Purpose: DNA double-strand breaks (DSBs) are considered the most critical determinant of the fate of the cells or organisms exposed to radiation. DSBs are mainly repaired through homologous recombination (HR) or non-homologous end-joining (NHEJ). DNA-PKcs and XRCC4 are thought to play central roles in NHEJ. The present study aimed to clarify the interrelationships between XRCC4, DNA-PK and ATM, which shows a structural similarity to DNA-PKcs.
Methods: 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) and XRCC4 cDNA mutations on the phosphorylation sites by DNA-PK (M10-S2,3A). NU7026, a DNA-PK inhibitor, KU55933, an ATM inhibitor and wortmannin, inhibiting both of DNA-PK and ATM were added to culture media 1hr prior to irradiation at the concentration of 10microM. Nuclear reactor UTR-KINKI or X-ray generator were used as radiation source. Radiosensitivity was evaluated by colony forming ability in media containing 0.16% agarose.
Results: None of the inhibitors altered the radiosensitivity of M10-CMV cells. On the other hand, all the agents enhanced the radiosensitivity of M10-XRCC4 and M1-S2,3A cells. However, the extent of radiosensitization was greater in M10-XRCC4 than M10-S2,3A and wortmamnnin was more effective than NU7026 and KU55933, which showed similar effects.
Conclusions: These results collectively suggested that DNA-PK and ATM might complementarily regulate XRCC4 via phosphorylation in DSB repair. The existence of additional phosphorylation site(s) was also indicated.

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Cells exposed to Ionizing radiation, UV light and some chemotherapeutic compounds are prone to the formation of DNA-protein crosslinks (DPCs) in their genome. DPCs are detrimental or even lethal to cells. Recently, we have shown that homologous recombination (HR) and nucleotide excision repair (NER) collaborate to tolerate or repair DPCs. Here we further characterized the repair and tolerance mechanisms of DPCs. We found that the damage tolerance mechanism involving HR and subsequent replication restart (RR) provides the most effective means of cell survival against DPCs. Translesion synthesis does not serve as an alternative damage tolerance mechanism for DPCs in cell survival. Elimination of DPCs from the genome primarily relies on NER, which provides a second and moderately effective means of cell survival against DPCs. Interestingly, Cho rather than UvrC seems to be an effective nuclease for the NER of DPCs. Independently of the repair of DPCs, DNA glycosylases mitigate azaC toxicity, presumably by removing 5-azacytosine or its degradation product from the chromosome. Finally, topA may have a role in the repair of FA-induced DPCs.

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In the previous study we have detected the nuclear gH2AX/53BP1/ATM foci even after one or two months culture of in vitro X-irradiated normal human diploid fibroblasts (NHDFs). The persistence of such nuclear foci appears to be attributable to the existence of unrepairable double strand breaks (DSB) in non-apoptotic cells. We here report that the generation of such unrepairable foci is strictly dose dependent but not dependent on growth condition; equivalent frequency of unrepairable foci production was observed in quiescent and serum stimulated cells if the radiation doses were the same. The unrepairable foci were larger than those of vast majority of repairable foci, which were generated immediately after irradiation. However, the unrepairable foci still contained proteins of gH2AX, 53BP1, phosphorylated ATM, as well as they retained polyubiqutinated forms of chromatin components and BRCA1, indicating that repair and checkpoint factors were kept activated at the foci.

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CHO-K1 cells and HeLa cells were irradiated with low energy iron ions (0.93 MeV u-1) at the HIMAC in the NIRS. Cells were immunostained against γ-H2AX, as an indicator of DNA double-strand breaks (dsbs), to clarify the correlation between the expected average number of ion traversals in the nucleus and the observed number of γ-H2AX fluorescent spots. The correlation between the number of γ-H2AX spots observed and calculated ion traversals was approximately 1, indicating that dsbs were produced at each ion traversals. The cell inactivation cross-section (σ) obtained from the survival curves was 98.0 γm2, which was 0.45 of the average area of nucleus of HeLa cells, indicating that one lethal required 2.2 ion traversals. Further investigation with lighter ions, such as C, N, O, Ne, and Ar ion were also performed and the σ showed no less than half the size of nucleus area. Pulse field gel electrophoresis was also performed to measure the dsbs induction per ion track by FAR assay, and was roughly estimated be more than 20 dsbs per track. The amount of fragmented DNA did not decrease even with 6 hr incubation in culturing condition after irradiation. From these experimental results, three possibilities can be considered. One is, DNA may not be the sufficient factor for cell killing, and secondary lethal factor, yet not clarified, exist. Secondly, radiation sensitive site in the cell nucleus are approximately half the size of nucleus. Thirdly, contrary to the production of dsbs along the particle traversals, the area of the cell nucleus are not equally sensitive to radiation, and perhaps some kind of radiation sensitive distribution exists in the cell nucleus.

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Purpose: We have studied the effect of ascorbic acid on radiation protection and our results have revealed that ascorbic acid cannot suppress the lethal effect induced by radiation, for example, chromosomal aberration, but can effectively suppress the non-lethal effect leading to mutation and cancer (Koyama et al. Mutat. Res. 1998). Now we focused on the induction of DNA double strand breaks, which is the main cause of the lethal effect, induced by radiation and examined whether ascorbic acid can suppress the induction of DNA double strand breaks.
Method: Human embryonic fibroblasts (HE17) cells were used. HE17 cells were cultured and the confluent cultures were treated with ascorbic acid (AsA, 5mM) or dimethyl sulfoxide (DMSO, 2%) for 2 hours and then were irradiated by X-ray. To visualize the site of DNA double strand breaks, immunofluorescence on p53-binding protein-1 (53BP1) foci, which are known to accumulate on this site, was performed. We counted the number of foci formed in nuclei at 15 minutes, 2 hours, and 24 hours after 1.8 Gy X-irradiation, and examined whether treatment with AsA or DMSO can decrease the number of foci. We counted the number of foci per 200 cells from each treatment group and evaluated using the average number of foci. Moreover, we examined the effect of AsA and DMSO on radiation protection by colony formation assay to confirm the effect of the same treatment as described above on survival fraction.
Results: In non-irradiated groups, there is no effect of AsA or DMSO treatment on the formation of 53BP1 foci at any time after X-irradiation. While in irradiated groups, we observed the number of 53BP1 foci at 15 minutes after X-irradiation as follows: non-treatment, 37.8; AsA treatment, 35.2; DMSO treatment, 27.0, and at 2 hours after X-irradiation as follows: non-treatment, 26.6; AsA, 24.8; DMSO, 17.0. Besides, we observed the number of 53BP1 foci at 24 hours after X-irradiation as follows: non-treatment, 4.0; AsA treatment, 3.6; DMSO, 2.2. These results indicate that treatment with AsA can little suppress the induction of 53BP1 foci formation by X-irradiation, while treatment with DMSO can. In other words, this suggests that AsA cannot suppress the induction of DNA double strand breaks, while DMSO can. In colony formation assay, we also observed that AsA cannot decrease lethal effects, while DMSO can. Taken together, these results suggest that AsA can little induce DNA double strand breaks and cannot decrease lethal damage.

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When the genome DNA of the eukaryotes is damaged and caused double strand breaks (DSBs), H2AXs nearby the damaged region are phosphorylated by the protein kinases such as ATM, NBS1, and DNA PKcs. Therefore, it is possible to assume that localization of γ-H2AX reflects the location and quantity of DSBs in the nucleus.
Effects of the ion-beam to the cell are thought to be partially attributed to the localized energy deposition, especially in the case of a heavy ion-beam, that deposites large energy along the particle track. When such beams are irradiated to the cell nucleus, it is predicted that concentrated DSBs are evoked along the beam track. Actually, there have been some reports, including ours, that indicates γ-H2AX foci formations reflecting the beam tracks after the irradiation of heavy ion-beam.
However, there is no detailed information about the morphological changes of γ-H2AX foci according to the loss of beam energy during the penetration. We report the morphological changes of γ-H2AX foci along the path of ion-beam, such as hydrogen, carbon, and argon ions, in the culture medium and cells.

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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. Ataxia-telansiectasia mutated (ATM) is activated by double strand breaks (DSB), and phosphorylates various signaling molecules involved in checkpoint regulation, apoptosis, and DNA repair. However, it is unclear whether ATM is modified with O-GlcNAc and how O-GlcNAcylation regulates the functions of ATM. We have reported that ATM derived from primary cultured neurons in mice was modified with O-GlcNAc. Therefore, in the present study we investigated the regulation of O-GlcNAc modification in the function of ATM.
Primary cultured neurons and Hela cells were exposed to X-irradiation at the dose of 5 Gy. After irradiation, whole cell lysates were immunoprecipitated using anti-ATM and anti-O-GlcNAc antibody, followed by analyses of O-GlcNAcylation of ATM and protein interactions of ATM with other signaling molecules.
From the results of immunoprecipitation with anti-ATM or anti- O-GlcNAc antibody, ATM derived from Hela cells was also modified with O-GlcNAc. After 5 Gy-irradiation, phosphorylated ATM (Ser 1981) (p-ATM) was increased significantly, while p-ATM was not detected in the immunoprecipitated samples with anti-O-GlcNAc antibody. It was suggested that O-GlcNAc modification was decreased in p-ATM.

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Base excision repair (BER) is one of the dominant pathways to repair altered DNA bases, and is well conserved from bacteria to mammals. Single nucleotide BER (SN-BER) is a simple pathway to remove a single nucleotide. We have investigated several protein–protein interactions and their coordination in SN-BER initiated by methylpurine-DNA glycosylase (MPG). MPG recognizes and excises damaged purines, such as hypoxantin or methylated bases, from DNA. With activity assays using radio-labeled duplex oligonucleotide substrates, we concluded that the downstream enzymes increase the upstream enzyme activity in SN-BER. For example, AP endonuclease 1 (APE1) increased MPG activity, and DNA polymerase β increased MPG and APE1 activity. Since the stimulations resulted from increases of kcat values, a downstream enzyme would accelerate the disassociation of the upstream enzyme from the product. Intracellular X-ray repair cross complementing protein 1 (XRCC1) is thought to make complexes with 80 percent of DNA Ligase 3 (LIG3) molecules under physiological conditions, and interacts with almost all of BER enzymes to stabilize the configuration. In the presence of recombinant XRCC1, kcat/Km value of MPG and APE1 activity increased 1.8-fold and 7.1-fold, respectively, suggesting the involvement in a repair complex during the early step of SN-BER pathway. The studies on the effects of XRCC1-LIG3 complex to MPG and APE1 activities are in progress.

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Base excision repair (BER) is highly conserved among various organisms as one of the major repair mechanism against altered bases in DNA. In single nucleotide BER pathway, a specific DNA glycosylase removes a damaged base. Resulting an apurinic/apyrimidinic site (AP site) is removed by DNA polymerase I, which fills the gap with a correct nucleotide to leave a nick to be sealed by DNA ligase III. X-ray repair cross complementing protein 1 (XRCC1) is known to interact physically with not only DNA ligase III but also other BER related proteins as a scaffold protein, suggesting that XRCC1 is involved in the whole BER pathway. To study the role of XRCC1 in the BER pathway against methyl methanesulfonate (MMS)-induced damages, we prepared XRCC1 knockdown cells with shRNA. We designed a shRNA against human XRCC1 (NCBI: NM 006297), and constructed the shRNA expression plasmid with psiRNA-hH1GFPzeo G2 (InvivoGen). We transfected the plasmids into HeLa RC355 cells with a liposome transfection agent, and selected the transfectant clones by zeocin resistance. With western blotting analysis, XRCC1 protein level in a knocodown (KD) clone was 44% of that in the negative control. Using colony-forming assay, we detected a significant decrease in survival of the KD clone at 1.5 mM MMS, as compared with the control. To reveal the effect of XRCC1 knockdown on methylpurine DNA glycosylase activity, ARP assay of the KD clone is in progress.

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DNA is exposed continuously to various exogenous or endogenous damaging factors. Most of simple altered bases emerged under the physiological condition are repaired by base excision repair (BER). BER is initiated by removal of the altered base by specific DNA glycosylase leaving apurinic/apyrimidinic site (AP site). This process leads to two repair pathways, short-patch BER (SP-BER) and long-patch BER (LP-BER) in which polymerase β (polβ) and Flap endonuclease 1 (Fen1) take major roles, respectively. We constructed the RNAi inductive plasmid that using the shRNA expression vector to clarify how SP-BER and LP-BER were selected for the repair of alkylated base damages. By introducing the plasmid into mouse embryonic fibroblast (MEF), the knockdown (KD) cells were obtained. The KD efficiency was 87% in polβ, and 92% in Fen1 KD cells when the protein levels were examined by western blotting analysis. Despite the almost same suppression levels, the polβ KD cell showed significantly higher sensitivity to methylmethanesulfonate than the Fen1 KD cell. Detailed kinetic study is in progress to reveal the relative contribution of two BER subpathways to the repair of MMS damages.

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Fractionated radiation (FR) is used in radiotherapy to allow the recovery of normal tissues from sublethal damage. To elucidate the biological effect of FR exposure, we studied the DNA damage response of FR in human tumor cell lines, HepG2 and HeLa. We found that cells with long-term FR exposure to 0.5 Gy X-rays twice per day for more than 31 days (31FR cells) overexpressed cyclinD1. Cyclin D1 overexpression was also observed in 31FR-31NR cells, 31FR cells cultured without FR for further 31 days. Interestingly, 31FR-31NR cells exhibited gamma-H2AX foci (phosphorylated H2AX), the marker of DNA double strand brakes (DSBs), without FR. Thus, these cells induced DSBs for a long time after FR exposure. Gamma-H2AX positive cells were also positive for PCNA, the marker of S-phase of the cell cycle, indicating that cells with DSBs were in S phase.
In this study we analyzed the correlation between cyclin D1 overexpression and DSBs formation. Gamma-H2AX was decreased in 31FR cells by down-regulation of cyclinD1 expression using siRNA targeting cyclinD1. This study is first to demonstrate that cyclin D1 overexpression is associated with induction of DSBs.
The level of cyclinD1 is tightly controlled for the normal progression of the cell cycle and its deregulation is linked to the development of various types of cancer. CyclinD1 overexpression caused by long-term FR exposure may lead to genomic instability due to the induction of DSBs.

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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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A compound having a relatively heavy atom would be a target molecule of radiotherapy, because of its large X-ray absorption coefficient. We have investigated the mechanisms of enhancement of the radiation biological effect by such a radiosensitizer, mainly using a platinum-containing molecules, chloroterpyridine platinum II (PtTC). The spatial distribution of the compoinds within the cell, is a very important factor for radiosensitization. We found that PtTC was not incorporated in the cell nucleus, although it showed a certain level of enhancement of the cell death. Recently, we started to use platinum-nanoparticle as the one of the candidate of an effective radiosensitizer. The metallic nanoparticle, can be modified its function by changing of the surface molecules. Solution of plasmid DNA, mixed with platinum nanoparticle, was irradiated with carbon ions (HIMAC, NIRS) or monochromatic soft X-rays (Photon Factory, KEK). The enhancement of the DNA strand breaks was almost same level as the PtTC.

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Asymmetric cell division is highly unique in stem cells. In the present study, we examine the possibility that the selective chromosome segregation is responsible for the asymmetric cell division in stem cells. We previously demonstrated that 3% of mouse neurosphere cells segregated chromosomes selectively by labeling newly synthesized DNA with 5-bromo-2-deoxyuridine (BrdU). Here, we enrich neural stem cells of neurosphere cells derived from mouse embryonic forebrain and examine the selective chromosome segregation in the enriched stem cell populations. We used mouse embryonic fibroblasts (MEFs) as a control. 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. To examine the selective chromosome segregation, cells were cultured in the medium containing BrdU for 48 hr to label de novo synthesized DNA. After removal of BrdU, the cells were cultured in the medium containing cytochalasin B for 24 hr to make binucleated cells. Then, the cells were fixed and immunostained with anti-BrdU antibodies. BrdU-labeled binucleated (BN) cells, which consisted of 91% of all BN cells examined, were classified into two types, i.e., homogeneously stained cells and heterogeneously stained cells. The former is that both nuclei are BrdU positive, and represents the cells that experienced the random chromosome segregation. The latter is that only one nuclear is BrdU positive, and represents the cells that experienced the selective chromosome segregation. The result revealed that 10% of BN cells were the heterogeneously stained cells in the enriched neurosphere cells. In contrast, only 0.7% of BN cells were the heterogeneously stained cells in MEFs. Thus, the present results strongly support the idea that neural stem cells segregate chromosomes selectively.

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To elucidate the mechanism for the transmissible trait of genomic instability induced by ionizing radiation, we hypothesize that the transmission of genomic instability to the progeny is mediated by an irradiated chromosome, and examine this possibility using the chromosome transfer experiment. In the present study, we transferred a human chromosome 8 exposed to 4 Gy of soft X-rays into unirradiated mouse recipient cells by microcell fusion and examined the numerical and structural changes of the transferred human chromosome in the microcell hybrids detected by fluorescence in situ hybridization (FISH) using a fluorescent probe specific for the whole human chromosome 8. In unstable microcell hybrids, we investigated the instability of telomere and subtelomere of human chromosome 8 by telomere FISH and subtelomere FISH, respectively. A human chromosome 8 was structurally stable in all microcell hybrids examined in case of no irradiation, suggesting that the chromosome transfer itself dose not make the transferred chromosome unstable. In contrast, the irradiated chromosome 8 was unstable and involved in multiple chromosome rearrangements in one of seven microcell hybrids. The telomere FISH analysis indicated that the translocations between an irradiated human chromosome and a mouse chromosome might be formed by reunion of chromosome breaks but not by telomere-telomere fusions. The subtelomere FISH analysis indicated that the subtelomere signals of short arms were lost in 90% of cells and that the subtelomere signals responsible for short and long arms localized on two different chromosomes in 10% cells. These results suggest that the irradiated chromosome has a trait to interact with an unirradiated chromosome and act as a driving force for accelerating instability.

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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 and leads to enhanced access by proteins, including NBS1 and others, that participate in homologous recombination-mediated repair However, it is likely that another histone modification could be involved in this DNA repair pathway because H2AX knock out 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, 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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Double strand breaks (DSBs) occur in mammalian 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. Ataxia telangiectasia mutated (ATM) is a central kinase in these responses. Upon indction of DSBs, ATM phosphorylates various proteins involved in cell cycle checkpoint, DNA repair, and apoptosis, such as Nbs1, 53BP1, p53 and ATM itself. To identify a new ATM target protein, we performed 2D electorophoresis analysis. Cell lysates from IR-exposed or control cells were resolved by 2D electorophoresis. Proteins were transfered to a nylon membran, and subjected to immunobloting analyses with antibody specific to the phosphorylated form of Nbs1, 53BP1, or ATM. We identified a protein that is cross-reactive to anti-phospho ATM specific antibody. The phosphorylation of this protein was completely dependent on exposure of cells to IR, suggesting that this protein is a participant of cellular responses to IR. We also used antibody specific to mono-, or poly-ubiqutinated proteins in our 2D electorophoresis analysis. We will present recent results of the 2D electorophoresis analysis.

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DNA precursors, i.e., dNTPs in the nucleotide pool, are oxidized during aerobic metabolism as in the case of DNA. Oxidized dNTPs could be incorporated into the genome DNA by DNA polymerases. We have shown possible involvement of Y-family DNA polymerases in the incorporation of oxidized dNTPs. Interestingly, the DNA polymerases incorporate 8-oxo-dGTPs opposite template dA much more frequently than dC. In this study, we assessed the amino acids involved in the preferential incorporation of 8-oxo-dGTP by human DNA polymerase κ (hPolκ) and η (hPolη). In hPolκ, the substitution of Y112 with alanine resulted in the ratio of incorporation of 8-oxo-dGTP opposite template dA:dC from 11:1 to 1:1. It suggests that Y112 may stabilize the 8-oxo-dGTP in the syn formation by aligning the sugar, because Y112 distinguishes rNTPs from dNTPs by recognizing 2'-OH of rNTPs. In hPolη, the substitution of F18, which corresponds to Y112 in hPolκ, did not change the preference. However, substitution of R61, which is not conserved among other Y-family polymerases, with alanine or lysine substantially changed the preference for incorporation of 8-oxo-dGTP opposite dA and dC; the ratio (dA:dC) of 660:1 in the wild-type hPolη was changed to 63:1 in R61A and 7:1 in R61K. The structural analysis of the incoming 8-oxo-dGTP in the active site of yeast Polη suggest that C8 oxygen of 8-oxo-dGTP in the anti formation may cause a sterical clash with R61, which lead to destabilization of 8-oxo-dGTP in the active site, although 8-oxo-dGTP in the syn formation could not be affected because of the lack of the structural interference. These results suggest that the amino acids affecting the specificity for incorporation of 8-oxo-dGTP are different between hPolκ and hPolη.

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Intracellular distribution of 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been examined by the immunostaining technique of human lung carcinoma A549 cells exposed to X-rays, carbon and iron ions. The LETs of carbon and iron ions were approximately 13 and 440 keV/μm, respectively. After fixation of irradiated cells, intracellular 8-OHdG was reactivated with diluted hydrochloric acid and detected by the immunostaining technique with the use of anti- 8-hydroxy-guanine antibody-FITC conjugates (Kamiya Biomedical. Co., USA). We also used the Fenton-reaction system to generate OH radicals as a positive control. The intracellular 8-OHdG exposed to carbon ions at a dose of 5 Gy was significantly detected and was distributed uniformly over the whole area of a nucleus. Furthermore, X-ray-induced 8-OHdG showed the same distribution as the case of carbon ions. In contrast, the intranuclear distribution of 8-OHdG by iron ions with a dose of 5 Gy seemed to be somewhat heterogeneous and the fluorescence intensity was less than those of X-rays and carbon ions. The distributions in other LETs in carbon and iron ions are under examination.

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The genomes of all living cells are continuously under attack by a variety of genotoxic agents. Translesion DNA synthesis (TLS) is one of the pathways to bypass DNA lesions with low fidelity of nucleotide insertion. TLS is believed to be the major source of DNA damage-induced point mutations and to have a close association with cancer. Many of TLS enzymes belong to the Y-family DNA polymerase, which can replicate various DNA template lesions. Member of Y-family in higher eukaryotic cells include polη, ι, κ, Rev1. Rev1 is highly conserved from bacteria to vertebrate and assumed to be an essential regulator of TLS events. Disruption of Rev1 is sensitive to a wide range of DNA-damaging agents, such as UV light, hydrogen peroxide, cisplatin, and X-ray.
To determine the role of Rev1 on radioresponse in a mouse model, Rev1 transgenic mice carrying murine Rev1 cDNA under the metallothionein promoter were constructed. We quantified radiation-induced somatic mutant frequencies by measuring T-cell receptor (TCR) in Rev1 Tg mice both before and after exposure to irradiation. TCR mutation assay is a sensitive indicator of exposure to ionizing radiation. We also measured the number of micronucleated reticulocytes in peripheral blood of Rev1 Tg mice in order to evaluate the radiation sensitivity. Both assays indicated the different radioresponse in Rev1 Tg mice compared with wild-type mice, which is supposed to the involvement of Rev1 on radioresponse.

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Mismatch repair (MMR) is the major pathway to improve the fidelity of DNA replication by removing mispairs from newly synthesized DNA. Especially, Msh2 gene is one of the key genes of MMR system. Studies on mice deficient in MMR genes such as Msh2, Mlh1 and Pms2 have shown that they have high levels of spontaneous mutation and high incidence of cancer indicating a close relationship between mutation and cancer. These studies, however, were done on adult mice and never examined at an earlier stage of life. Since DNA replication starts soon after fertilization, we have wondered if the spontaneous mutation increases in early life when the MMR system does not work. Here, we studied mutational burdon in Msh2-KO mice at embryonic and young stage of life. Spontaneous mutation level was judged on lacZ transgene in Muta^TM mice missing Msh2 gene. They were created by mating Muta^TM and Msh2(+/-) mice. Mutations in Msh2(+/-) were elevated at 9.5 days embryo and increased further till newborn age. Mutation levels in spleen and liver at 2 months of age after birth were similar to the levels at newborn stage. In brain, on the other hand, the mutation level increased continuously in the same period. These age- and tissue-dependencies could be explained by difference in gene expression level of Msh2. Preliminary study on mRNA levels by qRT-PCR seemed to support the idea. We are also examining the malformation frequencies in Msh2-deficient mice. So far, we do not observe any increase, suggesting that high level of somatic mutation might be compatible with normal embryonic development.

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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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Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutation of WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Werner syndrome cells show the sensitivities to several DNA damage, suggesting that WRN mights works in DNA damage response. Here, we investigated the role of WRN in several kinds of DNA damage responses using Werner syndrome cells and NBS and AT cells. We also examined the role of DSB repair and TLS DNA synthesis.
WRN rapidly formed discrete nuclear foci dependently on NBS1 following DNA damage. NBS1 physically interacted with WRN through its FHA domain and important for the phosphorylation of WRN. WRN formed DNA damage-dependent foci during S phase, but not in G1 phase. However, Werner cells showed the homologous recombination activity at normal level, although HR repair functions preferentially during S phase. WRN also interact with PCNA without DNA damage, but the generation of DNA damage let WRN dissociate from PCNA and caused the ubiquitination of PCNA, which is essential to tlanslesion DNA synthesis. WS cells showed constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase without DNA damage. Taken together, WRN might mediate the interaction of PCNA with Rad18 and the subsequent ubiquitination of PCNA, and could be involved in the regulation of TLS to prevent mutation.

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AT (ataxia-telangiectasia) is an autosomal recessive human genetic disorder characterized by radiosensitivity, chromosomal instability, and radioresistant DNA synthesis. The ATDC (AT group D complementing) gene was identified by its ability to restore the radiosensitivity of AT group D cells. Several sizes of ATDC mRNAs have been detected in various cell lines. The ATDC expression is elevated in various cancer tissues and is thought to promote tumor cell proliferation. ATDC is known to be a member of tripartite motif (TRIM) family, which contains a leucine zipper motif and a zinc finger domain. Because the molecular function of ATDC is still unknown, we are trying to establish the cells with ATDC overexpression or the ATDC knockout cell lines.
Overexpression of ATDC partially complemented radiation sensitivity of ATM knockout DT40 cells. This result suggests that ATDC might function in a pathway downstream ATM in response to DNA damage. To examine the role of ATDC in DNA damage repair in ATM deficient cells, ATDC was overexpressed in AT fibroblast carrying SCneo reporter, which can detect homologous recombination repair at a site specific DNA damage. Homologous recombination frequency was not affected by ATDC overexpression, suggesting that ATDC is not directly related to homologous recombination repair of DNA damage. Because ATDC+/- cells showed a significant delay in their cell growth, ATDC might be required for efficient cell proliferation.

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Reactive oxygen species (ROS) are generated in living cells during normal cellular metabolism as well as by exogenous stimuli such as ionizing radiation and various chemical oxidants. DNA carrying vital genetic information of cells constantly suffers from spontaneous oxidation by ROS. ROS produce a wide variety of oxidative damage to DNA, including various types of base modifications. Oxidatively damaged bases in DNA cause deleterious effects such as mutation, cell death and aging. Recently, relationship between oxidative damage to DNA and aging has been suggested, but the molecular mechanisms remain unsolved. Base excision repair (BER) is the main pathway to repair oxidative base damage. DNA glycosylase and AP endonuclease are essential to maintain genome stability by excising various types of DNA lesions. It is necessary to examine whether the mutation in the BER enzymes affects the life span of cells and organisms. The nematode C. elegans is a useful model organism for studying aging because its lifespan can be determined easily and quickly. However, there are little reports on BER enzymes of C. elegans. In this study, we identified and analyzed DNA glycosylases and AP endonucleases of C. elegans. CeNTH- and Ung-1-deficient C. elegans showed no significant difference in lifespan compared to the wild type N2. The results suggested the existence of other DNA glycosylase(s) that functions to repair oxidized pyrimidines and uracil in DNA. That is, the deletion of one DNA glycosylase could be complemented by other DNA glycosylases that are able to excise the same types of damaged bases. In addition to BER enzymes, we are studying roles of the OXR protein in relation to aging in C. elegans.

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DNA pyrimidines are continuously oxidized in physiological condition. Because the productive damaged pyrimidines can result in tumor formation, they are thought to be repaired majorly with single nucleotide-base excision repair (SN-BER) pathway. The mammalian homologs of endonuclease III and endonuclease VIII, Nthl1 and Neil1, are 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. As mouse spleen, stomach and lung, showed higher TGG activity than liver, it is suggested that TGG activity may play a role mainly in proliferative organs. This suggestion is supported an idea that strong apurinic/apyrimidinic (AP) lyase activities accompanied with Nthl1 and Neil1 may be deleterious in a nuclear of a proliferating cell. It is reasonable that the TGG activity exists in various mammals, including mouse. The similar genome sizes of mouse, monkey, human are 3.3x10⁹, 3.0x10⁹, 3.0x10⁹ base pairs, respectively. On the other hand, the life spans are very difficult; about 3, 30 and 90 years, respectively. The level of TG in urine is correlated with life span (Adelman R. et al., 1988, Proc. Natl. Acad. Sci. USA), suggesting that the difference of life span is associated with that of TG removal activity and a repair mechanism. Recently, with an elongation of dog life span, the incidence for canine cancers has been increasing. It is expected that the level of TG removal activity in canine is lower than that in mouse. In this report, we show that the specific activity of TGG in canine liver is low to 67% of that in mouse liver.

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Although radiation has been used as the mutagen in mutation breeding, little is known about which types of mutation are induced in plant DNA. In this study, we irradiated carbon ion-beams (220-MeV C : LET=112 keV/μm) and γ-rays to Arabidopsis carrying rpsL gene to analyze mutational effects of these radiations. G:C to A:T transition and > 2 bp deletions/insertions were the major type of mutation by the irradiation of 220-MeV C and γ-rays to Arabidopsis dry seeds. The incidence of −1 and −2 frameshift mutations was also higher in γ-ray-irradiation group, suggesting that γ-rays might induce smaller size of deletion mutation compared to 220-MeV C. Our observation also suggested that induction of oxidized guanine by 220-MeV C and γ-rays was low in dry seeds because of low incidence of G:C to T:A and A:T to C:G transversion in irradiated group. We irradiated γ-rays to Arabidopsis seedling to assess the mutational effect of oxidative DNA damage in plant. Water contents and cell proliferating activity of Arabidopsis seedling are higher than dry seed. Therefore we thought the effect of water radiolysis and misincorporation of oxidized dNTPs during replication could have been investigated. Our result of γ-ray-induced mutation analysis in Arabidopsis seedling was similar to that in dry seed. G:C to T:A and A:T to C:G transversion were not increased, suggesting the mutational effect of radiation used here in higher plant might be different from that in other organisms.

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It has been reported that the frequency of tumors in X-irradiated mice is dependent upon the age at exposure to radiation. Since mutation in the cancer-related genes is supposed to be one of the critical causes which lead to tumorigenesis, we analyzed the fraction of somatic mutations in vivo at the Aprt locus, encoding adenine phosphoribosyltransferase, after exposure of mice to X-rays at different ages. As the Aprt gene resides on the mouse chromosome 8, it could be a good model to analyze the mutagenesis at autosomal genes. We used mice which are heterozygous for the Aprt locus with an APRT-defective allele as well as a normal allele. Mice were irradiated at the age of either 1 week or 7 weeks, with X-rays of 1Gy once, of 4Gy once, or of 1Gy for 4 times with 1 week intervals. At the eighth week after irradiation, spleens were removed and lymphocytes were separated for the cell culture. The fraction of mutations were assessed following the microtiter plate protocol using 8-aza-adenine as the selection drug for APRT-deficient mutants. Generally speaking, lymphocytes from the mice irradiated at the age of 1 week showed increased mutant fractions to a certain extent, in contrast to the lymphocytes from mice irradiated at the age of 7 weeks which did not show any increase in the mutant fractions. These results suggest that mice irradiated at neonatal age are more sensitive to the radiation-induced mutagenesis at the autosomal loci than mice irradiated at adult age, which might have some impact on radiation-induced tumorigenesis.

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It is well known that high LET radiation such as heavy ions is more effective than low LET radiation such as X and gamma rays for various biological effects. The RBE- LET curves had a peak in the LET range of 100 to 200 keV/µm even if the biological endpoints were different. To clarify the qualitative and quantitative difference in cellular and molecular effects between high- and low-LET radiations, we investigated the difference in mutation induction on the hprt locus in normal human fibroblasts irradiated with X rays (200kV, 20mA) and iron ions (500 MeV/n) with LET ranging from 200 to 400 keV/µm.
First, we showed the dose-response curves of mutation induction at hprt locus detected with 6-thioguanine resistant clones for X rays and iron ions, and calculated the RBE for iron ions relative to X rays. The RBE of the mutation frequency per total number of surviving cells for iron ions with LET ranging of 200 to 260 keV/µm were higher than 1, and the RBE for iron ions with LET ranging of 300 to 400 keV/µm were nearly 1. These results indicated that the mutation induction for iron ions decreased with increasing LET, and the mutation frequency of high LET region (over 300 keV/µm) was similar to X rays. Next, the deletion spectrum of 9 exons in hprt locus of mutant colonies induced by X rays (1.5 - 2.4Gy) and 260 keV/µm iron ions (0.2 - 0.8Gy) were analyzed using the multiplex PCR. About 70% of mutant induced by iron ions showed deletion of the entire exons, while about 70% mutant induced by X rays showed partial deletion, which one or more exons are missing. The results indicated that iron ion induced mutants sustained severer damage than X rays induced mutant even if the dose response curves for mutation induction were similar between X rays and iron ions.

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Aneuploidy—the presence of an abnormal number of chromosomes— occurs in ~90% of solid human tumors and ~75% of hematopoietic cancers and was recognized as a common characteristic of tumor cells 100 years ago. Although the role of aneuploidy in cancer has been at the center of debate for almost a century, whether it is a cause or a consequence of the malignant transformation (oncogenesis) has not been clear. So we examine the role of aneuploidy in tumorigeneous.
In this study, to determine the effects of an additional chromosome on diploid cell character that caused by spontaneousness, abnormality of centrosome, abnormality of chromosome segregation gene, or orther, we generated aneupolid cells that carried an additional chromosome. With immortalized human mesenchymal stem cells that carried an additional chromosome, we will report the result of mutation assay, anchorage-independent growth assay, micronuclei assay. And with nomal human cells, we will report the result of same assays.

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[Purpose] It has been hypothesized that persistent inflammation causes radiation-induced genomic instability through elevation of inflammatory cytokine levels. In the present study, we evaluated relationship between inflammation and genomic instability in the murine hematopoietic system in vivo, using two inflammation models.
[Method] Persistent inflammation was induced with graft-versus-host disease (GVHD) in (C57BL/6 x DBA/2) F1 (BDF1) and (BALB/c x C57BL/6) F1 (CBF1) mice that received hematopoietic cell transplantation from parent mice. Acute inflammation effects were assessed in C57BL/6 (B6) and BALB/c mice treated with different doses of Poly (I:C). Reticulocyte MN frequencies and inflammatory cytokine levels were measured by flow cytometry with CD71/CD61/PI staining and cytometric bead array, respectively.
[Result] MN frequencies significantly increased in both the BDF1 and CBF1 mice that received transplantation from parental B6 mice (p = 0.001, and p = 0.015, respectively). However, there was no significant change in MN frequencies in the F1 mice that received transplantation from the other parental mice. In addition, the increase of circulating TNF-α levels was profound in F1 mice receiving transplantation from B6 mice compared with those receiving transplantation from the other parental mice, and positively correlated with MN frequencies (r = 0.48 and r = 0.67 in B6-into-BDF1 and B6-into-CBF1 models, respectively). In mice with acute inflammation induced by Poly (I:C) treatment, dose-dependent increases in levels of inflammatory cytokines were observed, especially for TNF-α, as well as in frequencies of reticulocyte MN. Furthermore, the dose dependencies differed between these mouse strains.
[Conclusion] We conclude that inflammation becoming manifest with increased TNF-α level may be involved in genomic instability and that there may be a mouse strain difference in sensitivity to inflammatory response affecting genomic instability.

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It is well known that incorporation of halogenated pyrimidines, such as bromo-and iodo-deoxyuridines (BrdU,IdU) incorporation into DNA to replace thymidine is sensitive to cells to ionizing radiation. However, a relation about radio-sensitization between halogenated pyrimidines and heavy-ion beams is not well known. We examined a radio-sensitization effect of cells incorporated BrdU when irradiated by carbon-and iron-ion beam. Single and double cell cycle BrdU incorporated cells were irradiated at G1-phase after synchronization. After exposure to various radiation, we tested colony formation to cell survival, DNA double strand break formation and repair by gamma-H2AX focus formation assay, and chromosome aberration formation for first post irradiation metaphase. Although two cell cycles BrdU incorporated cells presented most severe radiosensitization effects, fewer radiosensitization was observed to after higher LET radiation. Our results suggested that incorporation of halogenated pyrimidines lead to produce complex DNA damages after radiation exposure. The reason of small BrdU effects in high LET exposure may be explained that high LET radiation has already produced such damages.

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DNA is considered to be the critical target for cell killing by ionizing radiation. Therefore it is expected that the photoionization of phosphorus in DNA may cause some specific biological effects. However, at the 45th Annual Meeting of the Japan Radiation Research Society, we reported the similar enhancement of cell lethality of photoionization of phosphorus for both normal and DNA repair deficient cell lines including non-homologous repair deficient or homologous recombination repair deficient cell lines. Here, we will report the gamma-H2AX induction by k-shell photoionization of phosphorus in wild type mammalian cells.
Exponentially growing L5178Y cells (derived from mouse leukemia) were irradiated by monochromatic X-rays at 2.153 keV (K-shell resonance absorption peak of phosphorus) and 2.146 keV (off peak) at the Beam Line-27A in KEK-PF. The amount of gamma-H2AX per cell after 20 min incubation, detected by flowcytometry increased linearly with increasing dose for the both energy of X-rays. The dose enhancement ratio was about 1.5 and was similar to that for cell inactivation.

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It is well known that reactive oxygen species (ROS) are generated in the cells by radiation and ROS induced cellular damage is a major cause of the indirect effect of radiation. We are focused on the oxidation of nucleic acid to understand the cause of genetic instability. We have suggested that 8-oxoguanine (8-oxoG, major oxidized form of guanine) causes base substitution and chromosome recombination in the mammalian genome. To elucidate the influence of 8-oxoG on germline mutations, we analyzed the expression of each gene (Ogg1, Mth1, Mutyh) in germ cells and the effects of their deficiency. We found that each gene was highly expressed in meiotic cells of the testis. We also observed some cytogenetic abnormalities of chromosomes in the mutant cells. Moreover, triple-mutant mice lacking Ogg1, Mth1 and Mutyh exhibited increased frequency of hereditary congenital abnormalities as well as cancers, suggesting that the accumulation of 8-oxoG in germ cells increases their mutation frequency, thereby generating germline mutations.

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We used budding yeast S. cerevisiae and analyzed influence in it by two kinds of different radiations, a carbon beam and the proton beam. A survival rate was less than 10% by irradiation of 50Gy by the carbon beam and less than 10% by irradiation of 300Gy by the proton beam. Interestingly, a survival rate was not 0% even if we irradiated 2000Gy by the carbon beam. Furthermore, we compared the survived yeast by carbon beam irradiation of 2000Gy with a wild type strain, and a survival rate increased 8-10 times with a carbon beam of 300Gy, and a survival rate increased more when we irradiated once again. This result suggested that the yeast got radiation resistance. Therefore we performed single colony isolation to analyze specific having radiation resistance yeast or not. Concretely, we analyzed change of growth speed, high temperature sensitivity, low temperature sensitivity, MMS sensitivity, Bleomycin sensitivity and the change of the cell cycle by the checkpoint defect. In addition, we report a difference of the protein by the two-dimensional electrophoresis and a abnormality of the chromosome by a pulse field electrophoresis

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Aldehyde compounds are known to induce DNA-protein crosslinks (DPCs) at high frequency in the genome. It has been shown that aldehyde compounds also induce other types of DNA lesions such as inter- and intra-strand crosslinks, strand breaks, and base adducts in vitro and in vivo. However, the spectra and yields of DNA lesions induced by aldehyde compounds have not been compared on a quantitative basis. This type of information is crucial for understanding the molecular mechanism of genotoxic effects of aldehyde compounds. In the present study, we analyzed the cell-killing efficiencies of aldehyde compounds and DNA lesions induced by the compounds. HeLa cells were treated with formaldehyde (FA), trans-2-pentenal (PEN), crotonaldehyde (CRA), glutaraldehyde (GA), acrolein (ACR), and chloroacetaldehyde (CAA). Cell survival was analyzed by colony formation. The concentrations that gave 10% cell survival were FA (220 μM), PEN (200 μM), CRA (105 μM), GA (22 μM), ACR (17.5 μM), and CAA (10.5 μM). HeLa cells were treated with these concentrations of aldehyde compounds, and genomic DNA was isolated. The amount of DPCs was measured by Western blotting. The result showed that FA, GA, and PEN induced DPCs very efficiently, whereas ACR, CRA, and CAA induced DPCs with poor to negligible efficiencies. We will analyze the amount of DNA double strand breaks and DNA strand crosslinks. These data will be presented in the meeting.

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Malignant mesothelioma (MM) is a highly aggressive tumor arising from serosal surfaces of the pleura/peritoneum caused by asbestos exposure. MM has three histological types, epithelioid, sarcomatoid and mixed, and its incidence is expected to increase. Since the prognosis of patients remains poor after combined treatment with surgery, chemotherapy and radiation, it is important to develop more effective treatments. Thus, we evaluated the efficacy of carbon-ion radiotherapy (CIRT) and X-ray radiotherapy (XRT) for MM in mouse models. We inoculated human epithelioid and sarcomatoid MM cells subcutaneously into nude mice. Xenografts were irradiated with 2-30 Gy of carbon ions (290 MeV/u, a 6-cm SOBP), or 5-60 Gy of X-ray (200 kVp, 20 mA). Although the sizes of xenografts with CIRT (30 Gy) or XRT (60 Gy) increased until 2 weeks after irradiation in both tumor types, those turned to reduce thereafter. Pathological analysis showed increased cell death at 7 days after irradiation and increased fibrosis at 14 days. To determine whether the efficacy of treatment is evaluated before size reduction, we measured tumor uptake of ¹⁴C-FDG and ³H-FLT after CIRT (30 Gy) or XRT (60 Gy). In epithelioid tumors, FLT uptake decreased at 3 h and 1 day after CIRT and XRT. In sarcomatoid tumors, FLT uptake decreased with time after XRT, but it did not change after CIRT. FDG uptake was not correlated with efficacy of both treatments. These results indicated that the therapeutic efficacy of CIRT was twice as high as XRT in both types of mesothelioma. FLT-PET would be useful for the early assessment of treatment response in epithelioid tumors, but suitable tracer remains to be determined for sarcomatoid tumors.

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[Introduction] Quorum Sensing has recently been recognized as a type of signal transduction that senses the cell-population density and responds to changes in the cell number between bacteria cells. Its mechanism is controlled by the hormone-like low molecular autoinducer (AI). There have been numerous investigations of the regulation of bacterial using AI inhibitors, and synthetic analogs. On the other hand, little is known regarding the effects of AI inhibitors on the growth of human cells. The present study, examined the possibility of 96 synthetic autoinducer analogs of Pseudomonas aeruginosa quorum sensing having both suppressive and radiosensitizing effects on the growth of human oral cavity carcinoma cells.
[Materials and Methods] Synthetic 96 AI analogs of Pseudomonas aeruginosa quorum sensing were provided by Otsuka Chemical. The suppressive and radiosensitizing effects were tested by using human oral cavity carcinoma cell lines derived from tongue cancer cells (SAS) and gingival carcinoma cells (Ca9-22). Each cell line was cultured in liquid culture media containing 10 % fetal bovine serum. The clonogenic potential was tested by the plasma clot technique using platelet-poor human plasma. X irradiation was administered with an X-ray generator using 150 kV, 20 mA, 0.5-mm aluminum plus 0.3-mm copper filters at a dose of 90—100 cGy/min.
[Results and Discussion] A couple of compounds showed a significant suppressive effect on the growth of the SAS and Ca9-22 cell lines. The IC50 (50 % inhibition concentration) was in the range of 1.5—3.5 μg/ml in SAS and 2.2—5.4 μg/ml in Ca9-22. The radiosensitizing effect was observed with a 1 μg/ml compound and 2 Gy irradiation. These results demonstrated that some AI analogs showed anti-proliferative activity in human tumor cell lines. Currently, more detailed studies are underway to elucidate the structure activity relationships and suppressive mechanisms.

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Because of ongoing terrorist activity and dissemination of nuclear materials, the possibility of military or civilian personnel being exposed to ionizing radiation is a continuing threat. As yet, no drugs have been approved by the US Food and Drug Administration for treatment of hematopoietic or GI injury from penetrating ionizing radiation. Our research program aims to identify and develop pharmacological radiation countermeasures to prevent, mitigate, and treat the acute radiation syndrome. In light of the logistical realities of likely nuclear disaster scenarios, much of our current focus is on drug candidates with extremely low toxicity and ease of administration, suitable for use outside the clinic without physician supervision. Several promising radiation countermeasures are currently in various stages of development. Among the efficacious drugs, CBLB502 (truncated flagellin, a toll-like receptor-5 agonist and NF-kB activator), the steroid 5-AED (5-androstenediol), CBLB612 and CBLB613 (synthetic lipopeptides triggering activation of NF-kB through TLR2 receptor complexes), and tocopherol succinate showed radioprotection and radiomitigation activity in mice. 5-AED and CBLB502 were also tested in nonhuman primates (NHP) and found effective. We are investigating the ability of these countermeasures to modulate blood cytokine levels with the objective of identifying cytokines as biomarkers of drug efficacy against radiation damage. Mice and NHP serum/plasma samples from irradiated and drug treated animals were evaluated using multiplex Luminex for quantification of cytokine concentrations. Our results in mice indicate that all radioprotective compounds stimulated G-CSF production. CBLB502 and CBLB612 induced maximum levels of G-CSF within 2-8 h, while the effect of tocopherol succinate was maximal 24 h post-injection. In addition to G-CSF, 5-AED stimulated production of IL-6; CBLB613, CBLB612 and CBLB502 stimulated production of both IL-6 and KC (keratinocyte derived chemokine). The induced cytokine spectrum of CBLB502 and CBLB612 was tested in NHP and found to be very similar to that in mice. In both species, G-CSF, IL-6, and KC levels were correlated with drug dose. The CBLB502 dose dependence of cytokine levels coincided with CBLB502 dose-dependent radioprotection in mice. In conclusion, our results suggest that specific cytokines may serve as biomarkers for efficacy of radiation countermeasures, and may prove useful as a predictor of outcome.

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In situations of medical and occupational exposure, the radiological protection can be planned in advance. One possible method for protection, typically for dermal tissues, is a topical application of a kind of sunscreen which contains radioprotective agent with less adverse effects. Here we examined the radioprotective effects of five different natural plant-derived antioxidants, Caffeic acid (CA), Rosmarinic acid (RA), trans-Cinnamic acid (TCA), Hydroxy phenyllactic acid (HPA) and p-Coumaric acid (PCA) on cultured human keratinocytes (HaCaT). Among the compounds, the pretreatment of cells with CA, RA and TCA at the doses showing no cytotoxity (0.1-10μg/ml) resulted in approximately 10% recovery of clonogenic survival following gamma-ray exposure from Cs-137 source at 4Gy and 8Gy (1Gy/min). In addition to the intracellular antioxidative effect of these compounds against radiation-induced ROS generation as revealed by using CM-H₂DCFDA as an ROS indicator, the reduction of DNA double strand break by these compounds was also observed at 15min and 6h following the irradiation by immunofluorescent detection of gamma-H2A.X and 53BP1. These findings suggest that natural antioxidants CA, RA and TCA are the candidate ingredients of radioprotective agents through the mechanisms involving the inhibition of ROS generation as expected, and possibly, the promotion of DNA damage repair.

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Heavy particle radiotherapy is a useful means to eradicate solid tumors because of its physical, chemical and biological properties. It is, however, needed to accumulate the more evidence for the combined therapy between heavy particles and chemical agents for overcoming intractable tumors. In this study, we investigate a combination effect of carbon-ion irradiation and the telomerase inhibitor MST-312 treatment in tumor cells. Human cervical cancer HeLa cells were used here. Firstly, cells were cultured for 24 h at various concentrations of MST-312. After treatment, cells were reseeded in fresh medium without MST-312. Colonies having more than 50 cells were counted at day 14 to determine the survival. The survival decreased with increasing MST-312 concentration and cells could not form any colonies at 5 μM or higher concentration. Secondly, cells were cultured for 24 h with or without 1 μM MST-312, and then, irradiated with carbon ions (LET=110 keV/μm) and gamma-rays to evaluate the combined effect of irradiation and MST-312 treatment. After irradiation, cells were reseeded in fresh medium without MST-312 and colonies were counted as described above. The survival of the irradiated cells additively decreased in combination with MST-312 treatment. Irradiation and MST-312 treatment might inactivate the clonogenic potential of tumor cells through independent mechanisms. The doses needed to reduce the survival of irradiated cells to 10% were decreased by MST-312 treatment from 1.2 Gy to 0.5 Gy (carbon-ion irradiation) and from 5.4 Gy to 4.0 Gy (gamma-ray irradiation), respectively. MST-312 pre-treatment may reduce the dose needed for a curative effect in heavy-particle radiotherapy.

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Purpose: In this study, we reviewed an immunological enhancement effect, radiation protection effect and anticancer action for β-glucan (EF 2001) which was quality of natural product.
Methods: We divided an experimental group into control group, β-glucan treated group and 2Gy irradiation group and β-glucan + 2Gy irradiation group in total four groups. We used an SOD activity detection kit as an antioxidation experiment and tested it. We performed CD4, CD8 and CD16 analysis by flow cytometry as analysis of T lymphocyte. In addition, we examined activity of natural killer cell.
Result: The measurement of number of leukocytes revealed increase of leukocytes of a β-glucan treated group in comparison with control group. In addition, a radiation protection effect was seen in β-glucan+2Gy irradiation group after irradiation with lymphocyte counts. In the SOD-like active measurement, a rise was seen in a β-glucan treated group. In CD4, CD8 and CD16 measurement, remarkable increase of a β-glucan treated group was seen for control group. A killer cell and increase of suppressor T cell are thought about in β-glucan in this study.
Conclusion: Therefore, it is thought that there is anticancer action by a β-glucan intake by activation of a factor about attack to cancer cell. Immune therapy will attract attention in treatment for cancer in future. Therefore, we think that we must do a search of the target which is specific for cancer in treatment for cancer and basic researches of immune therapy from the genetic side.

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An FGF1:FGF2 chimera (FGFC) was created previously and showed greater structural stability than FGF1. FGFC was capable of stimulating epithelial cell proliferation much more strongly than FGF2 even in the absence of heparin. Therefore, FGFC was expected to have greater biological activity in vivo. This study evaluated and compared the protective activity of FGFC and FGF1 against the radiation-induced intestinal injuries. FGFC and FGF1 were administered intraperitoneally to BALB/c mice 24 h before or after total body irradiation (TBI). The surviving numbers of crypts per circumference were determined 3.5 days after TBI with γ-rays at doses ranging from 8 to 12 Gy. When 10 μg of each FGF was administered without heparin 24 h before irradiation, FGFC was more effective in promoting crypt survival than FGF1, although the effect of FGFC was equal to or slightly superior to FGF1 with heparin. FGFC was effective in promoting crypt survival even when it was administered without heparin at 24 h after TBI at a dose of 10, 11, or 12 Gy, whereas FGF1 did not increase crypt survival under these conditions. FGFC post-treatment promoted prominent BrdU incorporation into crypts, epithelial differentiation, and increased crypt depth. However, the number of apoptotic cells in FGFC-treated mice decreased to almost the same level as in FGF1-treated mice. These findings suggest that FGFC strongly enhanced radioprotection with the induction of epithelial proliferation without exogenous heparin after irradiation and is useful in clinical applications for both the prevention and post-treatment of radiation injuries.

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Malignant pleural mesothelioma (MPM) is an aggressive tumor arising from serous surfaces. Eighty percent of patients will have a definite asbestos exposure history, often with 20- 40 year latency between asbestos exposure and development of malignancy. Due to asbestos was widely used in the world between the 1940s and 1979, it has been estimated that the number of men dying from MPM increase each year until a peak is reached in about 2020. MPM is resistant to radiotherapy, surgery or chemotherapy, and only slightly improve prognosis. So far, no effective therapeutics has been established for the disease-advanced case. We examined the radiosensitivity of MPM cell lines by X rays and carbon-ion beams last year and this year we investigated the relationship between cell-killing effect and the biological character, such as the modal chromosome number. Six kinds of MPM cell lines were used and irradiated with X rays or carbon-ion beams (13keV/μm and approximately 80keV/μm) at the Heavy Ion Medical Accelerator in Chiba (HIMAC). Cell-killing effect was detected using a colony-formation assay. The number of chromosomes was observed in a metaphase spread. The results indicated there was a good correlation in the D₁₀ values of MPM cell lines between X rays and carbon-ion beams. Different MPM cell lines showed a great variation in numbers of chromosome, and the distribution of the number of chromosomes in the same cell line is also great different. The mean numbers of chromosomes of those cell lines are to be 48-95, and the D₁₀ values are not clearly correlated to the chromosome number of each cell line except for the MESO-4. The results suggest that the radiosensitivity of MPM cell lines correlate to LET of radiation but did not clearly depend on the modal chromosome number.

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Up to March 2009, over 4500 patients have been treated in HIMAC, and some kinds of tumor, for example the malignant melanoma showed a decrease of survival rate in contrast to high local control rate. It is an important subject to control tumor distant metastasis for future radiotherapy. The effects of radiation to metastasis must be clarified fundamentally. The aim of this study is to clarify the radiosensitivity of mouse melanoma cell line having high-metastatic potential to heavy-ion and photon beams, and to examine the inhibitory effects of radiations to metastasis. B16/BL6 cell line was used as high metastatic cell line. Carbon-ion beams (C-ions) accelerated with 290 MeV/u as high LET and X-rays or γ-rays as low LET beams. <in vitro> Survival curve of B16/BL6 to X-rays showed large shoulder, and RBE value of C-ions was 1.6. Horizontal migration was enhanced by radiations using wound-healing assay. In contrast, matrigel invasion assay showed that vertical migration was suppressed by C-ions. <in vivo> Survival of cells in a tumor was obtained using in vivo-in vitro assay, and the survival curve of even C-ions showed big shoulder. The RBE was 1.9 bigger than in vitro assay. In addition, RBE obtained from tumor growth delay analysis was too large, 3.7. The effect of radiations to metastasis was examined using spontaneous lung metastasis model. The lung metastatic nodules were decreased by C-ions compared with X-rays. Furthermore, survival of mice was extended by C-ions compared with non-irradiation and X-rays irradiation groups. It was suggested that high LET beams like C-ions have a potential to inhibit metastasis significantly more than low LET beams as X-rays and γ-rays.

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Purpose: Human tumors are heterogeneous in various biological features including radiosensitivities. We here studied significance of tumor heterogeneity in RBE of carbon ion therapy by using artificially mixed and transplanted experimental tumors in syngeneic mice. Materials and Methods: Three fibrosarcomas (#6107, #8697 and #9037) with similar growth rates were transplanted into C3H male mice. Cell suspensions prepared from 2 fibrosarcomas were mixed at the time of transplantation into hind legs of mice. Grew to 7-8 mm in diameter, tumors were locally irradiated with single doses of 74 keV/μm carbon ions or Cs-137 γ rays. Tumor diameters were measured after irradiation by calipers and tumor growth delay (TGD) time was calculated from growth curves. Five mice were used for each dose group. RBE values of carbon ions relative to γ rays were calculated by comparing isoeffect doses on dose-response curves. Results: Mixture ratios varied from 0:100, 10:90, 50:50, 90:10 and 100:0. Three combinations, i.e., #6107+#8697, #6107+#9037 and #8697+#9037,of the various mixture ratios provided 12 tumors with different heterogeneity. An isoeffect dose to produce TDG times of 15 days was used to calculate an BRE value on a dose response curve. Plotting all 12 RBE values on either γ-ray doses or carbon ion dose, the followings were obtained (1) Isoeffect doses distributed between 25 and 44 Gy for γ rays while between 6.5 and 25 Gy for carbon ions. (2) RBE values depended on carbon-ion sensitivities, and were larger for sensitive tumors than resistant tumors. RBE was independent of γ-ray sensitivities. (3) Any combinations of carbon-ion sensitive tumors (#6107 and #8697) showed RBE values either similar to or larger than parent tumors. (4) Any combinations of carbon-ion sensitive and resistant tumors showed RBE values larger than that of the resistant parent. Namely, combinations of carbon-ion sensitive (#8697) and resistant (#9037) tumors showed RBE values similar to the RBE of parental #8697 tumors while combinations of carbon-ion sensitive (#6107) and resistant (#9037) tumors showed RBE values between those of two parental tumors.Conclusions: RBE of carbon ions depended on carbon-ion sensitivities but not γ-ray sensitivities of tumors. It was carbon-ion sensitive tumors but not resistant tumors that determine an RBE of a heterogeneous tumor.

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Mucous membrane of small intestine is radiation-sensitive and damaged severely by local or systemic exposure of high dose of ionizing radiation via apoptosis in the crypt or the vascular endothelial cells. To find the therapeutic methodology, we examined drugs which contribute to regeneration of mucous membrane following lethal intestinal damage by radiation in mouse.
Damaging of whole small intestine was induced by abdominal exposure of 15.7 Gy of x-ray to anesthetized C3H/He mice. Parenteral nutrition and drugs to be examined were concomitantly injected to the mice from Day-1 to 10 after the irradiation. Damages by x-ray and effects of drugs were determined by the survival rates as well as histochemical analysis and quantification of mRNA in mucous membrane of small intestine. Mucous tissue with BrdU-incorporated cells were compensated from Day-5 to 8. Before the regeneration was evidently observed at Day-4, c-myb mRNA level was promptly increased. Mice in which the most of mucous tissue was covered with the regenerated tissue at Day-8 increased its body weight and survived until Day-28. Survival rate of approximately 60% were observed without introduction of drugs.
Among various frequently-used drugs, post-irradiational introduction of anabolic steroid, 19-nortestosterone is significantly effective in improvement of the survival rate. Both the level of c-myb mRNA at Day-4 and the area of compensated tissue at Day-5 were increased in mucous membrane. While estrogen decreased these parameters, the results show that the androgenic hormone accelerates regeneration of mucous membrane damaged by radiation in the small intestine.

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Glioblastoma is one of the most aggressive tumors in human malignancy. It generally shows resistance to radiotherapy. Recently, a novel concept of cancerous tissue has been proposed in several tumor models. A tumor contains a small fraction of ‘tumor initiating cells’, which could maintain proliferation and activate survival responses. In the latest study, it has been shown that stem cell marker CD133 was expressed in glioblastoma under hypoxic condition (Griguer E. et al, 2007). In the present study we investigated the expression of CD133 in mtDNA-deleted ρ0 glioblastoma cells.

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Objectives: The present study was performed to investigate whether dose and dose-rate effects are observed on dicentric chromosome aberrations in splenocytes of mice exposed to wide ranges of doses (100~8,000mGy) at different dose-rates, i.e. low (LDRs: 1 mGy/22h/day and 20 mGy/22h/day:0.91 mGy/h) to medium dose-rates (MDRs:200 and 400 mGy/22h/day:18.2 mGy/h). Methods: SPF C3H female mice were continuously exposed to gamma-rays with LDRs and MDRs for different periods from the age of 56 days to a maximum of 615 days. Lymphocyte chromosome spreads were prepared from splenocytes from these mice. Results: Incidences of dicentrics (Dics) plus centric rings detected by conventional Giemsa staining and those of Dics detected by FISH increased almost linearly up to a total accumulated dose of 8000 mGy at LDR (20 mGy/22h/day). The values of linear term for linear or linear quadratic regression lines for these aberrations were obtained by multiple linear regression analysis adjusted for age-related differences. The values were significantly decreased as the dose rate was lowered from MDRs to LDRs, suggesting that chromosome aberrations are varied at dose rates ranging 1 to 400 mGy/22h/day, which is discrepant from the recommended formula by ICRP to estimate dose and dose-rate effectiveness factor (DDREF). DDREF of 4.5 for Dics was obtained by comparing chromosome aberration rates at the same total dose of 100 mGy, at high-dose-rate (HDR:890 mGy/min) and LDR (20 mGy/22h/day). DDREF was varied from 4.5 to17.8 as accumulated doses were increased from 100 to1000 mGy. Further, ratio of values of linear terms of the LDR to HDR was 3.0. Conclusions: DDREF for Dic decreased as dose-rates were lowered in lower dose range. These results are useful information for risk assessment of low-dose radiation effects in human. This study was performed under the contract with the Aomori Prefectural Government, Japan.

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We previously showed that lifespan of human premature-aging syndrome model mouse (klotho) was prolonged by consecutively for the low dose-rate (0.63 mGy/h) gamma ray irradiation. We also found that antioxidant activities in various tissues of the mouse were enhanced by the irradiation, and maintained the level of calcium in the blood. Some researchers reported that the effects of lifespan prolongation were due to improve antioxidant functions or asthenia of insulin resistance. In addition, the role of klotho gene was contributed with calcium metabolisms, and glucose metabolisms. Therfore we used female klotho mouse as primary study of the action that the low dose-rate irradiation gave to lifespan prolongation effects, and measured glucose and insulin level in the blood. We also measured the level of TNF-alpha as one of the inflammation marker.
The kloho mice irradiated from 28 days old at a source of gamma ray (¹³⁷Cs). The level of TNF-alpha in blood of the irradiated group decreased after the longer irradiation. This result was thought the inflammation was suppressed by the irradiation. The glucose level in the irradiated group was decreased and the insulin level didn't change the longer irradiation. On the other hand, the level of glucose didn't change, and the insulin level was decreased in the non-irradiated control group. These results might be suggested that the enhancement of pancreatic antioxidants protected oxygen damages.
We thought that the prolongation effect of lifespan of klotho mouse by the irradiation might be not only the suppression of inflammation, but also increase in antioxidants, and controlled calcium metabolisms.

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