We reported that the yields base lesions which are recognized by base excision repair enzymes such as Nth and Fpg (glycosylases) strikingly decreased with increasing level of LET value of radiation (this conference in 2006). Multiply induced DNA damages composed of SSBs and base lesions (clustered damage) by high LET radiation are thought to compromise the enzymatic activities. One of the goals of our study is to clarify the structure of clustered DNA damage. Many studies using plasmid DNA, however, does have a technical limitation. The number of SSBs in a plasmid DNA molecule would be underestimated since these SSBs including enzymatically induced ones will not cause additional conformational changes if they are on the same strand or on the opposite strand but separated each other sufficiently (>6 bp) so as not to induce a DSB. In order to observe these secondary invisible SSBs, we have developed a novel technique using DNA denaturation by which irradiated DNA is analyzed as single strand DNA (SS-DNA)(this conference in 2007). Fully hydrate pUC18 plasmid DNA was exposed to He ion beam from (82 keV/m) TIARA (JAEA-TAKASAKI). After denaturation treatment at 37 C using formamide (50% v/v) for 5 min, remaining fraction of intact SS-DNA was analyzed by gel electrophoresis (1% agarose) and quantified by staining with EtBr. Obtained result shows that multiple SSBs in both DNA strand are hardly induced by high LET irradiation. This indicates that clustered DNA damage would be mainly composed of one SSB and one (or multiple) base lesion(s).

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An X-ray microbeam irradiation system using synchrotron radiation has been developed at the Photon Factory, KEK, Japan. By using this system, localization of the proteins related DNA repair in cells irradiated with subcellular-sized X-ray beam was investigated. GFP (green fluorescent protein) is useful to visualize the localization of the protein in "living" cells. We constructed CHO cells carrying GFP-tagged Rad51, which is involved in the homologous recombination, one of the main pathways for DNA double strand break repair. After microbeam irradiation, GFP-Rad51 foci were observed with a laser confocal microscope.
Within 1 hour after irradiation of X-ray microbeam, the dose of which was equivalent to 2-5 Gy, fluorescent foci of GFP-Rad51 could be observed at the microbeam-irradiated site in the significant proportion of the irradiated cells. After microscopic observation at 1hr after irradiation, cells were fixed and stained by propidium iodide (PI) to quantitate DNA contents for cell cycle determination. Vast majority of the cells without foci at 1 hour after irradiation was in G1 phase.
Time course of GFP-Rad51 foci formation and disappearance was observed. At 40min after irradiation, GFP-Rad51 foci started appearing in S/G2/M cells. The numbers of the foci increased (~2 hrs) and then decreased (~8 hrs). At 12 hrs after irradiation or later, the foci in G1 cells started appearing.

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In eukaryotes, the MSH2-MSH6 hetero dimmer initiate the repair of base-base and small insertion or deletion mismatches while the MSH2-MSH3 hetero dimmer recognize larger insertion or deletion mismatch. By disrupting MSH2, MSH3 or MSH6 in chicken DT40 cells we generated msh2^-/-, msh3^-/- and msh6^-/- cells respectively. Msh2^-/- cells exhibit hypersensitivity to nucleotides damage by UV irradiation similar extent to translesion DNA synthesis DNA repair (TLS) deficient rad30^-/- cells. Conversely, msh3^-/- or msh6^-/- deficient cells exhibit similar sensitivity as wild type cells to UV. The MSH2 deficient hypersensitive phenotype was synergistically increased by concomitant deletion of major NER gene, Xpa. Interestingly, in contrast to the XPA deficient cells, the removal of UV damages, pyrimidine dimmers or 6-4 photoproducts in MSH2 or RAD30 deficient cells were not reduced after 6hrs. These results suggest that MSH2 protein recognize UV damages in the process of post replication like the TLS pathway.

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Ionizing radiation induces oxidation of DNA precursor dNTPs in the nucleotide pool. The oxidized dNTPs can be incorporated into nascent DNA during DNA replication, resulting mutations and cell death. Previously, we have shown that incorporation of oxidized dNTP may be involved in Y-family polymerases. In particular, human polymerase η (hPolη) exhibited the activity to incorporation of 8oxodGTP comparable with normal dNTP. To elucidate the mechanism for incorporation of oxidized dNTP by hPolη, the enzymes replacing R61 were prepared and their activities were assessed. R61 might lie adjacent to the dNTP in hPolη structure inferred from the crystal structures of yeast Polη and DNA-Dpo4, an archaeal homologue of DinB. R61K exhibited the different specificity from wild-type for incorporation of oxidized dNTP; R61K elevated the activity to incorporated 8oxodGTP opposite dC and 8oxodATP opposite dT, although the specificity for incorporation of 2OHdATP was not different from wild-type. The activity bypassing 8oxodG or 8oxodA in the template strand did not differ between R61K and wild-type. These results indicated that R61 in hPolη is liable to induce the mutation by restricting to incorporate C8-oxidized dNTP.

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Homologous recombination is an essential mechanism in mitosis and meiosis. Meiosis-specific proteins are not expressed in normal somatic cells. However, accumulating evidence suggests that some meiosis-specific proteins, including those involved in homologous recombination, are expressed in tumors of non-germ cell origin. Although these meiosis-specific proteins regulate chromosomal dynamics in meiotic cells, their role in somatic cells is unknown. Here we show that the meiosis-specific synaptonemal complex protein SYCP3 was ectopically expressed in human cancers from various tissue origins, and induced aneuploidy and hypersensitivity to ionizing radiation in immortalized epithelial cells. In somatic cells expressing SYCP3, increases in gamma-H2AX foci and a decrease in radiation-induced Rad51 foci formation were observed. These observations suggest that aberrant expression of SYCP3 in somatic cells might induce chromosome instability and hypersensitivity to ionizing radiation by prohibiting the intrinsic homologous recombination pathway.

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DNA double-strand breaks (DSB) are a serious damage, which can lead to genomic instability and/or cell death. However, mammalian cells are able to immediately recognize and repair DSBs, once they are generated in the genomic DNA. Since genomic DNA is compacted into chromatin structure together with histone proteins, in higher eukaryotic cells, this structure might be remodeled to recognize and repair the damaged DNA site, with specific modifications of histones. It has been shown in the budding yeast , S. cerevisiae, that histone H2B is ubiquitinated by Bre1 E3 ubiquitin ligase, and it is required for cell cycle checkpoint and DNA repair. However, the function of RNF20, the human homolog of Yeast BRE1, as well as H2B monoubuquitination by it, in DNA damage response, is poorly understood. Herein, it was found that H2B monoubiquitination by RNF20 is important for DNA repair. Reduction of RNF20 by RNAi showed MMC and IR sensitivity. Yeast two hybrids experiment showed the direct binding of RNF20 with NBS1. This is confirmed by observation that RNF20 is present in immunoprecipitates from cell extracts with NBS1. These result indicated that ubiquitination of histone H2B by RNF20 is required for Homologous recombination after exposure to IR.

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Clustered DNA damage, defined as two or more lesions within one to two helical turns of DNA by a single radiation track, is a unique feature of ionizing radiation. We are currently focusing on the biological effects of non-dsb type of clustered damage in vivo, whose effect has remained largely unknown. We have chosen an experimental approach that utilizes synthetic DNA containing base damage as a model of radiation induced clustered damage. One of the advantages of this strategy is that the effect of specific configurations of clustered damage could be examined in detail. Using a bacterial plasmid-based assay, we have investigated the biological consequences of bistranded clustered damage sites which consist of a single strand break (SSB) and an apurinic/apyrimidinic (AP) site. Firstly, plamids were ligated with oligonucleotides with clustered lesions. Following transformation of the ligated plasmids into wild type strain of Escherichia coli, both the transformation efficiency and the mutation frequencies were measured. We found a significantly lower transformation frequency for the clustered SSB + AP lesions than that for either a single SSB or a single AP site, which implies that a double strand break is formed during the processing of the cluster. Also, significantly higher mutation frequencies were observed for the clusters compared to those for singly damaged sites. When the two lesions were very close to each other, almost every plasmid possessed a mutation. The major types of mutation induced by the cluster were AP:C to C:G and a 1-bp deletion at the position of the AP:C pair. These results show a clear contrast to the results from clusters comprised of base lesions. We suggest that the biological consequences of clustered DNA damage strongly depends on the type of the comprising lesions.

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[Purpose]
Reactive oxygen species formed endogenously and by mutagens as ionizing radiation produce damaged DNA precursors (deoxyribonucleotides). The hydrolysis of the damaged DNA precursors before their incorporation into DNA by nucleotide pool sanitization enzymes would prevent the mutagenesis by the damaged DNA precursors. Mammalian MTH1, MTH2, and NUDT5 hydrolyze 8-hydroxy-dGTP or 8-hydroxy-dGDP in vitro, and are expected to function as nucleotide pool sanitization enzymes. However, it is unclear whether they actually prevent the mutagenesis by 8-hydroxy-dGTP/8-hydroxy-dGDP. In this study, we examined roles of MTH1, MTH2 and NUDT5 in suppression of the mutations induced by 8-hydroxy-dGTP in living cells by their knock-downs using siRNAs.
[Methods]
siRNAs against MTH1, MTH2, and NUDT5 and shuttle plasmid DNA containing the supF gene were transfected into human 293T cells. After 24 hr, 8-hydroxy-dGTP was introduced by means of osmotic pressure. After 48 hr, the plasmid amplified in the cells was recovered and transfected into E.coli KS40/pOF105 cells and supF mutant frequencies were calculated.
[Results]
Triple knock-downs of MTH1, MTH2, and NUDT5 increased frequency of the mutation induced by 8-hydroxy-dGTP by two-fold. The knock-downs promoted occurrence of A:T  C:G transversion. We are analyzing effects of knock-down of each protein on the mutation.
[Conclusion]
MTH1, MTH2, and NUDT5 are involved in the suppression of the mutations induced by 8-hydroxy-dGTP as nucleotide pool sanitization enzymes in mammalian cells.

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Post-replication repair pathway (PRR) protects cells from a wild variety of DNA damage. Translesion DNA synthesis (TLS) in one of sub-pathway of PRR, in which a number of non-essential DNA polymerases is recruited to the 3'-ends and extends it beyond the lesions, rescues stalled replication. In eukaryotes, RAD6-RAD18 dependent mono-ubiquitination at the lysine 164 residue of proliferating cell nuclear antigen (PCNA) is deemed to play a key role in regulation of TLS. Indeed, RAD18 deficient human cells exhibit sensitivity to ionizing radiation, indicating a functional role of PRR for protection against ionizing radiation. In this work, we analyzed molecular mechanism for PCNA mono-ubiquitination using reconstituted system in vitro. The results demonstrated that PCNA molecules loaded on DNA by RFC are target of RAD6-RAD18 for ubiquitination.

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[Objective] Aprataxin is a protein causative for the autosomal recessive ataxia EAOH. This protein binds to XRCC1, a scaffold of DNA single strand break (SSB) repair proteins. We intended to clarify the functional involvement of aprataxin in SSB repair (SSBR), a repair pathway for oxidative DNA damage, and to assess interaction to PCNA, another scaffold repair protein. We also studied susceptibility of EAOH fibroblasts to oxidative stress and protective effects of various antioxidants. [Methods] We used XPA-UVDE cells that repair ultraviolet (UV)-induced DNA damage exclusively via SSBR. XPA-UVDE cells were transfected with aprataxin, XRCC1, and PCNA cDNAs and were focally irradiated by UV. Accumulation of these repair proteins was observed with a fluorescent microscope. Repair of DNA damage was also observed in XPA-UVDE cells with siRNA-mediated aprataxin knockdown. Binding of aprataxin to XRCC1 and PCNA was assessed by immunoprecipitation and GST-pulldown assay. EAOH fibroblasts were treated with l-buthionine-(S,R)-sulfoximine, a glutathione synthesis inhibitor that increased endogenous reactive oxygen species. Cell viability was then assayed with and without various antioxidant treatments. [Result] Aprataxin accumulated with XRCC1 and PCNA in SSB. Knockdown of aprataxin impaired DNA damage repair. Aprataxin was interacted with XRCC1 and PCNA, but N-terminal-deleted aprataxin markedly lost the binding to PCNA. EAOH fibroblasts were hypersensitive to oxidative stress and protected by various antioxidants. [Conclusion] Aprataxin together with XRCC1 and PCNA is functionally involved in SSBR, a repair pathway for oxidative DNA damage. Our results suggest that pathophysiology of EAOH mainly involves repair of oxidative-stress-induced DNA damage and that this disease can be treated with antioxidants.

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Nucleotide excision repair (NER) is a versatile repair pathway which counteracts the deleterious effects of various DNA lesions such as UV-damage and numerous bulky chemical DNA adducts that interfere with base pairing and obstruct DNA replication and transcription. There are two pathways in NER: global genome repair (GGR) that surveys and removes the DNA damage on entire genome and transcription-coupled repair (TCR) that focuses on damage that blocks RNA polymerase IIo in transcription elongation. XAB2 (XPA-binding protein 2), consisting of 15 tetratricopeptide repeats (TPR), has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A (XPA) protein, which has a crucial role at an early stage of NER. To further analyze the function of XAB2, we purified XAB2 as a multimeric protein complex from HeLa cells. The XAB2 complex consists of six subunits (hAquarius, XAB2, hPRP19, p50, hISY1 and PPIE) which appear to be involved in pre-mRNA splicing based on the function of yeast orthologues. Knockdown of XAB2 with siRNA in HeLa cells resulted in a hypersensitivity to killing by UV or mitomycin C, and a decrease of recovery of RNA synthesis after UV-irradiation, RNA synthesis itself and pre-mRNA splicing. The amount of XAB2 protein bound to RNA polymerase IIo or XPA was increased in the cells treated with DNA damaging-agents that are subjected to NER, indicating a DNA damage-responsive activity of XAB2. These results suggested that the XAB2 complex is involved in transcription elongation and pre-mRNA splicing, and in TCR by the recruitment of CSA, CSB and XPA to the DNA damage sites where RNA polymerase IIo is stalled.

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Oxidative stress is believed to increase the risk of lifestyle-related diseases, such as cancer and heart disease. To assess oxidative stress in vivo, 8-hydroxydeoxyguanosine (8-OH-dG) in DNA and urine is usually measured, as the most popular biomarker. Recently, 8-OH-Gua has also been measured, as a new oxidative stress marker in vivo. In this research, the utility of 8-hydroxyguanine (8-OH-Gua) as a radiation-induced oxidative stress marker was studied. Aqueous solutions of guanine and deoxyguanosine were irradiated with X- or γ-rays, and the levels of 8-OH-Gua and 8-OH-dG in the irradiated solutions were measured by HPLC-ECD. 8-OH-Gua and 8-OH-dG levels were increased dose-dependently by γ-ray irradiation up to 300 mGy. However, the dose-dependent increase of 8-OH-dG was suppressed at doses higher than 300 mGy. The 8-OH-dG in the aqueous solution was mostly decomposed by 10 Gy of X-ray irradiation. Furthermore, the formation of a new oxidation product was observed dose-dependently with X-ray irradiation of the dG solution. This new product eluted faster than 8-OH-dG on HPLC. On the other hand, the generation of 8-OH-Gua continuously increased up to 10 Gy of irradiation. In addition, very little decomposition of 8-OH-Gua was observed with 10 Gy of X-ray irradiation. These results suggest that 8-OH-Gua may be a useful marker of radiation-induced oxidative stress in vivo.

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Reactive oxygen species (ROS) are generated within cells by ionizing radiation via primary ionizing events as well as through secondary amplification systems including metabolic synthesis. ROS not only cause dysfunction of the target molecules but also perturb intra- and inter-cellular signal transduction pathways. To elucidate the contribution of ROS to the generation of DNA damage, elicited by ionizing radiation, we compared cellular responses to hydrogen peroxide (H₂O₂) and X-rays in an XRCC4-deficient mutant cell line generated by gene targeting and its parental human colon tumor cell line HCT116. XRCC4-deficient cells exhibited lower survival rates and more frequent induction of chromosomal aberrations than XRCC4-proficient parental cells after exposure to either H₂O₂ or X-rays. Since XRCC4 is a key component of the non-homologous end-joining, a predominant repair pathway for DNA double-strand breaks (DSBs), the increase in susceptibility of XRCC4-deficient cells to these insults must be attributed to the induction of DSBs. Consistently, formation of γH2AX foci, a marker of DSBs, was observed in both cells exposed to H₂O₂ as well as X-rays. Curiously, most foci of γH2AX colocalized with ATM[pS1981] foci in X-irradiated cells, but did not in cells exposed to H₂O₂. In addition, sensitivity of cell cycle phases to the induction of chromosomal aberrations after the insults showed remarkable differences between X-irradiated and H₂O₂ treated cells. These results suggest the presence of different processes for DSB-induction by H₂O₂ and X-rays.

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Clustered DNA damage consists of multiple DNA damage in the limited region of DNA strand scratched by the beam of ionizing radiation, as a specific form of radio-induced DNA damage. For its complex structure, clustered DNA damage is thought to strongly inhibit DNA replication and persistently resist the repair activity. However, there are little knowledge about its biological impact in radiation effect. Therefore, we analyzed the yields of clustered DNA damage in the isolated DNA molecule and the chromosomal DNA in Chinese Hamster Overy cell irradiated with various accelerated ion particle beams including carbon (13 keV/μm), silicon (55 keV/μm), iron ions (200 keV/μm), and also gamma-rays (0.2 keV/μm) as a control. The yield of clustered DNA damage were estimated by conventional and pulse-field gel electrophoresis with appropriate DNA repair enzymes for detecting base damage clusters. The results showed that the yields of clustered DNA damage decreased in both irradiated isolated DNA and chromosomal DNA in irradiated cells with an increase in LET (J. Radiat. Res., 49: 133-136, 2008). The additional experiments with binary filters indicated that the inverse correlation between the yield and LET was attributed to the LET but not to the radiation quality. Thus, the quantitative factor of clustered DNA damage seems to be not important to the effect of higher LET-radiation. We need to consider another aspect of clustered DNA damage such as implication of the quality factor for severity of higher LET radiation.

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DNA-protein crosslinks (DPCs), where proteins are covalently crosslinked to DNA, are ubiquitous genomic lesions. DPCs would impede the progression of replication and transcription machineries and also would impair the binding of repair proteins to the DPC site, thereby leading to lethal events of cells. Aldehyde compounds, alkylating agents, and platinum compounds are known to induce DPCs, but they also simultaneously induce other types of DNA lesions such as interstrand and intrastrand crosslinks, strand breaks, and base modifications to various degrees. Accordingly, the extent to which cell death by these agents was dependent on DPCs remains unknown. To elucidate the relationship between the efficiencies of DPC formation and cell killing by these agents, we have assessed the cytotoxic effects of selected aldehyde compounds (formaldehyde (FA), acrolein (ACR)), alkylating agents (mitomycin C (MMC), melphalan (L-PAM)), platinum compounds (cisplatin (cis-Pt), oxaliplatin (L-OHP)), a DNA methylase inhibitor (5-aza-2'-cytidine (azadC)), and a topoisomerase II inhibitor (etoposido (VP-16)). HeLa cells were treated with these agents, and cytotoxic effects were measured by colony formation. The cytotoxicity was compared on the basis of 10% cell survival. The cytotoxicity of the agents increased in the following order: FA (230 μM) < CAA (11 μM) < L-PAM (3 μM) = L-OHP (3 μM) < VP-16 (0.9 μM) < azadC (0.6 μM) < cis-Pt (0.3 μM) < MMC (0.02 μM) < ACR (0.016 μM). We are currently analyzing the amount of DPCs produced by these agents. The data will also be presented in the meeting.

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Intracellular distribution of 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been examined by the fluorescence-microscope observation of human lung carcinoma A549 cells exposed to X-rays or carbon-ions with an LET of approximately 13 keV/um. 8-OHdG was detected by the fluorescence antibody technique with the use of anti- 8-hydroxy-guanine antibody-FITC conjugates (Kamiya Biomedical. Co., USA). First, we used the Fenton-reaction system to determine the experimental conditions for the fluorescence antibody observation. Bouin's fixative solution gave the best results without a loss of antigenicity of 8-OHdG among other fixatives tested. Then we applied the experimental protocol to X- or carbon-ion irradiated cells. The results obtained from X-irradiation experiments demonstrated that 8-OHdG was significantly detected at a dose as low as 25Gy, which was drastically lower than that required for the case of the HPLC detection coupled with the electrochemical detector. The intracellular distribution of 8-OHdG seemed to be nearly uniform over the whole area of a nucleus. The preliminary experiments with carbon-ions showed the similar 8-OHdG distribution as the case of X-rays. Further experiments are planned to obtain dose and LET dependence of the 8-OHdG distribution.

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In cells that do not undergo apoptosis, unrapairable DNA double stand breaks (dsb), once generated by irradiation, might perpetuate for a long time. We wanted to see the dynamics of such unrepairable dsb. For this purpose, we used quiescent and exponentially growing normal human diploid fibroblasts (NHDFs) that were X-irradiated, and measured formations and clearance of gamma-H2AX/53BP1/ATM foci (repair centers). In the NHDFs, the repair centers were immediately formed in irradiated nuclei and faded away in a time-dependent manner within a few days, however, a few of them remained and perpetuated even in a long-term culture up to 8 weeks. Formations of such H2AX foci (unrepairable dsbs) increased with X-ray doses and they continued to retain 53BP1 and ATM proteins, indicating that the repair and check point signalings have been kept activated. The relationship between the number of unrepairable foci and X-ray doses seemed well correlated with cell clonogenic survival, which implies that the unrepairable damages indeed play important role in cell survival.

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Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are known as major DNA repair mechanisms involved in elimination of DNA double strand breaks (DSBs) induced by ionizing radiation. While NHEJ operates efficiently throughout the cell cycle, HR is efficient only in the S and G2 phases of the cell cycle. Recently, the existence of several pathways in NHEJ was revealed, and it was found that there are the DNA-PKcs/Ku-dependent pathway and the pathway requires artemis. Since roles of these two repair mechanisms have not been elucidated yet, we examined participation of these pathways to the repair of DSB induced by an introduction of restriction enzymes creating different types of broken ends. Normal human cells synchronized in G0 phase were introduced by restriction enzymes (Pvu II, 100 U) by electroporation, and DSBs were determined by immunofluorescence staining for anti-53BP1 antibody. We found the foci of 53BP1 in 90% of the nuclei at 1 hour after introducing restriction enzyme. Among those foci-positive cells, 60 % of them had plenty of foci that were uncountable, and 20 % had 3-20 foci, which were called Type III foci. The number of Type III foci increased with time after introducing restriction enzyme in accordance with the decrease of cells harboring multiple foci, indicating repair of DSB. To distinguish artemis dependent-repair cells were treated with ATM inhibitor KU55933(KU). Although the ratio of cells showing Type III foci was not different, the foci number per nuclei were more in cells treated with KU. Thus, it was indicated that blunt-end type DSBs require the activity of artemis.

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Various nucleocytoplasmic proteins are modified with O-linked N-acetylglucosamine (O-GlcNac). It is known that the post-translational modification is involved in localization, activation, and stabilization of proteins and regulates the phosphorylation of proteins at serine/threonine residues. Ataxia-telansiectasia mutated (ATM), which plays an important role in checkpoint regulation and apoptosis, is activated by double strand breaks (DSB) caused by ionizing radiation, through autophosphorylation at Ser1981. However, it is unclear whether ATM is modified with O-GlcNAc and if O-GlcNAc-ATM exists, how O-GlcNAcylation influences the functions of ATM. Therefore, in the present study we investigated the modification of ATM with O-GlcNAc and the effects of O-GlcNAc on the function of ATM.
Primary neurons were cultured from fetus cerebral cortex (E16) and exposed to X-irradiation (5Gy) on 7 DIV. After irradiation, whole cell lysate were analyzed by immunoblot or immunopresipitaion using anti-ATM, anti-phospho-ATM, and anti-O-GlcNAc antibody.
Since immediately after irradiation, phospho-ATM (Ser1981) increased significantly, it was suggested that ATM was activated by X-irradiation. However, the expressions of O-GlcNAc proteins were changed slightly compared with that of phospho-ATM by X-irradiation. We performed immunoprecipitation with anti-ATM antibody, and concluded that ATM is modified with O-GlcNAc.

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While ATM-dependent nuclear foci formation of checkpoint factors has been thought to be indispensable for the activation of DNA damage checkpoint, biological significance of the foci formation requires a quantitative analysis using the parameter which integrates foci number, foci area and density, and thus, reflects molecular number of foci forming factors. So far, no such foci study has been performed. Therefore, the aim of this study is to conduct a quantitative analysis of the foci parameter, which is necessary and sufficient for G2/M checkpoint induction. In the present study, the parameter is represented based on the digital images acquired from fluorescence microscopy, as the Sum Of Integrated Density (SOID), which is the sum of the Integrated Density (= foci area x average intensity) of each foci in each cell calculated by image analysis software. First, we determined the minimum dose of X-rays at which foci formation could be observed but G2/M checkpoint was not induced in normal human diploid cells. To evaluate G2/M arrest mitotic index was compared, and we found the minimum dose was 0.08 Gy. The average and maximum of SOID of phosphorylated H2AX foci in the cells which progressed to prophase at two hours after irradiation of 0.08 Gy of X-rays were 3846 and 10987, respectively. The minimal Integrated Density of each focus was about 1000. These results showed SOID could be quantitative index to evaluate inducibility of checkpoint by foci. Our results also indicated that SOID of foci have threshold for induction of G2/M checkpoint, which is approximately 11000.

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(Purpose) The thermal therapy is recognized widely as one of the cancer therapy. However, it is not known about a molecular mechanism of cell death by thermal stress. Recently, it is said that a main cause of thermal cell death is a DNA double-strand break by protein denaturation. ATM plays a central role in a DNA double-strand break repairing process. Therefore we performed examination about thermal sensitivity in normal cells and cancer cells used specific ATM kinase inhibitor, KU55933. (Methods) We used confluent normal human fibroblast cell; AG1522, and human osetosarcoma cell; MG63. Cells were soaked into hot water of 45 degrees for 20 minutes. We drew the surviving curve of irradiation only, irradiation and thermal stress, irradiation and KU55933, and irradiation and thermal stress and KU55933. On the other hands, we changed soaking time from 20 minutes to 60 minutes, and drew a surviving curve of thermal stress only, and thermal stress and KU55933. (Result and Discussion) In both examinations, the thermal sensitivity of normal cells and cancer cells used KU55933 was rose significantly. These results meant that ATM was associated with hyperthermia closely, and it seemed that it supported the opinion which assumed that cell death by hyperthermia was a DNA double-strand break.

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DNA-protein crosslinks (DPCs) generated by radiation and other genotoxic ‎chemicals are formidable obstacles to DNA replication and transcription, and ‎their persistence would lead to mutations and cell death. Thus, the repair or ‎tolerance of DPCs is essential for cells. Recently, we have shown that ‎nucleotide excision repair (NER) and homologous recombination (HR) are ‎involved in the repair and tolerance of DPCs, yet much remains to be learned ‎about how cells deal with these superbulky lesions. In this work, we utilized a ‎genetic approach to obtain further insight into the mechanistic aspects of NER ‎and HR of DPCs and to assess the roles of other repair mechanisms such as ‎base excision repair (BER) and translesion synthesis (TLS). We measured ‎the sensitivities of a panel of E. coli mutants to DPCs-inducing agents ‎including formaldehyde (FA) and 5-azacytidine (azaC). The priA mutant ‎displayed a marked sensitivity to FA and azaC, indicating a crucial role of the ‎PriA helicase in the restart of stalled replication forks following HR. In ‎contrast, other helicase mutants including priB, priC, and rep showed no to ‎only marginal sensitivities. The mutants of recJ and recQ which are involved ‎in the RecFOR-dependent HR pathway also showed marginal sensitivities to ‎FA and azaC. This observation is consistent with our previous finding that the ‎HR of DPCs proceeds exclusively through the RecBCD pathway. With respect ‎to TLS polymerase mutants, polB, dinB, and umuDC exhibited no sensitivity ‎to FA and azaC, eliminating their role in tolerance of DPCs. Regarding to the ‎excision repair, the mutant deficient in the Cho endonulease (a UvrC ‎homolog) showed moderate sensitivity to FA, suggesting a role of Cho in the ‎excision of DPCs. The mutants of TCR (mfd) and BER (nth/nei and xth/nfo) ‎were not sensitive to both DPC-inducing agents. The present study further ‎confirms the critical role of NER and HR and eliminates the roles of BER and ‎TLS in the repair and tolerance of DPCs. ‎

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The ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases, which are members of phosphatidyl inositol 3-kinase-like kinase family, play central roles in DNA damage checkpoints. ATM is primarily activated by DNA double-strand breaks (DSBs) caused by ionizing radiation or radiomimetic drugs. Activation of ATM is stimulated by the MRE11-RAD50-NBS1 (MRN) complex. On the other hand, in response to UV damage or other replication blocking stress, ATR is activated by TopBP1 and phosphorylates several substrates, including Chk1. In the NBS1 deficient cells, Chk1 phosphorylation after UV irradiation is impaired, suggesting that activation of the ATR-mediated pathway also requires NBS1. However, its precise mechanism is unknown. In this study, we investigated whether NBS1 is required for ATR localization to DNA damage sites after UV irradiation. Roles of NBS1 in the ATR pathway will be discussed.

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Most of the oxidized bases generated in mammalian genome under a physiological condition are repaired by base excision repair (BER) pathways. DNA glycosylases remove altered bases and form apurinic/apyrimidinic sites at the first step of BER. Mouse endonuclease VIII like glycosylase 1 (mNEIL1) is a DNA glycosylase against various oxidized pyrimidines, and also plays a key role during DNA replication. Two types of mouse variant mRNAs, NCBI AAH43297 (variant1) and NCBI BAC30707 (variant2), are detected in mammary gland tumors and in aorta/vein, respectively. Interestingly, there is no report about such variants in human tissues. To study significance of these variants, we prepared recombinant proteins of mNEIL1and these variants using the pET22b expression system. When we examined the DNA glycosylase activities against thymine glycol, a typical oxidized damage, no activity was detected. With polyclonal antibody developed against recombinant mNEIL1, the variants as well as NEIL1 were detected in liver extract. To study the expression in mouse tissues, we prepared primer sets to amplify specifically the cDNAs of mNEIL1 or variants and performed RT-PCR using total RNAs prepared from Jcl:ICR mouse kidney and liver. Since apparently more abundant expressions of variant mRNAs were observed in kidney, suggesting that there are differences among the mRNA levels in normal mouse organs. The analyses of the mRNA levels in various mouse organs and the enzymatic activities against a series of altered pyrimidines are in progress.

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Ionizing radiation, UV light, and a certain class of mutagens irreversibly trap proteins on the DNA strand and induce DNA-protein crosslink (DPC) lesions. DPCs are extremely bulky as compared to oxidative base damage and UV-induced pyrimdine dimers so that they would impede the progression of replication as well as transcription machineries, thereby exerting adverse effects on cells. The presence of DPCs as DNA lesions has been long known, but it has been largely unknown how they are repaired in cells. Recently, we have analyzed the repair mechanism of DPCs in vitro and in vivo using E. coli as a model system and shown that nucleotide excision repair (NER) and homologous recombination (HR) work cooperatively to cope with the genotoxic effect of DPCs. However, it remains elusive whether DPCs are also processed in a similar manner in eukaryotic cells. In the present study, we analyzed the repair mechanism of DPCs in vitro and in vivo using mammalian cells. When model DPC substrates were incubated with cell-free extracts from HeLa cells, a dual incision indicative of NER occurred on the 5´ and 3´ sides of DPCs. The incision efficiency decreased with increasing the size of crosslinked proteins (CLPs). The upper size limit of CLPs amenable to NER was 8-10 kDa, which was smaller than that observed for E. coli NER. We also isolated chromosomal DNA from cells treated with formaldehyde, a typical DPC-inducing agent, and analyzed the removal of DPCs by SDS-PAGE. The rates of removal of DPCs were similar for wild type and NER-deificient XPA cells. These in vitro and in vivo data suggest that unlike in bacteria cells, NER is not involved in the repair of DPCs in mammalian cells. We are currently assessing the role of HR and will present the result in the meeting.

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Ionizing radiation induces DNA double strand breaks (DSBs). Activated ATM phosphorylates H2AX, one of a chromatin protein, and phosphorylated H2AX forms focus at DSBs site. Phosphorylated H2AX foci are regarded as a recruiting mediator of repair factor of DSBs. Although most of phosphorylated H2AX foci disappear with repair of DSBs, a few foci are growing to large and remaining a few days after radiation. However, biological significance of residual foci is not clear. Therefore, we examined correlation between residual large foci and cellular radiosensitivity after ionizing radiation.
We examined dynamics of number and size of phosphorylated H2AX foci in X-irradiated human normal diploid fibroblast cells (HE49 and BJ cells) and human tumor cells (HeLa, U251, T24 and H1299). As a result, unirradiated cells had few large foci. On the other hand, most of cells, which were irradiated with a 0.1% survival dose, had phosphorylated H2AX foci 5 days after irradiation. And these cells stopped their cell division after irradiation.
These results indicate that remained large phosphorylated H2AX foci show loss of proliferating potential after irradiation. Therefore remained foci and cellular radiosensitivity had a close correlation. This suggests that we can use residual phosphorylated H2AX foci to estimate therapy effect of radiotherapy treatment of cancer.

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There are two pathways, in which DNA double strand breaks (DSBs) are rejoined by non-homologous recombination and homologous recombination (HR).Nijmegen breakage syndrome(NBS) is genetic disease characterized by ionizing radiation sensitivity and genome instability. NBS1, gene responsible for NBS, is reported that it is important factor for HR repair. NBS1 forms complex with Mre11 and Rad50 (MRN complex) and involved in sensor, signaling, and repair of DSBs. On the other hand, SbcC/SbcD/Xrs2 complex, homolog of the MRN complex, is also involved in a wide variety of DNA repair and has sensitivity to alkylating agent, suggesting the role of MRN complex in repair of base damage. Using NBS1 mutant cell line, we have examined whether NBS1 functions in a response to alkylating agents.

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Nijmegen breakage syndrome (NBS), characterized by high sensitivity to ionizing radiation and predisposition to lymphoid cancer, is phenotypically similar to Ataxia telangiectasia. It is well known that NBS1, the protein responsible for NBS, is cooperative with ATM for IR-induced DNA damage response such as double-strand break repair and cell cycle checkpoints. Recently, it was also reported that NBS shares common clinical signs with ATR-defective Seckel syndrome, such as microcephaly and NBS1 activates ATR. Since ATR functions in stalled replication fork after HU treatment or UV exposure, NBS1 might have potential roles in a response to UV damage. However, the function of NBS1 at S phase is unknown. Our result showed that NBS cells were high sensitivity to UV and NBS1 accumulated in the stalled replication fork. Moreover UV-induced PCNA mono-ubiquitination and pol eta foci were not observed in NBS cells. These results indicated that NBS1 might promote the UV damage response.

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It has been accepted that most of oxidized pyrimidines formed in mouse genome were exclusively removed by two bifunctional DNA glycosylases, mNTH1 and mNEIL1. Since EGFP-tagged mNTH1, unlike the human homolog, was localized mostly in mitochondria, it was considered that mNEIL1 had a major contribution toward the repair of oxidized pyrimidines in mouse nuclei. Recently, a monofunctional DNA glycosylase, SMUG1, was revealed in nuclei of mammalian cells. The DNA glycosylase removes a group of oxidized pyrimidines, such as 5-hydroxyuracil and 5-formyluracil (fU). Since many monofunctional DNA glycosylases remove a variety of altered bases in mammalian cells, the presence of SMUG1 seems to be reasonable. However, SMUG1 does not remove thymine glycol (TG), 5,6-dihydrothimine(DHT), 5-formylcytosine (fC), and no monofunctional DNA glycosylase against these altered bases has been discovered so far. We found a novel monofunctional TG-DNA glycosylase (TGG) activity in nuclei from several mouse organs. We prepared a nuclear extract without most of mNTH1 and mNEIL1 from mouse organs by a modified high-salt extraction method. We further separated the monofunctional TGG activity from most of apurinic/apyrimidinic (AP) lyase activities with column chromatography. The monofunctional TGG activity within the fraction was determined using radiolabeled oligonucleotide substrates containing TG or AP site. The monofunctional TGG activity removed not only fU, but TG and DHT, which are not removed by SMUG1. Under our reaction condition, an activity against fC was not detected.

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The abnormal radiosensitivity of Ataxia Telangiectasia cells is ascribed to the results of defective double-strand DNA breaks (DNAdsb) repair or enhanced misrepair. DNAdsb are repaired by homologous recombination, Ku-mediated nonhomologous end joining and 53BP1-mediated nonhomologous end joining. Although ATM is considered to contribute to homologous recombination, there are controversial reports on ATM involvement in nonhomologous end joining and also that ATM does not contribute to homologous recombination of I-SceI induced DNAdsb. Thus, ATM association with DNA repair pathways still remains unclear. In this study, we examined ATM relation to three pathways using a selective ATM inhibitor and RNAi knockdown of 53BP1 in Ku70 defective cells.
The increased radiosensitivity was observed after treatment with ATM inhibitors perhaps due to the decreased DNA repair capacity. 53BP1 knockdown cells showed the higher clonogenic survival presumably due to the increased homologous recombination. 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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To analyze mutagenic effects of ionizing radiation in higher plant, seeds of transgenic Arabidopsis carrying rpsL gene were irradiated with carbon ions (LET=112keV/μm) and gamma-rays. The doses were adjusted to give the same effect on survival reduction. Both carbon-ion and gamma-ray irradiation resulted in a 2.6 times increase of mutant frequency. The mutant frequency per unit dose was higher in carbon ions than in gamma-rays. Mutation spectrum showed that the carbon ions and gamma-rays frequently induced G:C to A:T transition and deletions. In addition, the carbon ions induced complex-type mutations and the gamma-rays induced frameshift mutations with relatively higher frequency. The G:C to T:A and A:T to C:G transversions, which are caused by oxidized guanine, were hardly found in our experiment, although those are major mutations induced by gamma-rays in many other organisms. The difference in cellular environment between seeds and proliferating cells may affect radiation-induced mutation spectrum.
Carbon ions with shallower penetration depth that give a maximum LET in the seeds had higher lethal effect pet unit dose than the carbon ions that penetrates the seeds. However, mutant frequency was remained at control level. High-LET radiations are known to induce chromosomal rearrangement such as large deletion, inversion and translocation. Since the rpsL mutation analysis system only detects intragenic mutations, our result suggests that the carbon ions near the Bragg peak less frequently induce small intragenic mutations in the Arabidopsis dry seeds.

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Mutagensis plays an important role for carcinogenesis and aging. Mutation is introduced into genome DNA through two different pathways; damage-induced mutagenesis and spontaneous mutagenesis. The former type of mutation occurs by replication through damaged DNA and the latter occurs by replication error. Mismatch repair (MMR) pathway is involved in both pathways by repairing mispairing bases. Furthermore, mispairing base is recognized by MMR protein in a process that is a potent signal for apoptosis. To investigate the molecular mechanism of spontaneous and damage-induced mutagenesis in vivo, we tried to generate MMR gene knockout mutant Medaka using TILLING method. We have done screening of mutations in Exo1 and Msh2 genes and have identified 2 non-sense mutations in Exo1 gene and 1 non-sense mutation in Msh2 gene. Summary of the screening and the phenotype of these mutants will be discussed.

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The ion beam is expected to increase the mutation frequency and wide spectrum, since it has the high linear energy transfer (LET), and the breeding technology using the mutations induced and cancer therapy by the ion beam has been greatly developed. However, the detailed molecular mechanism has not been proven considerably. S. cerevisiae strains used in this study are S288c (RAD+), ogg1, msh2. OGG1p is a DNA glycosylase / AP lyase that excises guanine lesions such as 8-oxoguanine (8-oxoG). MSH2p is the mismatch repair (MMR) protein that mediates DNA repair through the recognition of 1 and 2-bp mismatches. The yeast strains were irradiated with carbon ions (¹²C⁵⁺; 220 MeV) with the dose 10 to 100 Gy, and LET is 107 keV/µm. Carbon ion beams were generated from AVF cyclotron in JAEA. The mutation sites of ura3 mutants were determined by DNA sequencing. Our results show that the types of base changes in the carbon-ion induced mutants in wild type cells included GC to TA transversions (41 %), the other type of base substitution (41 %) and deletions/insertions (18 %). In the case of ogg1, GC to TA transversions were largely observed (70 %), but the deletion or insertion mutations were not obtained. GC to TA transversions are caused by misincorporation of 8-oxodG to DNA. These results in S. cerevisiae strongly suggest that the endogenous oxidative stress, due to the carbon-ion irradiation, induces the formation of 8-oxodG in DNA. In the msh2 mutant strain, the mutational spectrum exhibited a predominance of small deletions, with the mutations unequally distributed over the URA3 gene region.

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DNA double-strand breaks (DSBs) are repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) mechanisms. It has been revealed that NHEJ is the main repair pathway, and gene mutation and deletion are induced in the processes of DSBs repair (DSBR) in mammalian cells. However, these knowledge was mainly obtained by the studies in cultured cells, and the in vivo studies using individual animals are limited. The aim of this study is to establish the procedures to visualize DSBR using GFP as a repair indicator in medaka, Oryzias latipes. Here, we examined the two systems in medaka cultured cells as the first step for in vivo studies in medaka: 1) HR detection system using DR-GFP [Pierce et al, 1999], and 2) gene deletion detection system using Tetracyclin controllable expression vectors [Noda et al, The 49th, 50th Annual Meeting of JRRS]. We propose that it is possible to use both systems and visualize DSBR in medaka cultured cells from comparison of wild type and mutant medaka. By using the HR detection system, it revealed that DSBR via HR pathway was hardly caused in medaka cultured cells in similar way to other mammalian cells. A radiation sensitive mutant medaka cells showed remarkable decrease in HR repair, compared to wild type cells. And we are investigating the DNA sequence of the DSB sites after repair process. On the other hand, the stable cell lines were established, which showed increase in GFP fluorescence depending on the exposed dose using the gene deletion detection system. These systems would allow us to visualize DSBR and gene deletion in vivo by production of transgenic medaka.

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Isolated DNA damages such as single strand break (SSB) and base damage are restored efficiently in cells. These damages are thought to have little influence on cell death or mutation, and thus seen to have little relevance to biological consequences of radiation. However, it is proposed that dense ionizations with higher LET radiation induce closely spaced DNA damage, and results in clustered DNA damage sites including several with SSB and base damage. It has been reported that high LET radiation effectively induces cell death and mutation, most likely resulted from incompletely or incorrectly repaired DNA lesions. It is possible that the non-DSB clustered damage including SSB or base damage contribute to high biological effects, such as mutation. In this study, we investigated the mutagenicity of clustered DNA damage containing SSB and base damage in Escherichia coli. We used plasmid based assay in E. coli to measure the mutation frequency induced by bistranded clustered damage. As a model of clustered damage, we used synthesized oligonucleotides carrying an SSB and/or 8-oxo-7,8-dihydroguanines (8-oxoGs) at a restriction enzyme recognition site. Damaged DNA was transfected into wild-type or glycosylase-deficient strains (fpg, mutY and fpg mutY) of E coli and mutation frequency was assessed by the inability to cut by the restriction enzyme. The mutation frequency of clustered damage carrying a 8-oxoG opposite to a second 8-oxoG was the highest in all types of clustered damage tested. However, when a SSB was added to bistranded 8-oxoGs, the mutation frequency became lower in every E. coli strain. These results suggest that SSB located on the same strand to 8-oxoG reduces mutagenic potential of 8-oxoG and that the repair of the SSB or replication is involved in the process. Our studies demonstrate that the mutagenic potential of clustered damage including 8-oxoG depends on existence and position of SSB in clustered damage.

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We have previously found that hematopoietic cells of mice irradiated in utero or soon after birth showed surprisingly low translocation frequencies when mice were examined at age of 20 weeks. The results were based on 2-color FISH for detection of translocations involving only chromosomes 1 and 3. To exclude a possibility that the bone marrow cell pool is derived from a very small number of fetal stem cells after irradiation, and hence such partial FISH painting of the genome might have overlooked the true figure including clonal translocations, we proceeded to multi-color FISH (M-FISH) to confirm that the finding also holds true for the entire genome. Mice were irradiated at fetal stage (15.5th day p.c.) or 4-days of age with 2 Gy of X-rays, and metaphases were obtained from spleen T lymphocytes at the age of 18 - 21 weeks. M-FISH was applied to paint all chromosomes (#1 – #19, X and Y) in the genome for detection of translocations. Mice irradiated at fetal or neonatal stage showed lower translocation frequencies than that of their mother (0 - 9% vs. 25%). Clonal translocations were observed in mice irradiated as fetuses or neonate, but not in their mother carrying even much more translocations. The correlation between the genomic translocation frequencies measured by the M-FISH and 2-color FISH for the same animals showed good concordance. M-FISH analysis validated our previous findings based on 2-color FISH. We speculated that fetuses or neonates were capable of quickly eliminating cells carrying radiation-induced chromosomal damage, but survived hematopoietic stem cells or their progenitor cells, which happened to hold translocations, could clonally propagate in vivo during rescue period after irradiation.

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Ataxia telangiectasia-like disorder (ATLD) is the genetic disorder, which is characterized by high sensitivity to radiation, chromosome instability and aberrant cell cycle checkpoint. MRE11,the gene responsible for Ataxia telangiectasia-like disorder ,forms a protein complex with hNBS1 and hRAD50 , and functions in homologous recombination (HR) repair from DNA double strand breaks, which are elicited by ionizing radiation or other stresses. Therefore, the chromosome instability in MRE11 cells is considered to be due to defects in both DNA repair and cell cycle checkpoints. Now, we find MRE11 co-localize with centrosome and involved in centrosome regulation. Centrosome is the complex organelles comprising two microtuble-based centrioles surrounded by a protein matrix and other structural elements, a key regulator for chromosome separation in mitosis. Centrosome duplication and spindle formation are crucial for prevention of chromosomal instability. Therefore, the normal function of centrosome is essential for maintenance of genome stability. Recent studies suggest that BRCA1 is HR repair protein ,localizes to the centrosome in mitosis. Therefore , BRCA1-defective cells showed the abnormal duplication of centrosome . When we examined the localization of MRE11 by using a protein antibody, they showed to be accumulated in centrosomes. Moreover, MRE11 cells showed defect in centrosome amplification, suggesting an indispensable role of MRE11 in centrosome maintenance. We further discuss this novel role of MRE11 in centrosome maintenance.

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Nonhomologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1 phase of the cell cycle. Acute lymphoblastic leukemia cell line Nalm-6, like a chicken DT40 cell line, allows for high-efficiency gene targeting by homologous recombination. Thus, the Nalm-6 cell line has been used in many gene-knockout studies in human cells. Although Nalm-6 cells have been applied to establish multiple NHEJ factor-deficient cell lines, a major disadvantage is that Nalm-6 cells do not respond well to cell cycle synchronization using chemical agents, such as nocodazole and mimosine. This has prevented researchers from determining whether NHEJ-deficient Nalm-6 cells show increased IR sensitivity during the G1 phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by phosphorylated-gamma-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.

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It is well known that there is a cell-type-dependent apoptosis phenomenon in radiation induced apoptosis, like as the thymocytes and lymphocytes, which are known to be hypersensitive to radiation and exhibit interphase cell death. Contrary to those cell lines, many other human tumor cell lines which generally undergo necrotic cell death and show a delayed apoptosis following exposure to radiation. In order to identify the signaling regulators controlling apoptotic cell death, Jurkat (human leukemia) and HeLa S3 (human uncial carcinoma) were irradiated with 20 J/m2 of UV, after several hours post-incubation. Their cytosolic extracts were resolved with two dimensional gel electrophoresis (2DE). The different spots appeared in apoptotic and non-apoptotic cells in Jurkat and HeLa S3 cells cells were analyzed using a MALDI-TOF/TOF mass spectrometry. 12 proteins were identified, one of them was acidic ribosomal protein P2 (P2) which is known to be mainly located within the 60s ribosomal subunit and phosphorylated forms of P2 were detected in UV-irradiation Jurkat cells. Based on these observations, now we are trying to use in vitro and in vivo experiments to confirm the function relevance of the dephosphorylation of P2 in apoptosis sensitivity. All the data suggested that there is a regulator for the P2 phosphorylation and dephosphorylation and its function is associated with the activition of UV induced apoptosis.

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After the nuclear accident in Chernobyl, environmental effects of radiation have appeared remarkably in conifers such as pine trees in the surrounding forests. Depending on the exposed radiation levels, the conifers have showed growth inhibition, malformation of needles, reproductive loss and withering. For investigations of cellular mechanisms relating to the radiation damages, we assessed radiation–induced cell death in suspension cell culture of Japanese native conifer, Japanese cedar (Cryptomeria japonica). Histological examination of the irradiated cell culture with TUNEL method indicated development of cell death with nuclear DNA fragmentation, which is a typical feature of apoptosis in mammalian cells. The DNA fragments, however, was not detectable in conventional gel electrophoresis. Meanwhile, pulsed-field gel electrophoresis showed high molecular weight DNA fragments, larger than 5 Mb in size. This suggested that the apoptosis-like cell death in plant proceeded without oligonucleosomal DNA cleavage but with large chromatin breaks. The involvement of the apoptosis-like cell death in the radiation damages in plant organs will be also discussed in the presentation.

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We investigated the molecular mechanisms by which mouse pre-T ST4 and STH1a cells (gift of I. Radford) undergo the hyper-radiosensitive apoptosis. One pathway was the p53-driven upregulation of BH3-only Puma, Bim and Noxa, which activated Bax and Bak to release cytochrome c from mitochondria and activate the caspase-9—capsase-3/7 cascade. Another pathway relied on the caspase-2 activation via p53-mediated PPIDosome. Independent of the above pathway, caspase-2 was inactivated by VDVAD-CHO or caspase-2 siRNA. Rrather than IR-irresponsive caspase-8 in these cells, furthermore, active caspase-2 had a novel role for Bid cleavage to tBid, which plays a role in Cytc release for DNA damage-induced, p53-dependent apoptosis. Thus, the above two pathways are the molecular basis of hyper-radiosensitive apoptosis in ST4 and STH1a cells.

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We recently reported a novel suppressive effect of sodium orthovanadate (vanadate) on the DNA-binding activity of p53. In this study, we initially showed that vanadate had more potent antitapoptotic activity than other three chemical p53 inhibitors including pifithrin-alfa (PFT), a well-known inhibitor of p53. Although others inhibited the p53 transcriptional activity, they were not able to suppress p53-dependent apoptosis in irradiated MOLT-4 cells. To pursue the difference between the vanadate's effect and others, we chose PFT as a reference, and determined the effect of each inhibitor on p53-mediated apoptosis, especially focused on transcription-independent pathway. The following experiments revealed that vanadate showed more potent suppressive effect on p53-associated mitochondrial apoptotic events, such as the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the ubiquitilation and mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. In addition, vanadate was capable of suppressing the apoptosis-inducing activity of mitochondrially targeted p53 in temperature-sensitive SaOS-2 stable transfectants. Our data demonstrate that vanadate can also suppress the transcription-independent pathway, and suggest the possibility that inhibition of both the transcription-dependent and transcription-independent pathways is needed to sufficient suppression of p53-mediated apoptosis.

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Exposure to ultraviolet (UV) light causes several types of DNA damage in cells. Unrepaired lesions encountered by the DNA replication machinery during S-phase cause stalling of the replication fork, which may lead to cell death unless DNA synthesis resumes. Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at the UV irradiation-induced DNA damaged sites. Recently, it was demonstrated that RAD18-null HCT116 cells are sensitive to both X-ray irradiation and camptothecin, due to the defective repair of single-strand DNA breaks that arise during S- phase (Shiomi et al, 2007). These findings suggest that RAD18 is involved in the cellular response to DSBs via PCNA- and Polh-independent mechanisms. Therefore, in the present study, we investigated the regulation and function of RAD18 in response to DSBs. In cells exposed to DSB-inducing agents, many proteins involved in the DNA damage-response pathway, including g-H2AX, BRCA1, 53BP1, NBS1 and phosphorylated ATM accumulate in nuclear foci. These foci have been designated IR-induced nuclear foci (IRIFs). g-H2AX, which is immediately phosphorylated at its C-terminus in response to DSBs, plays a crucial role in regulating IRIF formation. IRIF formation is thought to be vital for cellular DNA repair and the activation of DNA-damage checkpoints. The function of RAD18 in IR-irradiated cells and the potential targets of its E3 ligase activity are unknown. Therefore, in the present study we sought to identify the role of RAD18 in the DSB response, and to determine the relationship between IRIFs and RAD18 foci.

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STK38 is a serine/threonine kinase, which is involved in the regulation of cell polarity and mitosis in yeast. However, the roles of STK38 is unclear in mammalian cells. We have recently found that STK38 plays an inhibitor of MAPKKK in stress-signal activated protein kinase cascades (Enomoto et al., Oncogene 2008). Furthermore, we have investigated the effects of various stimuli and the kinase inhibitor on the STK38 activity. As a result, wortmannin, a PI-3K inhibitor, suppressed oxidative-stress-induced activation of STK38. In this time, we provide a new cross-talk of stress-signal transduction pathways.

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Genetic studies have shown that under non-stressed conditions both MDM2 and MDMX are essential to maintain the tumor suppressor p53 in its latent form. It has also been shown that MDMX was rapidly degraded by MDM2 in response to DNA damage, a phenomenon which is critical for p53 activation. Thus, it is well documented that both MDM2 and MDMX play important roles for in the regulation of p53. However, the underlying molecular mechanisms of how these inhibitors cooperate and regulate p53 are still unclear. To gain insight into this question, we have studied using various chimeric proteins and demonstrated that MDM2 and MDMX can form a complex in vivo, and this heterocomplex had the ability to ubiquitinate p53 far more efficiently than MDM2 alone. Moreover, disruption of the binding between MDM2 and MDMX resulted in a marked increase in both abundance and activity of p53, emphasizing the functional importance of this heterocomplex in p53 control. In order to investigate further possible molecular mechanisms of the p53 regulation by MDM2 and MDMX, we are trying to develop an in vitro assay system in which ubiquitination studies of p53, MDM2 and MDMX can be performed. Here, we present data validating this assay system and report a novel mechanism by which the MDM2/MDMX heterocomplex regulates the p53 ubiquitination.

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Correlation of cellular radiosensitivity and cellular radiation responses with the levels of thioredoxin in HeLa cells A. Matsui, K. hashiguchi, Shuji Yonei, Konndo, Nomura and Q-M. Zhang-Akiyama Radiation and a variety of chemical agents generate reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide and hydroxyl radical within cells. ROS are also produced continuously during normal metabolic processes. ROS oxidize non-specifically various biomolecules, DNA, protein and lipid, giving rise to cellular oncogenic transformation and dysfunction. To protect cells against oxidative damage by ROS, cells have many types of defensive enzymes such as thioredoxin (TRX) and glutaredoxin (GRX), which eliminate intracellular ROS. These defensive enzymes also play important roles in maintaining cellular redox environment. Thioredoxins (TRXs) have a prominent function as an antioxidant by scavenging singlet oxygen or hydroxyl radical or through coordination with peroxiredoxin. TRXs also play regulatory roles in many kinds of cellular functions through protein-protein interaction. Human cells have two types of TRX; cytosolic TRX-1 and mitochondrial TRX-2. The expression levels of TRX-1 and TRX-2 well correlate with the cellular functions, and the different localization of these two TRX reflects the protection against different effects by oxidative stress. In this study, we investigate the correlation between the overexpression of TRX1 and TRX2 and the cellular effects of radiation and cellular radiation responses. Furthermore, we investigate the induction of TRX-1, TRX-2 and other cellular redox enzymes by radiation. We determined the levels of lipid peroxidation and DNA double strand breaks in irradiated HeLa cells with and without TRX-overproducing plasmid.

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Ionizing radiation (IR) is chosen as a primary treatment for various types of cancer. The popular protocol of cancer treatment consists of daily exposure to 2Gy of IR in total 50~90Gy. Thus, tumor cells receive multiple, fractionated and long-term exposures through the course of treatment. Although DNA damage response (DDR) after single-dose of IR is well documented, the biological effect of fractionated IR is not fully understood. Here we report that exposure of human tumor cell lines, HepG2 and Hela to fractionated 1 Gy/day of IR for long-term (more than 1 month) caused perturbation of G1/S checkpoint and accumulation of cells in S-phase. The expression level of the cyclinD1 protein was unchanged until day 14 of fractionated IR, whereas it was apparently elevated at day 31 of fractionated IR. Exposure to fractionated IR constantly activates AKT/PKB-glycogen synthase kinase-3beta (GSK-3beta ) pathway resulting in the inhibition of GSK-3beta-mediated cyclinD1 proteolysis. Persistence of the cyclinD1 protein causes nuclear accumulation of cyclinD1 during DNA replication, leading to induction of DNA double strand breaks detected by gamma-H2AX and activation of p53-dependent DDR. These observations suggest that constant activation of AKT-GSK-3beta pathway in response to exposure to fractionated IR achieves the tolerance to insults by IR in the absence of G1/S checkpoint and induces genomic instability in irradiated cells.

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Oxidative stress plays a critical role in the modulation of several important physiological functions. On the other hand, oxidative stress is accountable for development of many unphysiological changes, which could be deleterious for cells. Oxidative stress is a cellular or physiological condition due to elevated concentrations of reactive oxygen species (ROS) that cause molecular damage to vital structures and
functions. Several factors influence the susceptibility to oxidative stress by affecting the antioxidant status and/or oxygen free radical generation. These factors can be divided into those of endogenous, e.g. exercise and psychological stress or of exogenous origin, e.g. food, alcohol, cigarette smoke, environmental pollutants, UV and radiation. ROS have been shown to participate in the pathogenesis of many human diseases. However, the biochemical mechanisms by which ROS cause cell damage and ultimately organ dysfunction are not fully understood. One of the major cellular targets of ROS is protein. ROS can oxidize both aliphatic and aromatic amino acid residues of proteins, leading to irreversible structural changes. Redox environment is controlled by a variety of redox regulating systems that include superoxide dismutase, glutathione peroxidase, thioredoxin, thioredoxin reductase, glutaredoxin and peroxiredoxin. In this study, we examined whether and how cellular sensitivities to radiation and oxidative stress are modulated by overexpression of these antioxidant enzymes in cultured human cells.

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Here we have investigated bystander response of human fibroblasts to energetic heavy ions in confluent cultures. First, 0.0003% of cells were targeted with microbeams. Broadbeam irradiation was also conducted to see the effects in irradiated cells. Bystander cells manifested a more transient apoptotic response and delayed p53 phosphorylation compared with irradiated cells. More than half of the genes whose expression changed in bystander cells were downregulated, and most of the genes upregulated in irradiated cells were downregulated in bystander cells. Pathway analysis revealed serial activation of p21^Waf1 and NF-κB pathways in irradiated cells but G protein/PI-3 kinase pathway in bystander cells. These findings highlight the distinct response of irradiated and bystander cells. Moreover, interleukin genes were upregulated in irradiated cells but its receptor gene was upregulated in bystander cells, suggestive of the signal transmission from irradiated to bystander cells. Second, as regards chromosome aberrations in bystander cells treated with conditioned medium from X- or heavy ion-irradiated cells, we found the difference in the types of aberrations but little difference in its total yields, indicating that bystander responses occur independently of radiation types but are induced through different mechanisms. Altogether, these induced bystander responses could be a defensive mechanism that would minimize further expansion of aberrant cells. [Refs] JRR (2007) 48:87-95. IJRB (2007) 83:73-80. Mutat Res (2008) 637:190-196, 639:35-44, 642:57-67.

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Bystander effect is the phenomenon in which biological responses occur in cells that have not been traversed by charged particles, but located nearby the cells actually traversed. In experiments using a broad radiation field, bystander-type individual cellular responses cannot be distinguished. Therefore, the microbeam cell irradiation system, which enables the observation of cellular responses of both the non-irradiated individual cells and irradiated individual cells, become a powerful tool for elucidating the mechanisms underlying the bystander effect. We have been studying the differences in the lethal effects on V79 cells irradiated with microbeams of different sizes using an X-ray microbeam irradiation system developed at the KEK-PF. We have reported that low-dose hypersensitivity is more clearly observed in nucleus-irradiated cells than in those subjected to whole cell irradiation. Further, we reported that dose-dependent enhancement of cell death was observed in bystander cells in the absence of energy deposition in the cytoplasm, similar to the distinct hypersensitivity observed in irradiated cells. Recent studies have indicated the involvement of many factors in the signaling from irradiated cells to bystander cells. In particular, nitric-oxide (NO) is one of the promising mediators that induce bystander effects. In this study, we investigated the role of NO in bystander cell death in low-dose region and discussed the relationship between NO-mediated bystander cell death and intracellular energy-deposited sites.

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