In a living cell, a multiply damaged site in DNA is thought to be repaired by several different repair pathways simultaneously or sequentially. Under this situation the final cellular response to the lesion cluster might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of the initial repair protein, affecting the DNA-binding or lesion-excision activities of the latter repair protein. For example, a cluster comprised of an AP site or SSB and base lesions is formed after one of the base lesion in a base lesion cluster is exicesd by a glycosylase protein. Theoretical molecular dynamics simulation study showed that SSB proximately located 8-oxo-guanine could inhibit the binding activity hOGGI (Higuchi et al, 2010). In the present study, we investigate how the initial enzymatic repair affects the activity of the latter repair enzyme. Plasmid DNA (pUC18) irradiated with C6+ion is treated with two base excision repair enzymes, Nth and Fpg, which convert pyrimidine and purine lesions to a SSB. The enzymatic activities are quantified by measuring the conformational changes of the plasmid using agarose gel electrophoresis. Obtained results show that the amount of enzymatically induced SSB is slightly (about 5%) less in DNA sample treated with Nth first and then Fpg than that in the sample treated with Fpg first and then Nth, or with both enzymes simultaneously. The repairability of clustered damage induced by high-LET ions will be discussed.

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Tumor suppressor p53 Binding Protein 1(53BP1) is recruited rapidly to sites of DNA double-strand breaks (DSBs) and participates in the cellular response to DNA damage. Formation of 53BP1 foci is important for both DNA damage repair and signal transduction. We exploited property of 53BP1 that accumulate in DSB sites and developed biosensor detecting DNA damage using the specical form of 53BP1-GFP fusion protein that could express stably in the cell. Futhermore, we built a system that can identify the difference of cell cycle stage by intranuclear localization patterns of PCNA-DsRed. U2OS cell lines (U2RDP-LE53-21) expressing both these fluorescent fusion proteins make it possible to observe DSBs occurred in different cell cycle stage, these cells remain living. We will reveal that DNA damage and behavior of 53BP1 in the different cell cycle stage, and attempt screening chemotherapy drugs and radiosensitizers that induce DSBs in cancer cells. In this report, we will show how 53BP1 foci are observed in normal culturing conditions, and also how DSBs 53BP1 foci are induced by chemical agents such as Neocarcinostatine (NCS) or DNA Topoisomerase I inhibitor Camptothesin (CPT) that are well-known DSBs inducers. Also, 53BP1 foci induced by radiation as mono-DSBs were detected by confocal laser scanning microscope and its kinetics were revealed. We performed three-dimensional restructuring of DSBs 53BP1 foci and quantifying their steric volumes, and examined kinetics in specific cell stage using newly developed micro-imaging software.

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53BP1 accumulates at sites of DNA double strand breaks (DSBs), and plays a key role in repair of DSBs. We found that (1) 53BP1 plays a role in a novel pathway distinct from the Ku-dependent and Artemis-dependent non-homologous end-joining (NHEJ) pathways (Genes Cells, 11: 935, 2006), and that (2) RAD18 interacts with 53BP1 and is recruited at DSB sites in a 53BP1-dependent manner specifically during the G1-phase of the cell cycle. RAD18 monoubiquitinates 53BP1and enhances retention of 53BP1 in chromatin (Nucleic Acids Res, 37: 2176, 2009). Depletion of TRF2 (one of shelterin components) arises dysfunctional and unprotected telomeres. The unprotected telomeres, like DSBs, activate the ATM kinase, and are ligated by the NHEJ pathway to generate fused chromosomes (Nature Cell Biol., 7: 712, 2005). To ask whether 53BP1 and Rad18 are required for telomere-telomere fusions between unprotected telomeres, we removed TRF2 from telomeres with shTRF2 in wild type mouse embryo fibroblasts (MEFs), 53BP1-/-MEFs (a gift from Dr. Junjie Chen) and Rad18-/-MEFs. We assessed the frequencies of telomere-telomere fusions by FISH analyses with the telomere probe. We confirmed that the frequency of telomere-telomere fusions is reduced in 53BP1-/-MEFs. We will show roles of 53BP1, Rad18 and the interaction of these proteins in fusions of unprotected telomeres.

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Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, growth retardation, and cancer predisposition. Cells from NBS patients exhibit radiosensitivity, S-phase checkpoint defect, and chromosome instability. The gene product mutated in NBS, NBS1, forms a complex with MRE11 and RAD50 that is involved in the repair of DNA double-strand breaks (DSBs) and DNA damage checkpoints. DSBs are repaired by two major pathways, namely, homologous recombination (HR)-mediated repair (HRR) and nonhomologous end-joining (NHEJ). It has been reported that NBS1 functions in both HRR and NHEJ. However, the function of NBS1 in these processes has not been fully elucidated. We have been focused on the function of NBS1 in HRR. In the screening of NBS1-interacting proteins, we found that RAD51, a key player in HRR, interacted with NBS1. The mutant NBS1 that lacks MRE11-binding domain failed to interact with RAD51, suggesting that NBS1 bound to RAD51 through MRE11. Using an immunoprecipitation-based method, we searched RAD51-binding sites in MRE11, and found that MRE11 had two binding sites. We are now studying about these sites to elucidate the molecular function of them. We will report the results of these analyses.

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DNA base damage is generated continuously by various factors including oxidization, alkylation, and deamination. Reactive oxygen species (ROS) are generated as by-products of oxygen respiration in mitochondria, and also generated after exposure to radiation or ultraviolet. ROS induced oxidative DNA base damage leads to inhibition of replication, transcription, and mutation. Finally, those mutations induce cancer, aging, and neurological disorders. These damages are repaired primarily by base excision repair (BER) pathway. Endonuclease VIII-like 1 (NEIL1) possesses intrinsic AP lyase activity and cleave the DNA strand at the both sides of the apurinic (AP) site leaving a single nucleotide gap. Mouse NEIL1 has been reported to have two variants in mouse breast tumor or, aorta and vein but their physiological roles are unknown. We postulated the former as variant1, the latter as variant2. Previously, we have confirmed both variants' presence in normal tissue by RT-PCR. In this report, we initially extracted total RNA from each tissue of 8-week old male mouse followed by reverse transcription. Each primer sets recognizing NEIL1, variant1 and variant2, variant2 are designed for real time PCR. Beta-actin is used for internal standard of amount of mRNA. Because N-terminal tag fused NEIL1 had reported to lose its activity, we have examined the activity of C-terminal tag fused protein. Tag-fused variants had no activity. In this report, we attempted to assemble tag-free protein expression system to confirm the activity of purified protein toward damaged oligonucleotide.

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Reactive oxygen species are produced during the process of metabolism or by ionizing radiation, and they can damage the bases of DNA. The oxidatively damaged bases which arise from this induce cell death, and in higher organisms cancer formation, while also contributing to the evolution of organisms. All oxidized base lesions are removed from DNA by base excision repair (BER) initiated by DNA glycosylases. Endonuclease III (Nth), one of the DNA glycosylases, mainly excises damaged pyrimidine bases. In this study, we have been using the ascidian Ciona intestinalis. The aims of this study are to obtain an overview of the repair mechanism of oxidatively damaged bases and to elucidate the main functions of BER at various developmental stages. Firstly, we found that the amino acid sequence of the Ciona intestinalis Nth (CiNth) showed high homology with those of E. coli, S. cerevisiae, C. elegans and humans. We also found that two domains that are critical regions for activity, the Helix-hairpin-Helix domain and 4Fe-4S cluster, are conserved among these organisms. Secondly, we conducted nicking assays to determine whether CiNth has a DNA glycosylase activity and trapping assays to determine whether CiNth has an AP lyase activity. We used double-stranded oligonucleotides containing a thymine glycol, 8-oxoguanine or 5-formyluracil as DNA substrates. Our results revealed that CiNth is a bifunctional glycosylase/AP lyase, like human NTH1 and E. coli Nth. Finally, we performed a reporter assay to investigate the CiNth expression level at various developmental stages in Ciona intestinalis. We will make a spatial and temporal map of CiNth function during development.

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Radiosensitivity attributes to the defective repair of double-strand DNA breaks (DNAdsb) or the misrepair. DNAdsb are repaired by recombinational repair (HR) and nonhomologous end joining (NHEJ). NHEJ is dominant to repair of ionizing radiation induced DNAdsb in mammalian cells. HR pathway accelerates when NHEJ pathway is inhibited. ATM is contributed to HR. In this study, we examined the role of ATM in DNAdsb repair pathway using Ku70 defective cells and RNAi knockdown of 53BP1 in Ku70 defective cells. When cells were irradiated at higher dose, radiation resistant fraction was observed in survival curve. This resistant fraction was suppressed in ATM inhibited cells. This indicates that ATM dependent process was needed to repair lesions induced by higher dose irradiation. This result was confirmed as activation of ATM by western blotting. We are currently investigating the rate of homologous recombination in this system using a reporter gene and I-SceI induced DNAdsb repair.

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Among various types of DNA damages, DNA double-strand breaks (DSBs) are considered most critical determinant of the fate of the cells or organisms exposed to radiation. DSBs are repaired mainly through two pathways; non-homologous end joining (NHEJ) and homologous recombination. XRCC4 association with DNA-LigaseIV is considered one of pivotal molecule in NHEJ pathway. We have demonstrated that XRCC4 undergoes is phosphorylated by DNA-PK in response to radiation, although biological significance of phosphorylation remains to be elucidated. In this study, we examine the effect of nuclear reactor irradiation which contains neutron and gamma ray. We used murine leukemia L5178Y-derived, XRCC4-deficient cell line M10 as the host and introduced empty pCMV10 vector (M10-CMV), normal XRCC4 cDNA (M10-XRCC4) or mutant XRCC4 cDNA. Cells were irradiated in Nuclear reactor UTR-KINKI at Kinki University. Dose rate of neutron was 19 cGy/h and that of gamma ray was 19 cGy/h. Radiosensitivity was evaluated by colony forming ability in media containing 0.16 % agarose. To measure mutation frequency at HPRT (hypoxanthine phosphoribosyl transferase) locus cell were plated in media at containing agarose with 5 ng/ml of 6-thioguanine (6-TG). The fraction of mutant was increased by approximately 6 - 20 folds in multiple experiments. The mutant could not be recovered from irradiated M10-CMV. This frequency would be equivalent to 10 Gy of X-ray according to an earlier study by others on L5178Y (Shiomi et al., 1981). These results suggested that DNA damage induced by neutron might be hard to be repaired correctly and NHEJ repair of neutron-induced DNA damage might result in incorrect repair causing mutation. Further studies are ongoing to examine effect of DNA-PK inhibitors on mutation frequency and to analyze cells expressing mutant XRCC4 lacking phosphorylation sites.

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In mammalian cells, DNA constantly suffers from lesions such as strand breaks and damaged bases. Most damaged bases are repaired by base excision repair (BER) pathways. Human N-methylpurine DNA glycosylase (hMPG) excises various types of damaged purines generated by alkylation or deamination, and it is known to move over DNA and detect damaged bases. The abundance of hMPG in human cell is estimated to be about 2 x 10⁵ molecules per cell. On the other hand, it is estimated that about 1 x 10⁴ altered bases are formed in human genome every day. Thus it is possible that there is a very efficient system of damage sensing for rare lesions. hMPG interacts with various proteins including some transcriptional regulators, but it remains unclear what factors are associated with hMPG damage sensing. In this study, we performed pull down assay with purified GST-hMPG and nuclear extract from MMS-treated HeLa cells to identify the protein interacted with hMPG. The result showed that hMPG interacts with XRCC1, MBD1 and PCNA. The result was consistent with previous reports which suggested physical interactions of these proteins. Furthermore, by MMS treatment, the levels of XRCC1 and MBD1 in pull down fraction are increased 15.9 and 2.1-fold, respectively. To investigate the interactions in vivo, we prepared HeLa cell expressing GST-hMPG. The expression was confirmed by western blotting analysis with anti-hMPG and GST antibody. Now, we are investigating the effect of MMS on proteins interaction with hMPG through pull down assay.

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Ionizing radiation is known to induce mutations by forming oxidative damage on DNA. Previously, we investigated γ-ray-induced mutations in dry seeds of the model plant Arabidopsis. The result suggested that mutational effect of oxidative guanine, which is one of the major oxidative DNA damage, was low in Arabidopsis dry seeds. In this study, we analyzed γ-ray-induced mutation spectrum in Arabidopsis seedling, which has high water content and cell proliferation activity, in order to investigate the effect of oxygen radicals by water radiolysis and misincorporation of oxidized dNTPs during DNA replication on mutagenesis. Moreover, to evaluate the effect of nucleotide pool sanitization system on γ-ray-induced mutations, we analyzed a mutant deficient in the Arabidopsis NUDT1 gene that is a homologue of Escherichia coli mutT and human MTH. For mutation spectrum analysis, we utilized the rpsL-transgenic Arabidopsis to detect the mutations in γ-irradiated plants by plasmid rescue technique.
Background mutant frequency of the nudt1 mutant was comparable to that of wild type plant, suggesting that the deficiency of nucleotide pool sanitization enzyme hardly affects on mutagenesis under normal condition in Arabidopsis. The γ-ray sensitivity of the nudt1 mutant was slightly higher, but not statistically significant, than that of wild type plant. No difference in the extent of mutant frequency induced by γ-rays was found between the wild type and nudt1 plants. However, the mutation spectra induced by γ-rays were different between the wild type and nudt1 plants. From these results, it was suggested that the NUDT1 deficiency obviously affects on the mutagenesis but not the plant growth following γ-irradiation.

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Purpose: To investigate the LET and ion species dependence in PLDR-related mutation induction at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in normal human embryonic lung fibroblasts irradiated with high- or low-LET radiations. Materials and Methods: Normal human embryonic lung fibroblasts distributed by the RIKEN BRC Cell Bank were irradiated with X-ray or heavy-ion beams, such as carbon- (290MeV/u and 135MeV/u), silicon- (490MeV/u) and iron- (500Mev/u) ions with different LET ranging from 13keV/um to 200keV/um,generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC).Cell-killing efficiencies were measured using a colony-formation assay at either immediate plating (IP) method or delayed plating (DP) method keeping at 37⁰C for 24hours. Mutation induction was detected to measure 6-thioguanine resistant colonies. The cells were irradiated with D₂₀ dose, which was determined as the dose (Gy) required to reduce the surviving fraction to 20%, for PLDR-related mutation induction at the hprt assayed by IP or DP method. Results and Conclusion: The ratio for mutation frequency of DP/IP was ranging from 0.13 to 3.3. Then we calculated the radio of mutation frequency (IP or DP) against surviving fraction (IP or DP) and found that the ratio of mutation frequency (IP)/surviving fraction (IP) was higher than that of mutation frequency (DP)/surviving fraction (DP) in X rays, carbon, and iron-ion beams. However, the result for silicon-ion beams was ratio opposite. The result suggested that DNA repair fidelity in X-ray- and carbon-ion-induced damage in the DP method was more accurate than that in the IP method, although the results for silicon- and iron-ion beams were different. When the cells were irradiated with the same ion species, the higher LET beams showed the higher ratio of mutation frequency/surviving fraction in both IP and DP cases. These results showed that hprt mutation at IP and DP methods depended on LET and ion species.

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Clustered DNA damage, defined as two or more lesions within one to two helical turns of DNA induced by a single radiation track, is a unique feature of ionizing radiation. We have studied the biological consequences of bi-stranded clustered damage sites which consist of a combination of DNA lesions, such as a single strand break (SSB), an apurinic/apyrimidinic (AP) site, and an 8-oxo-7,8-dihydroguanine (8-oxoG), using a bacterial plasmid-based assay. We found significantly lower transformation frequencies for the clustered SSB + AP lesions than that for either a single SSB or a single AP site. We suggest that a double strand break (DSB) or a replication block is formed during the processing of the SSB + AP cluster. When the two lesions are placed farther apart (10-20bp), the transformation efficiencies are comparable to those of the single lesions. This recovery of transformation efficiency for separated lesions requires PolI activity. Similarly, the mutation frequency depends on the separation of the clustered SSB + 8-oxoG, although the SSB + 8-oxoG cluster, in contrast to the SSB + AP cluster, transforms at a comparable efficiency to the efficiencies of single lesions. PolI also seems to play an important role in avoiding mutations, as the lack of PolI enhances the mutation frequency of the separated lesions to the level of that of the closely spaced lesions. These results indicate that the biological consequences of clustered DNA damage strongly depend on the repair synthesis of the comprised lesion(s).

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Suppression of genetic mutation is expected to be an important method to inhibit the progression of disease such as cancer formation. Through studies on mechanisms of mutation events in cancer patients, we found an increased levels of glucose-regulated protein 78 (GRP78) expression in association with suppression of mutation in human cells irradiated with ultraviolet C (Pancreas, 36, e7-14, 2008). Then we were intrigued by the possibility that this carcinogen-induced mutation could be suppressed, via modulation of GRP78 expression, by agents such as foods. We focused on Japanese miso, which is a fermented food that has formed an important part of the Japanese diet for over 1300 years. Little is known about the ability of miso to modulate mutability in human cells. We here examined to determine whether miso treatment results in increased GRP78 expression and suppression of UVC mutagenicity in RSa cells. Supernatants of water extracts of miso products and their components were tested. In the samples-treated cells, the amount of GRP78 increased, as estimated by RT-PCR and immunoblotting analysis, and the frequency of UVC-induced ouabain resistant mutation (Oua^R) and K-ras codon 12-base substitution mutation decreased. This decrease was not observed in cells with downregulation of GRP78 by GRP78 siRNA transfection. The results suggest that miso suppresses UVC mutagenicity by enhancing GRP78 expression in human cells.

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The accelerated heavy ions deposit most of their energy close to the end of their range. Little is known about the mutational effects of ion beams near the range end, especially in plants. To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean LET: 425 keV/μm) were compared with the effects of carbon ions penetrating the seeds (mean LET: 113 keV/μm). We analyzed the mutations occurred in Arabidopsis GL1 locus with the emphasis on large deletions. Arabidopsis seeds obtained by crossing wild-type Col with the gl1-1 mutant were used as a material, and the polymorphic markers that can distinguish Col and gl1-1 sequence were used to detect deletions. While the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (> ~30 kb) were six times more frequently induced by carbon ions near the range end. The 352 keV/μm neon ions also showed a 6.4-fold increase in the frequency of large deletions compared with the 113 keV/μm carbon ions. These results suggest that the proportion of large deletions increases with LET in plants.

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  Cisplatin is a widely used anticancer drug and forms inter- and intrastrand DNA crosslink and monoaducts. While, 3-nitrobenzanthrone (NBA) is a carcinogenic air contaminant and forms several kind of bulky DNA adducts (ABA adducts) in vivo and in vitro. These DNA adducts can cause mutations through translesion DNA synthesis (TLS).
  A purpose of this research is to analyze the mutations caused by cisplatin and ABA adducts in human cells. We constructed six shuttle vector plasmids containing one of the following adducts: Pt-GG; Pt-GTG; dG-C8-N-ABA; dG-C2-C8-ABA; dG-N²-C2-ABA; and dA-N⁶-C2-ABA), site-specifically. We introduced these plasmids into human NER deficient XPA cells for replication, and then analyzed frequencies of TLS and the type of mutations of the replicated plasmids.
  We found that all adducts blocked DNA synthesis, but a portion of the adducted plasmids was replicated by TLS. The TLS frequencies were different among the adducts. In cicplatin adducts, Pt-GTG caused more mutations than Pt-GG, and the mutations were observed at the downstream sites from the addected bases. On the other hand, dG-C8-N-ABA was the most mutagenic adduct among the ABA adducts.

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Analysis of APRT-deficient mutations in neutron-irradiated mice Chisuzu Ooga, Tatsuya Esashi, Kazumi Yamauchi, Shizuko Kakinuma, Yoshiya Shimada, Akira Tachibana It has been reported that the frequency of tumors in mice is dependent upon the age at exposure to radiation. Since mutagenesis in the cancer-related genes is supposed to be one of the critical causes that lead to tumorigenesis, we analyzed the fraction of somatic mutations in vivo at the Aprt locus, encoding adenine phosphoribosyltransferase (APRT), after exposure of mice heterozygous for the Aprt locus to neutrons at different ages. Mice were irradiated at the age of either 1 week or 7 weeks, with fast neutrons of 0.25Gy once, of 1Gy once, or of 0.25Gy for 4 times with 1 week intervals. At the eighth week after irradiation, splenic lymphocytes were isolated, and the mutant fraction was assessed using 8-aza-adenine as the selection drug for APRT-deficient mutants. The results showed that lymphocytes from the mice irradiated with neutrons at the age of 1 week showed increased mutant fractions to a certain extent, in contrast to the lymphocytes from mice irradiated at the age of 7 weeks that did not show any increase in the mutant fractions. These results correspond to our previous similar studies on mutations in X-irradiated mice. Analysis of loss of heterozygosity (LOH) at the Aprt locus in mutants revealed that mutants from mice exposed to neutron at the age of 1 week showed higher fraction of LOH than those from unirradiated mice, while mutants from mice exposed to neutron at the age of 7 weeks showed lower LOH fraction than those from unirradiated mice. Further analysis of LOH at the D8Mit271 marker of chromosome 8 also showed that the fraction of LOH at this marker among mutants from mice exposed at the age of 1 week was higher than that from unirradiated mice. These results suggest that mice irradiated at neonatal age are more sensitive to the neutron-induced mutagenesis with higher frequency of LOH than mice irradiated at adult age.

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TThe analysis of chromosome aberrations in human peripheral blood lymphocytes is the most established method for the biological dosimetory of ionizing irradiation. Analysis of metaphase spreads with Giemsa staining, however, requires skilled person because of difficulty to identify abnormal chromosomes. FISH analysis of abnormal chromosomes makes it easy to identify chromosome abnormalities, but requires aging step for more than two days. Here, we established a FISH analysis using telomere and centromere PNA probes to obtain bright signals without aging for the analysis of abnormal chromosomes in the irradiated peripheral blood lymphocyte. Good agreement on the percentage of metaphases with dicentric chromosome after irradiation was observed between conventional Giemsa staining and FISH analysis. FISH detected more metaphase with multicentric chromosomes from lymphocytes irradiated at high dose than Giemsa staining analysis. Interestingly, percentage of metaphase with these complex abnormal chromosomes showed parallel increase with the irradiation dose. Since complex chromosome abnormalities could be missed by the analysis with Giemsa staining, the FISH technique may become a easy and reliable method for the biological dosimetory.

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Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots.

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Telomere is a unique loop structure that exists in the end of each chromosome and consists of (TTAGGG)n repeats. Telomeres progressively shorten through each DNA replication by the end replication problem, and finally result in the end-to-end fusion of chromosomes by the destruction of the telomere loop structure. Telomere FISH is a powerful tool to evaluate telomere lengths of each cell or chromosome. In the present study, to evaluate telomere instability, we developed a quantitative telomere FISH using normal human fibroblast (HE7) cells and mouse embryo fibroblast (MEF) cells. For telomere FISH, FITC-labeled 18mer peptide nucleic acid (PNA) probes specific for telomere repeats were applied. The intensity of telomere FISH (T-FISH) signals was quantified in relation to population doubling numbers (PDN). The result indicated that the intensity of T-FISH signals in MEF cells was about two fold stronger than that in HE7 cells, implying the longer telomeres in MEF cells than those in HE7 cells. In contrast to the prediction, however, both MEF cells and HE7 cells showed a similar 10% decrease in telomere lengths after 15~18 PDN, resulting in no difference in a telomere erosion rate between them. Then, we combined centromere FISH (C-FISH) with T-FISH in HE7 cells and used a ratio of the intensity of T-FISH to that of C-FISH as a T/C value to normalize the variation of analyses. The result revealed that HE7 cells showed a 30% decrease in telomere lengths, indicating a clear difference in the telomere erosion rate between them. Therefore, the present study recommends T-FISH normalized with the T/C value for evaluating telomere instability induced by telomere erosion.

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Background It is believed that the main cause of carcinogenesis is the accumulation of DNA damage and mutations in oncogene or tumor suppressor gene. However the mutation frequency is too low to explain the cancer development. On the other hand, it is well known that there are some aneupoid cells in tumor cells, suggestion the involvement of aneuploidy on cancer development. However we don't know whether aneuploidy is main cause of carcinogenesis or not. Here we established the aneupoid cells in which a human chromosome has been introduced artificially, and analyzed the cells focused on the phenotypes of the carcinogenesis. Material and Method We used immortalized adult human mesenchymal stem cells, and established two aneproid cell lines by microcell-mediated chromosome transfer method (each contains exogeneous human chromosome #1 and #7). We used the cells for carcinogenesis containing only drug resistance gene as a control cell line. We examined that 1) cell growth ratio, 2) anchorage-independent growth 3) ability of the cell growth in transplant for a mouse for index of the carcinogenesis in these cell lines. And we analyzed that 1) the abnormality of chromosome number, 2) structural abnormality of the chromosome and 3) micronuclei yields in aneuploidy cells for index of the chromosome instability. We examine the chromosome instability and carcinogenesis for aneuploidy cells in which a human chromosome #1 and #7 induced. Results The aneuploid cells which was #1 and #7 induced expressed characteristic of carcinogenesis. Though the cells induced #1 chromosome were redaction of the rate of cell growth, the anchorage-independent growth were 2-fold higher than tha in the control and #7 induced cells.

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As one of the measures of radiation effects, dose vs. frequency of DNA strand breaks (at the cellular level) or frequency of chromosomal aberrations (at the individual level) are commonly used. As we know, dose, DNA strand breaks and chromosomal aberrations are successive events, there are no common understandings for these relations. We have investigated many types of chromosomal dynamics using mathematically modeled whole human chromosome 17. Here, we will report on the relationship between physical properties of dynamics of chromosomal broken ends and concentration speed of typical repair enzymes.

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Effects of X-rays and ultraviolet radiation on the expression of telomere repeat-containing RNA (TERRA) Ziniu Xu, Kazunori SHIRAISHI, and Seiji KODAMA Laboratory of Radiation Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University. Telomeres can be transcribed into RNA called as telomeric repeat-containing RNA, TERRA, which consists of a combination of subtelomeric and telomeric sequences. To date, all individual telomeres examined in mammals produce TERRA transcripts and the expression of TERRA is conserved in several species. However, its biological function remains unknown. In the present study, we irradiated immortalized mouse CB/CB 09 cells with X-rays and ultraviolet radiation and examined the expression of TERRA transcripts post-irradiation to know how the TERRA expression could be influenced by irradiation. We detected the TERRA transcripts by the telomere RNA FISH using a FITC-labeled peptide nucleic acid (PNA) probe specific for telomere repeats. The telomere RNA FISH visualized the TERRA transcripts as foci in a nucleus, and we scored the foci per nucleus at 0, 6, 12, and 24 h after irradiation. To know the killing effect of radiation, we examined cell survival by X- or UV-irradiation and determined a dose for 10% survival; 5 Gy for X-rays and 13 J/m2 for UV. Average number of foci per nucleus was 5.1 in unirradiated mouse CB/CB 09 cells. In X-ray-irradiated cells, average numbers of foci per nucleus at 0, 6, 12, and 24 h after irradiation were 4.9, 4.3, 4.4 and 5.9, respectively, showing no significant difference between them. In contrast, in UV-irradiated cells, average numbers of foci per nucleus at 0, 6, 12, and 24 h after irradiation were 5.0, 6.8, 8.6, and 7.0, respectively, showing the significant increase compared with that of the unirradiated cells. This indicates that UV-irradiation rather than X-irradiation affects the expression of the TERRA transcripts.

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During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Medaka is a small laboratory fish, having several advantages as experimental model animal. We have established medaka TILLING library consists of 5700 F1. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing using capillary sequencer, nuclease-mediated screening of heteroduplexes or different melting temperature of mutation-bearing DNA fragment (High Resolution Melting (HRM) assay) are widely used for detection of mutations in TILLING. These methods are useful, but only one exon can be analyzed by one screening. Thus, there is a need for an alternative method that is of higher throughput. In this study, we developed a Giga-Base Sequencing (GBS) method using the Second Generation Sequencer and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We have previously identified many mutations in several genes by HRM assay. Therefore, we first tested the efficiency of the GBS assay by screening these known mutations. The GBS assay can detect mutation with efficiency more than 95%, and the percentage of false positive in the candidate clones is relatively low (less than 30%). These results indicate GBS assay is as useful as HRM assay. The most important feature of the GBS assay is the high-throughput screening of mutations. We can do screening of more than 100 exons by one shot sequencing by the GBS assay. Recent progresses in genome project have revealed the presence of multiple homologous genes (paralogue and orthologue) in our genome. These family genes are usually functionally-redundant, and thus knock-out of all family genes is necessary to know the function of their function. The GBS assay is useful to identify mutants in these family genes.

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Glioblastomas are known to the most lethal brain tumor and their resistance to anti-cancer therapies is elucidated by glioma stem cells (GSC). But it is still unclear how the molecular mechanisms provide the resistance to GSCs. In this study, we used GSCs characterized by Plagl1, which is a tumorigenesis factor, and Sox11, which is the negative regulator of Plagl1. We compared the cellular characteristics between GSCs (Plagl1 positive) and glioma differentiated cells (Sox11 positive) by using anti-cancer drugs and ionizing radiations. In consequence, we found that GSCs showed stronger resistance to DNA cross-link agents whose damages are repaired by homologous recombination (HR), delay of IR-induced DNA damage foci's decreasing, rapid escape from cell cycle arrest and repression of apoptosis. These results might suggest that HR repair is induced in GSCs and therefore this increases cellular survivability and tumor malignancy.

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Dysplastic glioblastoma is a radio-resistant brain tumor which is observed with the frequency of approximately 30%. Arsenite is one of the rare agents which enhance radio-sensitivity of dysplastic glioblastoma synergistically in vitro and in vivo. In order to clarify the molecular mechanisms for the anticancerous activity of arsenite, we have investigated the effects of arsenite alone on cultured glioblastoma cell line U87MG, and showed that induction of senescence-like growth arrest after treatment with arsenite was mediated by DNA damages accompanied by formation of the heterochromatin. Here we examined whether senescence-like growth arrest after treatment with arsenite depends on the p53-p21 signaling pathway. Signaling pathways containing either p16 or p21 have been shown to be involved in senescence-like growth arrest. As the functional p16 is lacked in dysplastic glioblastoma including U87MG cells, we examined involvement of p21 in senescence-like growth arrest in U87MG cells after treatment with arsenite. The cells irradiated with 5 Gy of X rays were used as the positive control because irradiation was previously reported to induce senescence in p53-proficient cancer cells. We used 1.25 microM of arsenite because this concentration is equivalent in toxicity (approximately 10% survival) to 5 Gy of X rays. First we treated the cells with 1.25 microM of arsenite, and observed induction of p21 protein 6 days later to a similar extent to that after irradiation with 5 Gy of X rays. Next we knocked-down p53 and p21 genes in U87MG cells by use of siRNA provided by Qiagen, where knockdown efficiency more than 70% could be confirmed by Western blotting. In these cells, formation of heterochromatin was not observed after treatment with arsenite, which is a similar observation to that after irradiation with 5 Gy of X rays. Based on these results, we conclude that senescence-like growth arrest after treatment with arsenite is mediated by the p53-p21 signaling pathway.

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  When sensitive cells are exposed by ionizing radiation, activations of growth arrest-related genes such as p21 and mdm2 and apoptosis-related genes such as bax and puma are observed. We previously established real-time RT-PCR methodology for accurate quantification of messages from these DNA damage-induced genes. When mice were externally exposed to 0.1 to 1.0 Gy of x-ray, these mRNA levels in peripheral blood and bone marrow were increased transiently and reached to the peak levels at 4 hours after the irradiation. The peak levels of mRNA were closely dependent on the exposed levels. To study molecular process following radiation, this model was applied for internal exposure.
  As internal exposure model, ³²P-phosphate from 0.5 to 5.0 MBq was injected into mice and hematocytes were collected for RNA quantification. The mRNA levels elevated 4 hours after the injection, and the peak levels were observed at 8 hours. In all the measured point, the levels of mRNA of apoptosis-related genes were closely related to the amount of administered ³²P-phosphate. These results indicate that molecular process of DNA damage can be studied by this experimental system and estimation studies of internal exposure can be reviewed more precisely.

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Radiation-induced adaptive response (AR) is the phenomenon that a priming low dose induced resistance against the subsequent challenging irradiation at higher doses. Study on AR is of great concern as it is expected to provide important scientific basis for risk estimates, offer significant insight into the biological defense mechanisms, and lead to possible novel radiotherapy for practical application. In a series of investigation, induction of AR with both low LET X-rays and high LET accelerated heavy ions was attempted in fetal and adult mice. The data have been successively reported in the last three JRRS annual meetings. In the present presentation, all the work on fetal mice was summarized. A priming dose from X-rays at either 0.05 Gy or 0.30 Gy on the 11th day of gestation (E11) could induce an AR (judged as the suppression of prenatal death and malformation) against a challenging high dose of X-rays at 3.50 Gy on E12. Using this AR model, 1) priming low doses of X-rays against high doses of high LET irradiations from accelerated heavy ions, and 2) priming low doses of high LET irradiation from accelerated heavy ions against the high dose of X-rays, were tested to verify if an AR could be observed. Accelerated heavy ion particles from mono beams of carbon, neon, silicon and iron generated by HIMAC, with the LET values of about 15, 30, 55, and 200 keV/micrometer respectively, were examined. Results showed that priming low dose of X-rays could induce an AR against the high challenging doses from carbon, neon and silicon ion irradiations while no AR was observed when the challenging dose was from iron ion irradiations. On the other hand, no AR was observed in the case that the priming low doses were from the high LET heavy ions and the challenging high dose was from the low LET X-rays. These findings indicate that under certain conditions the low doses of low LET X-rays could induce AR against the high doses of high LET heavy ion irradiations, and suggest that the successful AR induction by X-rays is dependent on the LET value and/or the particle species of challenging irradiation.

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We previously found that ionizing radiation (IR) commonly suppresses expression of ASPM gene, which are found mutated in a majority of human patients of familiar microcephaly (MCPH5). In pregnant mice, sub-lethal doses of IR drastically reduced the protein level of Aspm in their developing embryos, particularly in the neural stem cells that express nestins. To further investigating a cause of microcephaly, we developed a knock-out system where disruption of the Aspm gene can be controlled through Cre-loxP recombination systems. Aspm null homozygotes are obtained by mating Aspm +/- parental mice following Mendellian rule. They grew apparently healthy but had significantly small brains (85% in weight) and testes (20% in weight) compared to +/+ and +/- animals. HE staining has detected no pathological abnormality in the affected brains but loss of spermatogenesis in most seminiferous tubule. Next we destroyed the floxed Aspm genes selectively in CNS by introducing Cre expression under the murine Nestin gene promotors. A similar defect in the brain size was obtained in the homozygote animals of the floxed alleles only when Nes-Cre transgenes were inherited, while no testes were affected. Our results suggested that any synthetic suppression of Aspm can cause microcephaly phenotypes only if nestin expressing tissues are targeted during the CNS development.

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Several methods are known to isolate radiosensitive gene such as identification of responsible gene mutations in radiosensitive genetic disorder, homologue analysis from model organism, biochemical analysis of intermolecular interaction, differential display, and subtraction method. However, these methods have peculiarity in isolation, so we have designed more efficient methods to isolate radiosensitive genes. We call it RAZE (Radiosensitivity-related gene cloning using Zeosin vector) method, which enables to efficiently obtain DNA damage resistant cell strains. The hallmark of this method is the random insertion of zeosin vector, which leads to establish genetically modified cell strains. We first transfected our original zeosin vector into highly radiosensitive MOLT-4 cells by electropolation. The vector we constructed is specialized for inverse PCR (IPCR), which are used to specify vector's insertion sites in genomic DNA. It consists of zeosin resistant gene with multiple restriction sites in both ends. We then 10 Gy-irradiated those transfectants and evaluated its viability. We selected clones that are more than 2-fold viability to parental cell line. Since we proved that the amount of inserted zeosin-resistant gene has no correlation with its radioresistance, we concluded that acquired radioresistance is due to its DNA modification by zeosin vector. Thus, we identified zeosin vector's insertion sites in these radioresistant clones using IPCR. Multiple restriction sites enabled to create various DNA fragments, which were advantage to IPCR. We successfully isolated the disrupted genes of interest. Our efforts have been directed toward a functional analysis of the candidate genes.

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[Objective] Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), a transcription factor, is important for protection against stresses such as oxidative stress. Nrf2 rapidly translocates into the nucleus in response to reactive oxygen species (ROS) and then induces the expression of various antioxidant genes such as those encoding heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase, quinone 1 (NQO1). Low-linear energy transfer (LET) ionizing radiations such as X-rays generate ROS, which cause biological damage. Our previous report showed that HO-1 and NQO1 mRNA expression in human hematopoietic stem cells is upregulated after X-ray irradiation (Kato et al, Radiat. Res., 174, 177-84, 2010). However, it is not known whether this upregulation is induced by the transcriptional activities of irradiation-activated Nrf2. Therefore, this study investigated the response of the Nrf2 system after X-ray irradiation. [Methods] THP1 cells (human acute monocytic leukemia cells) were exposed to X-rays (1–10 Gy). Intracellular ROS were analyzed by flow cytometry. Nrf2 localization was estimated using confocal laser scanning microscopy. HO-1 and NQO1 protein expression was also examined. [Results] Although the ROS directly induced by irradiation increased in a dose-dependent manner, they disappeared immediately after irradiation. Nrf2 translocation into the nucleus was observed after 6 hr under 5- and 10-Gy irradiation conditions but was not observed under nonirradiation or 1–2 Gy irradiation conditions. HO-1 and NQO1 expression after 5-Gy irradiation was significantly higher than that under nonirradiated conditions, from 24 hr after irradiation. These results indicate that high-dose irradiation (>5 Gy) activates Nrf2 and that the Nrf2 protection system may function from 24 hr after irradiation.

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Reactive oxygen species (ROS) are generated in cells exposed to ionizing radiation. ROS oxidize DNA, proteins and lipids, and act as mediators of ionizing radiation-induced cellular damage. ROS have been shown to participate in various biological consequences of ionizing radiation. In this study, we examined whether and how cellular sensitivity to ionizing radiation and oxidative stress are modulated by the overexpression of superoxide dismutase (SOD) and glutaredoxin (Grx) in cultured human cells. SOD is a scavenging enzyme of superoxide anion and Grx repairs the oxidized macromolecules. In this study we examined whether the overexpression of SOD1 and SOD2 protects cells against cellular effect of gamma-rays. Overexpression of SOD2 in mitochondria enhanced the survival following gamma irradiation compared with control HeLa cells, while overexpression of SOD1 in the cytosol did not affect the cell survival. As the results of overexpression of SOD2, the levels of DNA double-stranded breaks and oxidized cellular proteins, the expression level of OXR1 protein, and morphologic change of mitochondria were suppressed in HeLa cells. These results indicated that mitochondrial antioxidant enzymes plays important roles in various cellular responses to the ionizing radiation and cellular homeostasis. Then, we examined the effect of overexpression of Grx in mitochondria. In the results, we observed the same effects as over-expressed SOD2.

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Radioadaptive response is a biological defense mechanism that is induced by low-dose ionizing irradiation for cellular resistance to the genotoxic effects of subsequent irradiation. Although radioadaptive response was first identified in human lymphocytes pre-treated with low concentration of 3H-thymidine by Olivieri et al., molecular mechanism of the induction of radioadaptive response by 3H-thymidine is still obscure. We have reported that the irradiation with 2 cGy X-rays prior to the challenging irradiation with 3 Gy on the induction of chromosome aberrations in quiescent mouse m5S fibroblasts. Similar effects have been reported by many others groups using other biological end-points, such as cell killing, mutation induction, the induction of micronuclei (MN), and single-cell gel electrophoresis (comet assay). We have suggested that the radioadaptive response is mediated through the pathways involving protein kinase C (PKC)  and p38 mitogen-activated protein kinase. Here, we examined the induction of radioadaptive response by 3H-thymidine in mouse m5S fibroblasts. Cells were treated with low concentration (3.7 kBq/ml) of 3H-thymidine for several days, according to the experimental conditions used by Olivieri and others. Cells were then irradiated with 5 Gy X-rays, followed by MN analysis. The results showed that radioadaptive response is induced in mouse fibroblasts by 3H-thymidine. Pretreatment of cells with other concentrations of 3H-thymidine (0.37 kBq/ml, 37 kBq/ml) was less effective to induce radioadaptive response. Investigation on the involvement of PKC in radioadaptive response is currently in progress.

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Activity of PKCα and p38 MAPK by treatment with low- and high-concentrations of hydrogen peroxide Aya Murase, Shuho Saotome, Mnami Matsunaga, Akira Tachibana College of Science, Ibaraki University The radioadaptive response is the induction of cellular resistance to moderate or high doses of radiation by previous exposure to low doses. Treatment with TPA or hydrogen peroxide (H2O2) mimicked X-rays in adaptation when applied at low doses. We have shown that the addition of PKC inhibitors or an inhibitor for p38 MAPK (p38) impaired the induction of the adaptive response, and that both PKCα and p38 were activated by irradiation with low dose X-rays, indicating the involvement of PKC and p38 in the adaptive response. In addition, it was also clarified that a PKC inhibitor reduced the p38 activation by low dose X-irradiation, and, in reverse, a p38 inhibitor reduced the PKCα activation, suggesting that the PKC-p38 signaling pathway are operating in the induction of the adaptive response. In order to clarify the relationship between PKCα and p38 in the adaptive response, we analyzed time course of activations of PKCα and p38 in cells treated with H2O2. Because the treatment with 1 μM H2O2 have been shown to induce the adaptive response, whereas the treatment with 100 μM H2O2 do not cause the adaptive response, we examined the phosphorylation of PKCα and p38 in the extracts of cells treated with either 1 μM or 100 μM H2O2 for various times. As a result, both p38 and PKCα showed relatively high phosphorylation for about 6 hours after the treatment with 1μM H2O2, whereas the phosphorylation levels of both PKCα and p38 were largely reduced for 1-6 hours after the treatment with 100 μM H2O2. We infer that these differences in the level of activation of both p38 and PKCα between the treatments with high and low concentrations of H2O2 may be involved in the induction of radoadaptive response. Analysis of p38 activation using cells with reduced expression of PKCα is currently in progress. Keywords: radioadaptive response, PKC, p38,

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The purpose of this study is to clear a time dependency of radiation-induced bystander cell-killing effect. Thus, we irradiated normal human fibroblasts with carbon- and neon-ion microbeam (LET=103 keV/μm and 380 keV/μm, respectively), carbon-ion broad beam (108 keV/μm) and γ-rays (0.2 keV/μm). Survival rate of bystander cells was measured after 6-24 h co-culture with irradiated cells. The ratio of irradiated and bystander cells was <0.0005:1 in microbeam irradiation and 0.5:1 in broad-beam and γ-ray irradiation, respectively. In microbeam-irradiated samples, the survival rate of bystander cells did not change at 6 h but decreased to about 85% of control at 24 h. In 0.13-Gy broad beam and 0.5-Gy γ-ray irradiated samples, the survival rate of bystander cells decreased to 80 to 85% of control at 6 h or later. From these results, it is found that bystander cell-killing effect is delayed when irradiated cells are extremely less than bystander cells.

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Target irradiation of individual cells using heavy-ion microbeam is a useful means for measuring exact hit effect of heavy-ion on cell. Moreover, heavy-ion microbeam is a powerful tool for analyzing mechanisms underlying radiation-induced bystander effect, which is induced on a cell population exposed to low fluence, low dose radiation. Thus we installed a focusing heavy-ion microbeam system under a vertical beam line of an AVF cyclotron of JAEA-Takasaki, and developed a method to irradiate individual cells using focusing heavy-ion microbeam. To improve a throughput of the cell irradiation using focusing microbeam, we developed a code that calculates voltages, which will be applied to a beam scanner for moving beam spot rapidly to the target positions. Using a developed code, a set of voltages was calculated from a pre-obtained cell image, and a film of CR-39 was irradiated by scanned beam according to the calculated voltages. After irradiation, hit positions of ion were visualized as etch pits, and the spatial distribution of them was observed. The distribution pattern of source cell image and etch pits on CR-39 film were well correlated, so that it was suggested that the developed code can finely convert detected cell positions to corresponding scanner voltages. Thus we concluded that using the code we will become able to irradiate cell rapidly with scanned heavy-ion microbeam.

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Immune cells accumulate in and around cancers and cooperate with each other using specific cytokines to attack the cancer cells. The heavy-ion beams for cancer therapy may stimulate immune cells and affect on the immune system. However, it is still poorly understood how the immune cells are stimulated by ion-beams. Here, we irradiated immune cells using heavy-ion beams and analyzed changes in production of tumor necrosis factor-α (TNF-α) that is important cytokine for the cancer treatment. THP-1 macrophages were irradiated using carbon-ion broad-beams (108 keV/μm). To examine the bystander response after heavy-ion irradiation, a very small fraction (approx. 0.5%) of the cell population was irradiated using heavy-ion microbeams. After irradiation, we examined the production of TNF-α. When cells were irradiated with 5 Gy that did not cause severe damage, TNF-α level was reduced after both microbeam irradiation and broad-beam irradiation. TNF-α production of macrophages with the nitric oxide (NO) inhibitor-treatment increased after carbon-ion broad-beam. NO was involved in the radiation-induced suppression of TNF-α production. The suppression of cytokine production arose after irradiation with heavy-ions, and may also be induced in the surrounding non-irradiated cells via the bystander effect.

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ATP (Adenosine tri-phosphate), one of ribonucleic acids, acts as an intracellular energy transfer. ATP is also used as a substrate to synthesized messenger RNA and as a ligand of inter-cellular signaling. To induce the specific molecular alterations in the molecule we used the monochromatic soft X-rays obtained from the synchrotron facility (SPring-8, BL23SU). In this study, we analyzed the soft X-ray-induced radiation damage of ATP by various biological assessments, such as energy donor activity, genetic information transfer activity and inter-cellular signaling activity. We analyze the luciferase activity to assess the energy transfer activity and the genetic information transfer activity. We also evaluate the change in activity as an inter-cellular signaling molecules by measuring ATP receptor-mediated phosphorylation of extra cellular signal-regulated kinase 1/2 (ERK1/2). In addition to the biological effect, we have also measured the molecular alterations using X-ray absorption and electro spray mass spectrometry (ESIMS). We observed attenuation of ATP receptor-mediated phosphorylation of ERK 1/2 and luciferase activity by the soft X-ray irradiations. Moreover, we observed the reduction of nucleotide from the X-ray absorption spectra and ESIMS. These results suggest that certain alteration of ATP structure induced by soft X-ray affects the biological function of ATP. We will discuss the correlation between the biological effect and the molecular alterations from these results.

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Understanding the mechanism (s) that underlie the bystander effects of low- or high-linear energy transfer (LET) radiations has been considered important for cancer therapy and a better understanding of the health risks associated with space exploration. We investigated the role of gap junction intercellular communication (GJIC) in propagation of events leading to amplification of stressful/bystander effects in confluent normal human fibroblasts (NB1RGB) cultures exposed to low doses/low fluences of low-or high-LET radiations using cell death and induction of DNA damage as endpoints. Using microbeam irradiation, confluent NB1RBG cells were exposed to 5.35 keV X rays (LET 6 keV/µm), 220 MeV carbon ions (LET 103 keV/µm), 260 MeV neon ions (LET 380 keV/µm), or 460 MeV argon ions (LET 1060 keV/µm) under condition by which only 0.04% of cells in the population was targeted in the presence or absence of a gap junction inhibitor (AGA). As expected, carbon-ions, argon-ions and neon-ions were more effective than X-rays at inducing cell killing. However, inhibiting of GJIC with AGA promoted increase survival following exposure to high, but not low LET radiation. The percent cell survival for carbon-ions after 3 h irradiation was around 90% in the absence of the inhibitor and almost 100% in the presence of the inhibitor. On the other hands, the cell survivals for neon- and argon ions were almost 100% irrespective of the presence or absence of the inhibitor. Although the incidence of micronuclei formation induced by X-rays showed no difference irrespective of irradiation with or/ without a gap junction inhibition, the frequency of micronuclei induced by carbon-ions was significantly higher in the absence of AGA than in its presence. Collectively, the results show that the propagation from irradiated to neighboring bystander cells, of clastogenic promoting effects depends on the LET and dose of the radiation, and that GJIC promotes the propagation of stressful bystander effects in high-LET-irradiated cell cultures. In conclusion, the expression of the above low dose/low fluence radiation-induced stressful/bystander effects are consistent with our earlier observation of a similar propagation of toxic effect among cells in cultures wherein all cells were targeted by densely ionizing radiations. Characterizing the nature of the propagated signaling molecules would have translational implications in radiotherapy and the formulation of countermeasures against toxic effects sustained by humans during space exploration. Supported by NASA grant NNJ06HD91G and NIH grant CA049062 in the USA, and the National Institute of Radiological Sciences in Japan.

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Bystander effect induced by radiation is a phenomenon which non-irradiated cells are affected by signals from irradiated cells. The mechanisms are still not fully understood. Mitochondria are organelle producing ATP and reactive oxygen species (ROS). A purpose of this study is to clarify the relationship between functions of mitochondria and bystander effect. We examined bystander effects by using human tumor cell lines (SF126 and HeLa), and normal human cells (NB1RGB, fibroblast-like cells derived from embryo HE35 and HE49). Bystander effect was investigated by the medium transfer method. Medium from irradiated cells was transferred to non-irradiated cells 24h after X-ray irradiation. We observed significant increases of micronuclei by bystander effect in all cell lines, and these were remarkable in 1 Gy and 0.5 Gy in SF126 and the other cell lines, respectively. Also, it was most effective that the period for the treatments of conditioned medium was 1 ~2 hours. When cells were treated with 0.2 mM vitamin C, the inductions of micronuclei by bystander effects were completely suppressed. Therefore, it is thought that oxidative stress is involved in this effect. In DCFH assay, bystander cells showed reduced levels of oxidative stress in normal cells but not in tumor cells. In addition, the levels of superoxide anion (O2-) measured by MitosoxRed dye increased in bystander cells of all cell lines. These results suggest that ROS in mitochondria play an important role in the expression of bystander effects such as micronuclei inductions.

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[Introduction] Hematopoietic stem cells (HSCs) have high radiation sensitivity because of their high proliferative potential. In addition, stem cells have relatively lower intracellular mitochondrial content, which is the source of reactive oxygen species (ROS) and plays a crucial role in cell death through apoptosis, than mature cells. However, the relationship between radiosensitivity of HSCs and mitochondrial function has not been reported. We evaluated the ROS production and mitochondrial function after exposing CD34 positive cells derived from human placental/umbilical cord blood to radiation.
[Materials and Methods] The CD34 positive cells were purified using a magnetic cell sorting kit and frozen at -150°C until the experiment. The thawed cells suspended in serum-free medium were exposed to X-irradiation. These cells were incubated at 37°C under 5% CO₂ for 0-7 days with or without cytokines (G-CSF, GM-CSF, IL-3, SCF, and EPO). Harvested cells were assayed for the progenitors by using methylcellulose culture stimulated with the same cytokines. Intercellular ROS, mitochondrial superoxide, and mitochondrial contents were analyzed using CM-H2DCFDA, MitoSOX Red, and MitoTracker, respectively, by flow cytometry.
[Results and Discussion] There was an approximately 20% reduction in the number of generated living cells and progenitors in the culture of 4-Gy X-irradiated cells for 3 and 7 days with cytokine as compared to non-irradiated cells. However, irradiated cells showed a similar increase in mitochondrial level as non-irradiated cells. There was a 4-fold increase in the intracellular ROS production peak on day 3, but no significant difference in mitochondrial superoxide level was observed. Our findings suggest that radiation-induced intercellular ROS causes serious damage to HSCs; however, mitochondrial involvement may be low.

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[Introduction] Hematopoietic stem cells (HSCs) are sensitive to extracellular oxidative stress. Exposure of the components of the hematopoietic system to ionizing radiation (IR) remarkably suppresses mature blood cell production in a LET- and/or a dose-dependent manner. However, information about how IR influences myeloid differentiation from HSCs is limited. This study investigated the effects of IR on myeloid hematopoiesis derived from human CD34⁺ cells exposed to heavy-ion beams and X-irradiation. [Methods] Human placental/umbilical cord blood CD34⁺ cells, which are HSCs, were separated using auto-MACS human CD34 selection kit (Miltenyi Biotech). Specific cell surface antigen expression was analyzed by direct immunofluorescence flow cytometry. Human CD34⁺ cells were exposed to monoenergetic carbon-ion beams (290 MeV/nucleon) and X-irradiation (150 kVp, 20 mA, 0.5 mmAl, and 0.3 mmCu filters). [Results and Conclusion] Total myeloid colony-forming cells (CFCs) among CD34⁺ cells exposed to heavy-ion beams, compared to those exposed CFCs among CD34⁺ cells exposed to X-irradiation, showed a significant decrease in D₀ and n, the parameters of radiosensitivity (heavy-ion beams: D₀ = 0.7 Gy, n = 1.1; X-irradiation: D₀ = 1.1 Gy, n = 1.9). On the stage of maturation, CD13⁺CD14^-/lowCD15⁺ neutrophil fraction derived from the X-irradiated CD34⁺ cells was significantly lower (7.7%) than that derived from non-irradiated control cells (22.2%). In addition, CD235a⁺ erythrocyte fraction increased to 64.1%, as compared to that derived from the non-irradiated control (41.9%). However, after heavy-ion beam irradiation, the amount of both these fractions was the same as that of the corresponding fractions derived from the control. These results suggest that the regeneration of myeloid hematopoiesis depends on LET and/or IR dose.

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A stem cell contributes to the maintenance of tissue homeostasis by retaining an ability to divide asymmetrically into a stem cell and a transit daughter cell. Several reports suggest that stem cells segregate their chromosomes selectively. On the other hand, the cells that lose the ability of the selective chromosome segregation may be involved in carcinogenesis. In the present study, we investigated the effect of ionizing radiation on the selective chromosome segregation in neural stem cells. Neurosphere (NS) cells that contain a fraction of neural stem cells and fibroblast cells (MEF) were obtained from embryos of ICR mice at day 14.5 of gestation. The cells were irradiated with 1 Gy, 2 Gy and 3 Gy of X-rays, and then their chromosomes were labeled with 5-ethynyl-2'-deoxyuridine (EdU) for 48 h. We determined the frequency of the selective chromosome segregation by scoring the unequal fluorescent intensity of the EdU-labeled nuclei in binuclear cells induced by inhibiting cytokinesis. In unirradiated NS cells, 1.2% of cells showed the selective chromosome segregation, while no cell segregated their chromosomes selectively in MEF cells, suggesting the selective chromosome segregation in neural stem cells. Irradiations with 1 Gy, 2 Gy and 3 Gy of X-rays reduced the frequency of the selective chromosome segregation in irradiated NS cells to 60%, 45%, and 15% levels, respectively, compared to that in the unirradiated NS cells. The present result indicates that exposure to ionizing radiation decreases the number of stem cells retaining the selective chromosome segregation, suggesting the implication for the radiation effect on tissue homeostasis.

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Ionizing radiation induces genomic instability in the progeny of irradiated cells that have divided several to a few dozen times. Previous studies suggest that DNA double-strand breaks (DSBs) are involved in the induction of genomic instability. Since it seems unlikely that DSBs persist through several cycles of cell division, we hypothesize that some DNA lesions, but not DSBs per se, are involved in inducing genomic instability. In this study, we examine whether non-DSB types of damage, particularly oxidative base lesions, induce genomic instability. Chromosomes were irradiated with UVA because it generates mainly oxidative base damage and only a small amount of DSBs. We transferred an UVA-irradiated human chromosome 21 into unirradiated immortalized mouse cells derived from fibroblasts using a microcell-mediated chromosome transfer. UVA-irradiated chromosomes were analyzed by the Whole Chromosome Painting Fluorescence in situ Hybridization (WCP-FISH) with specific probes for human chromosome 21. Most of the cells that were transferred with unirradiated human chromosome 21 remained diploid. However, we found that cells that were transferred with a UVA (4000kJ/m²) irradiated human chromosomes became polyploid and that structural aberrations occurred not only in the UVA-irradiated human chromosomes but also in the unirradiated mouse chromosomes. These results suggest that non-DSBs type of DNA damage induced by UVA irradiation induces genomic instability.

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Previously, we examined early events for lymphoma development in C57BL/6 mice after four consecutive irradiations of 1.8 Gy γ-rays at 1-week intervals, and it is concluded that the early stage of lymphomagenesis, induction of DNA double strand breaks, aneuploidy, delayed chromosomal instability, appearance of reactive oxygen (ROS) producing T cells, bystander effects, arrangements of cancer-related genes, and cell clonality were observed. Induction of these abnormalities might be associated with radiation-induced thymocyte atrophy. For a detailed analysis of early events of lymphomagenesis, the prelymphoma marker might be a useful tool. The thymic prelymphoma cells in sprit-dose irradiated B10 mice have been characterized phenotypically in relation to expression the marker defined by the mAb against TL-2 (thymus-leukemia) antigen. However in B6 mice, the expression of TL-2 antigens was limited. We found an monoclonal antibody against Ly-6C, which reacted at early stage of prelymphomas in irradiated B6 mice more strongly than TL-2. Also, Ly-6C positive cells were shown to be ROS-producing cells among the irradiated thymuses. Using an intrathymic infection assay after 4-10weeks of irradiation, it was demonstrated that some part of thymic lymphomas developed from cells highly reacted with anti Ly-6C antibody. It is concluded that Ly-6C surface antigen can be used as a marker of radiation-induced thymic prelymphomas.

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Because of rapid increase in the frequency of medical exposure to ionizing radiation, the effects of radiation on children have become a great concern in recent years. However, there is little information about this. We, therefore, started to study the radiation effects on children using mice model. The aim of this study is to demonstrate an age dependency of hepatocyte response to radiation in B6C3F1 mice. First, we investigated the proliferative response to radiation by means of immunohistochemistry for BrdU incorporation. Surprisingly, hepatocytes of 1-week-old mice did not stop proliferation even after irradiation. It suggests that high susceptibility of immature hepatocytes to radiation-induced hepatocarcinogenesis is ascribed to the expansion of damaged cells. Next, we investigated accumulation of mutations in hepatocytes at 10^th week, 10^th month, and 18^th month after birth by means of gpt assay. Although mutation frequency did not change significantly, the clonality increased slightly at 10 and 18 months of age.

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A neurosphere method makes it possible to culture neural stem cells in vitro. It is useful for stem cell research that mouse neurosphere cells can be immortalized with normal abilities of differentiation and radiation response. In the present study, to establish an immortal neural stem cell line, we immortalized several neurosphere cell lines and isolated a diploid cell line from them. Neurosphere cells that contain a fraction of neural stem cells and fibroblast cells were obtained from embryos of ICR mice at day 14.5 of gestation. To establish a stable neurosphere cell line, we examined three successive subculture protocols whereby the cells were plated at a density of 2X105 cells into a 25-cm2 flask and subcultured every 3 days (3T2), 5 days (5T2) or 10 days (10T2), with two additional trials (3T2 and 10T2) for the fibroblast cells. The results indicated that the neurosphere cells subcultured by the 5T2 and 10T2 protocols and the fibroblast cells subcultured by the 10T2 protocol immortalized (>100 PDN) whereas both cell strains subcultured by the 3T2 protocol senesced. Chromosome study demonstrated that about 50% of cells in 5T2 (75 PDN) and 10T2 (54 PDN) protocols were diploid whereas 70% of cells in 5T2 (145 PDN) and 10T2 (93 PDN) changed to be tetraploid. Fibroblast cells were immortalized when they were subcultured every 10 days (10T2). In contrast, all immortalized fibroblast cells showed to be triploid and tetraploid. We isolated three neurosphere cell clones retaining near-diploid karyotypes. These cells are expected to be useful for the study of radiation effects in neural stem cells.

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Background:It has been reported that large increase of leukemia among survivors of atomic bombs or thyroid cancer after the Chernobyl accident. The analysis of these cancers indicated that there was difference in incidence, causal gene or mutation mechanism with a relationship to age at exposure. We reported that there was no difference in incidence and latency in the T-cell lymphomas (TLs) that were induced by irradiation of female B6C3F1 mice weekly with 1.2 Gy X-rays. However, we observed high frequency of LOH on Chromosome 11 in young adult (4 or 8 weeks of age) group. According to the frequency of LOH, the mutation frequency of Ikaros (Ch11) was higher in 4, 8 weeks of age group. In contrast, in infant (1 week of age) group, we observed high frequency of LOH on Chromosome 19. In this report, therefore, we aimed to investigate that age dependency of radiation-induced mouse T-cell lymphomagenesis mechanism(s) by alteration analysis of Pten (Ch19) which was reported mutations in mouse TLs. Materials and Methods: We analyzed the mutation of Pten in mouse TLs, which developed after irradiation to female B6C3F1 mice weekly to 1.2 Gy X-ray for four consecutive weeks starting at infant (1 week) or young adult (4 and 8 weeks) age by sequencing, western blotting and array CGH. Result: We detected point mutations 22%(4/18), 31%(4/13) and 25%(2/8) in 1, 4 and 8 weeks of age group, respectively. In 1 week of age group, we observed no expression of Pten mRNA TLs (17%: 3/18), and array CGH was revealed that genome homo deletion in these TLs. Out of 44%(8/18) in 1 weeks of age group and 31%(4/13) in 4 weeks of age group TLs which observed LOH at Pten locus, 75%(6/8) and 75%(3/4) occur recombination without changing the number of genome, respectively. These Pten alterations and no expression frequency was 61%(10/18) in 1 week of age group higher than 46%(6/13), 38%(3/8) in 4, 8 weeks of age, respectively. Conclusion: We revealed that the recombination at the Pten locus occurred in TLs from 1, 4 weeks of age groups.High frequency of Pten mutations in TLs from 1 weeks of age groups indicated that T-cell lymphomagenesis in infant age depends on Pten more than Ikaros.

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Exposure to ionizing radiation (IR), in particular during childhood, is a well-known risk factor for thyroid cancers. Among a-bomb survivors, a vast majority of thyroid cancers are developed in survivors who have been adulthood at the bombing. There was little evidence of any effect among persons exposed as adult. In this meeting of the last year, we had reported that the level of phosphorylate p53 at Ser15 was increased, while no increase was found in the level of p21 and cleaved caspase-3 in immature (4 week-old rat) and adult (7 week-old rat) thyroid up to 24 h after IR. This result suggested that neither p21-dependent cell cycle arrest nor apoptosis was induced in spite of DNA damage response was occurred in thyroid epithelial cells after IR. This study aimed to investigate the participation of autophagy and the difference of age in thyroid follicular cells after IR. Immature and adult male wistar rats were exposed to X-ray at 8 Gy, then thyroid tissues were removed from 0 to 72 h after 8Gy IR. Histological examination revealed expansional alteration of follicular cells after IR. The level of alteration cells was peaked 24 h and increased significantly by 72 h in immature. In contrast, it was increased at 48 h and then decreased at 72 h in adult. The rate of Ki-67-positive cycling cells in immature and adult follicular cells was significantly decreased, and Cleaved caspase 3 positive cells were not detected until 72 h. Electron microscopy demonstrated the expansion of endoplasmic reticulum and the pool of secretion in the expansional immature follicular cells, and autophagosome was also observed at 6 h after IR. The expansion and collapse of mitochondria, stack of golgi, unusual form of lysosome and microautophagy were observed in adult. These observations were remarkable at 48 h. The level of LC3-II, which is a reliable marker for autophagic activity, increased until 24 h in immature and adult thyroid after IR. The present study revealed that IR induced autophagy but not apoptosis in immature and adult thyroid. Further study is required to examine the role of authophagy in radiation-induced thyroid cancer.

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Radiation-induced acute myeloid leukemia (AML) in C3H/HeN mice is an important animal experimental model to predict a human AML risk at low doses. The stem cells induced AML in mice usually carry a deletion of chromosome 2 and a mutation of the Sfpi1 gene on the retained homologue. These genetic changes result in downregulation of the transcript PU.1, which is crucial for normal myeloid differentiation. However the Sfpi1 gene mutations in AMLs are mainly point mutations. The C:G to T:A transitions which are the majority of them arise as a result of spontaneous mutations. Therefore, considering the effect of radiation for probability of the spontaneous mutations are required except the deletion of chromosome 2 to estimate AML risks induced by radiation. As spontaneous mutations occur in low probability with every cells divide, it is important that dynamics of hematopoiesis and effects of the dynamics changes with radiation are examined. In this study we use parameters which are obtained from experimental data using X-irradiated C3H/HeN mice to produce a mathematical model for calculating the dynamics of hematopoiesis. We focus the probability of spontaneous mutations of Sfpi1 gene and compare with the simulated AML risks in mice and experimental data.

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Previously, we reported significant life-shortening due to early neoplastic death in specific-pathogen-free B6C3F1 mice irradiated with continuous low dose-rate (20 mGy/22 h/day) gamma-irradiation accumulating to a high dose (8000 mGy). To understand the mechanisms of the life-shortening, we transplanted syngeneic tumor cells into both irradiated mice and age-matched, non-irradiated control mice to compare the transplantability of tumor cells. As a result, we found enhanced transplantabilities of tumor cells in irradiated mice. In this report, to understand the mechanisms of the enhanced tumor transplantability, we examined some factors relevant to tumor immunity of irradiated mice. Since chemokine network was known to be involved in tumor immunity, we focused on combinations of chemokine ligands and chemokine receptors. Using RT-PCR and ELISA, we found that the transplanted tumor cells expressed some chemokine ligands, Ccl2, Ccl5, Ccl25, Cxcl16, and Cx3cl1. These ligands were known to bind to chemokine receptors, Ccr2, Ccr5, Ccr9, Cxcr6, and Cx3cr1, respectively. We analysed expression patterns of 19 chemokine receptors by RT-PCR in both irradiated mice and non-irradiated control mice. Expressions of Ccr5, Ccr9 and Cxcr6 were reduced significantly in irradiated mice. These results indicated that the expressions of chemokine receptors against chemokine ligands expressed by tumor cells were reduced significantly in mice irradiated with low dose-rate gamma-ray continuously. The relevance of chemokine network to enhanced tumor transplantability in irradiated mice is needed to be investigated in detail. This study was performed under contract with the Aomori Prefectural Government, Japan.

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