Dependence of the yields of AP sites and AP clusters produced in pUC18 plasmid DNA on scavenging capacity and radiation quality The object of this study is to reveal whether the induction process of an abasic site (AP site) and the clustered DNA damage which contain AP sites (AP cluster) depends on ionizing density of radiation. Many studies using synthetic AP clusters incorporated into oligonucleotides reported that AP clusters retard base excision repair processes. Sutherland et al. (2002) reported that AP clusters are induced efficiently in human cells by low LET X-irradiation with the similar yields with those for the clusters which contain pyrimidine or purine base lesions. There, however, has been very little knowledge of AP sites and AP clusters induced by high LET radiation. In order to clarify the relation between track structure of C ions or X-rays and the induction processes of an AP or AP clusters, we measure the yield of AP sites visualized by the treatment of irradiated pUC18 plasmid DNA with the AP endoclease (Nfo), which converts an AP site to detectable single strand break. Several scavenging capacities of the samples are tested to estimate the effect of indirect action of diffusible OH radicals in the induction of AP site. These experimental data will be discussed with theoretical radiation track structure in the respect of repair susceptibility of the AP cluster.

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It is known that DNA lesions induced by ionizing radiation and chemicals can cause mutation and carcinogenesis. In particular, "clustered damage" site, that is a DNA region with multiple lesions within a few helical turns, is believed to hardly be repaired. This type of damage is considered to be induced around high-LET radiation tracks and at track-end of secondary electron. However, details of the clustered damage sites are not known. In order to get experimental information about the hypothetical damage, we have developed an analytical method for measuring the degree of dispersion of distances between DNA lesions using fluorescence resonance energy transfer (FRET). Complementary two 31-mer single-strand DNA with an AP (apurinic/apyrimidinic)-site at each center nucleotide were synthesized to obtain relationship between FRET efficiency and AP-site- AP-site distance. One DNA strand with an AP-site was labeled with a donor or fluorescent probe at the lesion, and another strand was labeled with an acceptor. As a result of fluorescence spectral analyses with annealing these strands, fluorescence intensity from acceptor increased with proceeding the annealing. D-A distance calculated from the FRET efficiency was 4 nm, which was reasonable with the estimated one taking the diameter of B-form DNA duplex and length of each probe into account. Now we have tried to apply the FRET method to plasmid DNA irradiated with some radiation qualities. The results and the perspective will be discussed.

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In general, survival curves of mammalian cells can be fit by linear-quadratic model. The quadratic term has been related to the lethal damage as a result of interaction of two damages in previous biophysical models. Unrepairable double-strand breaks (DSB) have been considered to be most responsible for cell lethality. However, it is still open question that what is the lethal damage and what kind of condition can be related to the linear and quadratic terms of the lethal damage. We have been obtained detail information on initial DNA damage by track structure simulation method. According to the simulation result, the increase of the complexity of initial DNA damage and cell lethality shows partially similar trend in their LET dependence. The purpose of this study is to elucidate and modeling the relationship between initial DNA damage and cell lethality. Here, the yield of lethal damage is estimated from experimentally obtained survival curves using linear-quadratic model. The estimated yields are compared with the simulated DNA spectrum and the spatial distribution such as the distance between damages in cell nucleus. From the comparison, the condition of initial damage related to the cell lethality is examined. In particular, LET and cell cycle dependent of survival curves are focused relating to the initial DNA damage pattern and repair pathways, respectively.

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Background: Micronuclei formation induced by X-irradiation is mainly thought to result from DNA double strand breaks. However, we think that DNA double strand breaks cannot lead to micronuclei formation following mitotic phase, because cell cycle progression of cells, which have DNA double strand breaks, is inhibited by the checkpoint mechanism. We now examined whether DNA double strand breaks during G1 phase can lead to micronuclei formation or not. Method: Human embryonic fibroblasts (HE17) cells were used. HE17 cells were cultured and their confluent cultures were maintained for a week to synchronize their cell cycle at G1 phase, and then were irradiated by X-ray. To visualize the site of DNA double strand breaks, cells were fixed by formalin at 15 minutes, 2 hours and 24 hours after X-irradiation, and immunofluorescence staining on p53-binding protein-1 (53BP1) foci, which are known to accumulate on this site, was performed. On the other hand, to visualize micronuclei formation, cells were fixed by formalin at 24 hours after X-irradiation and DAPI staining was performed. In this study, we examined the relationship between DNA double strand breaks and micronuclei formation by using 2 different radical scavengers in radioprotection effect. Dimethyl sulfoxide (DMSO) was used as an inhibitor for DNA double strand breaks, because DMSO can capture irradiation-derived reactive oxygen and inhibit cell death. On the other hand, ascorbic acid (Vitamin C) was used as a radical scavenger, which does not inhibit DNA double strand breaks, because ascorbic acid cannot inhibit lethal effect by X-irradiation. Results: First, we examined the effect of radical scavengers on radioprotection by using colony formation assay. The result showed that treatment with ascorbic acid (5 mM) before X-irradiation cannot cause a significant radioprotection effect, but treatment with DMSO (2%, 256 mM) can, by comparison of survival fraction with non-treatment controls. Moreover, in evaluation of DNA double strand breaks by using 53BP1 foci, we found that treatment with ascorbic acid cannot inhibit the number of 53BP1 foci at 15 minutes, 2 hours and 24 hours after X-irradiation at all, but treatment with DMSO can significantly. These results showed that DMSO can inhibit the generation of DNA double strand breaks via inhibition of reactive oxygen during X-irradiation and this inhibition can lead to decrease cell lethal effect, while ascorbic acid cannot. Next, we examined the effect of radical scavengers on inhibition of X-irradiation-induced micronuclei formation. The result showed that micronuclei formation is dose-dependently induced in the range between 0.5 Gy and 2 Gy, and treatment with DMSO before X-irradiation can hardly inhibit micronuclei formation, but treatment with ascorbic acid can.If DNA double strand breaks during G1 phase form micronuclei by cell cycle progression to mitotic phase, treatment with DMSO can inhibit micronuclei formation because of its inhibition of DNA double strand breaks. However, in fact, despite the result that ascorbic acid cannot DNA double strand breaks, it can significantly inhibit micronuclei formation. It is suggested that this effect of ascorbic acid on radical scavenging has something to do with the mechanism of micronuclei formation and this effect do not lead to lethal effect. Over all judging from these results, it is strongly suggested that DNA double strand breaks during G1 phase cannot mainly progress its cell cycle due to the perpetual cell cycle arrest and it does not lead to micronuclei formation.

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Ionizing radiation produces oxidized bases and strand breaks as major DNA lesions. These lesions are repaired by base excision repair and recombination mechanisms, respectively. Ionizing radiation also generates DNA-protein crosslinks (DPCs) under certain conditions. However, the repair mechanism of DPCs remains to be elucidated. Recently, we have assessed the repair mechanism of DPCs in E. coli, showing that nucleotide excision repair (NER) and homologous recombination (HR) collaborate to mitigate the toxic effects of DPCs. In this study, we have asked whether mammalian cells use a similar strategy to ensure survival in the face of DPCs. The activity assays with cell extracts revealed that the upper size limit of crosslinked proteins (CLPs) amenable to NER was very low (about 8 kDa). Consistent with this observation, NER-dependent removal of genomic DPCs was not observed in cells. Moreover, CLPs were not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. Conversely, HR-deficient irs1SF and 51D1cells were hypersensitive to DPC-inducing agents, indicating the involvement of the HR-dependent damage tolerance mechanism for DPCs. Upon treatment with DPC-inducing agents, cells accumulated nuclear RAD51 and γ-H2AX foci. We are also elucidating the effect of DPCs on transcription using a defined system.

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DNA-protein crosslink (DPCs) are ubiquitous genomic lesions where proteins are irreversibly trapped on the DNA strand via covalent bonds. DPCs are produced by ionizing radiation, ultraviolet light, aldehyde compounds, heavy metal ions, and anticancer drugs. Since trapped proteins are extremely bulky, steric hindrance conferred by DPCs inhibit DNA replication and transcription, thereby exerting adverse effects on cells. It has been shown that aldehyde compounds are useful agents to investigate the biological consequence and repair mechanism of DPCs since they are potent and fairly selective DPC-inducing agents. However, not so much comparative data are available as to how much DPCs are actually produced in the genome of cells upon treatment with individual aldehyde compounds. In the present study, we treated HeLa and MRC5 cells with a series of aldehydes including formaldehyde (FA), trans-2-pentenal (PEN), crotonaldehyde (CRA), glutaraldehyde(GA), acrolein (ACR), chloroacetaldehyde (CAA) and analyzed cell survival and DPC formation. Cell survival was analyzed by colony formation. The efficacy of cell killing decreased in the order of ACR, GA, CRA, PEN, and FA. Cells were treated with aldehydes at concentrations that gave 10% survival, and genomic DNA was isolated. The amount of DPCs was measured by Western blotting. These data will be presented in the meeting.

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Relative biological effectiveness is greater in high-LET heavy ions than in low-LET radiation. Heavy-ion-induced DNA damage has been focused to reveal the underlying mechanism. DNA double-strand breaks (DSBs) are believed to be the most serious DNA damage. Since 1990s, DSBs have been quantified with pulsed-field gel electrophoresis (PFGE) technology. In conventional studies, DNA preparation from irradiated cells was performed in agarose plugs for avoiding excess DNA fragmentation during experimental procedures. However, we have found that DNA fragments are partly lost from the agarose plug during DNA preparation. In our experiment, DNA molecular size standards were used as a substitute of genomic DNA. DNA was embedded in agarose plugs and they were incubated in 0.5 M EDTA for 24 h and in 0.5 X TBE buffer for 2 h before PFGE assay (the conventional DNA preparation). After PFGE, DNA was stained with SYBR Green I and the band pattern was visualized on a UV transilluminator. Gel image was recorded with a cooled CCD video camera and the fluorescence intensity of each band was measured. The fluorescence intensity of the bands smaller than several kilo base pairs decreased during the conventional DNA preparation. This phenomenon makes difficult to reveal the total DSB yield in irradiated cells. In the workshop, we will present the latest results.

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Our previous study suggested that the DNA damage induced by very low X-ray doses are largely due to bystander effects. The aim of this study was to verify whether DNA damage created by radiation-induced bystander effects are likely to be repaired. We examined the generation of DNA damage in cells by enumeration of phosphorylated ataxia telangiectasia mutated (ATM) foci, which are correlated with DNA damage repair, in normal human fibroblast cells (MRC-5) after X-irradiation at doses ranging from 1 to 1000 mGy. At 24 h after irradiation, 100% (1.2 mGy), 58% (20 mGy), 12% (200 mGy) and 8.5% (1000 mGy) of the initial number of phosphorylated ATM foci were detected. The number of phosphorylated ATM foci in MRC-5 treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X-irradiation was assessed; phosphorylated ATM foci were not observed at 5 h (20 mGy) or 24 h (200 mGy) post-irradiation. We also counted the number of phosphorylated ATM foci in MRC-5 co-cultured with 20-mGy-irradiated MRC-5. After 48 h of co-culture, 81% of the initial numbers of phosphorylated ATM foci remained. These findings suggest that DNA damage by the radiation-induced bystander effect persist for long periods, whereas DNA damage induced by direct radiation effects are repaired relatively quickly.

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We examined the protective effects of water-soluble derivatives of flavonoids,glucosyl hesperidin (G-HSP) and glucosyl rutin (G-Rutin), against DNA double-strand breaks induced by photo- and gamma-irradiation using a single-molecule observation. For photo-irradiation, we performed a real-time observation of double-strand breakage of individual DNA molecules stained with a fluorescent cyanine dye, YOYO-1, under focused illumination in solution by fluorescence microscopy, where YOYO-1 acts as a photocleavage agent by generating reactive oxygen species. To evaluate damage induced by gamma-ray irradiation, the length of DNA was measured after irradiation (0–650 Gy). The results indicate that G-HSP and G-Rutin protect against double-strand breaks caused by both photo- and gamma-ray irradiation. With regard to photo-induced double-strand breakage, G-Rutin provides twice as much protection as G-HSP. In contrast, the protective effect of G-Rutin against gamma-ray irradiation is almost the same as that of G-HSP. We concluded that the antioxidant potential of these compounds vary according the source of irradiation.

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Eukaryotic genome is the tightly packed into the chromatin, a hierarchically organized complex of DNA and histone and nonhistone proteins. This packing represents a common obstacle for most of the DNA functions. Concerning transcription, covalent modifications of core histone N-termini and ATP-dependent nucleosome remodeling plays a role in regulation of gene expression in the chromatin context. On the other hand, role of these chromatin modifications in other aspects of DNA metabolism, in particular, in the cellular response to DNA damage, remains largely unexplored. Recently, we have already shown that histone H2AX is acetylated by TIP60 histone acetylase complex upon DNA damage. The acetylation of H2AX is required for the eviction of H2AX from damaged chromatin. A bromodomain is a protein domain that recognizes acetylated lysine residues. This recognition is important for the association of histone with chromatin remodeling. We are now investigating the mechanism of H2AX eviction after induction of DNA damage focusing on BRDX. Here we will present our recent data about the role of BRDX in DNA damage response.

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Various nucleocytoplasmic proteins are modified with O-linked N-acetylglucosamine (O-GlcNAc). It is known that O-GlcNAc modification is involved in protein degradation, localization, protein-protein interaction, and regulation of protein phosphorylation. We have reported that ataxia-telansiectasia mutated (ATM) was modified with O-GlcNAc, and that the elevation of O-GlcNAc modifications suppressed the activation of ATM due to X-irradiation. In the present study, in order to clarify the mechanisms of the regulation of O-GlcNAc modification in the activation of AT, we examined the effects of O-GlcNAc modifications on the interaction of ATM with various signaling molecules and O-GlcNAc related proteins.
Hela cells were exposed to X-irradiation at the dose of 5 Gy. To elevate the levels of O-GlcNAc modified proteins in the cells, we added 100µM PUGNAc (O-(2-acetamido-2-deoxy-D- glucopyranosylidene)amino N-phenyl carbamate) to the cell cultures. After irradiation, nuclear fractions were obtained by cell fractionations and immunoprecipitated using anti-ATM antibody. Protein interactions of ATM with other signaling molecules and O-GlcNAc related proteins were analyzed by western blots.
From the results of immunoprecipitations, ATM interacted with O-GlcNAc transferase (OGT), which adds O-GlcNAc to proteins, but not O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. Furthermore, it was suggested that Rad50, Mre11, Tip60, and PP2A interacted with ATM and that the interactions was affected by the addition of PUGNAc.

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AP endonuclease 1 (APE1) is an essential enzyme in the base excision repair (BER) pathway. APE1 possesses DNA repair activity as an endonuclease that hydrolyzes the phosphodiester bond just 5' to an apurinic/apyrimidinic (AP) site. In addition to its role in DNA repair, APE1 regulates the expression of genes important for cellular viability, and cancer promotion and progression by stimulating the DNA binding activity of various transcription factors. Previous studies in which APE1 was knocked down using siRNA revealed that tumor cells were induced apoptosis and increased the sensitivity to chemotherapy agents. However, these knockdown studies did not fully elucidate the significance of APE1 in the BER pathway. In this study, we prepared APE1 knockdown cells which are responsive to Doxycycline using Tet-On® System (Clontech) to examine the effect of APE1 knocking down on the BER pathway. We initially prepared pSingle-tTs-shRNA (6,990 bp, Clontech) inserted the target sequence of human APE1 (NCBI:NM001641). Then we transfected the plasmid into HeLa RC355 cells and selected the transfectant on the basis of the resistance to G418. We are going to examine the sensivity to methylmethane sulfonate (MMS) and H₂O₂.

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

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Base excision repair (BER) pathways are conserved among a variety of organisms and thought to have a major contribution for repairing alterd bases emerged on the genome. A lesion specific bifunctional DNA glycosylase, which is accompanied with apurinic/apyrimidinic (AP) lyase activity via /-elimination, initiates AP endonuclease-independent BER pathway. Mouse endonuclease VIII like 1 (Neil1) is one of the major bifunctional DNA glycosylase accompanied with / -lyase and removes various altered bases, especially oxidized pyrimidines. Nth1 is known as mouse oxidized pyrimidines DNA glycosylase in mitochondrion and monofunctional thymine glycol DNA glycosylase activity in mouse nucleus was reported in our previous study. Rosenquist and co-workers reported that mouse ES cells depleted of Neil1 were hypersensitive to -irradiation (Rosenquist T.A. et al., DNA Repair, 2003). In this study, we developed Neil1 knockdown mouse cell system using shRNA technology and examined the sensitivity to oxidizing agent. Based on the registered base sequence of mouse Neil1 (NCBI: NM_028347.2), we constructed shRNA plasmid and transfected the plasmid into mouse embryonic fibroblast. Based on the resistance to Zeocin (Invivogen) and the expression of GFP, the transformant was selected and then cloned. The study on the repair of H₂O₂–induced base damage in these Neil1-knockdown cells is now in progress.

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Regulation of XRCC4 protein in DNA double strand break repair DNA double strand break (DSB) is considered the most fatal type of DNA damage induced by radiation. Eukaryotic organisms equipped two major DSB repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination. We focused on XRCC4 protein, which binds to and cooperates with DNA ligase IV in the joining of DSB at the end stage of NHEJ. XRCC4 protein undergoes a number of post-translational modifications, including phosphorylation by DNA-PK. We guess that NHEJ is regulated through post-translational modifications. In order to reveal these modification mechanisms, it is necessary to evaluate the function of XRCC4 mutant, especially in terms of radiosensitivity. However, colony formation assay following cloning takes one or two month to get the experiment result. We sought to establish a rapid assay using fluorescent reporter protein. A series of XRCC4 point mutants were created by PCR and then integrated into pIRES-DsRed vector, which drives expression of XRCC4 and DsRed from a single mRNA. These vectors were introduced into XRCC4-defective murine leukemia cell line M10. After irradiation, cells expressing XRCC4 with normal function would predominate because of increased survival. We can identify and measure XRCC4-expressing cells by fluorescence using flow cytometer.

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Tumor hypoxia contributes to resistance to ionizing radiation. It is considered that this radioresistance is due to oxygen effect. On the other hand, tumor hypoxia undergoes genetic change such as increased expression of hypoxia-inducible factor-1 (HIF1), which allows them to survive and proliferate, and now it is considered that HIF1 is related to radioresistance. In this study we analyzed the relationship between HIF1 and radioresistance. When cell surviving was compared with normoxic cells, hypoxic cells and hypoxic HIF1α knockdown cells, normoxic cells were most radiosensitive, hypoxic cells were radioresistant, hypoxic HIF1α knockdown cells were intermediate. This result indicates that HIF1α is relate to radioresistant. When DNA damage after irradiation was analyzed under the normoxic and hypoxic condition, DNA repair rate was more efficient under the hypoxic condition. When DNA repair proteins were detected by western blot after immunoprecipitation with HIF1α, Ku70 and Ku80 were immunoprecipitated with HIFα, but rad51 was not immunoprecipitated. These result indicate that HIFα contribute to radioresistant by being concerned in non homologous end joining repair.

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Radiosensitivity attributes to the defective repair of double-strand DNA breaks (DNAdsb) or the misrepair. DNAdsb are repaired by recombinational repair (HR) and nonhomologous end joining (NHEJ). NHEJ is dominant to repair of ionizing radiation induced DNAdsb. ATM is contributed to HR. Although relationships of repair pathways still remains unclear, it is reported that 53BP1 work to repair pathway choice. Namely, radiosensitivity increased in BRCA1 defective cells because of HR defective. Radiosensitivity was recovered by 53BP1 knockdown. This was because DNA bound 53BP1 inhibit BRCA1/CtIP. 53BP1 defect induce CtIP mediated resection. HR acceleration is ATM dependent in 53BP1 knockdown BRCA1 defective cells. In this study, we examined the role of ATM and 53BP1 in DNAdsb repair pathway using RNAi knockdown of 53BP1 in Ku70 defective cells. 53BP1 knockdown cells showed higher survival than that used to be in the parental Ku70 defective cells. Increased radiosensitivity was suppressed after treatment with ATM inhibitors. This indicates that ATM associated HR is accelerated. 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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When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint clamp component, is phosphorylated by Hsk1Cdc7, the S. pombe Dbf4-Dependent Kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation occurs after 9-1-1 chromatin loading, depends on the Rad9-associated protein Rad4/Cut5TopBP1 and disrupts interactions between Rad9 and RPA. rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and increased inviability following replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.

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Glioblastomas are known to the most lethal brain tumor and their resistance to anti-cancer therapies is elucidated by glioma stem cells (GSC). But it is still unclear how the mechanisms provide the resistance to GSCs. In this study, we used GSCs characterized by Plagl1, which is a tumorigenesis factor, and Sox11, which is the negative regulator of Plagl1. We compared the cellular characteristics between GSCs (Plagl1 positive) and glioma differentiated cells (Sox11 positive) by using anti-cancer drugs and ionizing radiations. In consequence, we found that GSCs showed stronger resistance to DNA cross-link agents whose damages are repair by homologous recombination (HR). Therefore, it might suggest that HR is induced in GSCs.

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Glioblastoma is one of most lethal human tumors because of radioresistance. Recent reports suggested that the reason for the radioresistance of glioblastoma was due to the existence of cancer stem cells that had high ability of DNA repair and DNA damage response. Although neural stem cells and brain cancer stem cells share common features including some stem cell markers, radiation-induced DNA damage response has not been examined in neural stem cells. In the present study, we investigated the repair kinetics of radiation-induced DNA double strand breaks (DSBs) by scoring phosphorylated histone H2AX (ganmma-H2AX) in mouse neural stem cell enriched (CD133 positive) neurosphere cells and the remaining (CD133 negative) neurosphere cells. To enrich neural stem cells, neurosphere cells were stained with phycoerthrin (PE) conjugated anti-CD133 antibodies, and mixed with anti-PE antibodies that were crosslinked to magnetic nanoparticles. Then, CD133 positive cells were enriched by using a magnetic column. The repair kinetics study indicated that the number of gamma-H2AX immediately after 1 Gy of X-irradiation was 31 per cell in mouse embryo fibroblast (MEF) cells whereas 23 per cell in mouse neurosphere cells, showing the significant difference between them. However, the significant difference was not evident between CD133 positive and negative cells. The significantly less focus number per cell was observed in neurosphere cells than in MEF cells until 1 hr after X-irradiation. The difference became small at 3 h and disappeared at 12 h. The present result suggests that the repair ability of DSBs in neurosphere cells is higher than that in MEF cells, implying that the high repair ability in cancer stem cells might be originated from stem cells.

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Even under physiological condition, a variety of base damages are created and these are mainly repaired by base excision repair (BER) pathway. Considering the paucity of damaged bases within the genome, the cell should have a very efficient system for searching them. Human N-methylpurine DNA glycosylase (hMPG) is a BER protein which initiates repair of a diverse group of alkylated and deaminated purines, and can diffuse along the DNA to find base lesions. And many proteins such as nucleotide excision repair (NER) related protein or transcriptional control elements interact with hMPG to regulate the activity. However, the exact damage detection mechanism of hMPG is not fully understood. In the present study, we have focused to identifying proteins interact with hMPG. We cloned an Escherichia coli strain expressing GST-fused hMPG. Now we are investigating HeLa cell nuclear proteins which interact with GST-hMPG using pulldown assay and mass spectrometry.

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Double strand breaks (DSBs) occur in chromosomal DNA as a result of exposure of cells to ionizing radiation (IR). DSBs induce cellular responses that lead to cell cycle checkpoint, DNA repair, and apoptosis. The ataxia telangiectasia mutated (ATM) kinase plays a centarl role in these responses, and the pathways in these responses form complicated networks through non linear relationships. Upon indction of DSBs, activeated ATM phosphorylates many proteins that are involved in DSB responses. In a process of our previous research to clarify functions of 53BP1 in DSB responses, we identified a few proteins which cross-react to anti-phospho ATM antibody after treatement of cells with IR. To identify a new ATM target, cell lysates from IR-exposed cells were subjected to immunoprecipitation with anti-phospho ATM antibody. The precipitated proteins were resolved by SDS-PAGE, and analyzed by LC-MS/MS. We would like to present a protein that is phosphorylated after exposure of cells to IR in a ATM-depnedent manner.

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Centrosome is an organelle that functions as a microtubule organizing center (MTOC) and regulates proper cell division. The centrosome consists of a pair of centrioles and the surrounding pericentriolar matrix (PCM). Overduplicated centrosomes cause multipolar spindle formation and unproper chromosome segregation. γ-tubulin, which is a component of PCM, provides microtubule anchoring machinery by forming a γ-tubulin ring complex (γ-TuRC). In our previous report, DNA damage proteins BRCA1 and NBS1 are involved in ubiquitination of γ-tubulin to regulate centrosome duplication. Meanwhile, Fanconi Anemia (FA) is a rare genomic instability disorder characterized by chromosomal instability, bone marrow failure, and cancer predisposition. FA pathway is regulated by 13 FA proteins. FA core complex that consist of 8 of those FA proteins monoubiquitinate FANCD2 and FANCI as a E3 ubiquitinligase. This monoubiquitination is intensified by DNA damage. Monoubiquitinated FANCD2 and FANCI localize to DNA damage site and promote DNA repair. Recently, it was reported that FANCD2 localizes to discrete sites of mitotic chromosomes. In this study, we investigated that whether FANCD2 is involved in centrosome maintenance in FA patients cells and FANCD2 knockdown cells.

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Previously we have reported that about 1% of DNA doubles strand breaks (DSB) induced by ionizing radiation formed unrepairable damages, and stayed in cells indefinitely, thereby induced permanent growth arrest and premature senescence in normal human diploid fibroblasts (NHDF). The foci at unrepairable DSB contained all of the previously reported proteins and appear to continue polyubiqutination of the substrate proteins, i.e., H2AX, H2B, gH2AX, MDC1, even 1 or 2 month after irradiation, indicating that repair and checkpoint signaling are kept activated. Cells carrying unrepairable DSB exhibited abundant repair activities against repairable damages induced by second irradiations, however, we found an accumulation of unrepairable damages. This result indicated that the measurement of unrepairable DSB in non-dividing cells and tissues in vivo make it possible to estimate individual radiation doses as an accumulated dose.

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A well-characterized early histone modification that is triggered by DNA is the phosphorylation and ubiquitination of the histone variant H2AX. This occurs minutes after the formation of DNA double strand breaks (DSBs) and leads to enhanced access by proteins, including NBS1 and others, that participate in homologous recombination-mediated repair (HRR) However, it is likely that another histone modification could be involved in this DNA repair pathway because H2AX-/- cells still display a normal localization of RAD51, a key protein in HRR, to sites of DSBs and exhibit significant HRR activity. We report here that, after exposure to ionizing radiation (IR), H2B is mono-ubiquitinated independently of H2AX modification and plays a critical role in RAD51-mediated HRR of DSBs. RNF20, an E3 ubiquitin ligase implicated in chromatin remodeling for transcription, forms IR-induced nuclear foci and promotes H2B mono-ubiquitination 1.5 hours post-irradiation, and this is followed by the release of H2B and H3 from chromatin. Surprisingly, the formation of IR-induced foci containing RAD51 and BRCA1 was substantially suppressed by decreased RNF20 expression or by a mutation of the ubiquitinated amino acid residue in H2B. Consistent with these data, depletion of RNF20 interfered with the accumulation of RAD51 and BRCA1 in chromatin and resulted in a pronounced reduction in HRR. As a result, RNF20 down-regulation significantly enhanced sensitivity to mitomycin C and IR. Moreover, RNF20-deficient phenotypes were corrected in part by chromatin decondensation using a histone deacetylase inhibitor, but IR-induced H2B ubiquitination was not affected by the addition of transcription inhibitors, suggesting the existence of transcription-independent chromatin reorganization activity at DSB sites by RNF20.

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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 Rad18,pol eta foci were not observed in NBS cells. These results indicated that NBS1 might promote the UV damage response.

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DNA pyrimidines are continuously oxidized in physiological condition. The productive damaged pyrimidines are thought to be repaired majorly by single nucleotide-base excision repair pathway. The mammalian homologs of endonuclease III and endonuclease VIII, NTH1 and NEIL1, are well known as oxidized pyrimidine-DNA glycosylases, and thought to have a major contribution to remove oxidized pyrimidines in mammals. We found an unidentified monofunctional thymine glycol (TG)-DNA glycosylase (TGG) activity in nuclei of mouse organs. Based on studies of comparison of the activity levels between organs and species, we concluded that higher level of the TGG activity is observed in more proliferative cells and in organisms which have shorter life-span. In this study, we purified the TGG activity protein from mouse cultured cell, L1210, which is derived from leukemia cell. L1210 has advantages; mouse genome database is fulfilled and the cell has high proliferative activity. We prepared whole cell extract, and performed hydroxyapatite column, ion-exchange column and gel-filtration column chromatography. The calculated molecular size of the TGG activity protein from L1210 cells was less than 25.7 kDa.

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UVB can damage DNA by causing formation of cyclobutane pyrimidine dimmer (CPD). Photoreactivation mediated by enzyme, photolyase, is the major pathway for repairing CPD in plant, and CPD photolyase is a crucial factor for determining UVB sensitivity in plants. We previously reported that CPD photolyase functions in nuclei, chloroplasts and mitochondria in rice. This result indicates that CPD photolyase, which is encoded by a single-copy gene in the nuclear genome, translocates to chloroplasts, mitochondria, and nuclei. Thus, CPD photolyase is subjected to "triple targeting" in rice cell. Furthermore, the subcellular localization studies indicated that the C-terminus of rice CPD photolyase [385-506 amino acid regions of CPD photolyase (PHR); 385-506-PHR] contains a functional targeting signal for transport into nuclei and mitochondria. In this study, in order to the identify the nuclei and mitochondrial-targeting amino acid sequence, we constructed expression vectors encoding protein chimeras of GFP and C-terminus partial amino acid of PHR under the control of the CaMV 35S tandem promoter. We found that 385-437-PHR-GFP or 438-506-PHR-GFP was detected only in mitochondria or nuclei, respectively. According to further studies in detail, 385-403-PHR-GFP was detected in mitochondria, and three amino acids, 487-489, were functional targeting signal for transport into nuclei. In this congress, we discuss the acquired mechanisms of transport into mitochondria and nuclei in higher plants.

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A DNA site that has no base is called an AP site (APurinic/apyrimidinc site). AP sites arise from both spontaneous depurination/depyrimidination (about 10,000 per cell per day) and removal of a damaged base in the base excision repair (BER) pathway as an intermediate. AP sites are among the major DNA lesions and, if not repaired, inhibit replication and transcription or induce deletional mutations. AP endonuclease is an enzyme that recognizes AP sites and then excises the DNA strand at the site. The resulting nick in DNA is resynthesized and the repair is completed. The accumulation of such damage in DNA is thought to be related to aging, but this is still uncertain. In order to examine the relationship between DNA damage accumulation and aging, we are studying the DNA repair system in the worm C. elegans. C. elegans is very useful for the study of lifespan.
In this study, we focused on the apn-1 gene, which is a putative AP endonuclease in C. elegans. To confirm that APN-1 does have an AP endonuclease activity, we first cloned the apn-1 gene from a C. elegans cDNA library and purified APN-1 protein expressed as a GST-fusion protein in E. coli. The purified APN-1 protein indeed showed an excision activity toward AP sites in vitro. Then we used an E. coli mutant that is deficient in AP endonucleases and therefore hypersensitive to DNA-damaging agents that produce AP sites. The E. coli mutant was rescued by expression of C. elegans APN-1. Next we produced a rabbit polyclonal antibody against APN-1 protein. We also produced transformed C. elegans that is expressing APN-1-GFP fusion protein and now we are investigating when, where, and how APN-1 functions in C. elegans.

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Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, growth retardation, and cancer predisposition. Cells from NBS patients exhibit radiosensitivity, S-phase checkpoint defect, and chromosome instability. The gene product mutated in NBS, NBS1, forms a complex with MRE11 and RAD50 that is involved in the repair of DNA double-strand breaks (DSBs) and DNA damage checkpoints. DSBs are repaired by two major pathways, namely, homologous recombination (HR)-mediated repair and nonhomologous end-joining (NHEJ). It has been reported that NBS1 functions in HR-mediated repair of DSBs. More recently, it has also been reported that NBS1 is required for NHEJ. However, the function of NBS1 in these processes has not been fully elucidated. To understand the function of NBS1 more clearly, we are trying to identify NBS1-interacting proteins using a proteomics approach. At the last meeting we reported that RAD51 and BRCA2 interacted with NBS1. This interaction requires the C-terminal region of NBS1. More detailed analysis revealed that MRE11-binding domain of NBS1 on its C-terminus was required for this interaction. Moreover, NBS1-binding domain of MRE11 was not required for the interaction between MRE11 and RAD51, suggesting that NBS1 interacts with RAD51 through MRE11. The results of a more detailed analysis will be reported.

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Ionizing irradiation induces lethal genome damages like DNA double strand breaks (DSBs) in human cells. Reorganization of damaged chromatin, such as posttranslational modification and/or exchange of histones, has been shown to play a role in the regulation of DNA damage response. Histone variant H2AZ in yeast is deposited close to the DSBs early but transiently and directs DNA resection, single DSBs-induced checkpoint activation, and DSBs anchoring. However, the role of H2AZ in the reorganization of damaged chromatin in human cells is still unclear. To investigate the role of H2AZ in DNA damage response, we analyzed the dynamics of H2AZ upon DNA damage induced by a UVA-laser microirradiation. Living cell imaging techniques revealed that H2AZ is released from damaged chromatin from just after induction of DSBs by UVA-laser microirradiation. This suggests that human H2AZ is involved in the regulation of DNA damage response at the very early stage via reorganization of damaged chromatin.

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Reactive oxygen species (ROS) generate various types of oxidative deoxyribonucleotides, such as 8-oxo-dGTP and 8-oxo-dGDP, in the nucleotide pool. 8-oxo-dGTP is generated by both direct oxidation of dGTP and phosphorylation of 8-oxo-dGDP. It is often incorporated into DNA by DNA polymerases during replication, which could result in mutagenic consequences. Many organisms have enzymes for eliminating these abnormal nucleotides in the nucleotide pool as well as repair enzymes for damage in DNA. For example, Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP, while MutT and human NUDT5 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP, thereby preventing mutations caused by misincorporation of 8-oxo-dGTP into DNA. However, whether there is homolog of these enzymes in the nematode C. elegans, a multicellular eukaryote frequently used as a model for the study of development and aging, has remained uncertain. To clarify the mechanism for eliminating oxidized dNTPs and its critical roles in maintaining the genome stability in C. elegans, we have been studied MutT homolog(s) of C. elegans and recently found that the NDX-1 protein had 8-oxo-dGDPase activity. Although 8-oxo-dGDPase was found in C. elegans, 8-oxo-dGTPase has remained to be identified. As direct oxidation of dGTP also occurs in C. elegans, NDX-1 is not sufficient to fully suppress the generation of 8-oxo-dGTP. Therefore, there must be some mechanism for eliminating 8-oxo-dGTP from the nucleotide pool. So we are investigating enzyme(s) involved in the mechanism. We will show the data obtained in the study.

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[Introduction] Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. We have found that FANCD1 could be the best target for sensitization of ACNU or TMZ among FANCA, FANCC, FANCD2 and FANCG. Since homologous recombination repair (HRR) follows the FA repair pathway through the step involving FANCD1 (BRCA2), it was of interest to know if HR in SPD8 cells was specifically induced by ACNU or TMZ. [Materials and methods] We used Chinese Hamster Ovary cell line, SPD8 cells. We examined the reversion frequency by reversion assay after each dug treatment. [Result]HRR was induced by both ACNU and TMZ. After 10 μM and 30 μM ACNU treatments, the HR frequencies were 7.2-fold and 13.9-fold high, respectively, as compared with the control cells. After 100 μM and 300 μM TMZ treatments, on the other hand, the HR frequencies were 8.4-fold and 22.8- fold high, respectively. [Conclusion] We elucidated that HRR was induced by both ACNU and TMZ. Next, we would like to investigate how HRR, especially FANCD1, effects on the therapeutic results following ACNU- or TMZ- treatment using quantification of FANCD1 protein in brain tumor.

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Genomic stability is crucial to the survival and proliferation for all organisms. The genome is subject to various types of DNA damage and chromosome aberration, caused by exogenous and endogenous agents, such as ionizing radiation, alkylating agents and reactive radicals. In Saccharomyces cerevisiae, two members of DNA damage checkpoint pathway, MEC1 and TEL1 (ATR and ATM homolog), have functionally redundant roles in DNA repair. A stress response pathway, HOG1-SWE1 (p38 and WEE1 homolog) MAP kinase pathway, is involved in chromosome integrity caused by unscheduled G2 cell cycle arrest. In this study, we characterized genome stability properties in mec1, tel1, chk1, hog1, and swe1 null mutants. Spontaneous loss of heterozygosity (LOH) increased about 10-fold and 100-fold in the mec1Δ and mec1Δtel1Δ strains, respectively, compared with the wild-type strain. In the chk1Δ, tel1Δ, hog1Δ and swe1Δ strains, the frequency of spontaneous LOH was as low as the wild-type strain. In the chk1Δ strain, however, the rate of chromosome loss was higher than the wild-type strain. On the other hand, in the hog1Δ and swe1Δ strains, chromosome loss frequency was exhibited low levels compared with wild-type strain. We further showed that mec1Δ, mec1Δtel1Δ mutants did not increased rates of UV-induced gene conversion. These results suggest that G2 checkpoint activated by MAP kinase pathway was involved in the formation of spontaneously and UV-induced chromosome aberrations, such as chromosome loss, while MEC1 damage checkpoint pathway activated by UV-induced DNA damage was involved in DNA damage repair. It was reported that LOH was not induced by MMS treatment and x-ray irradiation in the mec1Δ strain. However, UV irradiation could induce LOH in the mec1Δ strain. We therefore assume that UV-induced LOH elevation was caused by the recombination through the RAD51 and RAD52, activated by UV-induced pyrimidine dimers.

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N-ethyl-N-nitrosourea (ENU) is an alkylating agent, which shows carcinogenic and mutagenic potentials. In order to clarify the mutagenesis mechanism of ENU in vivo, we examined mutations at the gpt gene in B6C3F1 gpt-delta mice treated with ENU. Mice at 4 weeks of age were exposed to ENU (200 ppm) in the drinking water for 4 weeks, and were sacrificed immediately (8 weeks old), four weeks (12 weeks old), or eight weeks (16 weeks old) after the termination of ENU treatment. Genomic DNA was prepared from thymus, lung and small intestine, and the mutant frequency at the gpt gene was determined by selecting bacterial colonies that were resistant to 6-thioguanine. The mutant frequencies in thymus decreased from 4 to 8 weeks after treatment, although mutant frequencies in lung and small intestine remained unchanged. Sequence analysis of the gpt gene revealed that the mutation spectrum was different among the tissues. These results indicate that there may be a tissue-specificity of ENU-induced mutations in vivo, which might reflect the difference of cell types, such as cell turnover rate and repair capacity.

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DNA double-strand breaks (DSBs) are considered most critical determinant of the fate of the cells or organisms exposed to radiation. DSBs are repaired mainly through two pathways non-homologous end joining and homologous recombination. However, especially in the latter, some part of the repair events would lead to mistake, causing mutations and chromosome aberrations. In the last meeting, we reported the effects of nuclear reactor irradiation. The results suggested that DNA damages induced by neutron are difficult to be repaired correctly. Therefore, in the present study, we sought to examine the nature of misrepair of neutron induced DNA damage. We used murine leukemia L5178Y-derived, XRCC4-deficient cell line M10 as a host and introduced empty pCMV10 vector (M10 -CMV), normal XRCC4 cDNA (M10-XRCC4). Nuclear reactor UTR-KINKI in Kinki University or X-ray generator was used as radiation source. Radiosensitivity was evaluated by colony forming ability in media containing 0.16% agarose. The mutant of HPRT (hypoxanthine phosphoribosyl transferase) was detected as 6-thioguanine (6-TG) resistant cells. The mutation frequency was increased by approximately 10 - 20 folds. Now we are investigating the structure of the HPRT mutations in each clone.

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Radiation dose, frequency of DNA strand breaks and frequency of chromosomal aberrations are commonly used for the assessment of radiation effects. As we know, dose, DNA strand breaks and chromosomal aberrations are successive events, there seems to be no common understanding for these relations. We are modeling chromosome using a kind of "toy-model", and extensively studied many dynamical features. Here, we will report on the physical properties of dynamics of chromosome by computer simulation of our mathematical model.

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It is well known that delayed chromosome aberrations are induced in the progeny of cells exposed to radiation. To elucidate the mechanism for induction of delayed chromosome aberrations, we transferred a human chromosome 8 exposed to 4 Gy of soft X-rays into unirradiated mouse recipient cells by microcell fusion, and examined structural changes of the transferred human chromosome by whole chromosome FISH. In the present study, we asked whether an intrachromosomal rearrangement is involved in inducing delayed chromosomal instability by examining the stability of subtelomere region of the transferred human chromosome 8. Subtelomere FISH was applied for detecting intrachromosomal rearrangements of both short and long arms of chromosome 8. A transferred chromosome was structurally stable in four microcell hybrids transferred with non-irradiated chromosome 8. The subtelomere FISH revealed that a frequent intrachromosomal rearrangement, isochromosome 8q (i8q), was observed in one out of four hybrids. In contrast, the 4 Gy-irradiated human chromosome 8 was unstable in 3 out of 19 hybrids (16%), showing multiple aberrations in high frequencies (88~98%). Although a frequent intrachromosomal rearrangement, i8q, was evident in one unstable hybrid, no such rearrangement was observed in the other two unstable hybrids. Therefore, the present study indicates that the intrachromosomal rearrangement that is detected by subtelomere FISH is not strongly correlated with induction of delayed chromosome aberrations by radiation and that the pre-acquisition of the intrachromosomal rearrangement does not precede radiation-induced chromosomal instability.

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To reveal the characteristics of radiation-induced mutations in higher plants, we examined intragenic mutations induced by gamma-rays and carbon ions using transgenic Arabidopsis that carries Escherichia coli rpsL gene and demonstrated that gamma-rays and carbon ions (Mean LET 112 keV/μm) induced different mutations in respect to the size of deletions. Carbon ions that have a shorter range within the diameter of Arabidopsis seeds (Mean LET 402 keV/μm) showed a higher cell killing effect than the carbon ions (LET 112), but the mutation frequency in the rpsLgene was unchanged from background mutation frequency. This suggests the possibility that large structural alterations, which can not be detected by this method, are frequently induced by the carbon ions (LET 402), because this experimental system detects intragenic mutations via plasmid rescue. The Arabidopsis mutant plants defective in the Ligase IV gene, a major determinant in the double strand break repair pathway, showed higher radiation sensitivity than the wild type. We compared the difference in Dq values between the two kinds of carbon ions (LET 112 and 402) and found that the difference in the Ligase IV mutant was much lower than that in the wild type. This suggests that the carbon ions (LET 402) induced much irreparable DNA lesions. To further examine the characteristics of mutations induced by carbon ions (LET 402), mutation detection using Arabidopsis GL1 gene and E. coli codA gene as marker genes have been carried out. We will show the characteristics of mutations detected with these marker genes.

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A single track of ionizing radiation is considered to induce closely spaced DNA lesions (clustered DNA damage) including single strand breaks (SSBs) AP sites, and base lesions. It has been hypothesized that a clustered damage site is highly relevant to biological effects of radiation, such as lethality or mutagenesis. Previous studies have mainly focused on the biological effect of two-lesion clusters. In this study, we have investigated on the mutagenic potential of three-lesion clusters containing SSB and 8-oxo-7,8-dihydroguanine (8-oxoG), to see how an additional lesion will affect the biological consequences. We used plasmid based assay in E. coli to measure the mutation frequency induced by clustered damage sites. Briefly, we constructed clustered damage sites carrying an SSB and bistranded 8-oxoGs. 8-oxoG was placed within a restriction enzyme (Alw26I) recognition site. Damaged DNA was transfected into wild-type or glycosylase-deficient strains (fpg, mutY and fpg mutY) of E coli. The mutation frequency was assessed by the inability to cut by the restriction enzyme, and the nature of mutation was determined by sequence analysis. The mutation frequencies of three-lesion clusters were lower than those of two-lesion bistranded 8-oxoGs, and was similar to those of two-lesion bistranded clusters consisted of an SSB and an 8-oxoG. These results suggest that an SSB lowers the mutagenic potential of the 8-oxoG located on the same strand. Consistent with this prediction, sequence analysis of mutations induced by three-lesion clusters made clear that the mutation is mainly, if not all, derived from the 8-oxoG that does not accompany an SSB on the same strand. Our study demonstrates that the mutagenic potential of clustered damage is influenced by the presence of an SSB and that there is a hierarchy of repair of lesions in a clustered damage site.

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Radiation is a well-characterized carcinogen and mutagen. Radiation has also recently been shown to increase the frequency of mutations at tandem repeat loci. Microsatellite (Ms) DNA loci are short tandem arrays of simple DNA sequences with core repeat units of 1 - 6 bp. Mutation rates at 30 expanded Ms loci were studied in the germ line of mismatch repair deficient msh2 or p53 deficient medaka. Spontaneous mutation rate in homozygous msh2 males were significantly higher than those in isogenic wild-type or p53 deficient fish. In contrast, the irradiated msh2 fish did not show any additional increases in their mutation rate compared to wild-type fish, whereas significant increase in mutation induction was observed in the irradiated p53 fish.

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Translesion DNA synthesis (TLS) takes place at the DNA-damaged sites where DNA replication fork migration is stalled. The TLS-specific DNA polymerase often inserts incorrect nucleotide on the opposite side of the damaged nucleotide, leading to a mutation. We established a TLS assay system capable of measuring the TLS and mutation rates at the sites of aryl hydrocarbon-dG adducts in p53 sequences of shuttle vector plasmids. An environmental carcinogen 4-aminobiphenyl (ABP) is metabolically activated and forms a major DNA adduct, N-(2'-deoxyguanosine-8yl)-4-aminobiphenyl (dG-ABP). The ABP adduction hotspots coincide with the mutation hotspots in p53 gene sequences in bladder carcinomas of ABP-exposed people. Codon 248 (guanine) is one of these hotspots, but codon 249 (guanine) is the site of neither adduction nor mutation. To investigate mutations and TLS across dG-ABP, we constructed two kinds of the shuttle vector plasmids containing a single dG-ABP site-specifically in the codon 248 or 249 of p53 gene sequences. These plasmids were introduced into nucleotide excision repair deficient human cells (XP2OS(SV)) and their derivative cells overexpressing TLS polymerase eta. The TLS and mutation rates and mutation types of the plasmids propagated in these cells were analyzed. The result showed that the TLS rate of codon 248 was about a half of that of codon 249, whereas, the mutation rate of codon 248 was about twice higher than that of codon 249 in XP2OS(SV) cells. By overexpression of pol eta in XP2OS(SV) cells, the TLS rate of codon 248 was decreased to about a half of that in XP2OS(SV) cells, whereas the mutation rate of codon 248 was increased to sevenfold higher than that in XP2OS(SV) cells. Most frequent mutations were G to T substitutions in both cells. No significant change in the TLS and mutation rates of codon 249 was caused by the pol eta overexoression. These results suggest that mutation proneness of the p53 hotspot codon may be ascribed to surrounding sequences of the guanine, and pol eta plays an important role to induce mutations at the guanine with a bulky adduct.

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Calorie restriction (CR) means a reduction of calories by 30-40% compared with animals fed ad lib without changes in essential nutrient intake and it is well known that it can reduce the pathological symptoms of aging and extend lifespan in multiple species. However, little is understood for its mechanism. Many studies have shown that genomic mutations increase with age in an organ and tissue-dependent manner. Thus, the study of the effect of CR on mutation in vivo could be an interesting challenge. Here, by using Muta^TM mice harboring the lacZ reporter gene as part of a bacteriophage lambda vector, we have investigated that if the age-dependent accumulation of mutation could be affected by CR. Mice were divided into two cohorts: the control group (95kcal/week/mouse) and the CR group (65kcal/week/mouse). At 18 month, in the control group, the mutant frequencies in liver, spleen and small intestine were 14.3, 8.08 and 33.2x10^-5, respectively, whereas in the CR group, they were 15.9, 9.39 and 22.6x10^-5. In liver and spleen, there was no significant difference in mutant frequency between control and CR groups, while in small intestine, the mutant frequency in CR group was significantly lower than that in the control group. We also determined the spectra of mutations in small intestine. Interestingly, mutation type of G:C to T:A was high in both control and CR groups. The frequencies of the other types of mutations also showed no significant difference. The results suggest that CR suppresses all kinds of mutations in small intestine but not in liver and spleen.

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We have reported that translocations and dicentric chromosomes were increased almost linearly up to a total accumulated dose of 8000 mGy, in female C3H mice continuously irradiated by gamma-ray in SPF condition at a low-dose-rate (LDR) of 20 mGy/22h/day (0.91 mGy/h) from the age of 8 weeks.
Translocations in human lymphocytes induced by high-dose-rate (HDR) irradiation persist for a long time without elimination, on the while dicentric chromosomes decrease to about 50% at 3 years after irradiation. In similar, dicentric chromosomes decrease to background level at 112 days after HDR-X-ray irradiation in mice.
In present experiment, mice were kept for up to 400 days, after the LDR-irradiation for 200 days (total dose is 4,000 mGy), and chromosome aberration rates were examined periodically. Translocations and dicentric chromosomes were slowly decreased up to 200 days after the LDR-irradiation. Frequencies of dicentric chromosome were still higher than that of age-matched non-irradiated control mice, which was different from HDR-irradiation. The slight reduction of dicentric chromosomes after LDR-irradiation was not only caused by life span of splenic lymphocytes, but also by turn-over of splenic lymphocytes supplied from immature stages in bone marrow or other organs. The persistence of lymphocytes with dicentric chromosomes for more than 200 days after LDR-irradiation without rapid reduction might be closely related to the increase of dicentric chromosomes almost linearly depending on accumulated dose during the continuous LDR-irradiation. This study was performed under contact with Aomori Prefecture Government, Japan.

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One of the unique biological effects caused by ionizing radiation is the persistent occurrence of pleiotypic endopoints, including mutagenesis, chromosomal aberration and cancer predisposition. To search factors associated with the persistent property, the behavior of DNA binding proteins as possible candidates were assessed in human cells, which had overcome DNA disorders and restored proliferous status. Interestingly, we found that irradiation to cells introduces inheritable changes into a higher order protein-DNA interaction, which is accompanied by DNA-dependent protein kinase components, DNA-PKcs and Ku heterodimer. Notably, this response maintained over several decades of cellular generation without quantitative changes of the factors. The altered state of the protein-DNA interaction may heritably affect somewhat nuclear processes linked to DNA organization, resulting in the persistent degradation of genetic integrity.

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Hematopoietic stem cells (HSCs) that have a potential to differentiate into all lineages of hematopoietic cells are present in the hematopoietic niche, which maintains HSC stemness. Cytokine stimulation of HSCs promotes the cell cycle, and HSCs proliferate and differentiate thereafter and mature into functional blood cells. However, a lack of cytokine stimulation prevents the survival of HSCs, finally leading to apoptosis and cell death. Therefore, HSCs exposed to radiation under cytokine-free conditions causes an increase in cell death-related factors, but little information has been obtained to date regarding the precise mechanisms involved in this process. In the present study, we investigated the effect of ionizing radiation on the survival and clonogenic potential of human hematopoietic stem/progenitor cells under cytokine-free conditions. After informed consent was obtained from the legal guardian of the patient, hematopoietic mononuclear cells were separated from placental/umbilical cord blood, and CD34⁺ cells were enriched using magnetic cell sorting. The purified CD34⁺ cells were irradiated by X-rays with a dose of 0.5 or 2 Gy at a dose rate of 85–90 cGy/min, and were thereafter cultured in serum-free medium with no cytokine stimulation. After 12, 24 or 48 hours in culture, the cells were harvested from the culture and the surviving cells were quantified. Changes in cell-surface antigens during in vitro culture and the cell cycle status were also analyzed by flow cytometry. The clonogenic potential of harvested cells was analyzed using the methylcellulose culture technique. As a result, the surviving fraction cells decreased with the increasing period in cell culture, and the expression of early hematopoiesis-related antigens such as CD110 and Tie-2 were altered. In addition, the number of colony-forming cells was also decreased with increasing culture period and radiation dose. In the cell cycle analyses, treated cells shifted into the subG1 phase from the G0/G1 phase. Because CD34⁺ cells are a heterogenous population, their radiosensitivity may vary among subtypes. Currently, the subsequent analyses are undergoing investigation in our laboratory.

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Human T-cell leukemia cell line MOLT-4 is highly radiosensitive, and used as a model of p53-dependent radiation-induced apoptosis in many studies. On the other hand, several researchers have reported a novel apoptotic function of p53, i.e., transcription-independent apoptogenic role of p53 in mitochondria. Moreover, recent studies have shown that codon 72 polymorphic variants of p53 show different sensitivities to apoptosis. Arg72 variant of p53 has more potent apoptosis-inducing activity in mitochondria than Pro72 variant. In this study, we initially investigated the codon 72 polymorphism of p53 in MOLT-4 cells. Analysis of p53 exon4 genomic DNA sequences encoding codon 72 revealed that MOLT-4 was homozygous with respect to the allele encoding Arg72. We next investigated the involvement of the transcription-independent function of p53 using a RNA synthesis inhibitor, actinomycin D (ActD), and a protein synthesis inhibitor, cycloheximide (CHX). As a result, the apoptosis was suppressed by CHX but not by ActD. We also revealed that the suppressive effect of CHX on apoptosis is specifically mediated by p53 using p53-knockdown MOLT-4 transformants. Furthermore, the suppressive effect of CHX on the apoptosis was highly correlated with that on the p53 accumulation, and less correlated with that on the expression of p53 target genes. These results indicate that p53 transactivation is not necessary to induce the apoptosis, and that p53 accumulation by itself is sufficient to do this. The MOLT-4 apoptosis is probably useful for studying the transcription-independent pathway.

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Recent studies have revealed that there are two branches in p53-mediated apoptotic pathway: one is mediated through transcription of p53 target genes, another is initiated by direct interactions between p53 and mitochondrial Bcl-2 family members, which are so-called respectively “transcription-dependent and transcription-independent pathways.” However, the relative contributions of each in different types of cells and tissues are not yet clearly defined. To clarify the contributions, it is essential to use some chemical inhibitors that could selectively inhibit the different pathways. Pifithrin-μ (PFTμ) is so far the only reported p53 inhibitor specifically suppressing the transcription-independent pathway in the cultured cells. Though it was also reported to protect death from radiation-induced hematopoietic syndrome in mice, our recent study did not demonstrate any reproducibility of such a protective effect. Thus it makes the significance of the transcription-independent pathway in radioprotection be worthy of further verification. Some zinc chelators are known to inhibit p53 by inducing dissociation of a zinc ion from the metal ion binding site of p53. In the present study, 5, 7-bis(N,N-dimethylaminosulfonyl)-8-quinolinol (bis(DMAS)-QOH), a zinc chelator, was found to suppress the transcription-independent pathway without affecting the expression of p53 target genes. Moreover, bis(DMAS)-QOH showed lower toxicity and higher cell death inhibitory effect than PFTμ. These results suggest that bis(DMAS)-QOH can be a superior inhibitor to PFTμ against the transcription-independent pathway to reveal the role of the divergence pathway.

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Caenorhabditis elegans is a good in vivo model system to examine radiobiological effects. Recently, we found that locomotion caused by body-wall muscles was reduced in a dose-dependent manner after gamma-ray irradiation, and that the locomotion was eventually restored. However, it is not known whether the same effects are observed in other types of movements in C. elegans. The combination of muscles and motor neurons used for locomotion is different from that used for the other movements such as chewing and swallowing (pumping motion). Therefore, a study of radiation effects on different types of movements might help to identify the ionizing radiation-affected regions among muscles and the nervous system and here we examine the radiation effects on pumping of pharyngeal muscles in particular. In the experiments, 50 or more well-fed adult C. elegans were placed on an agar dish with a bacterial lawn (food) and irradiated with a graded dose of ⁶⁰Co gamma rays. Pharyngeal pumping in 5 or more animals was recorded using a high-speed camera every 2 h from 0 h to 8 h post-irradiation. The frequency of pharyngeal pumping was counted using 60 continuous recording images of 3 s duration. We found that irradiated animals could be classified into 2 groups. One group stopped pumping immediately after irradiation and the other showed normal pumping activity. This tendency of the 2 groups was distinctly different from that of locomotion using body-wall muscles, wherein the motility of the irradiated animals decreased uniformly in a normal distribution wholly in a dose-dependent manner. In addition, the pumping activity was completely restored within 2 h and the restoration rate was higher than that of locomotion. Our findings indicate that the whole body irradiation reduced the pumping and locomotion in a different way, although both movements were restored within several hours. This suggests the existence of multiple action mechanisms of radiation effects on C. elegans' motility.

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The radioadaptive response is the induction of cellular resistance to moderate or high doses of radiation by previous exposure to low doses. Treatment with TPA or hydrogen peroxide mimicked X-rays in adaptation when applied at low doses. We have shown that protein kinase C (PKC)-specific inhibitor abolished the adaptive response and that low dose of X-rays induced an alteration in the localization of PKCalpha protein. Although these data suggested that PKCalpha is involved in the radioadaptive response, it has not bee directly proved yet. In this study, we examined the involvement of PKCalpha in the induction of radioadaptive response by using RNAi of PKCalpha to reduce the expression of the protein. We introduced siRNAs into mouse m5S fibroblasts, which target the mouse PKCalpha gene, Prkca. The decrease in the PKCalpha expression was confirmed since both the amount of PKCalpha protein detected by Western blot and the Prkca cDNA quantity analyzed by RT-PCR were reduced. Radioadaptive response in the m5S cells were examined using cells which were pre-treated with low concentration of hydrogen peroxide for 5 hours before the irradiation with 5Gy X-rays, and analyzed the number of micronuclei. The radioadaptive response was suppressed in the cells with reduced amount of PKCalpha protein. In addition, the amount of phosphorylated PKCalpha protein was increased by the treatment with low concentration of hydrogen peroxide, indicating the activation of PKCalpha. These results suggest that the activation of PKCalpha might play an important role in radioadaptive response.

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