and following recombination intermediates are necessary for appropriate chromosome segregation in the reductional nuclear division. To be efficiently repair ed, the DSBs formation must be strictly regulated, and it is known that in Saccharomycs cerevisiae, at least 10 gene products are required, such as Spo11, Rec102, Rec103, Rec104, Rec114, Mei4, Mer2, Mre11, Rad50 and XRS2, although the precise molecular function has not been figured out. Here, I will present recent data about targeting meiotic DSBs to new sites, using GAL4BD fusion protein.

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Recently, CR39, solid state track detector, was frequently used to compare the positions of ion tracks traversing mammalian cells and of the cluster of DNA damage or DNA repair related enzymes. The procedure employed, however, was laborious. Thus, we have tried to develop a new method which can visualize simultaneously the position of ion tracks and the image of cells on the surface of CR39, using the fact that UV enhances etching speed of CR39. The plate of CR39 bearing mammalian cells were irradiated by UV and then etched in 7N NaOH at 70 degrees. The reliefs of cells were embossed on the surface of CR39 according to the absorbance of corresponding part of the cell. The measurement by AFM of the surface of CR39 provided well-defined images of nucleus and the cell as well as the etchpits of the irradiated ions. We applied this cell imaging method to detect whether the position of DSB cluster visualized by immuno-fluorescent staining against gamma-H2AX obtained by confocal microscope and etched pits measured by AFM. The images of the gamma-H2AX foci, showed agreements in position of gamma-H2AX foci with the etch pits as well as the shapes of the cells.

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This study is intended to elucidate the mechanism of the mutagenesis by ion beam at molecular level. At first, survival rate and mutation frequency for the ion beam were obtained using yeast S288C. Next, mutation spectrum of mutants obtained by ion beam irradiation was measured by sequencing the mutation sites. S. cerevisiae strains used in this study are S288C(RAD+),rad3, rad18, and rad52.
The yeast strains were irradiated with carbon ions (12C⁵⁺; 220 MeV) with the dose 10 to 300 Gy, and LET is 107 keV/µm. Carbon ion beam was generated from AVF cyclotron in JAERI. The results indicated that rad52 strain showed high sensitivity to ion-beam, and that the optimum dose for mutagenesis was 100 Gy, and at the dose, mutation occurred 168.5 times more than spontaneous mutation. Moreover, The ura3 mutants were amplified by PCR, and the sequence analysis was carried out.
The result shows that mutations caused with ion beam irradiation have remarkable features and hot spots. Mutations of spontaneous generation tend to take the type of transition, and transversion is rare compared with transition. It was observed in this experiment that transversion was occurred by 5 times more than transition.

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We developed the system for detection and analysis of mutations occurring on chromosomal DNA in plant. Plasmid carrying E. coli rpsL gene, a target gene for mutagenesis, was inserted into pCGN5138, a plasmid for transformation of plant, and introduced into tobacco (Nicotiana tabacum BY-2). The plasmid sequence containing rpsL gene was rescued by PCR several days after treatment of transgenic tobacco with mutagen. Amplified fragment was circularized to form plasmid and introduced into streptomycin-resistant E. coli DH10B to isolate mutant clone. In this system, clones carrying mutated rpsL gene produce colonies on LB agar plate supplemented with streptomycin. AT to GC base change mutations and frameshifts were observed in UV-irradiated transgenic tobacco. The assay system developed here is quite useful for detection and analysis of mutations arising on chromosomal DNA in plant and may be valuable to evaluate mutagenicity of environmental mutagens.

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The p53 gene plays a key role in the cellular response to genotoxic stress. It is thought that p53 deficiency results in increased survival of cells with DNA damage, either by failure of DNA repair, or by failed deletion of mutation-bearing cells. The in vivo T-cell receptor (TCR) assay detects somatic mutation with a high sensitivity. Mutant CD4⁺ lymphocytes, are found to be CD3^-CD4⁺ by flow cytometry. The number of these mutant cells increases in humans and mice following exposure to radiation. At 8 weeks of age, wild p53(+/+) mice, heterozygous p53(+/-) mice and null p53(-/-) mice were given a whole-body acute dose of 3Gy X-rays. TCR assays were done at various time intervals between 9 days and 40 weeks after exposure. In p53(+/+) mice, the TCR-MF reached a peak level 9 days after irradiation, and TCR-MF decreased and reached background levels within 8 weeks. In p53(+/-) mice, the TCR-MF reached a peak level 9 days after irradiation, and TCR-MF decreased and reached background levels within 28 weeks. However, TCR-MF increased again 36 weeks after irradiation. In p53(-/-) mice, the TCR-MF reached a peak level 9 days after irradiation, and high frequency followed. These findings show that loss of p53 function becomes relevant to the acquisition of delayed mutations.

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We have analyzed T-cell receptor (TCR) mutations as radiation-induced somatic mutations in mice. In these studies, we found that the TCR assay may be useful for estimating the effect of genotoxic effect of radiation. To evaluate the risk of radiation during pregnancy, the TCR mutation frequencies (TCR-MF) of pregnant mice and newborns were analyzed.
C57BL/6N mice were each exposed to 3Gy of gamma-rays at 2.5, 6.5, 10.5 14.5, 16.5 and 18.5 days gestation and at 1, 7, 14, 21 and 28 days post-delivery. Mothers and their litters were sacrificed 28 days after irradiation and the TCR assay was performed. The pregnancy itself didn't influence the TCR-MF. In the exposed group during pregnancy and post-delivery, the TCR-MF was 2 and 5 fold higher than that of irradiated non-pregnant mice and the background frequency of TCR-mutant cells, respectively. On the other hand, the TCR-MF was not increased in the litters at any time of the pregnancy period.
These results suggest that pregnancy may cause increased sensitivity to radiation in mother mice. Besides, the child may have high repair ability to radiation.

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Germ-line mutation is detected in mice but not in humans. To estimate genetic risk of humans, a new approach to extrapolate from animal data to humans or to directly detect radiation-induced mutations in man is expected. We have developed a new method to detect germ-line mutations by directly comparing DNA sequences of parents and offspring. C3H male mice of 8 weeks of gestation were irradiated with gamma rays of 3, 2 and 1 Gy, and after 3 weeks, they were mated with the same aged C57BL female mice. The nucleotide sequences of 150 STS markers with 300-900 bp and SNPs of parents and offspring DNA were determined by a direct sequencing. At each radiation dose, about 5 Mb DNA sequences were examined to detect radiation-induced mutations. We could find 7 deletions and 1 point mutation in 3 Gy irradiated mice, 1 deletion in 2 Gy, 1 deletion in 1 Gy and no mutation in control mice,indicating that the maximum mutation frequency was 2.2x10^-4/locus/Gy at 3 Gy. These results suggest the non-linear increase of mutation with dose and that it is applicable for humans having many SNPs.

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Ku70 is one of the proteins involved in non-homologous end joining (NHEJ) repair of DNA double strand breaks. Its absence is known to make cells sensitive to radiation. Here we studied a possible involvement of NHEJ in a formation of radiation-induced mutations in vivo. Ku70-deficient and lacZ-containing mice were created by mating Ku70(+/-) and Muta^TM mice. At 2 months of age, they were exposed to 20 or 50Gy of X-rays, and analyzed for mutation 3.5 days after the irradiation. The tissues examined were spleen, liver and brain. Although the spontaneous mutant frequencies in Ku70(-/-) mice were similar to those in Ku70(+/-) and Ku70(+/+) mice, the levels of mutant frequency after irradiation were suppressed in Ku70(-/-) mice when compared to those in Ku70(+/+) and Ku70(+/-) mice. Preliminary study on the molecular nature of the mutations induced by radiation in Ku70(-/-) mice indicates that the deletion type mutations was not as frequent as those found in wild type mice. The data suggests that the major process which is responsible for production of radiation-induced mutation is NHEJ.

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Recent studies have showed that the sharp of the LET-RBE curves of biological effects was different, when using different ion species even if similar LET values. In this study, we investigated LET- and ion-species dependence on mutation induction and deletion spectrum of exons in hprt locus. Normal human skin fibroblasts were irradiated with carbon-, neon-, silicon- and iron- ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS). Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies, and deletion spectrum of exons was analyzed by multiplex PCR. The LET-RBE curves of mutation induction for carbon- and neon-ion beams showed a peak around 75keV/micrometer and 155keV/micrometer, respectively. On the other hand, there observed no clear peak for silicon-ion beams. The deletion spectrum of exons was different in induced mutants among different ion species. These results suggested that quantitative and qualitative difference in mutation occurred when using different ion species even if similar LET values.

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