An X-ray microbeam irradiation system using synchrotron radiation has been developed at the Photon Factory, KEK, Japan, and is now being fully operated for various biological experiments. The minimum beamsize by the slit is 5-micrometer-square, and the size of the microbeam can be changed quickly and arbitrarily by the high-precision slit installed in the system. This specification might be ideal to investigate localization of the damage induction and subsequent cellular responses in cells irradiated with subcellular-sized beam.
To visualize the DNA damage induction, the microbeam-irradiated cells were immuno-stained by phosphorylated histone H2AX (gamma-H2AX) antibody. Most DNA double strand breaks were observed in localized area in cell nuclei, the size of which was almost the same as the beam size.
GFP-tagged protein enables us to visualize cellular responses in living cells. We constructed Chinese hamster ovary (CHO) cells having GFP-tagged Rad51, one of the protein related homologous recombination. Within 1 hour after irradiation, significant number of foci of GFP-Rad51 could be observed at the microbeam-irradiated site in the majority of the irradiated cells, and remained at least 5 hours after irradiation. The foci were appeared in the restricted area in the cell nuclei, which was corresponded to the damage induced area visualized with gamma-H2AX. In some of the cells, the foci could not appeared at all even after 12 hours, and the percentage of the population was almost same as the estimated population of G1-phase cells at the irradiated time.

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Non-homologous end joining (NHEJ) is the principal mechanism in human cells for repairing DNA double strand breaks (DSBs). The aim of this study is to clarify the biological roles of NHEJ-related genes on DNA damage induced by ionizing radiation (IR) through the generation and characterization of NHEJ-related gene deficient human cell lines. We have generated 3 cell lines that defective in XRCC4, Artemis and MDC1, respectively, by gene targeting in human colon cancer cell line HCT116. Chromosomal aberrations induced by X-ray irradiation were significantly higher in all deficient cell lines than those in parental HCT116. Survival rate after X-irradiation was the highest in the parental HCT116 and the lowest in XRCC4-/- cells, while MDC1-/- and Artemis-/- cells exhibited intermediated radio-sensitivities between the parental and XRCC4-/- cells. Generation of g-H2AX foci increased in a dose-dependent manner of X-rays and the number of foci reached the maximum at 30 ~ 60 min after X-irradiation in all cell lines. The foci then disappeared gradually and returned to the basal level within 4 hr in the parental HCT116, while a number of foci still remained at 4 hr after X-irradiation in XRCC4-/- and MDC1-/- cells. MDC1 might affect the formation of 53BP1 foci, since many g-H2AX foci did not colocalized to 53BP1 foci in the MDC1-/- cells after X-irradiation. These rersults suggest that defects in NHEJ-related genes cause a deterioration of g-H2AX foci-associated event, probably a DNA DSB repair process and in turn become a serious reason for the radio-sensitivity of theses cells.

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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

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DNA double strand break (DSB) is one of the most critical damage which is often induced by ionizing radiation. The damage responding pathways are regulated by sensors (damage recognition), mediators (signal modification and amplification), transducers (signal transduction), and effecters. In this presentation, a brief overview for regulating factors for these damage responding pathways will be given for further discussion.

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To investigate a role of 53BP1 in repair of X-ray induced DNA damage in vertebrate cells, we established, by using hyper-recombinogenic chicken B cell line DT40, a 53BP1 knockout cell line and double knockout cell lines deficient in 53BP1 and either Ku, DNA ligase IV, ATM or Artemis. 53BP1-/- cells showed intact intra-S and G2/M phase checkpoints for X-ray induced DNA damage. We carried out an epistasis analysis, using colony formation assay with cells irradiated in the G1 phase, and revealed that there are at least four sub-pathways in repair of X-ray induced DNA damage: 1) the core non-homologous end-joining (core NHEJ) which is dependent on Ku/DNA-PK, 2) Artemis-dependent pathway which includes ATM, 3) 53BP1-dependent pathway, and 4) DNA ligase IV-independent pathway. In contrast to the core NHEJ and Artemis-dependent pathways, the 53BP1-dependent pathway was resistant to PI-3 kinase inhibitor Wortmannin. Furthermore, Ku70-/-53BP1-/- double mutant cells were more sensitive than Ku70-/- cells to low dose rate X-ray irradiation (0.3 or 0.5 Gy/day), providing further evidence that 53BP1 plays a role in a different pathway from the core NHEJ pathway.

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Nijmegen syndrome (NBS) is a radiation-hypersensitive genetic disorder. NBS and AT (Ataxia-Telangiectasia syndrome) show the similar cellular phenotype such as radiation-hypersensitivity chromosomal instability and radiation-resistant DNA synthesis. So far, it has been clarified that the responsible gene product of NBS, NBS1 interacts with ATM (the responsible gene product of ATM) and this interaction is indispensable for the recruitment of ATM to DNA double-strand break (DSB) sites and activation of ATM kinase. Hence, the functional interaction between NBS1 and ATM is important for regulation of cell cycle checkpoint. Previously, we reported that NBS1 forms the complex with phosphorylated histone H2AX (gamma-H2AX) in response to DSB damage and this interaction is essential to the recruitment of NBS1 to DSB sites. Thus, NBS1 might have important roles in extensive DNA damage response through binding with ATM and H2AX. Therefore, we investigate the functional interaction among these factors in DSB repair. NBS1 has BRCT and FHA domain in N-terminus, ATM-phosphorylated sites in central region and hMRE11 and ATM-binding sites in C-terminus. Hence, we investigated the role of these domains for homologous recombination (HR) repair using DR-GFP assay. The mutation in BRCT, FHA or MRE-binding domains decreased HR activity, but the mutation in ATM-phosphorylated or ATM-binding sites did not influence HR activity. Moreover, AT cells showed the HR activity in normal level, suggesting that ATM might be dispensable for HR repair. As gamma-H2AX interacts with NBS1 through the FHA/BRCT domain, we examined the role of H2AX in HR repair. H2AX-knockout ES cells showed the decrease in HR activity and the mutation into acetylated sites of H2AX influenced the IR-induced foci formation and HR activity. Further, the repression of acetylation at common sites between H2A and H2AX by a specific inhibitor also decreased IR-induced foci formation and HR activity. Taken together, both NBS1 and H2AX might function in HR repair and the acetylation of histone H2A may be important for DNA damage response.

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The Mus81-Eme1 complex is a structure specific endonuclease that belongs to the XPF-ERCC1 superfamily, preferentially cleaving aberrant fork structures. Because ERCC1 is now considered to be a potential target of cisplatin-based chemotherapy and radiochemotherapy, it is of great importance to understand the role of the complex in cancer. In order to investigate the role of the complex in human cancer cells, the genes were knocked out in the colon cancer cell line HCT116. Deficiency of both genes led to decreases in sister chromatid exchanges and gene targeting frequency, indicating a role of the complex in homologous recombination. Hypersensitivity to mitomycin C and cisplatin but not to other DNA damaging agents was observed in the knockout cells, suggesting that Mus81-Eme1 is specifically involved in the rescue of stalled replication fork generated by DNA cross-linking. Consistent with its role during DNA replication, the intra-S phase checkpoint pathways mediated by Chk1 and Chk2 were activated in the mutant cells. These findings support the biochemical evidence that Mus81-Eme1 plays a critical role in processing stalled replication forks by the endonuclease activity.

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Fanconi anemia (FA) is a rare hereditary disorder characterized by progressive bone marrow failure, compromised genome stability, and increased incidence of cancer. FA is caused by genetic defects in altogether 13 genes, which include components of the FA core complex (FancA/B/C/E/F/G/L/M), a key factor FancD2, breast cancer protein BRCA2/FancD1, BRIP1/FancJ helicase, and just recently identified FANCI. In the DNA damage response, FancD2 is targeted to chromatin and forms nuclear foci following its monoubiquitination (mono-Ub), a process catalyzed by the FA core complex. This mono-Ub is critical for regulating nuclear dynamics of FancD2 as well as DNA repair through homologous recombination. We reported the core complex also has function in chromatin targeting of monoubiquitinated FANCD2 and DNA repair. However, the newest member of the FA pathway, FANCI, is found to be monoubiquitinated, and to interact with FANCD2 (called ID complex), raising a possibility that these additional functions are actually mediated by monoubiquitinated FANCI. We investigated roles of FANCI phosphorylation and mono-Ub using mutant proteins and ubiquitin fusions expressed in DT40 knockout cell lines. Our results indicate that FANCI is an essential co-factor for FANCD2 regulation and its function.

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The ubiquitin-conjugating enzyme Ubc13 is implicated in Rad6/Rad18-dependent post-replication repair (PRR) in budding yeast, but its function in vertebrates is not known. We show here that disruption or siRNA depletion of UBC13 in chicken DT40 or human cells confers severe growth defects due to chromosome instability, and hypersensitivity to both UV and ionizing radiation, consistent with a conserved role for Ubc13 in PRR. Remarkably, Ubc13 deficient cells are also compromised for DNA double strand break (DSB) repair by homologous recombination (HR). Recruitment and activation of the E3-Ub ligase function of BRCA1 and the subsequent formation of the Rad51 nucleoprotein filament at DSBs are abolished in Ubc13 deficient cells. Furthermore, generation of ssDNA/RPA complexes at DSBs is severely attenuated in the absence of Ubc13. These data reveal a critical and unexpected role for vertebrate Ubc13 in the initiation of HR at the level of DSB processing.

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Radiation-induced DNA double strand breaks are well known to initiate ATM-dependent DNA damage checkpoint pathway. The factors involving this pathway form discrete foci at the sites of DNA double strand breaks, which amplify DNA damage signals. DNA double strand breaks are repaired by two major repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Although NBS1 involving HR has been shown to form foci, which is important for ATM-dependent signallin, little is know about a role of RAD51 foci. Present study examined spatiotemporal relationship between ATM foci and RAD51 foci. We successfully detected RAD51 foci in normal human diploid cells 30 minutes after X-irradiation with 0.5 Gy. These foci were mainly observed in the S phase cells, and most of the foci were colocalized with those of phosphorylated ATM. Interestingly, significant change in size of phosphorylated ATM was observed thereafter, and grown foci were colocalized with NBS1 and phosphorylated BRCA1. Three dimensional analysis revealed that RAD51 foci were included in a part of large colocalized foci, indicating that RAD51 foci detected chromatin regions processing HR. These results suggest that DNA damage checkpoint pathway is activated at the sites of HR, and the secondary changes in chromatin structure coupled with HR may be involved in the growth of foci of DNA damage checkpoint factors.

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Non-chromatin nuclear domains are formed by proteins involved in nuclear functions such as transcription, replication and repair. During repair of DNA double-strand breaks (DSBs), a fraction of repair proteins form nuclear domains (Radiation Induced repari Foci, RIRF) at sites containing DSBs. RAD51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DSB in yeast and is conserved from yeast to human. RAD51 forms nuclear foci, and the percentage of cells containing RAD51 foci increase after induction of DNA damage. We have reported that RAD51 foci are formed specifically during S phase, and RAD51 proteins accumulate at sites containing DSBs in human cells.
To study the biological significance of RAD51 nuclear domains, we analyzed the changes of RAD51 nuclear domain components following ionizing irradiation using multicolor immunofluorescence technique. A fraction of RAD51 nuclear foci did not show collocalization with gammaH2AX, even after ionizing irradiation, suggesting the presence of DNA damage-independent RAD51 nuclear domains. Accumulation of ubiquitinated proteins were observed from immediately after induction of DNA damage. 53BP1, which interacts with a deubiquitination enzyme, was recruited to RAD51 nuclear domain in the late steps of DNA repair. These results suggest that RAD51 forms heterogenous nuclear domains after ionizing irradiation.

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Nijmegen breakage syndrome(NBS), characterized by high sensitivity to ionizing radiation (IR) and predisposition to lymphoid cancer, is phenotypically similar to Ataxia telangiectasia. It is well known that NBS1, the protein responsible for NBS, is cooperative with ATM, mutated in AT, 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 NBS cells is similarly defective in response to hydoxyurea treatment. Since ATR, a family gene of ATM kinase, functions in stalled replication fork after HU treatment or UV exposure, NBS1 might associate with ATR during stalling replication forks. Our result showed that UV-induced TopBP1 foci were abolished in NBS cells, suggesting that TopBP1 foci formation is regulated by NBS1. Co-immunoprecipitation assays showed that NBS1 interacts with TopBP1 in a manner that depends on UV-induced damage. These results indicated that TopBP1 functions downstream of NBS1 and mediates DNA damaging signal to ATR.

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In order to investigate function of NBS1, the protein responsible for Nijmegene breakage syndrome (NBS), we established Nbs1 knockout DT40 cell lines. The Nbs1-knockout cells showed not only the defect in homologous recombination repair of DNA double strand breaks, but also marked suppression of apoptosis induction following irradiation. Because DT40 cells lack p53, Nbs1 mediating apoptosis seemed to be independent of p53 pathway. The suppression of apoptosis was also observed in lymphoblastoid cells derived from NBS patients with functional p53. Because NBS lymphoblastoid showed further reduction of apoptosis compared to ATM deficient cells, NBS1 might function in ATM-p53 independent pathway. To determine the apoptotic pathway mediated by NBS1, expression of pro-apoptotic factors in NBS fibroblast immortalized with SV40 or in its NBS1 complemented cells was analyzed. Although Chk2 activation after irradiation was impaired in NBS cells, any differences in E2F-1 stabilization between NBS and the complemented cells were not observed. In contrast, Bax activation was obviously decreased in NBS cells, and it was well correlated to complementation of apoptosis induction, suggesting the involvement of NBS1 in Bax activation process. In this presentation, the role of NBS1 in Bax activation and critical domains of NBS1 for apoptosis regulation will be discussed.

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DNA-PK is now considered a pivotal enzyme in the repair of DNA double-strand breaks and there are several lines of evidence indicating that the catalytic activity to phosphorylate protein is essential for the repair function of DNA-PK. Nevertheless, it has been unclear which protein, and for what sake, should be phosphorylated by DNA-PK. Several recent studies have shed light on the importance of the autophosphorylation of DNA-PKcs itself. Then, does DNA-PKcs exist solely to phosphorylate itself?
We have sought to find the true substrate and the physiological significance of phosphorylation by DNA-PK in DNA double-strand break repair. In 2000, we demonstrated that XRCC4 is phosphorylated in response to ionizing radiation in a manner dependent on DNA-PKcs. Now we identified the phosphorylation sites on XRCC4 by DNA-PK, which are really phosphorylated in living cells in response to ionizing radiation and the loss of which lead to elevated radiosensitivity with deficient DNA repair capability. These results in the aggregate would indicate that XRCC4 phosphorylation by DNA-PK is an essential event in DSB repair. Further studies are warranted to clarify what consequence, e.g., change in conformation, property or activity, will be brought by the addition of phosphate group on XRCC4 protein.
The above results not only found a missing link in our understanding of DSB repair mechanism but also may provide us with a new approach for the development of radiosensitizers and the prediction of radiosensitivity and cancer susceptibility, as will be discussed. Furthermore, the phosphorylation sites identified above did not conform to the SQ TQ rule. Thus, the present results would also give us an important lesson in expanding our research on DNA-PK and related kinases ATM/ATR.

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The biological importance of nucleotide excision repair (NER) in humans has been suggested by studies of autosomal recessive human genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD), in which NER activity is impaired. XP patients are hypersensitive to sunlight and show an increased incidence of UV-induced skin cancers. Although CS patients are sensitive to sunlight, they have no predisposition to sunlight-induced skin cancer, but instead show severe developmental and neurological abnormalities as well as premature aging. Seven NER-deficient complementation groups have been identified in XP (XP-A to XP-G), and two in CS (CS-A and CS-B). In addition, XP-B patients and certain patients with XPD or XPG gene show features of CS in addition to symptoms of XP (XP-B/CS, XP-D/CS and XP-G/CS). The clinical features of CS in XP-G/CS patients are difficult to explain on the basis of a defect in nucleotide excision repair (NER). We found that XPG forms a stable complex with TFIIH, which is active in transcription and NER. Mutations in XPG found inXP-G/CS patient cells that prevent the association with TFIIH, also resulted in the dissociation of CAK and XPD from the core TFIIH. These results provide an insight into the role of XPG in the stabilization of TFIIH and the regulation of gene expression, and provide an explanation of some of the clinical features of XP-G/CS.

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DNA double strand break (DSB) is one of major DNA damage caused by irradiation. The impact of this damage including the biological responses such as its repair has been extensively described. Since formed as chromatin in vivo, histones, major component of chromatin, has to be released to make naked DNA before the repair of DSB. It has been also known that histones like H3 or H4 could be modified to control chromatin status. To understand if the chromatin status could be maintained through the DSB repair, we are establishing an system to analyze the modification of histones around DSB site. We will present the current data.

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The risks of exposure to low dose ionizing radiation (below 100 mSv) are estimated by extrapolating from data obtained after exposure to high dose radiation, using a linear no-threshold model (LNT model). However, the validity of using this dose-response model is controversial because evidence accumulated over the past decade has indicated that living organisms, including humans, respond differently to low dose/low dose-rate radiation than they do to high dose/high dose-rate radiation. In other words, there are accumulated findings which cannot be explained by the classical "target theory" of radiation biology. The radioadaptive response, radiation-induced bystander effects, low-dose radio-hypersensitivity, and genomic instability are specifically observed in response to low dose/low dose-rate radiation, and the mechanisms underlying these responses often involve biochemical/molecular signals that respond to targeted and non-targeted events.
Recently, correlations between the radioadaptive and bystander responses have been increasingly reported. We found that nitric oxide is an initiator for radiation-induced adaptive and bystander responses (Int. J. Radiat. Biol., 76: 1649-1657, 2000; Radiat. Res., 155: 387-396, 2001; Cancer Res., in press. 2007). This presentation focuses on the two phenomena, radioadaptive and bystander responses, by summarizing observations supporting their existence, and discussing the linkage between them from the aspect of production of reactive oxygen and nitrogen species.

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Low-dose hyper-radiosensitivity (HRS) is the increased sensitivity of mammalian cells to acute radiation doses below ~40 cGy. As the dose increases above 40 cGy, increasing levels of DNA damage produce increased radioresistance (IRR). The transition from HRS to IRR is determined by cell-cycle related events involving the ATM-dependent G2-phase cell-cycle checkpoint (ATM-G2-check). Specifically, G2-phase cells that receive low-dose radiation exposure in the HRS region evade the ATM-G2-check, exhibit reduced fidelity of DNA repair and have an elevated risk of dying when they proceed unchecked into mitosis. HRS is associated with an increase in DNA double-strand breaks in G2/M phase cells and slower repair, and there is a correspondence between HRS and post-mitotic caspase-3 mediated apoptosis. At higher radiation doses, in the IRR region, the ATM-G2-check is activated in response to radiation-induced DNA double-strand breaks via ATM-mediated events, allowing cells irradiated in G2 to more effectively repair DNA damage before entering mitosis, which is reflected by an increase in net radioresistance per unit dose. HRS also manifests as a strong inverse dose rate effect in cycling cell populations exposed to continuous irradiation at less than ~60 cGy h^-1. Also in this case, the lack of an ATM-G2-check can be demonstrated. These observations provide a compelling case for exploring radiotherapeutic strategies based on HRS, and for building low-dose hypersensitivity into the radiotherapy treatment planning process, and for re-evaluating low-dose radiation risk estimates.

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Cellular exposure to DNA damaging agents like ionizing radiation can result in direct (targeted) damage to the genetic material and a number of non-targeted indirect effects that can manifest in the progeny of a damaged cell. Both targeted and non-targeted effects can result in DNA mutations, gene amplifications, chromosomal rearrangements, carcinogenesis, and even cell death. The paradigm for understanding how induced damage results in these cellular endpoints dictates that cellular responses to the induced damage, e.g., DNA repair, and cell cycle arrest fix the damage and thereby seal the fate of the irradiated cell. However, non-targeted effects cannot be accounted for by this paradigm. This presentation will focus on delayed genetic effects occurring in the progeny of cells after exposure to ionizing radiation. We will describe how the cellular micro-environment can perpetuate instability in clonally expanded populations of cells surviving irradiation. The emphasis will be on gene expression analysis, the persistently elevated levels of reactive oxygen species and the role of mitochondrial dysfunction that characterize many of our chromosomally unstable clones. These results will be discussed in terms of non-targeted bystander like effects where by cells that themselves were not irradiated exhibit many of the same detrimental effects as irradiated cells. In addition, other non-targeted effects associated with radiation exposure including clastogenic factors, the death inducing effect, hereditary effects, and abscopal effects of radiation and how these might impact on human disease will be discussed.

This work was supported by the Biological and Environmental Research Program (BER), U.S. Department of Energy.

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In a variety of in vitro and in vivo models, pre-exposure to low priming dose of ionizing radiations is well known to decrease the biological effects of a subsequent higher challenging dose. However, very few data exist concerning the possibility of inducing this phenomenon, called radiation-induced adaptive response (AR), when using high-LET accelerated heavy-ion radiation. In this study, cultured human lymphoblastoid cells (with different p53 status: TK6, AHH-1, NH32) were exposed to X-rays, carbon-ion and neon-ion radiation at various LETs (ranging from 20 to 150 keV/micrometer). Mutation frequency at HPRT locus, radiation-induced cell death, cell cycle effects after irradiation, as well as estimation of DNA double-strand breaks induction and repair by measuring gamma-H2AX phosphorylation kinetics were compared in primed and unprimed cells. Our results suggest that the biological effects resulting from exposure to priming and challenging radiations vary according to irradiation type and LET. An AR to mutagenic effects of heavy-ion radiation at higher LET was observed when cells were exposed to priming doses of X-rays. The ability of low dose and low dose-rate heavy-ion radiation to trigger an AR was also assessed.

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Phosphorylated ATM immunofluorescent staining was used to investigate the dose-response relationship for the number of DNA double strand breaks induced in non-dividing primary normal human fibroblasts cells irradiated with doses from 1.2 mGy to 100 mGy. The induction of DNA double strand breaks showed a supralinear dose-response relationship; the number of breaks per unit dose in the range of 1.2-5 mGy exceeded that at higher doses. Radiation-induced bystander effects, which can enhance cell damage, may explain these findings. To test this hypothesis, the number of DNA double strand breaks in cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X-ray irradiation was assessed; a supralinear dose-response relationship was not observed. Moreover, the number of DNA double strand breaks obtained by subtracting the number of phosphorylated ATM foci in lindane-treated cells from the number of phosphorylated ATM foci in untreated cells was proportional to the dose at low doses (1.2-5 mGy) and was saturated at doses from 10-100 mGy. Thus, the increase in the number of DNA double strand breaks per unit dose in the range of 1.2-5 mGy was largely due to radiation-induced bystander effects, while at doses >10 mGy, the DNA double strand breaks might be induced mainly by dose-dependent direct radiation effects and partly by dose-independent radiation-induced bystander effects. The findings of our present study provide a direct evidence of the dose-response relationship for radiation-induced bystander effects from broad X-rays not microbeam.

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We have investigated genomic instability in normal human fibroblasts induced by low fluences of different types of radiation, such as gamma rays, neutrons and high-LET heavy ions, focusing on the radiation-quality dependence in both cell-killing effects and mutation induction. The cells were pre-treated with low-fluence irradiation (~1mGy/7-8h) of ¹³⁷Cs gamma rays, ²⁴¹Am-Be neutrons, helium (LET=2.3keV/μm), carbon (LET=13.3keV/μm) and iron ions (LET=200keV/μm) before following irradiation with an X-ray challenge dose (1.5Gy). No difference was observed in cell-killing effects, which was detected with the colony-forming assay for reproductive cell death, using pre-treatment with different types of radiations. For mutation induction at hprt locus detected as 6-thioguanine resistant clones, there was no difference in X-ray-induced mutation frequency at 1.5Gy of X-ray challenge dose between un-pretreated and gamma-ray pre-treated cells. In the case of the pre-treatment with heavy ions, mutation frequency was around 4.0 times higher in carbon-ion pre-treated cells and 1.9 times higher in helium-ion pre-treated cells than that in un-pretreated cell. However, X-ray-induced mutation frequency in cells pre-treated with helium and carbon ions was reduced at the control level, when using a specific inhibitor of gap-junction mediated cell-cell communication (40μM lindane). There is evidence that gap-junction mediated cell-cell communication play an important role of inducing genomic instability by pre-treatment with heavy ions.

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The time of transfer of a signal required for a bystander effect expressed in an adjacent cell of an X-irradiated cell via gap junction (GJ) has been estimated by using pulse-like treatment of lindane (γ-BHC), an inhibitor of the GJ function. X-ray microbeam with a diameter of 100 micrometer was available from a micro-capillary tube installed in a table-top X-ray fluorescence analyzer for local specimen area (XGT-2700, HORIBA Ltd.). Human neonatal dermal fibroblasts were used for their normal GJ function judged by the Lucifer yellow transfer method. First, the concentration and the treatment time of lindane were determined to exert a maximal inhibitory effect in a minimal treatment time. The treatment of 25 μM for 30 minutes was adopted for the cells used. The complete recovery from the inhibition by removing lindane was observed after 2 hrs. Therefore the GJ function can be inhibited for a period of about 2.5 hrs. Second, time dependence of the transfer of a bystander signal was estimated by changing the time of lindane administration after irradiation. The detection of a bystander effect was performed by p53 expression around an irradiated cell after 24 hrs. The bystander effect was detected at 1, 2 and 4 hrs after irradiation, while in the case of 3 hrs significant reduction of the p53 expression area was observed. These results suggest that the time of the transfer of a bystander signal for the p53 expression is around 4 hrs after irradiation.

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Gap junctional intercellular communication (GJIC) is an important function of metazoan cells and is believed to play a role of enhancing irradiation-induced bystander responses and have beneficial effects in anti-tumor therapy. This study found that, when primary fibroblast AG1522 cells on confluent were irradiated with a 100 keV/μm carbon-ion beam, the cell responses of micronuclei (MN) formation and G1-phase arrest had a low dose sensitivity effect that was positively regulated by the GJIC. Meanwhile, when neoplastic human salivary gland (HSG) cells were irradiated under the condition of confluent but with poor internal GJIC, MN and G2-phase arrest were induced. Unexpectedly, when GJIC between HSG cells were enhanced by treating of cells with 8-Br-CAMP, these radiation-induced cellular damage was reduced so that cell surviving fraction was slightly enhanced, suggesting that increased GJIC protects HSG cells from lethal radiation damage. Further investigation demonstrated that ROS were involved in the GJIC-enhanced radiation effect on AG1522 cells and nitric oxide contributed to the GJIC-reduced radiation effect on HSG cells.

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We have investigated effects of low-dose-rate radiation (LDR) on cells derived form normal human and patients of ataxia-telagiectasia (AT), NBS and Artemis deficient. Non-proliferative cells were irradiated by LDR (0.3 mGy/min) continuously up to 2 weeks or high-dose-rate radiation (HDR; 1 Gy/min). While the survival in normal cells after LDR radiation was significantly higher than that of HDR radiation, AT cells showed almost similar survival after both LDR and HDR. In contrast, the survival in Artemis deficient cells after LDR radiation was resistance than that in AT cells. Surprisingly, the survivals in NBS cells after LDR radiation showed almost similar resistance to that of normal cells. Although only few γH2AX foci was observed in LDR-irradiated normal cells, in AT cells significant numbers of γH2AX foci remained after LDR irradiation. However, in Artemis deficient cells numbers of γH2AX foci after LDR irradiation were about 50% of those in AT cells. In contrast, only a few γH2AX foci was observed in LDR irradiated NBS cells. These results suggest that ATM played an important role in repairing the double-strand break (DSB) induced by LDR radiation. Moreover, it is suggested that ATM might activate not only Artemis but also other factors in DSB-repair pathway after LDR radiation. On the other hand, function of NBS in cells irradiated with LDR might be differ to that HDR-radiated.

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Radioadaptive response is a biological defense mechanism that is induced by low-dose ionizing irradiation for cellular resistance to the genotoxic effects of subsequent irradiation. We also showed that the pre-treatment with low concentration of TPA or hydrogen peroxide mimics the pre-irradiation with low dose of X-rays. We have suggested that the radioadaptive response is mediated through the pathways involving protein kinase C (PKC) and p38 mitogen-activated protein kinase. Especially, we found that one isoform of PKC, PKC alpha, is activated by irradiation with 2 cGy X-rays, suggesting that PKC alpha is playing an important role in the induction of radioadaptive response. However, the molecular role of PKC is still largely elusive. Here, we examined the involvement of PKC alpha in radioadaptive response by the use of RNAi for PKC alpha. We introduced three kinds of siRNA into m5S cells, each of which targets defferent site of the mouse PKC alpha gene, Prkca. One of them greatly reduced the amount of Prkca mRNA, indicating that RNAi is effective for suppressing the expression of the Prkca gene. We examined the radioadaptive response in the m5S cells in which the Prkca mRNA was reduced. The radioadaptive response was suppressed in the cells with low amount of the Prkca mRNA. This results indicate that PKC alpha is one of the critical factor in the signal transduction pathway of the radioadaptive response.

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It is extremely important to investigate the genetic alterations in human cultured cells exposed to low-dose/low-dose rate ionizing radiation (IR) from the aspect of estimating human health influences. Here, we used LOH (loss of heterozygosity) analysis system in human lymphoblastoid cell TK6, which has been established as a radiation-sensitive detection methodology. The frequency of mutations (MF) induced in thymidine kinase (TK) gene was increased from (3.0 +/- 0.5) X 10*-6 to (4.4 +/- 0.8) X 10*-6 after the exposure to gamma-rays (1.2 mGy/hr, total 30 mGy) in NIRS. The further lower-dose/ lower-dose rate gamma-irradiation (0.125 mGy/hr, total 12 mGy) rather showed a tendency of decrease in MF of TK, (3.0 +/- 0.15) X 10*-6 to (2.1 +/- 0.75) X 10*-6. LOH analyses for the TK- mutants recovered after 30 mGy exposure demonstrated an induction of interstitial deletions of the small region restricted to TK locus. Taking into consideration the fact that the probability of DNA double strand breaks (DSBs) formation in this restricted region can be estimated to be ~7 X 10*-11, indirect effects of IR is suggested to be involved in this induction of small deletions. An LOH event caused by the chromosome recombination in this restricted region was also induced at very low frequency. We would like to discuss the observed phenomenon together with the results indicating that the above gamma-irradiation enhanced the efficiency of DNA homologous recombination repair for the site-specific DSB introduced by expressing the restriction enzyme, I-SceI (~50% and ~80% enhancement for 30 mGy and 12 mGy, respectively).

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Astronauts are known to receive approximately 1 mGy/day of ionizing radiation (IR) in the space environment. Although protons are the most prevalent radiation in space, low levels of heavy ion particles become important due to their severe biological effects. We irradiated normal human fibroblasts (HFL III) with carbon ions (290MeV/n, 70 keV/um) as well as gamma-rays at 1 mGy/6-8 h once and the cell growth was observed up to five months by continuous culturing. The carbon irradiated cells went to senescence much earlier than non-irradiated control cells, while there was no growth disadvantage in gamma-irradiated cells under the same conditions. Since not all the cells actually were hit by carbon irradiation at this dose level, this phenomenon seems to be a by-stander effect. DNA double strand break (repair) markers (gammaH2AX and phosphorylated DNA-PKcs) were studied by immuno-staining method in these cells. The numbers of foci for these markers increased several passages before senescence and the increase was more pronounced in carbon irradiated cells than control or gamma-irradiated cells. Of special interest, the number of foci was significantly reduced after cells became fully senescent. Studies with non-homologous end joining (NHEJ) deficient cells (180BR) showed accelerated senescence significantly sooner than normal cells. Our studies indicate that a single low dose heavy ion irradiation can affect the genome stability many weeks after irradiation, and that the protein(s) necessary for NHEJ appear to be involved in the cell senescence process.

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Chronological changes of chromosome aberration rates along with accumulated doses in specific pathogen free (SPF) mice continuously exposed to low-dose- rates (LDR) gamma- rays were studied by centromere FISH and M-FISH methods. Chromosome aberrations in mouse splenocytes were serially observed following irradiation for about 400 days (total dose of 8,000 mGy) at 20 mGy/22hr/day and for about 615 days (total dose of 615 mGy) at 1 mGy/22hr/day. For comparison, high-dose-rate (0.89 Gy/min) gamma-irradiation was also performed. Both dicentrics and translocations increased almost linearly up to 8,000 mGy at 20 mGy/22hr/day, accompanied by complex aberrations and clonal aberrations. Incidences of dicentrics at 1 mGy/22hr/day were lower than 20 mGy/22hr/day and increased up to 412.5 mGy. The incidences of dicentrics and translocations slightly increased with age. The dose and dose-rate effectiveness factor (DDREF) for dicentric chromosomes was estimated from the ratio of slope in fitting regression lines of acute and low-dose rate irradiation data. The most fitting lines were obtained in each different dose-range. DDREF changed from 1.9 to 3.6 in each dose-range at 200 mGy after irradiation at 20 mGy/22hr/day, which indicates variable DDREF depending on dose-range and dose-rates. These results are important to evaluate biological effects of chronic exposure to low-dose radiation. This study was supported by a grant from Aomori Prefecture, Japan.

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We report a novel method of rapid and quantitative evaluation of the degree of radiation-induced apoptosis in the developing brain, using medaka (Oryzias latipes). We also show the radiation effects on the hisogenesis of medaka brain. Embryos at stage 28 were irradiated with 1-10 Gy of X-rays. Twenty to twenty-four hours after the irradiation, living embryos were stained with a vital dye, acridine orange (AO), for 1-2 h, and whole-mount brains were examined under an epifluorescence microscope. The numbers of AO-stained apoptotic bodies and single nuclei increased in a dose-dependent manner, and AO-stained large apoptotic bodies (rosette-shaped clusters of nuclear fragments) were found in the optic tectum. We used the number of rosette-shaped clusters/tectum as an index of the degree of radiation-induced brain cell death. The results showed that the number of rosette-shaped clusters/tectum in the irradiated embryos exposed to higher than 2 Gy was highly significantly different from that in the nonirradiated control embryos, whereas no difference was detected at 1 Gy. Thus, the threshold dose for brain cell death in medaka embryos is about 1 Gy and this dose is probably comparable to those for rodents and humans. The dead cells disappeared thereafter, and the irradiated embryos continued to develop apparently normally. The grown irradiated (10 Gy) embryos, however, had smaller brains and eyes than the nonirradiated control embryos. At hatching, the irradiated (10 Gy) embryos exhibited histological abnormalities in the brain and retina, although most of them hatched normally and survived. The results show that medaka embryos are useful for studies of the developmental neurotoxic effects of radiation.

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High linear-energy-transfer (LET) radiations such as alpha particle brought by radon are included in environmental radiation. Therefore, to consider about effects of environmental radiation on ecological systems and their members, LET dependencies of their radiation effects should be elucidated. However, there are a few findings of radiation effects and its LET dependencies in plants, although plants are a majority of ecological systems and occupy an important position of them. We have been thus estimated cell killing effects based on colony formation and been quantified initial yields of DNA double-strand breaks (DSBs) by pulsed-field gel electrophoresis assay in protoplasts that were isolated from dicot tobacco cultured cell line (BY-2) and irradiated with high-LET (8.9-440 keV/μm) heavy ions. As a result, both effects of cell killing and DSB induction depended on LET, and indicated peak effectiveness per dose by irradiation of carbon ions having LETs of 247 keV/μm and 124-241 keV/μm respectively. These dependencies are similar to those of survival reduction reported in dicot dormat seeds. In contrast, it is reported that carbon ions having LETs of 100-150 keV/μm maximize the cell killing effects per dose in mammalian cells. They are also reported that the killing effects of heavy ions peak at around 100 keV/μm in yeasts and Bacillus subtilis, and that there is no peak of the effects in Deinococcus radiodurans. From these facts, it was cleared that LET dependencies of radiation effects are not always common among various organisms and the LET values giving the maximal lethal effects are higher in plants than in the other organisms. We must take care to compare the LET dependencies of DSB induction among organisms because different types of methods for quantifying DSBs can lead different results. In this workshop, we will also discuss about the relationships between strategies of genome maintenance and LET dependencies of radiation-induced biological effects in plants.

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Soil bacteria play a vital role in the functioning of ecosystem such as nutrient cycling, organic matter formation and decomposition, soil structure formation, and plant growth promotion. In general, the effect of ionizing radiation on bacteria has been studied by conventional methods based on cultivation of isolated bacteria. Nowadays it is well known that more than 90% of the bacteria existing in soil cannot be cultured under laboratory conditions. A vast portion of soil bacterial communities, therefore, remains unstudied. To overcome the drawbacks of these culture-dependent methods, molecular techniques are widely used approach in the last decade. In the present study, denaturing gradient gel electrophoresis (DGGE) based on the 16S rRNA gene sequence was applied to estimate the effect of ionizing radiation on soil bacterial community.
Gray lowland soil was collected from a paddy field and was flooded with well water. The flooded soil samples were exposed to chronic gamma radiation for 10 days. The dose rate was approximately 1 Gy/day. For a control, samples were shielded from exposure to gamma radiation by lead blocks 10 cm of thickness. After the end of the exposure, DGGE analysis was carried out.
DGGE analysis showed the presence of bacterial species grown specifically in the irradiated samples, suggesting that changes in bacterial community structure occurred by the exposure of gamma radiation. Radiation sensitivity of bacteria in natural soil may be higher than previously thought.

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We are accumulating physiological information after exposure organisms to environmental stress using OMICS (Genomics, Proteomics, and Metabolomics) technology. Environmental stress includes radiations, high temperature, low temperature, high pressure, chemicals and so on. As the model organisms, we selected yeast, rice, medaka, rat, and human cell lines. In this workshop, we will focus on the stress response of yeast cells caused by radiation comparing with other stress response. Further, we also talk the possibility of medaka, microorganisms and rice as the target of OMICS technology. the irradiations, heavy-ion (C) (6.0,12.0,25.0 Gy, National Institute of Radiological Sciences), thermal neutron (4.0, 6.9, 20.8 Gy, Kyoto University), fast neutron (3.3, 9.7, 21.4 Gy, Hiroshima University), gamma ray (3.3, 5.8, 17 Gy, Hiroshima University). The yeast Saccharomyces cerevisiae, strain S288C was used as the indicator strain for DNA microarray analysis. Under irradiation conditions, yeast cells were kept in a resting state at low temperature. Cell suspensions in cuvets or culture flasks were irradiated. After irradiation, cells were resuspended in YPD medium (50 ml) and cultured for 40 min. We analyzed mRNA expression profiles from the cells exposed to radiation with approximately 20 Gy, as we could confirm biological effect to yeast cells. In 6000 yeast genes, we could detected induced genes with the number of 92 genes (heavy-ion), 222 genes (thermal neutron), 37 genes (fast neutron), and 220 genes (gamma ray) and repressed genes with that of 38, 131, 73, 151 genes. We could also selected specifically induced or repressed genes by each irradiation with 3, 162, 31, 231 genes and 12, 88, 34, 321genes. We will characterize these genes on site.

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HiCEP (High-coverage expression profiling) is a novel comprehensive analysis method which is based on DNA finger printing and PCR amplification. It enables to detect any altered gene expression among 60-70% of all the actually transcribed genes in any eukaryotic cells and tissues. We discovered interesting response to X-rays at very low dose by using HiCEP. The strategy successfully detected the very small alteration of the gene expression, thus promising as a useful tool to develop risk assessment for environmental toxic reagents. Another advantage of HiCEP over other prevailing methods is that it can be applied to most animals and plants on the earth regardless of the availability of their genome information, which will promote us to explore many useful biomarkers.

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Although the importance of radiological protection of the environment based on scientific principles is increasingly recognized internationally, the relevant scientific data are extremely limited. This paper summarizes research activities in National Institute of Radiological Sciences (NIRS) for evaluation of the effects of radiation on representative terrestrial and aquatic organisms as well as the ecosystems. Among organisms, conifers, fungi, earthworms, collembolans, algae, daphnia and Medaka are presently selected to study. Transfers of radionuclides and related elements from medium to organisms are estimated for dose estimation. Dose-effect relationships of acute gamma radiation on the survival, growth, and reproduction of selected organisms have been studied. Studies on the effect of chronic gamma radiation at low dose rate were also started. The High Coverage Expression Profiling (HiCEP) is applied as on of the indicators of the radiation effects. The effects of radiation on ecosystems were estimated by using a microcosm, i.e. an experimental model ecosystem consisting of Euglena, Tetrahymena and Escherichia coli. An index for the holistic evaluation of effects on various ecological parameters, such as sizes of population or community was proposed. The population dynamics, and mass and energy budgets, of the microcosm have been simulated as a computer simulation code. Developments of more complicated and practical model ecosystems have been started.

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The radiation effects on mutation at tandem repeat loci in plant genome were investigated. A tandem repeat locus similar to mammalian unstable loci, such as ESTR in mouse, was identified by BLAST searches of the arabidopsis genome database. The locus consists of an array of 6 bp repeat units, ATGGGG. The length of the array varied greatly between ecotypes of arabidopsis, which indicates high frequency of mutation resulting from gain or loss of the repeat units in the locus. The change in the length of the locus was analyzed in the descendants of the plants irradiated with 137Cs gamma rays at 50 mGy/h (50-59 Gy in total) during their growth. Among 328 descendants of the irradiated plants, four plants showed changes in the length of the locus, whereas no changes were observed in 516 descendants of non-irradiated plants. Among the four mutated plants, two plants showed large loss of the repeats (nine and ten repeats, respectively) in the locus. The occurrence of the large deletions implies that a simple model of replication slippage cannot account for mutation events at this locus. These results suggest that the mutation in the tandem repeat locus is unique not only in the high mutation frequency but also in the mode of mutation.

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Because of their ecological importance, the soil invertebrates, especially earthworms are increasingly used for ecological impact assessment of chemical substances for the terrestrial ecosystem. Earthworm toxicity test guideline was already adopted in OECD, and earthworms are considered to be reference organisms by ICRP. Among earthworms, Enchytraeus japonensis discovered in 1991 by a Japanese investigator has a peculiar reproductive manner. The earthworms reproduce asexually by fragmentation and subsequent regeneration in the normal breeding condition, but develop reproductive organs and achieve sexual reproduction in a particular breeding condition. There is only one report that investigated the effect of radiation on the earthworms. Miyachi et al. reported the induction of sexual reproduction by extremely low dose of beta-irradiation. In the present study, we investigated the effect of gamma-irradiation on the population increased by asexual reproduction. The population of the earthworms 30 days after irradiation at a dose of 20Gy was decreased to 50% of sham control. On the other hand, ³H-thymidine incorporation into DNA from 24hr to 48hr after irradiation decreased to a half of control by irradiation at a dose range of 3-4Gy. It was concluded that the radiosensitivity of the earthworms at the cellular level was slightly lower than that of mammalian cells, but the radiation effect on the population of the earthworms was not easily observed due to the vigorous proliferation of only a few cells survived irradiation.

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Ecological effects should be evaluated at the community-level, considering indirect effects through interspecies interactions. We therefore began to investigate effects of ionizing radiation on microcosms mimicking aquatic microbial communities.
The microcosm used in this study consisted of green algae (Chlorella sp. and Scenedesmus sp.) and blue-green alga (Tolypothrix sp.) as producers; oligochaete (Aeolosoma hemprichi), rotifers (Lecane sp. and Philodina sp.) and ciliate protozoa (Cyclidium glaucoma) as consumers; and four or more species of bacteria as decomposers. The developmental-stage microcosm was acutely irradiated with ⁶⁰Co gamma-rays at 100, 500, 1000 and 5000 Gy, and population changes were observed over 160 days.
At 100 Gy, all consumers decreased compared with controls, while Tolypothrix sp. increased. At 500 Gy, growth of green algae was inhibited. Lecane sp. decreased compared with controls, and bacteria decreased temporarily. Tolypothrix sp. increased. At 1000 Gy, Scenedesmus sp. and A. hemprichi died out. Growth of Chlorella sp. was inhibited, and bacteria decreased temporarily. Tolypothrix sp., Lecane sp. and C. glaucoma increased. At 5000 Gy, Scenedesmus sp. and all consumers died out. Growth of Chlorella sp. and bacteria was inhibited, while Tolypothrix sp. increased. These effects were not necessarily depended on radiation doses, and some higher population densities were observed in the irradiated microcosm. It is thought that these unexpected results arose from indirect effects. The effects of gamma-rays will be quantitatively compared with those of insecticides, herbicides and copper.

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In the study of DNA repair and the damage response, the UV damage played an important role as the model damage. Organisms are always exposed to the various type of DNA damage. Studies in this decade have clarified the outline of DNA repair mechanism and damage responses including cell cycle checkpoint, apoptosis and the acquisition of resistant mechanism. UV damage played an important role in these studies both for elucidation of the excision repair mechanism and for the establishment of a new concept in which the common intermediates derived from the process of damage processing, rather than the damage itself, are recognized as the initial signal for damage response. The photoproducts induced by UV is extremely clear. This is one of the reason why the UV damage served as a central role. Another reason is that UV has been a threat for all organisms on the earth from birth of the life to present. The continuing threat is the reason why living organisms completely possesses means against UV, and also suggests that the analysis of the biological response for the UV damage is connected for elucidation of the basic strategy of the organisms for the damage response. The ultraviolet photobiology plays a part in the photobiology, showing another characteristic. Organisms utilize light energy in various ways, as an outside environmental signal and as an energy source. UV is not only the environmental stress but also an important environmental signal. I will summarize the situation of the ultraviolet photobiology from these two viewpoints and want to think about the future prospects.

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The harmful effects of UVB in the sunlight are unavoidable problems for higher plants that live on photosynthesis. To keep the genomic information intact, plant cells remove the UV damage formed on the DNA by photoreactivation. Plants also have dark-repair and damage tolerance pathways to prevent growth defect caused by DNA damage. Here we report about AtREV3 and AtREV1 genes, which encode specific polymerases to bypass DNA damage. The AtREV3- or AtREV1-disrupted plants were more sensitive to UVB, γ-ray, and DNA crosslink agents than the wild type, suggesting that these REV proteins are required for plant damage tolerance. Although bacterially expressed AtREV1 protein inserted a dCMP at opposite the AP site, it failed to bypass two major UV damages in vitro. This inconsistency makes us propose a novel function of AtREV1 in the UV-tolerance pathway. To evaluate the function of AtREV3 and AtREV1 in bypassing UV-damage, we have detected replication errors that often emerged during damage bypass process. The point-mutated, non-functional uidA genes were introduced into Arabidopsis plants and reversion events (mutations) were detected by blue GUS+ sectors on the somatic tissues. We found that a disruption of AtREV3 or AtREV1 reduced the mutation frequency to 1/4 of the level of the wild type. These results suggest that the AtREV3 and AtREV1 are bypassing the DNA damage in error-prone manner.

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Chromosomal double-strand DNA breaks (DSBs) are produced by ionizing radiation, oxygen free radicals, DNA cross-linking reagents, and DNA replication failure. There are two major repair pathways for DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR). HR is also involved in meiotic recombination process. Although plants have long played a key role in building our understanding of genetics, relatively little is known at the molecular level of HR processes of plants.
The completion of the Arabidopsis genome sequence has greatly facilitated the search for genes involved in HR in this model plant. In addition, the huge collection of T-DNA /transposon tagging mutant lines in Arabidopsis has helped identify the function of some of these genes in plants. Several Arabidopsis mutants involved in HR show hypersensitivity to DSBs-inducing agents. However, in contrast to the case in vertebrates, with a few exceptions these mutants do not show embryonic lethality or severe growth defects. This could represent a distinct advantage of plant systems for the study of DSB repair and recombination. In addition, several genes involved in HR that are unique in plants have been identified by forward genetic approaches.
In this presentation, we focus on recent advances in our knowledge of the process of HR, in higher plants

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The ultraviolet component of sunlight induces two major DNA photoproducts linking two adjacent pyrimidines on one nucleotide strand: Cyclobutane Pyrimidine Dimers (CPD) and (6-4) Photoproducts. These photoproducts interrupt various life activities taking place on the DNA strands, such as transcription and replication. Photolyase is a unique DNA repair enzyme that uses blue light to reverse this UV-induced damage and restore the two parental pyrimidines. Many eukaryotes employ two functionally distinct photolyases, Class II CPD and (6-4) photolyases, to maintain genomic integrity, whereas most prokaryotes have a unique photolyase or photolyase-like gene, designated Class I CPD photolyase. Interestingly, despite their substrate differences, (6-4) photolyase is evolutionally closer to bacterial CPD photolyase, than either is to the eukaryote CPD photolyase. Studies on the prototypical bacterial CPD photolyase from Escherichia coli have been used to interpret the related functions of eukaryote photolyases, but many questions remain regarding the respective roles of these two photolyases in DNA repair in eukaryotes. To better understand the repair mechanisms and functional roles of photolyases in UV-tolerance in eukaryotes, we are applying and combining NMR, X-ray, structural modeling, phosphoramidite synthetic chemistry and molecular biochemistry to both eukaryotic photolyases. Here, we contrast the distinct substrate recognition and repair mechanisms of the two eukaryotic photolyases and discuss their potential roles in the DNA repair networks.

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Rice cultivars vary widely in their UVB sensitivity and this has been correlated with CPD photolyase mutations that alter the structure/function of this photorepair enzyme. Here, in order to test whether CPD photolyase function determines the UVB sensitivity of rice, we constructed transgenic plants bearing the CPD photolyase gene of the UV-resistant rice cultivar Sasanishiki (japonica) in the sense or the antisense orientation using the Sasanishiki (sense; S-B and S-C lines, antisense; AS-D line), UVB-sensitive Norin 1 (japonica) (SN-F line) and UVB-sensitive Surjamkhi (indica) (SSu-B line) rice as wild-type plants. The sense transgenic plants had higher photolyase activities than the each wild-type plant, were significantly more resistant to UVB-caused growth inhibition, and maintained significantly lower CPD levels in their leaves during growth under elevated UVB radiation. Conversely, the antisense transgenic plant had little photolyase activity, was severely damaged by elevated UVB radiation, and maintained higher CPD levels in its leaves during growth under UVB radiation. These results strongly indicate that CPDs are one of principal cause of UVB-induced growth inhibition in rice, and that increasing CPD photolyase activity can significantly produce alleviation of UVB-caused growth inhibition.

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We previously investigated that the supplementary UVB radiation on the growth and yield of Japanese rice cultivar in paddy field in Japan, and found that the supplementary UVB radiation inhibited growth, yield and grain development. One of principal causes of such UVB-induced damage is UVB-induced cyclobutane pyrimidine dimer (CPD), and increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition. By the way, how degree of growth inhibition was caused by UVB radiation in natural sunlight? In this work, effects of solar UVB radiation on growth and yield of rice under outdoor conditions were investigated. For this study, UV-resistant Koshihikari (japonica) and the UV-sensitive chromosomal segment substitution line (SL-229) were used as experimental plant; chromosomal 10 region, on which CPD photolyase gene located, in SL-229 was homozygous for Kasalath (indica) allele, whereas all other chromosomal regions were homozygous for Koshihikari alleles. CPD photolyase activity of Kasalath was lower than that of Koshihikari, resulting from the alteration of a single amino acid in the photolyase sequence. The grain size and weight of SL-229 under current outdoor conditions in 2006 were reduced. These results mean that growth and yield of rice grown under current environmental conditions would be inhibited by UVB radiation in natural sunlight.

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Cyclobutane pyrimidine dimer (CPD) is a major type of UV-induced DNA damage. CPD photolyase is a crucial factor for determining the sensitivity of plant to UVB. CPD photolyases have been reported in many organisms and classified into two classes, class I and class II, based on their similarity of amino acid sequence. Class I CPD photolyases have been studied well. However, little is known about class II CPD photolyase. To characterize rice class II CPD photolyase, the enzyme from rice leaves was partially purified by 4 purification steps. At the final purification step, the enzyme bound to CPD-containing DNA-conjugated magnetic beads were released by blue-irradiation. Specific activity of the purified enzyme was 8,100-fold higher than that of crude extract. SDS-PAGE of the purified enzyme showed existence of two proteins of about 54- and 56-kDa. Western blot analysis using anti-rice CPD photolyase antibody and TOF-MS analysis showed that both proteins were CPD photolyase. In the transgenic plant bearing the cDNA of rice CPD photolyase in the sense orientation, the revel of both 54- and 56-kDa proteins were significantly higher than those in wild-type plant. It is thus speculated that the difference between 54- and 56-kDa protein is due to post-translational modification. Furthermore, the CPD photorepair activity by the native rice CPD photolyase was significant higher than that of E. coli-expressed rice CPD photolyase, which showed one band about 55-kDa on SDS-PAGE. These results suggested that native CPD photolyase is different from E. coli-expressed protein.

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Translesion DNA synthesis (TLS) is one of the damage tolerant mechanisms avoiding detrimental effects of DNA damages due to exposure to several mutagens. REV1 protein is a component of error-prone TLS in yeast and mammals. Previously, we isolated and characterized AtREV1, which is an Arabidopsis homolog of the REV1 gene. The AtREV1 disrupted mutants, rev1, showed hypersensitive to UV-B and DNA cross-linkers suggesting the importance of AtREV1 to be tolerant to several DNA damaging agents. To clarify the functions of AtREV1, we overexpressed AtREV1 in E.coli cells and obtained a functional recombinant protein. The activity of AtREV1 recombinant protein was measured by primer extension assay. The AtREV1 recombinant protein transferred one or two nucleotides to the primer end. Especially, it efficiently inserted a dCMP regardless the opposite base. The AtREV1 also inserted a dTMP or a dGMP opposite the guanine. These results indicate that the AtREV1 has a deoxynucleotidyl transferase activity. The AtREV1 inserted a dCMP or other nucleosides opposite the apurinic/apyrimidinic (AP) sites. This result suggests that AtREV1 is involved in the bypass of AP sites that provide a damage-tolerance in plant cells. However, AtREV1 showed no insertion or extension activities against UV-inducible DNA lesions, cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct. We speculate that AtREV1 has unknown function(s) other than the transferase activity and the second function is involved in the bypass of CPDs and 6-4 photoproducts.

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Accelerated ion beam is an excellent mutagen in plant breeding which can induce higher mutation frequencies and wider mutation spectrum than those of low linear energy transfer (LET) irradiations, such as X-rays, gamma-rays and electrons (Okamura et al. 2003, Yamaguchi et al. 2003). Mutation breeding operation of two Saintpaulia ionahta cultivars using the method combining plant tissue culture technique and carbon ion beam irradiations were set out at Institute of Modern Physics from 2005 (Zhou et al. 2006). As a result, a chlorophyll-deficient (CD) mutant of Mauve cultivar, which could transmit the character of chlorophyll deficiency to its progeny through three continuous tissue culture cycles, was obtained (Zhou et al. 2006). This year, we gained another mutant of Indikon cultivar from those samples irradiated by 80MeV/u ¹²C⁶+ provided by Heavy Ion Facility in Lanzhou (HIFRL) at the dose of 25 Gy in 2005, whose flower-color has changed from violet to light pink, simultaneously, among the petals the androecia and gynoecium have disappeared. Two of the five sepals have mutated to malformed petals and the number of petals also varied from eleven to fourteen. Moreover, the edge of leaves has become smooth while the normal type is jagged and the color of the abaxial side of leaf also varied from aubergine to light green. On the other side, we could not obtain any regenerated adventitious shoots from the leaf explants of this mutant using the normal Saintpaulia ionahta tissue culture methods unless the recipes of callus or shoot inducing medium was adjusted to appropriate proportion. It is particularly because the entogenous plant hormone level or the hormone receptor in this mutant have changed. In addition, DNA sequence analysis of such ion beam induced mutations and the measuring of entogenous hormone level or receptor should also be executed in the near future to analyse and identify the genetic alterations of these regenerated plantlets at the molecular level.

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To investigate how the XPA mutations influence the temporal and spatial interactions between NER proteins and DNA damage, detergent-insoluble PCNA was visualized in XP-A cells using micropore UV irradiation combined with fluorescent antibody labeling. Interestingly, the PCNA foci appeared at UV damage sites as late as 9 h and 24 h post-irradiation, even though XP-A cells could not repair CPDs and 6-4PPs at all. The following explanations may underlie the delayed recruitment of PCNA to DNA damage sites. Firstly, it might be due to DNA repair that occurs normally in XP-A cells. Defects in NER do not affect the global genome repair of oxidative DNA damage or single strand breaks, which are minor types of DNA lesions caused by UV. Secondly, it might relate to DNA fragmentation resulting from apoptosis. The UV irradiation level (100 J per m2) used in this study produces a high density of DNA damage at localized nuclear areas and might cause apoptotic DNA breaks, which are possibly associated with PCNA dependent repair. Thirdly, it might reflect blocked or delayed DNA replication. When partially irradiated cells enter the S phase, DNA replication in damaged nuclear areas may be slowed down more than in undamaged areas because of the block by DNA damage. Finally, it might be attributable to an incomplete or stalled NER of CPDs or 6-4PPs. If the lack of functional XPA interrupts the normal NER process and keeps it stalled for more than several hours, other NER proteins including PCNA might be recruited slowly to the sites of DNA damage. Our results support the last explanation.

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Ultraviolet light (UV) in the sunlight induces skin cancers. Although the UVB component of the solar UV is known as a major contributor for the skin carcinogenesis, the relative importance of the longer wavelength component UVA has not been determined clearly. Since in vitro studies showed that UVA induces oxidative damage like 8-hydroxy-2'-deoxyguanine (8-OH-dG) in cellular DNA, some relevance of the UVA-mediated oxidative stress has been insisted. To clarify this point, we studied an in vivo mutation spectrum in skin induced by a monochromatic UVA light within the UVA1 range (340-400 nm), using a laser emitting a highly intense 364-nm beam. LacZ-transgenic mice developed for mutation research were exposed to the 364-nm light from an Ar-ion laser irradiator (National Institute for Basic Biology, Okazaki, Japan) at the intensity of 300 W/m². Dose-dependent induction of 8-OH-dG and cyclobutane pyrimidine dimers (CPD) were observed in the skin genome. The mutant frequency of the transgene also increased along with the irradiation dose. The induced mutation spectrum in the exposed epidermis was determined: more than 90% of the mutations were base substitutions, and 70% of them were C -> T transitions, 90% of which occurred at dipyrimidine sites. Oxidative stress-specific G -> T transversions were only recovered at a background level. These results indicate that genotoxic effect of UVA1 is mainly mediated through formation of CPD, not through oxidative DNA damage produced with the UVA-mediated oxidative stress. (This study was carried out under the NIBB Cooperative Research Program for the Okazaki Large Spectrograph; 4-507, 5-507, 6-511 and 7-509.)

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...Sonodynamic effect and photodynamic effect of rhodamine derivatives were compared on free radical formation and cell killing. When electron paramagnetic-resonance spectroscopy (EPR) was used to detect 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) after photo-irradiation or sonication with 2,2,6,6-tetramethyl-4-piperidone (TMPD), the order of TAN formation in the photo-irradiated samples was as follows: rhodamine 6G (R6) > sulforhodamine B (SR) > hematoporphyrin (Hp) > rhodamine 123 (R123) > rose bengal (RB) > erythrosine B (Er) = 0; although there was time-dependent TAN formation when the samples were sonicated, no significant difference among these agents were observed. All these agents suppressed ultrasound-induced OH radical formation detected by EPR-spin trapping. Sensitizer-derived free radicals were markedly observed in SR, RB and Er, while trace level of radicals derived from R6 and R123 were observed.
...Enhancement of ultrasound-induced decrease of survival in human lymphoma U937 cells was observed at 1.5 W/cm² (less than inertial cavitation threshold) for R6, R123, SR and Er, and at 2.3 W/cm² for R6, R123, Er, RB and SR. On the other hand, photo-induced decrease of survival was observed for R6, Hp and RB at the same concentration (10 μM).
...These comparative results suggest that 1) ¹O₂ is not involved in the enhancement of ultrasound-induced loss of cell survival, 2) OH radicals and sensitizer-derived free radicals do not take part in the enhancement, and 3) the mechanism is mainly due to certain mechanical stress such as augmentation of physical disruption of cellular membrane by sensitizers in the close vicinity of cells and/or cavitation bubbles.

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