DNA-dependent protein kinase (DNA-PK), composed of DNA-PKcs and Ku86/70, is thought to play a critical role in DNA double-strand break (DSB) repair via non-homologous end-joining (NHEJ) pathway. DNA-PK can phosphorylate in vitro a number of proteins and there are lines of evidence indicating the requirement for kinase activity of DNA-PKcs in its DNA repair functios. Nevertheless, it is poorly understood what is/are the in vivo phosphorylation target(s) and why the kinase function of DNA-PK is essential for DNA repair. Several studies have indicated that DNA-PK can phosphorylate XRCC4 in vitro and we have demonstrated that DNA-PK mediates DNA damage-induced phosphorylation of XRCC4 in living cells. Now we identified three serines of XRCC4 as the sites of phosphorylation by DNA-PK in vitro and two of them as the sites of phosphorylation in living cells, requiring DNA-PKcs. Furthermore, the replacement of these serines with alanine substantially reduced the ability of XRCC4 to confer increased radioresistance and proliferation rate to XRCC4-deficient cell line M10, indicating that the phosphorylation by DNA-PK of XRCC4 at these sites might be important for the cooperation of these molecules to sustain cell proliferation in the face of radiation-induced and spontaneous DNA damages. We would also discuss the implication of this phosphorylation event in DSB repair and its possible application in cancer therapy.

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We have been investigating effects of heavy-ion beams on immortal human cells established by introducing the hTERT gene. G0/GI phase arrested normal and AT cells were irradiated with carbon ions, iron ions and X-ray. In normal cells, the survival after irradiated with iron ions was significantly lower than those after irradiation with carbon ions and the induction of micronuclei was significantly higher after iron ions than carbon ions. In contrast, significant difference between carbon and iron ions was not observed in AT cells. In normal and AT cells after irradiation with heavy-ion beams, induction of micronuclei was significantly high compared with those irradiated with X-ray. Almost similar number of γH2AX foci formation was observed in both normal and AT cells after irradiation with carbon or iron ions. After 40 hours in AT cells significant number of γH2AX foci were observed. In normal cells, DSBs induced by iron ions repaired significantly slower than those induced by carbon ions, while after 40 hours most of γH2AX foci disappeared induced by both carbon and iron ions. These results suggest that AT cells might not be able to repair the DSB induced by heavy-ion beams and severely affected by them.

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Clustered DNA damages induced by high LET heavy ions are thought non-repairable or difficult to repair. However, much less is known about the reparability of clustered DNA damages. The major DNA repair pathway in mammalian cells is non-homologous end joining (NHEJ). In this study we investigated NHEJ pathway by exposure to high LET heavy ions, by analyzing responses of Ku to DNA damage induced by high LET heavy ions.
pGFP and pGFP-Ku80 were transfected into xrs-5 cells (mutated in Ku80). Cells were irradiated with γ rays and ion beams (LET=2.7-1610 keV/µm) at TIARA JAERI-Takasaki. Survival rates were measured by colony formation assay. To examine DNA damage and NHEJ pathway induced by heavy ion beams, γH2AX and GFP signal on the nuclei were observed.
Xrs5-GFP-Ku80 cells were radioresistant to γ rays and all ion beams, in comparison with xrs5-GFP cells. When the inactivation cross section was calculated to evaluate lethal effects per one particle, the difference of inactivation cross section between xrs5-GFP cells and xrs5-GFP-Ku80 cells became smaller with increasing LET. GFP and γH2AX foci on the nuclei co-localized from 10 to 30 min after C (108 keV/µm) and Ne ion (321 keV/µm) irradiation. While GFP foci were observed for 10 min after Ar ion (1610 keV/µm) irradiation, were not observed for 20 min. The difference of LET of ion beams influenced NHEJ responses.

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Ionizing radiation causes various types of DNA lesions, such as strand breaks, oxidative base lesions and, in particular, biologically relevant complex damage known as cluster damage sites, which consist of two or more elemental lesions within one or two helical turns of DNA. The clustered DNA damages would be less readily repaired than isolated lesions and finally induce serious genetic change of a living cell. We present here the evidence that the yield of DNA lesions and clustered damage strongly depends on the LET of He ion beam. Hydrated plasmids were irradiated with He ions at LETs of 19, 63 and 121keV/µm. The yield of prompt single strand breaks (ssbs) gradually decreased with increasing LET, whereas the yield of prompt double strand breaks (dsbs) showed a peak at 63 keV/µm. Base lesions and clustered damage were revealed as strand breaks by postirradiation treatment of the DNA with base excision repair proteins endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). Fewer enzymatically-induced ssbs and dsbs were observed as the LET increased. These results suggest that the clustering of damage becomes more intense with increasing LET. Studies with carbon ions are now in progress.

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In order to clarify the molecular mechanism of congenital teratogenesis, establishment of an appropriate animal model for the investigation of the effects of exposure to environmental factors, the relationship between dosage and external malformations, as well as visceral, is essential.Historically, knowledge of the embryogenesis of any cardiovascular defects has been difficult to attain because of the lack of models in which the pathogenesis of the defects could prospectively be studied.We focused on embryonic deaths, external malformations, and visceral malformations of cardiovascular origin caused by maternal exposure to 60</SUP<Co gamma rays and excess Tretinoin. Following maternal exposure to Tretinoin, we observed a high incidence of 100% teratogenesis, especially cardiovascular conotruncal defects and craniofacial anomalies, such as transposition of the great arteries, dextroposition of aorta, riding aorta and aortic arch anomalies including interruption of aortic arch and aortic coarctation, along with craniofacial anomalies were micrognathia and a cleft palate. These results indicate the high Tretinoin sensitivity of mouse fetuses to conotruncal anomalies and neurocristopathy syndromes. In humans, these conditions are termed DiGeorge-Velocardiofacial Syndromes.The Tretinoin treatment of mice could be a suitable animal model to study the mechanism of human complex congenital abnormalities including cardiovascular and craniofacial anomalies, especially DiGeorge-Velocardiofacial Syndromes.

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Ionizing radiation generates free radicals along its track and produces locally multiply damaged sites in DNA molecules. Among these sites, those containing base lesions and/or strand breaks (SSBs) within a few helical turns of DNA across both strands are referred to as clustered damage. Clustered damage is important to elucidate the biological consequences of radiation since it is believed to be repair-resistant or to lead to error prone repair. Clustered damage containing base lesions (clustered base damage) is repaired by the base excision repair (BER) pathway. During the initial step of BER, SSB is formed by the AP lyase activity of DNA glycosylase or AP endnuclease. Therefore, processing of clustered base damage by BER could result in the formation of fatal DSB lesions.
In the present study, we have examined the activity of hOGG1 for clustered base damage containing two 8-oxoguanine (8-oxoG) residues and analyzed the formation of DSBs. In substrates, the 8-oxoG residue in the top strand is 1 to 5 bp away from the second one in the bottom strand in the 3' or 5' direction. Substrates were incubated with hOGG1, and the amounts of SSBs and DSBs induced by hOGG1 were analyzed by PAGE. The excision of the first 8-oxoG was not influenced significantly by closely opposed 8-oxoG. The subsequent excision of 8-oxoG was retarded moderately and resulted in DSBs. Interestingly, some 8-oxoG clusters were converted to DSBs very inefficiently. We are also examining the activity of hOGG1 in the presence of hAPE1.

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Nijmegen breakage syndrome(NBS), characterized by high sensitivity to ionizing radiation (IR) and predisposition to lymphoid cancer, is phenotypically similar to ataxia telangiectasia (AT). NBS1, the protein responsible of disease, is cooperative with ATM, mutated in AT, in response to IR-induced DNA double-strand breaks (DSBs) and has crucial roles in DNA repair and cell cycle checkpoints. Recently, it was reported that when NBS cells are treated with hydroxyurea (HU), they show the phenotype similar to cells from Seckel syndrome, which is mutated in ATR and shares common clinical signs to NBS, such as microcephaly and growth retardation. Although ATM and ATR, a family of PI3-kinase, are key regulators for the checkpoint mechanisms, ATR is indispensable at stalled replication fork when cells are treated with HU or exposed to Ultraviolet (UV). To investigate the role of NBS1 for maintenance of replication fork, the cellular localization of NBS1 and the related proteins after exposure to UV-C were analyzed by immunostaining. NBS1 formed discrete foci after UV exposure and they co-localized with g-H2AX foci, which are formed at stalled replication forks. Interestingly, NBS1 mutant lacking FHA domain formed UV-induced foci. This is quite different from IR-induced foci, since FHA domain is essential for foci formation after IR irradiation, suggesting distinctive recruitment mechanism of NBS1 to replication fork. We will discuss more involvement of NBS1 in ATR-signaling at our poster session.

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There are at least two pathways, in which DNA double strand breaks (DSBs) are rejoined: non-homologous end joining and homologous recombination (HR). Defect of HR can result in genomic instability and cancer predisposition. Previously, we have demonstrated using NBS1-deficient DT40 cells that NBS1, protein responsible for radiation sensitive disorder Nijmegen breakage syndrome, functions in HR repair. We also showed that hMRE11 is important for NBS1-dependent HR activity, while the ATM kinase is dispensable. Here, we investigate the relationship between NBS1 and BRCA1/BRCA2, which are known to function in HR. Cells expressing either a tandem BRCT domain of BRCA1 or BRC4 repeats, binding region to Rad51, of BRCA2, inhibit radiation-induced foci formation of endogenous respective BRCA1 and Rad51, due to dominant-negative effect. When homologous recombination was assayed by DR-GFP reporter gene, these cell lines with dominant-negative effect showed the reduced HR frequency regardless the presence of NBS1 protein. This additive effect of HR suggests that NBS1 functions in HR independently of BRCA1 and BRCA2. To confirm these results, we are also investigating the dominant-negative effect of BRCA1, BRCA2 on MMS-induced lethality and chromosome instability in NBS cells and the complemented cells.

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The structural maintenance of chromosome (SMC), family proteins that plays a central role in chromosome dynamics and stability, is categorized to three groups in eukaryotes: SMC1-3 as a core protein of the cohesins which regulates the chromosome synapsis, SMC2-4 as a core protein of condensins which plays a role in chromosome condensation during miosis and SMC5-6 complex possibly involved in DNA repair and chromosome segregation. Yeast SMC5 or SMC6 mutants showed increased sensitivity to DNA damage-inducing agents such as methyl methanesulfonate (MMS) and hydroxyurea (HU), suggesting the potential involvement in homologous recombination repair and stabilization of replication fork. Moreover, high sensitivity to MMS and ionizing radiation is reported in the mutants of yeast Rad62, non-SMC proteins formed complex with SMC5-6.
To clarify the function of SMC5-6/non-SMC protein complex in higher eukaryotes, we identify human Rad62 gene and analysis the role in a response to radiation damage. Two orthologs of Rad62, Rad62A and Rad62B, were identified when human cDNA was screened. The modification of Rad62A protein was observed after exposure to radiation (IR). We are presently preparing Rad62A knock-down cells to examine the sensitivity to DNA damage agents, such as UV and radiation.

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Ionizing radiation produces a unique form of DNA damage called clustered damage, which contains two or more lesions induced within the one or two helical turns of DNA. Clustered damage would be less readily repaired than isolated lesions. Therefore clustered damage might be biologically significant. In this report we showed that HeLaS3 cells transfected by hOGG1 type1a or type2a plasmid were more sensitive to gamma-rays than HeLaS3 cells without plasmid. Clustered damage produced by ionizing radiation might be converted to lethal double-strand breaks during attempted base excision repair. hOGG1 type1a protein localized in nuclei and hOGG1 type2a in mitochondria. The present results that overexpression of hOGG1 type2a protein enhanced the sensitivity to gamma-rays suggest that double-strand breaks are also induced by abortive base excision repair in mitochondrial genome. We are currently investigating the biological effects of clustered damages in human cells.

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Reactive oxygen species cause a wide variety of oxidative modifications to purines and pyrimidines in DNA. Bacteria and eukaryotes have evolved base excision repair mechanisms for oxidative base damage in DNA. E. coli has three kinds of DNA glycosylase, MutM,Nth and Nei, that recognize and remove oxidatively damaged bases from DNA. These DNA glycosylases are able to recognize and remove 5-formyluracil and 5-hydroxymethyluracil in DNA. However, several evidence showed that there are residual activities in crude extract from E. coli mutM nth nei triple mutant. In this study, we identified the abilities of novel enzyme(s) to recognize double-stranded oligonucleotides containing 5-foU. We are currently investigating the nature of the enzyme(s).

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Various oxidative damages, such as thymine glycol (TG), are spontaneously created in DNA. These damaged bases are mainly repaired by base excision repair pathway. It has been reported that mNTH1 and mNEIL1 may participate actively in the repair process. Although both enzymes possess bifunctional activities of TG-DNA glycosylase and AP lyase, we have found a novel monofunctional DNA glycosylase activity toward TG-DNA in the nuclei of various mouse organs. We have previously reported the purification of the activity from murine stomach with hydrophobic, hydroxyapatite and ion exchange column chromatography. In the experiment, the monofunctional activity could not separate completely from minor bifunctional activity. We have developed a new method to differentially extract the intranuclear target activity from murine lung. The prepared nuclear extract fraction showed the target activity with little bifunctional activity. In the previous experiment, we detected mNEIL1 as one of the candidate proteins having the target activity. At present, two mNEIL1 isoforms and one putative one are known. mNEIL1 itself (NCBI : NP082623) may not be the target protein because of the reported bifunctional activity and the chromatographic behavior. The investigation on the activity of the other isoforms is in progress.

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TopBP1, which has been isolated as an interacting protein with DNA topoisomerase II beta, has eight BRCT domains, and shares sequence homology with yeast Cut5/Rad4. It has been reported that TopBP1 forms foci at the sites with DNA damages, which were co-localized with those of gamma-H2AX, NBS1, PCNA, and BRCA1. TopBP1 is phosphorylated by ATM, and is involved in Chk1 and Chk2 phosphorylation after DNA damage. TopBP1 knockdown cells using anti-sense oligonucleotide showed increase sensitivity to DNA damaging agents. Here, we show that TopBP1 physically interacts with NBS1, which is the underlying protein for Nijmegen breakage syndrome (NBS). The formation of TopBP1 foci was significantly inhibited in NBS cells after irradiation, whereas NBS1 foci formation was not affected in the cells with reduced TopBP1 expression by RNAi. Formation of TopBP1 foci was also inhibited in AT cells. Physical interaction between TopBP1 and NBS1 was increased after irradiation, and NBS cells transfected with S343A mutant showed reduced formation of TopBP1 foci. These results suggested that TopBP1 is a downstream protein of NBS1, and the physical interaction with NBS1 requires phosphorylation of NBS1 by ATM.

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Nijmegen breakage syndrome (NBS) is a rare autosomal recessive genetic disorder characterized by immunodeficiency and cancer predisposition. The NBS cellular phenotype includes chromosomal instability, hyper-sensitivity to radiation and abnormal S phase checkpoints which is similar to Ataxia-telangiectasia(AT). NBS1 protein, product of the NBS gene, forms a complex with MRE11 and RAD50, and the RAD50/MRE11/NBS1 (R/M/N) complex plays a crucial role in DNA double strands break (DSB) repair. The human NBS1 has three functional regions. N-tarminus includes a fork head associated (FHA) domain and a BRCA1 C-terminus (BRCT) domain. In the central region, there are two serine residues at 278 and 343 that are phosphorylated by the ATM or ATR kinases. The C-tarminus includes MRE11 binding domain. We have reported that the R/M/N complex with functional NBS1 is essential for homologous recombination (HR) repair pathway. This suggested that NBS1 regulates the function of the complex by regulating both their subnuclear localization and enzymatic activities. To analyze functional domains of NBS1 in HR repair pathway, an SCneo reporter was introduced into HeLa or NBS patient cells. Then, mutant NBS1 was expressed in these cells, and HR activity was analyzed by transient expression of I-SceI endonuclease. It was found that C-tarminus and FHA domain of NBS1 is essential for regulation of nomal HR function.

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Reactive oxygen spiecies cause a wide variety of oxidative modification in DNA, which include many types of base damage such as thymine glycol (Tg) and 7,8-dihydroxy-8-oxoguanine (8-oxoG). 8-oxoG mispairs without cytosine and Tg blocks DNA synthesis in vitro. Therefore, these base damages are cytotoxic and mutagenic. In Escherichia coli, Nth protein functions as a DNA N-glycosylase/AP lyase for these base damages. Nth protein removes 8-oxoG from 8-oxoG:A and 8-oxoG:G pairs, and the functions of Nth protein suppress G:C to T:A and G:C to C:G transversions. We recently revealed that the Nth protein also recognizes 5-hydroxymethyluracil and 5-formyluracil. In C. intestinalis, Nth homologues are not yet identified. In this study, we identified and characterized CiNth protein in vitro and in vivo.

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Abasic (apurinc/apyrimidic, AP) sites are a major type of DNA damage. They cause cell death and mutation. They are generated spontaneously or through hydrolysis catalyzed by DNA N-glycosylases, leaving AP sites in DNA. AP endonucleases recognize and incise the DNA strand at the AP sites. The nicked DNA is filled by DNA polymerases and DNA ligases. Recently, the genome of Ciona intestinalis was deciphered. In this study, AP endonuclease in Ciona intestinalis (CiAPE) was purified and characterized. The properties of CiAPE were compared with those of human APE1. The CiAPE protein was about 34 kDa. It efficiently cleaved tetrahydrofuran (THF) - containing duplex oligonucleotides. In addition, the transformation of E. coli, BW9093Δxth with CiAPE complemented the H₂O₂ sensitivity. These results demonstrated that CiAPE has AP endonuclease activity to prevent against oxidative stress in Ciona intestinalis.

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7,8-dihydro-8-oxoguanine (8-oxoG) is the most important product of oxidative base damage in DNA, and causes G:C to T:A transversions in bacteria and mammalian cells. 8-oxoG is repaired by MutM in E. coli and 8-oxoG-DNA glycosylase (Ogg1) in yeast and mammalian cells. In the present study, we identified and characterized an ascidian homolog of the human hOgg1 in Ciona intestinalis (CiOgg1). Introduction of the CiOgg1 gene significantly reduced the frequency of spontaneous G:C to T:A transversions in E.coli mutM mutY strain. purified GST-CiOgg1 fusion protein had 8-oxoG DNA glycosylase/AP lyase activity. It formed Schiff base intermediates with 8-oxoG-containing duplex oligonucleotides and removed 8-oxoG preferentially from 8-oxoG/C. The CiOgg1 cleaved 8-oxoG-containing duplex DNA via β-elimination reaction. Furthermore, the expression level of CiOgg1 was compared in various tissues in Ciona intestinalis. The highest expression level was observed in testis.

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Once double-strand breaks (DSBs) are generated within genome DNA, DSBs are immdiately recognized by sensor mechanisms and then activate ATM kinase for phosphorylation of the substrates, which leads to the cellular responses such as cell cycle checkpoints, apoptosis and DNA repair. Recently, it was reported that the C-terminus of NBS1 has an indispensable role for recruitment of ATM to DSB sites and possibly for the resulting phosphorylation of substrates through the interaction with ATM. Previously, we demonstrated that gamma-H2AX is involved in the recruitment of NBS1/hMre11/hRAD50 complex to DSB sites through interaction with NBS1 at the N-terminus. Hence, gamma-H2AX might function, as a sensor or initial factor, in ATM-dependent damage response. We showed here that gamma-H2AX forms the complex with ATM and NBS1 in irradiated cells. When the expression of H2AX was repressed by H2AX siRNA, chromatin-bound NBS1 in irradiated cells was significantly decreased. Early focus formation of NBS1 and phosphorylated ATM was also attenuated. As a result, H2AX siRNA reduced the phosphorylation of ATM substrates, which were present in gamma-H2AX complex in irradiated cells. However, these phosphorylations were restored a few hours after irradiation, indicating an altenative activation pathway for ATM-dependent damage response. Taken together, our results suggest that gamma-H2AX could mediate a rapid activation pathway of ATM through interaction with ATM and the substrates.

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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 fly. 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 and Epoxomicin, suggesting an involvement of ubiquitin-proteasome pathway on the regulation of dRAD30B stability. Another new finding is the lethal phenotype of RNAi over-expression transgenic fly. We have established transgenic fly lines, in which expression of Rad30A, B and Rev1 RNAi construct is regulated under control of GAL4-inducible UAS promoter. Although induction of the RNAi construct only in eye by tissue-specific driver show no phenotype, induction in whole body by Actin-driver show pupal lethality. This phenotype was relieved by crossing the RNAi fly with cDNA over-expressing fly.

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Helicases are able to catalyze the unwinding of duplex nucleic acid, therefore, helicases play crucial roles in essentially every function involving DNA and RNA, including DNA replication, repair and recombination, and RNA transcription, processing and translation. Helicases are classified into superfamilies, and most helicases are in superfamily I(SFI) and SFII. Members of SFI and SFII contain seven motifs. The S.cerevisiae RRM3, encoding a 5` to 3` DNA helicase, is a member of a helicase SFI that is conversed from yeast to humans. RRM3 was first identified because its absence increases recombination in rDNA genes. It has been suggested that RRM3 is needed for normal fork progression. As a result, the S.cerevisiae RRM3 can promote genome integrity by preventing replication fork stalling. We suppose that S.cerevisiae RRM3 might contributes to restart DNA replication stopped by replication fork stalling which is caused by ionizing radiation (IR). In our study, we have intended to get the human homolog of scRRM3 first, then identify its function in maintenance of genome integrity and resuming the DNA replication. We have identified a partial fragment of human homolog of scRRM3 by blastp analysis through GenBank database. Later we acquired several splicing variants. And we also determined the chromosomal location of the gene.

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In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients with characteristics such as growth retardation and short life span. Xpg null mice exhibited the same characteristics as human patients do. We generated four mutant Xpg mouse strains with different mutations in Xpg gene to identify the Xpg region that causes onset of the CS phenotype. We found that the deletion of C terminal 183 amino acids results in the CS phenotype. The primary embryonic fibroblasts isolated from Xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization. If chimera mice in which some tissues are replaced with Xpg-deficient cells or tissues could be generated, it seems to be very interesting to analyze the fate of such chimera mice and/or Xpg-deficient cells in such chimera mice. So we have generated bone marrow chimera mice in which normal blood cells were replaced with Xpg-deficient cells and examined their life span, shortening of life span by X-irradiation and class switching of immunoglobulin heavy chain. The life span of Xpg-chimera mice was not different from that of normal chimera mice. The life span of Xpg-chimera mice irradiated with X rays was shorter than that of normal chimera mice. Though in vitro studies indicate that Xpg functions as a nuclease for class switching of immunoglobulin heavy chain, class switching was normal even in Xpg-chimera mice.

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We previously reported a Japanese girl with the novel chromosomal instability syndrome. The clinical symptoms included severe microcephaly, short stature, combined immunodeficiency, and development of malignant lymphoma. The primary skin fibroblast from the patient showed increased spontaneous chromosome breakages and radiation hypersensitivity. The clinical and cellular phenotypes were similar to those of the patients with Nijmegen breakage syndrome (NBS). However, no mutation was detected in the NBS1 gene, and western blot revealed normal expression of NBS1, Mre11, and Rad50 proteins. In addition, the cells showed the irradiation-induced focus formation at the similar level to that of normal cells.
To identify the underlying gene for the patient, we utilized the technique of microcell-mediated chromosome transfer (MMCT) to introduce a human chromosome into the patient’s fibroblast cell line, and functional complementation assays were carried out. We found that a chromosome 13 complemented the radiation hypersensitivity of the patient’s cells. Since the chromosome 13 contains the DNA ligase IV (LIG4) gene, which is involved in non-homologous end joining pathway of DNA DSB repair, mutation screening was performed in the LIG4 gene of the patient, and biallelic mutations were detected in the LIG4 gene. These results demonstrated that this is the first Japanese patient with the LIG4 syndrome.

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We studied the effect of this mutation on UVB-induced mutagenesis in mouse skin, using a transgenic mouse harboring -phage shuttle vector-based bacterial lacZ genes as a mutational reporter. UVB increased the lacZ mutant frequency in the epidermis moderately but significantly higher in the homozygous mutant frequencies were not different appreciably among them. Ninety-eight lacZ mutant sequences isolated from the UVB-exposed epidermis of the Xpgex15-homozygous mice were analyzed and compared with mutant sequences from the wild-type mice. The spectra of the mutations were not significantly different and highly UV-specific: a high frequency of C->T transitions at dipyrimidine sites and several CC->TT tandem mutations, although the UV-specific mutations occurred more frequently at CpG sites in the mutant mice. The distreibution of the mutations observed in the lacZ transgene and the preferred sequence context of the UV-specific C->T mutations (5-TC-3> 5-CC-3>5-CT-3) in the Xpg mutant were similar to those found in the wild-type mice. Despite those similarities, we detected a previously unrecognized type of the UV-induced mutation only in the Xpg mutant, which is characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. The possible significance of this putative new class of mutation, which we defined as the triplet mutation, was discussed.

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Werner syndrome (WS) is an autosomal recessive disorder whose phenotype mimics premature aging. This syndrome is caused by mutations in WRN, a member of the RecQ DNA helicase family, of which biological function remains unclear. In the present study, we investigated the relationship between the dysfunction of WRN protein and chromosomal instability. Primary fibroblast cells (WS3RGB) derived from a WS patient showed chromosomal instability during cell culture with complex-type translocations including at least five chromosomes. In addition to this karyotype instability, the WS cells indicated high level (4.9%) of dicentric formation spontaneously. These findings imply that genomic instability accompanied by telomeric dysfunction is evident in the WS cells. Therefore, we asked telomeric stability of the WS cells by telomere-FISH technique. The result revealed that extra telomere signals (ETS) emerged more frequently (3%) than that in the control cells (0.8%). Interestingly, the frequency of ETS was elevated after exposure to 4NQO, a DNA damaging agent, suggesting that ETS was responsible for structural abnormality in telomeres by some DNA damage and that the complex-type translocations might be formed mediated by telomeric dysfunction. However, we did not have evidence to support telomere fusion in forming the translocations by subtelomere-FISH analysis. The present study demonstrates that telomeres in WS cells are constitutively unstable possibly due to WRN dysfunction, suggesting a crucial role of WRN protein in telomere maintenance.

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Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. The non-homologous end-joining pathway is required both for normal development and for suppression of tumors. In the non-homologous end joining pathway of DNA double-strand break repair, the ligation step is catalysed by a complex of XRCC4 and DNA ligase IV. To address the role of XRCC4 in human cells, we have used gene targeting human HCT116 colon cancer cells to functionally inactivate the XRCC4 locus. The homolozygously null XRCC4 cells showed a growth defects and hypersensitivity to ionizing radiation. To analyze functions of XRCC4, we attempted to introduce XRCC4 cDNA tagged with FLAG into the XRCC4 (-/-) cells. The clones transfected with the XRCC4 cDNA showed a partial correction of ionizing radiation sensitivity. Further analysis is in progress.

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We analyze the mechanisms of radiation tolerance found in plants ^{1), 2)}. We also developed the ion-microbeam system for irradiating single plant cells ³⁾. This is the first system for irradiating non-adherent cells with microbeam. In this meeting, we introduce our system and biological effects of ion-microbeam irradiation in single tobacco cells.
Single tobacco cells were plated in an agarose medium onto an irradiation vessel, in which CR-39 nuclear track detector was set on the bottom. The cells were irradiated with 17.5 MeV/amu Ne and 18.3 MeV/amu C ion microbeam that were accelerated by cyclotron at JAERI-Takasaki and collimated through a 20-μmΦ aperture. After irradiation, ion tracks were detected by alkaline etching of CR-39 and hit rate of ions (No. of ions that actually hit an aimed cell / No. of ions counted by a scintillator-photomultiplier assembly) was determined. Colony formation rate of the cells was also measured after a month.
Hit rates of Ne and C ions were 84±3 and 73±6%, respectively. Colony formation rate of tobacco BY-2 cells was reduced to half, as cells were hit by 25 and 100 particles of Ne (LET = 377 keV/μm) and C (116 keV/μm) ions, respectively. We will plan to obtain survival curves of single tobacco cells using this ion microbeam system and compare the results with those obtained by the broad-field irradiation.
References:
1) Yokota et al. (2003) Int. J. Radiat. Biol. 79
2) Yokota et al. (2005) Radiat. Res. 163
3) Yokota et al. (2003) Biol. Sci. Space 18

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Irradiation procedures of the microbeam and the luciferase detection system for p53 gene transcription activity in mouse fibroblast-cultured cells were established for the usage to evaluate low dose rate radiation-induced biological effects. Confluent cells were irradiated by either ion microbeam or broad-ion beam, and the transcription levels of p53 in irradiated cells were observed by this system. In broad beam irradiation of Ne ion from TIARA AVF cyclotron, the particle fluence was approximately one ion per cell. In microbeam irradiation of the same ion, the exact one ion was irradiated at each cross point of orthogonal lattice in a 3 mm square. In broad beam irradiation, the p53 transcription activities had 2-fold increase of control. In microbeam irradiation, the p53 transcription activities reversely decreased to 30-40% of control with increase of number of irradiation cross points of 1-16. The p53 transcription activities decreased with increase of density of ion irradiation in an area. Reduction of the p53 transcription activities in irradiated cell population by the microbeam irradiation, can be explained not by effect of secondary electrons but by bystander effects, because percent of cell population located within the range of secondary electrons in the penumbra around one ion track is corresponding to about 1/10,000 of all cells. Microbeam irradiation of targeting cells may reduce p53 transcription activities in non-irradiated cells by an unknown mechanism through bystander effects. This study was financially supported by Aomori Prefecture, Japan.

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A compact micro X-ray beam irradiation system has been developed to research the effects of radiation for single cell. A micro radiation beam experimental system is one of the most useful tools to research cellular damages such as chromosome aberrations and mutagenesis. The system was composed of a 50kV micro focus X-ray tube with X-ray guide-tube, an X-ray detector for fluorescent X-ray analysis and a sample stage with an inverted microscope. In the microscope observation, a single cell can be irradiated with the micro X-ray beam. It has been obtained from the beam profile measurement that the X-ray beam diameter was 14 µm[FWHM] and the flux was ~5.0×10³ [photon/100mm²/s]. Also, a Monte Carlo photon-electron transport simulation was performed to estimate the dose rate for the irradiated single cell and the maximum dose rate was 0.5 Gy/s. In a preliminary experiment, the single-cell irradiations were performed using the yeast cells. The budding yeast Saccharomyces cerevisiae was irradiated with the micro X-ray beam irradiation system, and the maximum dose for the samples was 60 Gy. After the X-ray irradiation, the irradiated yeast cells were incubated at room temperature, and the time-lapse images of the irradiated cells were collected during incubation. After incubation for 20 hours, cell growth and division were hardly observed. This result shows that the X-ray beam had been delivered to targeted cells, and the irradiated cells had the lethal dose of X-ray.

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We measured the frequency of xeroderma pigmentosum A (XPA) heterozygotes (carriers) in 1,000 Hiroshima and Nagasaki residents as a preliminary study for evaluation of excess risk of skin cancer among the XPA heterozygotes. The advantage of the study is three folds; the frequency of XP patients is over 10 times higher in Japan than in other countries, about half of them belong to group A (XPA), and 90% of these XPA cases have the same base-change mutation (a founder effect mutation). In this study, we used archival lymphocyte slides examined in the past at RERF for F1 cytogenetic studies. The DNA region that includes the founder mutation was amplified by PCR method and the amplified DNA was digested with a restriction enzyme. When the founder mutation was present, the amplified DNA was cleaved into two shorter fragments. In total, 9 founder mutations were found (4/512 in Hiroshima and 5/508 in Nagasaki, Χ²=0.12, p>0.50). The overall frequency was therefore 1/115, which is higher than the previous estimates (i.e., 1/150 to 1/240). It is likely that the latter estimates are less certain since they were based on theoretical extrapolation from small-scale clinical surveys on XP patients, and the present estimates are closer to the real.

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Phosphorylation of histon H2AX (termed g-H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). g-H2AX induced by ionizing radiation and anti-cancer drugs, forms nuclear foci that are microscopically visible by immunofluorescence staining. We have previously shown that coexposure to benzo(a)pyrene (BaP), a wide-spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In present study, we examined whether coexposure to BaP and UVA induces g-H2AX in CHO-K1 cells. Single treatment with BaP (10-9-10-7) or UVA (~2.4J/cm2) did not induce g-H2AX, however, coexposure drastically induced foci of g-H2AX in dose-dependent manner. Furthermore, the induction of g-H2AX by coexposure was efficiently inhibited in the presence of NaN3, indicating that induction was due to the production of singlet oxygen. This is the first visual evidence that coexposure to BaP and UVA, a one of the environmental combined pollution, induced DSBs, involving g-H2AX. Interestingly, we successfully detected the g-H2AX at very low concentration of BaP, which did not change the cell survival rates. Epigenetic change is recently considered to relate with cancer initiation. DSBs and accompanied g-H2AX might contribute to photocarcinogenesis of environmental concentration of polycyclic aromatic hydrocarbons.

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UVB irradiation is well-known to induce apoptotic cell death. However, we have found that low dose of UVB irradiation prevented apoptosis induced by both serum-depletion and detachement from the extracellular matrix. In this study, we examined the participation of PI3-kinase/Akt pathway in UVB-induced antiapoptosis. Treatment with PI3/kinase inhibitors, wortmannin and LY294002 partly eliminated the UVB-mediated inhibition of apoptosis. Furthermore, phosphorylation of Akt was observed 15min after UVB irradiation. These results suggested that UVB irradiation transduced a survival signal via PI3-kinase activation and phosphorylation of Akt. Three major isoforms of Akt, termed Akt1, Akt2 and Akt3, have been found in mammalian cells, which share a high degree of structural similarity. Experiments with isoform-specific short interference RNA (siRNA) revealed that Akt1, and Akt2 knockdown eliminated the UVB-mediated inhibition of apoptosis. At present, we are trying to confirm the participation of Akt3 in UVB-induced antiapoptosis using siRNA for Akt 3. Among the three Akt isoforms, Akt1 is the predominantly expressed isoform in most tissues. We consider that further analysis of mechanism of antiapoptosis induced by UVB using siRNA for Akt1.

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Radiation-induced Long-Lived Radicals(LLRs), assigned as sulfinyl radicals (SLF: R-S-O•), in mammalian cells are likely to be mutagenic because scavenging LLRs by addition of Vit. C AFTER irradiation is correlated to the reduction of mutation frequency. In this study, we have performed UVB radiation to human fibroblast (HE40) cells, and hprt- mutation is analyzed by 6-thioguanine (TG) selection and multiplex PCR methods. UVB-induced LLRs in Syrian hamster embryo cells were also measured by ESR. The result of the 6-TG method indicates that levels of HPRT- mutation by UVB radiation are significantly higher than that of control, and were reduced to 40% by addition of Vit. C at 20 min AFTER irradiation. PCR analysis indicates that most of mutants are point mutation-type and total deletion or partial deletion-type are rarely found, so that most of mutation types are point mutations. LLRs measurements and analysis indicate that there are two kind of radicals as methyne disulfide radicals (MDS: R-S-CH•-S-R') and sulfonyl radicals (SFO: R-SO₂•), with ESR parameters of hyperfine coupling constant by one proton = 1.76 mT, g = 2.003 for MDS, and maximum slope width = 2.0 mT and g = 2.010 for SFO, respectively. MDS radicals yielded in 88 % of all LLRs, have long half-lifetime of 4 h, and were scavenged by Vit. C., so that MDS radicals are likely to be mutagenic. Chemical species of mutagenic LLRs are different as MDS and SLF radicals for UVB and ionizing radiations, respectively.

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Lens alpha crystallin consists of large aggregates of two molecules, alpha A- and alpha B-crystallin. The present study focused on the differential sensitivity of these two crystallins to UV-B irradiation. Alpha A- and alpha B-crystallins were studied by circular dichroism spectrum analysis, analytical ultracentrifugation and Bis-ANS fluorescence spectra following UV-B irradiation at 10, 20, 30, 40 or 50 J/cm². Sedimentation coefficients of alpha A-crystallin aggregates increased and their distribution became heterogeneous after UV-B irradiation above 30 J/cm². In addition, the chaperone activity of alpha A-crystallin dramatically decreased following irradiation. However, the size of alpha B-crystallin aggregates did not change and retained its chaperone activity after UV-B irradiation even up to 50 J/cm². The present study indicates that alpha A-crystallin is more susceptible to UV-B irradiation than alpha B-crystallin. This may be one of the reasons why alpha-crystallin consists of both alpha A- and alpha B-crystallin molecules.

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UVB radiation can damage plants, decreasing growth and productivity. It also induces formation of pyrimidine dimers in DNA, which are the predominamt and biologically significiant UVB induced damages in DNA. Cyclobutane pyrimidine dimers (CPDs) constitute the major class of these damages (80%) with the remainder being mainly pyrimidine (6-4) pyrimidone dimers. Two major mechanisms of CPD repair are excision repair and photoreactivation. CPD photoreactivation is the major pathway in plants for repairing UV-induced DNA damage. Plant cell has different genome in each nuclei, chloroplasts, and mitochondria. CPD photoreactivation has been reported to be involved in nuclei, but not in the other two organelles. We previously found in rice that CPD photorepair was observed in nucleus-rich fraction, but not in chloroplast-rich fraction.
This study aimed to confirm whether or not CPD photorepair would be involved in mitochondrion. We measured UVB-induced CPDs and their photorepair in each organellar genome using intact leaves by Real-time quantitative PCR assay and Southern blot assay using CPD specific enzyme T4 endonuclease V. This enzyme cleaves the dimers quantitatively at CPD sites. Genes such as rbcS, cab, and, phr (nuclear genome-encoded genes), cox3, cob, and orf288 (mitochondrial genome-encoded genes) and atpB and rbcL (chloroplast’s genome-encoded genes) were targeted for analysis. As a result, we found that CPDs caused by UVB in mitochondria were photorepaired.

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Type II Diabetes model mice (BKS.Cg-+Lepr^db/+Lepr^db/Jcl, DB mice, 10 weeks old, female) were irradiated with gamma rays at 0.35, 0.70, or 1.2 mGy/hr for 12 weeks. Total superoxide dismutase (SOD) activity decreased both in control and irradiated groups; however, the decrease was less in the irradiated groups, especially in 0.70 mGy/hr group. In this group Mn-SOD activity increased after the 12-week irradiation. A pathological examination of the pancreas revealed that damage to beta cells responsible for the secretion of insulin was much less in the 0.70 mGy/hr group compared to that in the non-irradiated controls. The plasma insulin concentration decreased within the first 4 weeks in all groups, and the level was kept low in the control mice. On the other hand, the insulin level in the irradiated groups showed a tendency to increase. In the 0.70 mGy/hr group the increase was statistically significant after the 12 weeks of irradiation.
These results indicated that the low dose-rate irradiation increase the antioxidative capacity in the pancreas to protect beta cells from oxidative damage, and then to increase the insulin level. This mechanism would lead the mice to the recovery from the disease demonstrated in our previous report.

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Oxygen radicals produced by ionizing radiation cause a variety of oxidative damage into DNA and its precursors. Among such damages, 7, 8-dihydro-8-oxoguanine (8-oxoG) is known to be abundant and highly mutagenic. 8-OxoG can pair with adenine as well as cytosine, thus causing G:C to T:A and A:T to C:G tansversions, if not repaired. Enzymes that may prevent 8-oxoG-evoked mutagenesis were identified in mammalian cells. Mth1 hydrolyzes 8-oxo-dGTP, 2-OH-dATP and 8-oxo-dATP to their monophosphate forms in the nucleotide pool, thereby preventing occurrence of mutations caused by oxidation. Ogg1 is an 8-oxoG DNA glycosylase and prevents G:C to T:A tansversion by excising 8-oxoG from 8-oxoG:C pairs. Previously, we analyzed X-ray induced mutagenesis in mice and found a significant increase of A:T to G:C transitions in spleens of Mth1-deficient mice, probably caused by a failure to remove 8-oxo-dATP formed in the nucleotide pools. On the other hand, G:C to T:A tansversion caused by 8-oxo-dGTP did not increase in the animals. These results suggested that Ogg1 might prevent an occurrence of this type of mutation. To examine this possibility, we investigated X-ray induced mutagenesis in Ogg1-deficient mice. Ogg1^-/- mice were irradiated with 4 Gy of X-rays at the age of 6 weeks. Two weeks after irradiation, we analyzed mutations in spleens of Ogg1^-/- mice and found an increase of G:C to T:A tansversions. These results suggest that Ogg1 could play an important role in preventing the mutagenesis induced by ionizing radiation.

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Ionizing radiation (IR) gives the normal human fibroblasts the phenotype of Senescence-Like Growth Arrest (SLGA), which is a p53-dependent irreversible growth arrest similar to that observed in senescent cells. Recently, it has been reported that IR-induced chromatin alteration activates ATM, suggesting that the residual chromatin alteration may associate with persistent p53 activation. In this report, we examined whether p53 is activated by chromatin damage at the rejoining site of DNA double-strand breaks.
X-irradiation with 4 Gy induced SLGA in 40% and 60% of normal human fibroblasts 4 and 5 days after IR, respectively, while these cells rarely showed SLGA under an unstressed condition. In mitotic cells observed between 4 and 5 days after IR, chromosomal fragments and chromosomal bridges were observed. These mitotic cells with chromosomal aberrations had 2 or 3 phosphorylated histone H2AX foci, and the localization of the foci was both on the intact chromosomes and aberrant chromosome. Especially, frequency of the foci localized on those aberrations was more than 90 % in observed aberration. In the interphase cells 5days after IR, phosphorylated histone H2AX was observed about 4 foci per cell, and all the foci colocalized with phosphorylated ATM foci and phosphorylated p53 at Ser15. These results suggest that cause of persistent activation of p53 could be caused by residual chromatin aberration as well as DNA double-strand break itself.

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BRCT domain is a highly conserved domain found in many proteins involved in DNA damage checkpoint, DNA repair, and cell cycle control. PTIP protein carries 6 BRCT domains and forms nuclear foci after the irradiation with ionizing radiation (IR). To know whether PTIP acts in these pathways, we constructed chicken DT40 PTIP disruptants. Expression of PTIP was detected in DT40 cells. Chicken PTIP is located on chromosome 2, which is trisomy in DT40 cells. We disrupted three alleles of the gene and obtained two PTIP disruptants. Doubling time of these disruptants was 50% longer than that of wild type cells. This prolonged doubling time of disruptants was largely ascribed to the appearance of many disintegrated cells. Colony forming ability of these disruptants was less than 1 % of that of wild type cells. Expression of human PTIP complemented these phenotypes of the disruptants. Phosphorylation of CHK1 after the irradiation with IR, continued longer in the disruptant cells than in wild type cells. We also constructed conditional DT40 PTIP disruptant with Tet-off human PTIP expression system. At 24 hours after the addition of doxycycline (Dox), the conditional disruptant expressed human PTIP at slightly detectable level. These conditional disruptants with or without Dox treatment were exposed to IR and colony forming survivals were compared. LD10 of Dox treated cells was 0.8 of that of non-treated cells. These results indicate that PTIP protein is involved in DNA repair and signal transduction pathway of DNA damage.

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LyGDI inhibits the dissociation of GDP from Rho family GTPases and is found in abundance in hematopoietic cells (1, 2). Here we report truncation of LyGDI as a biological indicator for mice irradiated in whole body. Two LyGDI fragments, 21-kDa and 17-kDa truncated products, accumulated in thymus of the exposed mice at peak quantities between 5 and 12 h after irradiation at doses exceeding 1 Gy. The truncated 17-kDa fragment of LyGDI was constitutively expressed in thymus of non-irradiated mice, but its decreased expression levels were found in mice irradiated with extremely low doses. These data demonstrate that regulation of Rho family GTPases by LyGDI is perturbed during thymic apoptosis, suggesting that fragmentation of LyGDI is a useful biological indicator to detect animals who had exposed with ionizing radiation even at low doses.
This work was partly supported by the Japan Atomic Energy Research Institute. To Drs. Masao Inoue and Osamu Nikaido, we wish to express our deep appreciation of their encouragement. We are grateful for the expert patent application assistance of Mr. Kensuke Matsumoto.
1. Ota, T., Maeda, M., Suto, S., and Tatsuka, M. Mol Carcinog, 39: 206-220, 2004.
2. Zhou, X., Suto, S., Ota, T., and Tatsuka, M. Radiat Res, 162: 287-295, 2004.

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The mitogen-activated protein kinase(mapk) cascades, in which the major compornents are MAPK, MAPKK, and MAPKKK, are conserved in eukarytic signaling pathways. The MAPKs in each pathway are ERK, JNK, and p38. Recently, JIPs (JNK Interacting Protein) are identified as JNK scaffold proteins, through interacting with MAPKKK, MAPKK, and MAPK. We isolated and identified a novel protein kinase (STK-X)that interacts with JIP proteins. The role and substrates of STK-X is unknown. We examined the role of STK-X in JNK signaling and apoptosis, using dominant negative STK-X or siRNA transfected cells.

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Radiation has been reported to induce low-dose specific effects on cells. Adaptive response and low-dose hypersensitivity of some cells suggest that low-dose irradiation induces cell survival relating repair processes. However the relationship between cellular effects and molecular processes remains unknown in low-dose specific effects. Here we have tried to investigate low-dose specific cell survival induction using human breast cancer cell line (MCF7). 5 hour after the culture of MCF7 cells, the cells were irradiated at 0.1, 0.25, 0.5 and 1Gy. The cell survival rates of the irradiated cells decreased dose-dependently at 0.1, 0.5 and 1Gy. However, at 0.25Gy the cell survival rate increased significantly. Interestingly, irradiation 24 hour after the culture had no significant increase in cell survival at 0.25Gy. To study the molecular processes, phosphoproteome analysis was performed using the cells irradiated at 0.25Gy. Protein kinase C (PKC) is known to be involved in cell survival signaling cascade. Using a phospho-PKC substrate antibody, changes in PKC signaling cascade was studied as a phosphoproteome analysis in cell survival induction. Some of the substrates were phosphorylated after irradiation at 0.25Gy. Mechanisms of low–dose specific cell survival induction will be discussed with the data showing culture condition dependency.

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In mammalian cells, it is speculated that low LET radiation induces transcriptional regulators via signal transductions by diverse kinds of radicals, and that the regulators contribute to decrease cellular damages. The analysis of molecular mechanisms of the cellular responses is useful to develop the methodology for regulation of the damages. Seveal radiation responsive genes have been identified in human fibroblast by HiCEP method. Their radiation responsiveness were examined in RAW267.7 mouse macrophage cells using quantitative real-time RT-PCR. We found that fra-2 mRNA was increased 2.3-fold after x-irradiation at 4Gy as similar to that in the human cells. It has been reported that Fra-2, a member of Fos family protein, is ubiquitously expressed during the embryogenesis and also required for the chondrogenesis. For further investigation, we constructed a reporter gene that possesses the upstream region of fra-2 gene. RAW264.7 cells with transient introduction of the reporter gene were irradiated and the expression levels of reporter gene were measeured. The enzymatic activity and transcript level were increased at 6-fold and 1.9-fold, respectively. We are now studying the nucleotide sequence essential for X-ray response of fra-2 gene and the results will be discussed.

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Because of the accomplishment of the human genome project and development of molecular biological techniques including microarray analysis, it is now possible to investigate the common structure of radiation–responsive genes and genes involved in radiation sensitivity of mammalian cells. To reveal gene expression network after exposure to ionizing radiation, it is important to analyse and describe the common structure of the transcriptional regulatory region of ionizing radiation–resonsure genes promoter in the proximity of the mRNA start site. Jeong Sun Seo, Vivian G Cheung, Sally A Amundson, et al. have identified human genes responsive to gamma-rays by a microarray analysis. Here, we attempted to reveal a common structural character of the core promoter of 217 kinds of ionizing radiation–responsive genes by examining the frequency of TATA–box, initiator, GC–box, CCAAT–box element. We used the Match software with investigated the frequency of each factor under the same conditions (the optimum cutoff value and the search range). The frequency of TATA–box, initiator, GC–box and CCAAT–box were 35.50%, 94.90%, 88.94% and 44.70%, respectively, in the 217 genes responsive to gamma–rays. By comparing with promoters of randomly selected genes, TATA–box was ascertained that there isn’t a significant difference between two groups. However, the frequency of the initiator was 10% higher than that in usual genes. Frequencies of GC–box and CCAAT–box were 8% and 20% lower, respectively.

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The biological effects of ionizing radiation on learning have been studied over 50 years. However, it is still unknown how ionizing radiation affects a neuron network itself, although it has been reported that the radiation-induced learning and memory impairments are caused by suppression of hippocampal neurogenesis¹⁾. The nematode Caenorhabditis elegans is known as a model organism of which function is associated with individual neurons to some extent. The present study therefore aims to investigate responses of two functions of a neuron network induced by gamma irradiation, i.e. chemo-attraction to NaCl, and food-NaCl associative learning²⁾ of C. elegans in the adult stage when neurogenesis does not occur.
The animals were irradiated with ⁶⁰Co gamma-rays (0, 100, 500 and 1000 Gy). Chemo-attraction to NaCl was not significantly inhibited by gamma-irradiation (< 500 Gy). The decrease of chemo-attraction linked with food-NaCl associative learning was significantly enhanced than that of non-irradiated ones (100 – 500 Gy, i.e. at the dose range of no effects on chemo-attraction).
These results suggest that the sensitivity to gamma-rays is different between neural circuits related with chemo-attraction to NaCl and food-NaCl associative learning, even in the same neuron network.
References:
1) Raber et al., (2004) Radiat. Res. Vol. 162(1) 39-47
2) Saeki et al., (2001) J. Exp. Biol. Vol. 204 1757-1764

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Recent investigations have established several experimental models for bystander effect of radiation and have accumulated evidences that revealed responses of bystander cells as well as candidates for bystander signals. However, further studies are obviously necessary to clarify significance of bystander responses in low dose radiation effect. In this study, we aimed to examine the effect of low LET radiation on normal human cells, especially focusing on responses of signaling molecules in bystander cells. Bystander signals were conveyed to unirradiated cells by medium transfer method, i.e., conditioned medium of normal human diploid cells (HE49) following 4h post-irradiation with 1Gy of gamma-ray (¹³⁷Cs source, 1Gy/min) was transferred to unirradiated HE49 cells. In 4h after medium transfer, the number of phosphorylated ATM focus in nucleus, visualized by immunofluorometry, was increased to approximately 30% over unirradiated cells. On cell membrane, receptor tyrosine kinase microarray exhibited 20-30% up-regulation of ErbB, PDGF alpha, and HGFR families in 30min after medium transfer. Furthermore, among MAP kinases, a transient and slight increase of ERK, followed by sustained increases in JNK and p38 were observed.These findings suggested that both DNA damage-dependent and extracellular soluble ligand/receptor-dependent pathway were activated in bystander cells. The biological significance of such marginal responses of signaling molecules is to be determined.

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Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of extracellular signal-regulated kinase (ERK) as indicated by its phosphorylation at Thr202/Tyr204. ERK1/2 became activated 2-5 min and 6 h after 0.5-10 Gy irradiation. Enhanced phosphorylation of ERK1/2 observed 2-5 min after irradiation was at the same level in the dose range of 0.1-10 Gy. A specific inhibitor of the epidermal growth factor receptor (EGFR) AG1478 inhibited IR induced activation of ERK1/2. However, in response to IR, EGFR did not become phosphorylated at Tyr992 and Try1045, and phosphorylation of Tyr845 and Tyr1068 was decreased 2-30 min after irradiation with increase at 6 h after irradiation. Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2), which positively regulates EGFR/Ras/Raf/ERK signaling cascade, became activated 2 min and 2-4 h after irradiation as indicated by its phosphorylation at Tyr542. Src and protein tyrosine phosphatase α (PTP α) became activated 1-6 h after irradiation as indicated by their phosphorylation at Tyr789 or Tyr426 respectively. These results suggest that ERK1/2 activation by IR is mediated through activation of SHP-2 and EGFR.

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Angiogenesis is required for the growth and progression of malignancies. Recent studies have shown that the p53 gene is one of the factors for determining cellular radiosensitivity. Previously, we have reported that the cellular radiosensitivity of wild-type (wt) p53 cells was higher than that of p53-deficient or mutant (m) p53 cells. We have also found that the radiosensitivity of wtp53 tumors was higher than that of mp53 tumors. These findings suggest that the induction or suppression of tumor environmental factors due to a p53-dependent manner may contribute to the kinetics of tumor regrowth after irradiation. However, the correlation of p53 functions and the induction of VEGF by irradiation remains unclear. In the present study using human glioblastoma cells, we found that the accumulation of VEGF was induced by proton beam irradiation in the mp53 cells, but not in the wtp53 cells. We will discuss the contribution of p53 gene to anti-tumor effects of proton beam irradiation.

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In order to clear the characteristics of heavy-ion beams cells, the p53 induction by heavy-ion beam irradiation was compared to the results from reference irradiation with -rays. As well known, p53 is important protein at cell-cycle control or apoptosis in response to DNA damage or other stress. Recently protein CARF is identified as a protein having deep relation to p53. In this study, there is another interest on the interaction of p53 and CARF when the cells are irradiated with heavy-ion beams. U2OS cells were irradiated with 220 MeV 20Ne7+ beams at the beam line of TIARA AVF-cyclotron. The value of LET of the beam was 430 keV/m. As a reference experiment, UV irradiation was done with a germicidal lamp. Irradiated samples were kept in incubator for various hours after irradiation and analyzed, such as immunostainig, Western blotting and immunoprecipitation. The induction of p53 was clearly observed in both cases of irradiation with Ne-ion beams and UV. The amount of p53 induction was the maximum at 24 hours after irradiation. On the other hand, the induction of protein CARF was not clear in the case of irradiation with Ne-ion beams, while it was clearly shown with UV irradiation. Furthermore, the agreement of the localization of p53 and CARF was observed undoubtedly in the case of irradiation with ultraviolet, though the co-localization of these proteins was not seen in the case of irradiation with Ne-ion beams. Some differences in the interaction of p53 to CARF may exist between the case of irradiation with Ne-ion beams and UV.

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Recently radiation biology using microbeam is developing in the world. In this study, abnormal cell division in cultured human cells induced by X ray microbeam is investigated using synchrotron radiation as a microbeam radiation source. Irradiation with microbeam X rays on each cell enabled us to analyze the cell division in M phase minutely and in detail.
HeLa cells with EGFP Aurora B protein were used all through this study. The EGFP Aurora B protein can indicate the each stage in M phase of each cell. The irradiation was carried out at Beamline–B27 of PF in KEK. The energy of the beam was 5.35 keV, and the beam size was 10 µm square. The irradiation dose rate was about 20 Gy per min. Each cell was irradiated with 10 Gy X rays at various stages in M phase. The cells under fluorescent microscope were set at 35±1 °C using special heating system. Thus the cell cycle progress could be normally observed and progress of irradiated cell also could be observed easily.
Results was that 10 Gy irradiation to cells before metaphase induced the cell cycle arrest at metaphase. The elapsed time of each stage except for metaphase was not significantly different from unirradiatted HeLa cells. Cells irradiated after anaphase progressed their cycle at normal speed. From these results, it is suggested that the existence of mechanism to arrest the cell cycle at metaphase caused by irradiation of X rays. Now, we are researching about mad2 to evaluate the relation between mechanism of metaphase arrest and spindle assembly checkpoint.

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Objective: In order to elucidate the effects of radiation on centrosome hypeamplification, we examined the centrosome duplication cycle in KK47 bladder cancer cells following irradiation.Methods: KK47 cells were irradiated with various doses of radiation and were examined for centrosome hyperamplification by immunostaining for -tubulin.Results: Nearly all control cells contained one or two centrosomes, and mitotic cells displayed typical bipolar spindles. The centrosome replication cycle is well regulated in KK47. Twenty-four hours after 5-Gy irradiation, ~80% of irradiated cells were arrested in G2 phase, and at 48 h after irradiation, 56.9% of cells contained more than two centrosomes.The present study had two major findings: 1) continual duplication of centrosomes occurred in the cell cycle-arrested cells upon irradiation, leading to centrosome amplification; 2) cytokinesis failure was due to aberrant mitotic spindle formation caused by the presence of amplified centrosomes. Abnormal mitosis with amplified centrosomes was detected in the accumulating G2/M population after irradiation, showing that this amplification of centrosomes was not caused by failure to undergo cytokinesis, but rather that abnormal mitosis resulting from amplification of centrosomes leads to cytokinesis block.Conclusion: These results suggest that centrosome hyperamplification is a critical event leading to chromosomal instability following exposure to radiation.

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