We irradiated third instar larvae of fruit fly, Drosophila melanogaster with X-rays, and carried out the somatic mutation and recombination test (SMART). When wild-type flies were irradiated with 0.2Gy, mutation frequency was lower than in the control, and an U-shaped dose response relationship was suggested. In a mutant strain defective in DNA repair function, dose response was linear. Here, we introduced baculovirus p35 gene into wild-type flies to suppress apoptosis function in the posterior region of their wings. In these flies, mutation frequency increased in the posterior region of the wings. Especially, terminal deletion of the third chromosome and non-disjunction increased drastically, whereas somatic recombination was not influenced by the suppression of apoptosis. The dose-response curve was U-shaped when apoptosis was suppressed, suggesting that the contribution of apoptosis function to the formation of U-shaped dose-response relationship is not as large as DNA repair function.

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Multiple mutation meaning two or more mutations in a single gene or DNA is observed at unexpectedly high frequency in normal tissues. It is assumed to be one of the indices of genomic instability called mutator phenotype that might contribute in changing normal cells to cancer cells, because cancer cells contain many mutations. Here we report that the frequency of multiple mutation is elevated in Xpc-deficient mice when they get old. Age-dependent accumulation of spontaneous mutation in spleen, liver, heart and lung was accelerated in Xpc(-/-) mice as was judged by mutation on the transgenic lacZ gene. The acceleration was also observed in spleen and liver of old Xpc(+/-) mice indicating tissue-specific haploinsufficiency. Sequencing of the mutated lacZ genes revealed that the frequency of multiple mutation was elevated in old Xpc-deficient mice. The distribution of different types of mutations (spectrum) found in the multiple mutations was distinct from that of single mutations. The frequency of base substitution at A:T was higher in multiple mutations. The two multiple mutations among 20 found in old Xpc-deficient mice revealed as many as 4 alterations within a short stretch of DNA sequence. They might be produced by error-prone TLS DNA polymerases, because some of the TLS polymerases are reported to make mistakes more than once within a small range of DNA.

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NBS1 is the responsible gene for Nijmegen breakage syndrome which patients display cancer predisposition and hyper-sensitivity to ionizing radiation, NBS1 is known to from a complex with MRE11 and Rad50, and it regulates the precise repair of damaged DNA through homologous recombination. We reported that NBS1 activates a p53-independent apoptotic pathway in response to DNA damage through the regulation of the dissociation of Ku70-Bax complex. The phosphorylation at 343-serine residue of NBS1 was essential for this function, whereas FHA, MRE11-binding, or ATM interacting domains were not. The kinase toward this NBS1 phosphorylation could be ATR because ATR inhibitor suppressed the NBS1 phosphorylation at the time when apoptosis occurring. but ATM inhibitor did not.
To get insight of the interaction between NBS1 and Ku70, we successfully established a Nbs1-Ku70 double Knockout cell line. In this presentation, we will discuss about the phenotypes of the Nbs1-Ku70 double knockout cells and the function of the interaction between NBS1 and Ku70 in DNA damage response induced by ionizing radiation.

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DNA alkylating drug, temozolomide (TMZ), is a common drug of chemotherapy against brain tumors. However, the therapeutic efficacy of this drug is limited by the development of resistance. It has been reported that the mechanism of this resistance is complex and involves multiple DNA repair pathways. Fanconi anemia (FA) pathway was initially identified by virtue of its inactivation in a rare genetic disorder. In mammalian cells, the FA pathway is frequently activated in response to DNA strand breaks, replication arrest, and other cytotoxic secondary lesions induced by TMZ. The regulation of the FA pathway is highly complex, involving at least 12 proteins (FANCA, B, C, D1, D2, E, F, G, I, J, L, M). In this work, we tested the role of the FA pathway in mediating cellular resistance to TMZ. We used the cultured mouse embryonic fibroblasts; FANCA -/-, FANCC -/-, FANCD2 -/- cells, their parental cells (provided by Fanconi Anemia Cell Repository; Oregon Health and Science Univ., USA) and CHO cells; FANCD1mt, FANCGmt and their parental cells (provided by Dr. Larry H. Thompson; Lawrence Livermore National Laboratory, USA). We examined the cell survival after 3 h TMZ treatment by colony forming assay. The sensitivity of each cell line was assessed from its D₅₀ value, i.e. from the TMZ dose which reduced cell survival to 50%. In order to accurately compare TMZ sensitivities in the repair defective cell lines, the relative D₅₀ values were normalized using the D₅₀ values of the corresponding proficient cell lines. The relative D₅₀ values listed sequentially in the order in which they increase (reflecting decreasing sensitivities to TMZ) are: FANCD1mt cells < FANCGmt cells < FANCD2 -/- cells < FANCA -/- cells < FANCC -/- cells. The most effective target could be FANCD1 in TMZ treatment. Therefore, we are going to apply siRNA of FANCD1 against human glioblastoma cells in TMZ treatment.

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Ionizing radiations cause DNA double strand breaks (DSBs) which are potentially the most lethal lesions causing genomic rearrangements and cancer if left unrepaired. Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ) pathways are the major repair mechanisms observed in mammalian cells. HR is usually dominant in S and G2 phases of the cell cycle as it requires a pair of sister chromatids for error-free replication; whereas NHEJ is observed in all the stages.
According to the classical model, Ku70/80 heterodimer is recognized as a sensor to first bind the DSB site and recruit DNA dependent protein kinase catalytic subunit (DNA-PKcs) which brings the broken ends in synapsis. The nucleases like Artemis and DNA polymerases like λ and μ process the DNA ends to attain adequate homology before ligation. XRCC4-DNA Ligase IV complex along with the newly identified molecule, XLF/Cernunnos (XRCC4-like factor), plays an important role in the final end-joining step.
The dynamics of this repair machinery is yet to be clarified. We have been using detergent fractionation method to capture the radiation induced chromatin bound complex. A subpopulation of XRCC4 changed into an extraction resistant form that was liberated by micrococcal nuclease treatment, indicating that it had been tethered to chromatin DNA. This chromatin recruitment of XRCC4 could be seen immediately after irradiation and remained almost constant up to 4hr after 20Gy irradiation. Quantitative estimation revealed that a very small percentage of the XRCC4-Ligase IV complex was recruited to each DNA end; thus explaining the complications involved in the detection of the NHEJ machinery at the damaged site.
In addition, we are exploring the mechanism for recruitment of the ligation complex considering the plausible roles of protein modifications induced by radiation. We have also found some evidence for the requirement of DNA-Ligase IV for the recruitment of XRCC4 at the site of damaged chromatin.
Currently, we are also isolating a higher order chromatin bound complex associated with XRCC4. This complex suggestively congregates with some histone molecules. Such a striking association of histone molecules with XRCC4 on the damaged chromatin site leads to a speculation that they are structurally reorganized and may be involved in the recruitment dynamics.

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The nonhomologous end-joining (NHEJ) pathway is conserved in eukaryotes, from yeast to humans. Without requiring homologous DNA, NHEJ repairs DNA double-strand breaks produced by xenobiotic agents, such as topoisomerase II inhibitors and ionizing radiation, or by the cellular pathway for V(D)J recombination of the immunoglobulin genes. The key proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), the Ku70/80 heterodimer, the XRCC4-DNA ligase IV complex and recently found molecule XLF/Cernunnos. From all of these key proteins, DNA-PKcs plays an important role in mediating the repair process through NHEJ repair. Moreover, the protein kinase activity of DNA-PK is required for its in vivo function, as DNA-PKcs containing inactivating mutations in the catalytic domain does not complement the radiosensitive phenotype of DNA-PKcs-deficient cells. DNA-PKcs is known to phosphorylates tens of proteins in vitro but the true substrate in vivo and the significance of phosphorylation in NHEJ remains to be clarified. So, there is need to identify the genuine phosphorylation targets of DNA-PK and found physiological significance of these phosphorylation targets. In the present investigations we have identified several new phosphorylation sites in XLF and XRCC4 protein by DNA-PK in vitro. We have prepared phosphorylation-specific antibodies to respective sites and observed that some of these phosphorylation sites were indeed phosphorylated in the living cells following irradiation. To explore the biological significance of phosphorylation, we established cells expressing phosphorylation-defective mutants.

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A several lines of evidence such as animal experiments and epidemiological studies of A-bomb survivors indicated that radiation exposure in utero of the embryo or fetus induces growth retardation and microcephaly with mental retardation. However, the molecular and cellular mechanism of radiation-induced microcephaly remains unclear. We try to dissect the molecular pathology of human genetic microcephaly in order to understand the essential mechanism of radiation-induced microcephaly.
Seckel syndrome is an autosomal recessive and genetic heterogeneous disorder of severe microcephaly and short stature. So far, the ATR gene and centrosomal Pericentrin/ PCNT gene were identified as the responsible genes for Seckel syndrome. Here, we identified the mutations of the PCNT gene in two Japanese Seckel syndrome patients, and found that PCNT regulated cell cycle at some checkpoints. Mitotic index after UV treatment was significantly increased in PCNT-Seckel syndrome cells as compared with wild-type cells. In contrast, PCNT-Seckel syndrome did not exhibit radiation sensitivity. These results suggest that PCNT is a component of ATR signaling pathway and controls the G2/M checkpoint. Next, we found that the population of cells with primary cilia, a hair-like cell surface structure at G0/G1 phase, was significantly increased in PCNT-Seckel primary skin fibroblasts, and that the patient cells inhibited BrdU uptake despite of the presence of proliferation marker Ki-67. Taken together, we conclude that PCNT-Seckel Syndrome cells fall into G1 phase arrest. The control mechanism of G1/S phase transition by PCNT is currently under investigation.

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4-Nitroquinoline-1-oxide (4NQO) is a UV-mimic carcinogen but does not have activity of DNA-binding or mutagenicity. 4NQO is converted into 4-hydroxyaminoquinoline-N-oxide (4HAQO) by seryl-tRNA synthetase. 4HAQO binds to bases, producing adducts in DNA. It has been suggested that 4NQO has other activation mechanisms, which produce reactive oxygen species (ROS), increasing oxidative stress in cells and damaging lipids, proteins and DNA. But whether this mechanism is involved in mutagenesis is not yet known. mutM and mutY are involved in the base excision repair that is concerned with repair of the DNA damage produced by ROS. The uvrA gene is involved in the nucleotide excision repair and seems to be able to remove DNA adducts.
In this study, we examined the mutation spectrum of E. coli treated with 4NQO in the growth phase. The lactose reversion assay measuring Lac+ revertant colonies can independently detect each of the six possible base-pair substitution mutations. The results indicate that 4NQO induces specific mutations: G:C → A:T, G:C → T:A, and A:T → C:G. We further investigated which 4NQO mutagenesis depended on: the activity as a DNA adduct or generating ROS using a rifampicin reversion assay of mutM, mutY, mutM mutY, and uvrA mutant strains. The rifampicin reversion assay can analyze nonspecific mutation frequency. The results suggested that mutM and uvrA deficient mutants are sensitive to 4NQO, but there mutation frequencies are not increased by 4NQO.
We are currently further examining the survival and mutation frequency of a mutM mutY uvrA triple mutant and katG mutants (deficient for hydrogen peroxidase) treated with 4NQO in order to further clarify the relationship between ROS produced by 4NQO and mutagenesis. In addition, we hope to reveal which has a stronger effect; 4NQO activity as a DNA adduct or as an agent generating ROS.

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Reactive oxygen species (ROS) play critical roles in a wide variety of cellular functions. On the other hand, excess amounts of ROS generated within cells result in oxidative stress and are responsible for many deleterious changes, which can induce oxidative damage to DNA, proteins and lipids. Several factors can influence the susceptibility to oxidative stress by affecting the antioxidant status and ROS generation. These factors include endogenous factors (e.g., exercise and psychological stress), and exogenous factors (e.g., ionizing radiation, cigarette smoke, environmental pollutants, and UV light). ROS have been shown to participate in various biological consequences of ionizing radiation. However, the biochemical mechanisms by which ROS cause molecular damage and ultimately cellular dysfunction are not fully understood. One of the major cellular targets of ROS is proteins. ROS can oxidize both aliphatic and aromatic amino acid residues of proteins, leading to irreversible structural changes. The redox environment is controlled by a variety of redox-regulating systems that include superoxide dismutase (SOD), glutathione peroxidase (GPX), thioredoxin (Trx), thioredoxin reductase (TR), glutaredoxin (Grx) and peroxiredoxin (Prx). In this study, we examined whether and how cellular sensitivities to radiation and oxidative stress are modulated by the overexpression of SOD and Grx in cultured human cells. Specifically, we examined the change in survival, phosphorylation of H2AX, level of expression of OXR1 protein and oxidized protein after irradiation by overexpression of SOD. One of our results indicated that oxidized protein was decreased by the overexpression of SOD, which suggested that SOD and Grx play important roles in various cellular responses to radiation.

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Superoxide dismutase (SOD) serves as the primary defense against superoxide. It has been shown that SOD1 is present in the cytoplasm, nucleus and mitochondrial intermembrane space and SOD2 is present in the mitochondrial matrix in vertebrate cells. Disruption of SOD1 or SOD2 results in cell death and growth delay, respectively in DT40 cells. Sister chromatid exchange frequency and the number of apurinic (AP) sites in chromosomes were increased in concomitant with SOD1 depletion. Neither such DNA damages nor mitochondrial dysfunction were not observed in SOD2 deficient cells. All the phenotypes involved the elevation of intracellular superoxide level in SOD1 depleted cells were completely suppressed by APM treatment. In addition to APM, either NAC or TIRON were able to rescue the growth delay in SOD2 depleted cells. These results suggest that SOD1 is essential for cell viability and playing a crucial role in the scavenging around cytoplasm and nucleus and preventing from DNA damage. And APM might scavenge the superoxide with offset the absence of the SOD1.

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[Purpose] Recently, several studies have shown that reactive oxygen species (ROS) play an important role in the signaling pathway of apoptosis. We reported that ROS generation was increased several hours after irradiation and participated in cytochrome c release from mitochondria in A549 cells (Ogura et al., Cancer Lett., 277:54, 2009). In this study, to reveal the mechanism of radiation-induced ROS generation, we analyzed how irradiation modulates mitochondrial functions by using mitochondrial inhibitors.
[Materials and methods] Human lung carcinoma A549 cells were irradiated with 10 Gy X-rays. Intracellular ROS level was measured by flow cytometry using specific ROS fluorescence probe DCFDA. Cellular oxygen consumption and intracellular ATP level were measured to assess mitochondrial respiratory activity, and the effect of rotenone (a complex I inhibitor) and oligomycin (a F₀/F₁-ATPase inhibitor) on respective index was examined.
[Results] ROS generation was significantly increased from 6 hours after irradiation. The cellular oxygen consumption rate was risen 12 hours after irradiation, and it was suppressed by rotenone. Intracellular ATP level was elevated time-dependently up to 24 hours after irradiation, and it was suppressed by oligomycin. These results suggested that irradiation activated mitochondrial electron transport chain, and the increase of ROS generation. We investigate the further mechanisms of radiation-induced mitochondrial activation in tumor cells.

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Reactive oxygen species (ROS) are toxic substances produced during metabolic processes. ROS increase internal oxidative stress. ROS produced in living organisms are highly reactive and damage lipids, proteins and DNA. ROS-mediated DNA damage contributes to spontaneous mutagenesis. The oxidation of DNA results in various functional disorders, including premature aging and cancer. Recently, the OXR1 gene was identified in human cells and revealed to be involved in protection against oxidative stress. However, its function still remains uncertain. The nematode C. elegans is often used to clarify the mechanisms underlying aging and development. However, any homologs of human OXR1 have not been identified in the nematode. In this study, we first identified the C. elegans homolog CeOXR and examined the roles of CeOXR in protection against oxidative stress. We cloned the CeOXR gene and examined whether this protein could complement the phenotype of the E. coli mutM mutY mutant. The double mutant shows a high mutation frequency. We found that the E. coli mutM mutY mutant expressing the cloned CeOXR protein showed a decreased mutation frequency, indicating that the CeOXR suppress the oxidative stress in C. elegans. Furthermore, we compared the lifespan of C. elegans CeOXR mutant with that of wild-type strain N2. The lifespan of CeOXR mutant was shorter than that of wild-type N2. Thus, CeOXR may have a function to extend lifespan by suppressing the oxidative stress in C. elegans.

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In cells of aerobic organisms, reactive oxygen species (ROS) are continually generated during respiration. ROS are also produced by exposure to ionizing radiation and various chemical oxidizing agents. ROS induce various types of oxidative damage in DNA. Oxidative DNA damage occurs directly in DNA and in dNTPs in the nucleotide pool. Oxidized dNTPs such as 8-oxo-dGTP or 2-OH-dATP could be incorporated into DNA during replication, which could result in mutagenic consequences. Therefore, many organisms have enzymes for eliminating the abnormal dNTPs in the nucleotide pool as well as repair enzymes for damage in DNA. E. coli MutT and human MTH1 can hydrolyze 8-oxo-dGTP, and human MTH1 also hydrolyzes 2-OH-dATP. However, whether there is homolog of these enzymes in C. elegans, a multicellular eukaryote frequently used as a model for the study of development and aging, has remained uncertain. To clarify the mechanism for eliminating oxidized dNTPs and its critical roles in maintaining the genome stability in C. elegans, we selected three genes as players in sanitization of the nucleotide pool by homology search. Proteins encoded by the three genes were purified and their enzymatic activity was examined by using HPLC. None of the three enzymes could hydrolyze 8-oxo-dGTP or 2-OH-dATP, but one hydrolyzes 8-oxo-dGDP. The finding provides one possible mechanism of how the dNTP pool is sanitized in C. elegans. For this enzyme, divalent cation dependence were experimentally determined. We will show these data.

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High atomic number molecules, such as gold and platinum, are known to be able to enhance the biological effect of X-irradiation. It is generally thought to be due to the increase of DNA damage mainly by Photoelectric and Compton effects when cells are irradiated in the kilo-voltage range. Herold et al. (INT. J. RADIAT. Biol.,76:1357, 2000) demonstrated radiosensitization by the gold particles (3 μm diameter) in mouse tumor EMT-6 cells. However, for application of in vivo tumor cells, there are some problems such as their toxicity for normal tissues and delivery of the gold particle to tumor cells. To solve these issues, we newly synthesized gold-nanogel (GNG) consisting of polyamine, polyethylene glycol, and gold-nanoparticles (6 nm diameter). The diameter of this GNG was determined as 148 nm by transmission electron microscopy (TEM). In this study, we investigated whether GNG enhances the cell death induced by X-irradiation and studied the mechanism of the radiosensitization. Mouse squamous carcinoma SCCVII cells, human lung adenocarcinoma A549 cells, and Chinese hamster V79 cells were incubated without or with various concentration of GNG for 14 h. After X-irradiation at 200 kV, the cytotoxicity and the reproductive cell death were evaluated by colony formation assay. In all cell lines, GNG treatment increased the reproductive cell death after irradiation without a marked cytotoxicity. PI staining also revealed that GNG enhanced apoptosis in SCCVII cells induced by irradiation. Futhermore, results obtained by immunofluorescence analysis, western blotting and pulsed-field gel electrophoresis indicated that GNG decreased DNA double-strand break in cells irradiated. In addition, TEM images revealed that GNGs were distributed in cytoplasm after uptake by endocytosis. These results suggested that GNG induced radiosensitization not by enhancing the damage of nuclear DNA but by targeting other organellars.

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Recently, cancer treatment with a combination of radiation and anticancer drug has been widely applied to enhance cell death in solid tumor cells. A purine analog 8-amino-adenosine is converted into its triphosphate form, 8-amino-ATP, after uptake by cells. Intracellular accumulation of 8-amino-ATP is reported to induce the depletion of ATP level and the inhibition of RNA/DNA synthesis. In this study, we examined whether 8-amino-adenosine has an ability to enhance radiation-induced reproductive cell death and apoptosis. Human lung adenocarcinoma A549 cells were treated without or with 8-amino-adenosine and irradiated. Reproductive cell death was assessed by clonogenic assay and apoptotic cells were evaluated by propidium iodide staining. Expression of apoptosis-related proteins was examined by western blot analysis. 8-Amino-adenosine significantly increased reproductive cell death as well as apoptosis induced by X-irradiation. When peptide inhibitors against caspase-3, -8, -9 were utilized to evaluate the involvement of caspases in this increase of apoptosis, all inhibitors suppressed the enhancement of radiation-induced apoptosis. This result suggested that not only mitochondria-mediated apoptotic signal transduction pathways but also death receptor-mediated one, were involved in this radiation-induced apoptosis in the presence of 8-amino-adenosine. In addition, 8-amino-adenosine inhibited the expression of an anti-apoptotic protein, survivin, suggesting that this inhibition could contribute to the enhancement of radiation-induced apoptosis. We are currently examining if 8-amino-ATP instead of endogenous ATP acts as a promoting factor of apoptosis.

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Radiotherapy is widely used for cancer treatment. The standard fractionated radiotherapy consists of exposure to 2 Gy of X-ray per day for several weeks and total doses with 50 to 90 Gy. However, recurrence frequently occurs due to tumor radioresistance resulting in the failure of radiotherapy. Therefore, it is important to elucidate the underlying mechanisms of radioresistance for the improvement of radiotherapy.
We have studied DNA damage response to fractionated radiation (FR). Long-term FR exposure to 0.5-Gy X-rays twice per day, for more than 31 days conferred radioresistance on human tumor cell lines, HepG2 and HeLa, along with cyclinD1 overexpression. Radioresistance was due to less induction of apoptosis.
CyclinD1 overexpression was brought by down-regulation of proteolysis via the AKT/GSK3b pathway. To determine the role of AKT-mediated cyclinD1 overexpression in radioresistance, we treated long-term FR cells with an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2). API-2 suppressed AKT phosphorylation resulting in down-regulation of cyclinD1 expression. Under this condition, API-2 completely suppressed the radioresistant phenotype of long-term FR cells.
In conclusion, we can control radioresistance in combination in an AKT inhibitor. The present study suggests the importance of the AKT/ cyclinD1 pathway as a novel target to improve the outcome of radiotherapy, especially on recurrent radioresistant tumors

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[Introduction] In order to understand the mechanisms of tumor radioresistance and to develop more effective tumor radiotherapy, we established clinically relevant radioresistant (CRR) cell lines; these continue to proliferate under daily expose to 2 Gy of X-ray. Here, we studied whether CRR cells are cross-resistant to anti-cancer drugs or not using pairs of CRR and their sensitive parental cell lines.
[Method] Compared with parental cell lines, HepG2, HeLa, SAS, KB and H1299 were used, cross resistance of their CRR derivative cell lines, HepG2-R, HeLa-R, SAS-R1, SAS-R2, KB-R and H1299-R were used. Anti-cancer drugs used were cisplatinum (CDDP), docetaxel (DOX), bleomycin, fluorouracil (5-FU), vincristin, etoposide (VP-16) and adriamycin (ADM). Chemosensitivity was analyzed by the high-density survival assay. In addition, RT-PCR was carried out to analyze gene expressions of MDR1.
[Result] 5-FU was effective against all radioresistant cell lines. Six out of 7 (86%) CRR cell lines had cross-resistance to DOC and vincristin. Four (57%) CRR cell lines had cross-resistance to VP-16 and ADM. Overexpression of the MDR1 gene was observed in HepG2-8960-R.
[Discussion] This study suggested that anti-cancer drugs targeting beta-tuburin had little effect for the elimination of clinically relevant radioresistant cells. Not all radioresistant cell lines showed cross-resistance to bleomycin whose action mechanism was similar to X-rays, indicating that contribution of efficient repair of DNA double strand breaks for clinically relevant radioresistance is low.

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With Ptch1+/- mouse, which develop medulloblastoma, the most common childhood brain tumor, we analyzed dose-dependent and age-dependent effects of radiation during fetal and postnatal stages. We found here that the incidence of medulloblastoma increased and the latency shortened, with increasing dose, after whole-body exposure to seven different doses (0.05-3Gy) at postnatal day 1. Importantly, the significant increase in incidence and decrease of latency was evident for low-dose irradiations of 0.05Gy and 0.1Gy. In order to explore the molecular mechanisms for radiation-induced medulloblastomas, we analyzed loss of heterozygosity (LOH) status at 6 SSLP markers on chromosome 13, where Ptch1 locus is located. It was revealed that all 17 spontaneously developed tumors in non-irradiated mice showed LOH in broad regions including Ptch1 locus and distally-extending telomeric portion (designated as S-type), while all 19 tumors induced after 3Gy irradiation exhibited losses only at interstitial markers around Ptch1 locus (designated as R-type), indicating that the inactivation mechanism of a wild-type Ptch1 allele in radiation-induced tumors was different from that in spontaneous ones. This was consistent with a previous report in a different strain by Pazzaglia et al. (2006). Interestingly, both S- and R-types were observed in the irradiated-groups with lower doses, and that there was a clear dose-dependent increase in the ratio of R-type to S-type. Data on the age-dependent change in radiosensitivity to medulloblastoma in Ptch1 heterozygous mice will be also presented.

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X-ray irradiation at postnatal day 1 significantly increased the incidence of medulloblastoma in Ptch1 heterozygous mice on C3B6F1 genetic background in a dose-dependent fashion. Spontaneously developed tumors showed LOH in broad regions on chromosome 13, including Ptch1 locus and distally-extending telomeric portion (S-type), while tumors induced after 3Gy irradiation showed losses only at interstitial markers around Ptch1 locus (R-type), and tumors induced after lower doses exhibited a clear dose-dependent increase in the ratio of R-type to S-type (Ishida et al., at this conference). To explore the difference between S-typed and R-typed tumors, we performed integrated array-CGH and expression microarray analysis on 6 S-typed and 6 R-typed tumors. Array-CGH analysis revealed that copy-number reduction around Ptch1 locus occurred only in R-typed tumors, suggesting that wild-type Ptch1 allele was lost by deletion in R-typed tumors, while it was due to mitotic recombination in S-typed tumors. In addition, all 12 tumors had copy-number gains of whole chromosome 6 in some degree. In expression analysis, more than 1,000 genes showed statistically significant difference between groups of S- and R-typed tumors. Importantly, many of them were located within the commonly deleted region around Ptch1 locus and showed decreased expression by half in R-typed tumors as if they were influenced directly by the copy-number reduction there. On the other hand, many genes on chromosome 6 showed increased expressions with showing tight correlation with levels of aneuploidy, that were calculated based on results by array-CGH analysis. These data suggest that copy-number aberrations, some of which are typically caused by irradiation, lead to considerable changes in transcriptome possibly by both cis- and trans-acting manners.

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Ionizing radiation is a carcinogenic agent. Radiation carcinogenesis is the results of a series of somatic mutations. Translesion DNA synthesis (TLS) is known as error-prone DNA repair. Y family DNA polymerases which operate TLS can bypass a various damage lesions with low fidelity. Y family DNA polymerase is responsible for spontaneous and damage-induced mutagenesis.
Rev1 is one of Y family DNA polymerase and has a regulatory role in TLS. However, little is known whether Rev1 protein has the precise role in tumor formation in vivo.
We generated Rev1 transgenic mice (Rev1 Tg mice) that continuously express a Rev1transgene in various tissues. Both Rev1 Tg and wild-type (Wt) mice were subjected to Υ-irradiation of 1.6Gy four times at a weekly internal, starting at the age of 4 weeks. Rev1 Tg and wild type mice (C57BL/6) were treated with the mutagenic agent N-methyl-N-nitrosourea (MNU) through intraperitoneal injection and examined their development of tumor.
Most mice were died because of thymic lymphoma among all developed tumors deduced from MNU treatment. Compared with wild type mice, Rev1 Tg mice showed the rapid and high tendency of thymic lymphoma development. On the other hand, overexpression of Rev1 prolonged radiation-induced thymic lymphoma development.
These findings demonstrate that overexpression of Rev1 may be associated with chemical and raditation-induced tumorigenesis with the different mechanism.

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We have proposed a new route of in vitro cell transformation, which assumes aneuploid driving force. This route does not originated from DNA lesion origin, but it is thought that it is the main route of cell transformation. A main target of this route may be centrosome, and disorder of centrosome causes chromosomal non-disjunction and aneuploid. From the results of research with mouse embryo cell and Syrian hamster embryo cells, intracellular oxidation radical which an electron to leak from mitochondria generates may become a trigger of both natural- and radiation-induced cell transformation. Intracellular oxidation radical and the derivation radical of a high level induce dysfunction of centrosome, and centrosome dysfunction causes aneuploidy. These results suggest that the route of radiation-induced cell transformation may be the same as that produced by natural physiology action. Alternatively, this means that physiological threshold is present in low dosage area in cell transformation.

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There might be two types of radiation carcinogenesis. One type is targeted carcinogenesis through radiation-induced DNA damage in target cells. The other is untargeted carcinogenesis through changes in a microenvironment that might indirectly induce genetic changes in unirradiated cells. The existence of untargeted thymic lymphomagenesis was substantiated by the occurrence of untargeted lymphomas induced in unirradiated thymuses transplanted into irradiated mice. The cause of radiation-induced lymphomagenesis and abnormal events occurring during lymphomagenesis are, however, not clear. We examined causative events for lymphoma development during prelymphoma stage in C57BL/6 mice after four consecutive irradiations of 1.8 Gy γ-rays at 1-week intervals. Radiation-induced thymic atrophy was observed until 6 weeks after irradiation. γH2AX foci in thymocytes were formed 6 to 8 weeks after irradiation. Aneuploid cells comprising 41 chromosomes in thymocytes reached 34% in average during 4 to 6 weeks. Chromosomal instability was induced in culture in descendants of irradiated thymocytes, and 8 to 10 weeks after irradiation, 2.3-fold increase in chromatid-type aberrations was observed. When co-cultured with descendants of irradiated thymocytes, bystander effect on chromosomal aberration induction was observed in X-ray-sensitive XRCC4^-/- cells. The bystander effect was decreased by treatment with superoxide dismutase and catalase. Notch1 and Bcl11b rearrangements were increased at 10 to 1000-fold frequency in some mice after irradiation. Cell clones exhibiting identical V(D)J recombination in TCRβ locus were observed 2 to 10 weeks after irradiation. Thus, during the early stage of lymphomagenesis, induction of DNA double strand breaks, aneuploidy, delayed chromosomal instability, bystander effect, rearrangements of cancer-related genes, and cell clonality was observed. Induction of these abnormalities might be associated with radiation-induced lymphomagenesis.

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We summarize results on the experimental gene therapy of methylcholanthrene-induced tumors in transgenic mice with mutant p53. pTE50 transgenic mice were constructed with the mutant p53 cDNA carrying a 9 bp deletion in exon 6 obtained from a radiation-induced tumor. Fetus fibroblasts of the mouse exhibited the suppressed p21 induction after 5 Gy X-irradiation, indicating the dominant-negative activity of mutant p53 in the mice. Previously, the transgenic mice showed a 1.7 fold elevated tumor incidence (42% excess) as compared to wild-type mice after subcutaneous injection of 0.02 mg MCA. These tumors, after grown to 5 mm diameter, were treated by local injection of siRNA no.220 designed to suppress the promoter/enhancer of the mutant p53 with a delivery system using atelocollagen, 4 times with an interval of 2 days. The tumors responded with a frequency of 4 out of 23, including 3 cures without recurrence for 51-116 days, and 2 with growth suppression. These results indicate that the autochthonous tumors can be cured by the gene therapy. Combined with results on transplanted tumors, the total frequency of siRNA no.220-responsive tumors was 30%, corresponding to the estimated frequency of mutant p53-dependent tumors. Transplant line TT18 exhibited the induction of apoptosis after treatment with siRNA no.220. Tumors in wild-type mice did not respond to siRNA no.220. These results indicate that suppression of the mutant p53 with siRNA no.220 restored the apoptotic activity of wild-type p53 retained in the tumors. (Cancer Gene Therapy, in press; online June 26, 2009)

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Homologous recombination is critical for DNA double-strand break repair in mitotic cells, and deregulation of the pathway could cause carcinogenesis and alter sensitivity to DNA-damaging agents. Recently, some meiosis-specific proteins involved in homologous recombination are shown to be aberrantly expressed in cancer cells. We assumed that ectopic expression of such proteins in mitotic cells could modulate the intrinsic homologous recombination pathway and alter sensitivity to radiation or DNA-damaging anti-cancer drugs, and have assessed the role of such proteins in mitotic cells. Here we report on the role of the SYCP2 protein in mitotic cells. The SYCE2 protein is a component of the central elements of the synaptonemal complex, a meiosis-specific supramolecular proteinaceous structure that is essential for synapsis of the maternal and paternal homologous chromosomes. While it is not normally expressed in mitotic cells, reverse transcription-polymerase chain reaction analysis revealed that SYCE2 is aberrantly expressed in cells derived from breast cancer and hematopoietic malignancies. Forced expression of SYCE2 in normal epithelial cells conferred resistance to ionizing radiation. Radiation-induced focus formation of Rad51 was increased in SYCE2-expressing cells. These findings suggest that SYCE2 induces resistance to radiation by modulating the intrinsic Rad51-dependent homologous recombination pathway in cancer cells.

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Post-replication repair pathway (PRR) protects cells from a wild variety of DNA damage and it is regulated by RAD18 gene. Indeed, RAD18 deficient human cells exhibit sensitivity to ionizing radiation, suggesting a functional role of PRR for protection against ionizing radiation. Translesion DNA synthesis (TLS) is one of sub-pathway of PRR, in which a number of non-essential DNA polymerases is recruited to the 3'-ends and extends it beyond the lesions, rescues stalled replication. In eukaryotes, RAD6-RAD18 dependent mono-ubiquitination at the lysine 164 residue of proliferating cell nuclear antigen (PCNA) is deemed to play a key role in regulation of TLS. In this study, we demonstrated that the polymerase switching reactions were stimulated by mono-ubiquitination of PCNA.

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In order to verify the possibility of selective damage induction in DNA, the yields of base lesions as well as strand breaks have been measured in dry plasmid DNA films irradiated with highly monochromatized soft X-rays in the energy region of 270-760eV, which includes the carbon, nitrogen and oxygen K-edges. The experiments were performed at the beamline BL23SU in the SPring-8. Irradiated plasmid DNA was analyzed by agarose gel electrophoresis and the yields of strand breaks were determined by measuring the band intensity of the separated closed circular, open circular and linear forms of the plasmid DNA. The yields of base lesions were determined by the post-irradiation-treatment of the DNA with enzymatic probes (Fpg and Endo III) which excise base lesion. The yields of both pyrimidine and purine base lesions, observed as Nth-sensitive and Fpg-sensitive sites, respectively, are strikingly high (5-6x10^-11 lesions/Gy/Da) at the oxygen K-edge (560eV) but extremely low (2-6x10^-12 lesions/Gy/Da) at an energy just below the nitrogen K-edge (380eV) as compared with the yields observed at other photon energies. The yields at 560eV are enhanced 10-fold and 27-fold for Nth-sensitive and Fpg-sensitive sites, respectively, compared with those at 380eV. The yield of prompt single strand breaks is also enhanced at the oxygen K-ionization energy, but only 2-fold, as compared with that at 380eV. These results could potentially lead to new methods for selective induction of specific types of DNA damage through tuning the energy of soft X-rays.

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Xrs5(Ku80 deficient CHO mutant cells)are well-known for the DNA double strand breaks (DBSs) repair deficiency. The amount of DNA-DSBs following gamma irradiation in xrs5 cells was evaluated by the phosphorylated H2AX and 53BP1 foci using the immunofluorescence intencity. After the 72 hours irradiation, the number of H2AX foci per xrs5 or CHO-K1 cells was 35 and 7 respectively. Our results suggested that the DNA-DSBs in xrs5 cells were not repaired 72 hours after irradiation.

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The development of SPICE (Single-Particle Irradiation system to Cell), a microbeam irradiation system, has been completed at the National Institute of Radiological Sciences (NIRS). The beam size has been improved to approximately 2 μm in diameter. Cell targeting system can irradiate up to 24,000 cells per hour and enables one to irradiation cells from a single proton. This facility is now in operation for collaborative researches.
From this year we started a study of effects on neural stem cells irradiated with a proton microbeam (SPICE). Neural stem cells are a subtype of progenitor cells in the developing fetal brain that can self-renew and generate both neurons and glia, which form a central nervous system. In the developing stage, radiation exposure is said one of causes of inducing microcephaly. Radiation effects with neural stem cells are yet to be understood. In order to clarify the mechanism that neural stem cells have radiation damages in the both processes of proliferation and differentiation, we used a microbeam. We prepared homogenous neural stem cells differentiated from ES cells by NSS (; Neural Stem Sphere) method, original differentiation-inducing method. Each of the targeted cells was irradiated. First of all, in the process of proliferation, we started studying about survival fraction, DNA damage and its repair of neural stem cells to a number of irradiated proton particles. In this announcement, we also report about current status of the development of SPICE.

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We already reported our microbeam irradiation system using synchrotron monochromatic X-rays. Our system adopted a precise slit system to make X-ray microbeam, which enables us to change the beam size arbitrarily larger than 5 micron square. Using this advantage of our system, we have measured dose-survival relationships of V79 cell in two irradiation conditions with clonogenic assay method. One is to irradiate with 10-micron square beam aiming at nucleus only, the other with 50 micron square aiming at whole cell. This work revealed that hypersensitivity in low dose region is more enhanced in nucleus-irradiated cells than in whole-cell irradiated cells. We have observed also in bystander cells that bystander effect depends upon the energy-deposited area in the irradiated cells. These results suggest that intracellular communication between nucleus and cytoplasm plays an important role in determining the cell death in low dose region. For further investigation, we have developed a method to irradiate cytoplasm only without irradiating cell nucleus.
In order to shield the nucleus in the uniform irradiation field, we made a gold mask, 15 micron in diameter and 20 micron thick, on a very thin (200 nm thick) SiN film. The thickness of the gold was determined to decrease the intensity of 5.35 keV X-rays to less than one-thousandth. It was mounted on a small X-Y stage and set in the system between the slit system and the sample stage. Using a scintillater dish, we adjusted the position of the mask and the size of the beam. Finally, we got a 50 micron by 50 micron beam with uniform intensity, at the center of which located was the gold mask of 15 micron diameter. When we want to irradiate only cytoplasm of the cells, center of the mask was recognized as the beam position and cytoplasm of cells, nuclei of which were stained with Hoechst dye and recognized by its fluorescence, were irradiated with X-rays, leaving nucleus unirradiated. Survival curve of cytoplasm-irradiated cells will be presented.

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Radiation-induced bystander response is defined as a response in cells that have not been directly targeted by radiation but is in the neighborhood of cells that have been directly exposed. To elucidate the bystander response is important to evaluate the risk of low dose radiation. It has already reported many results of bystander response induced by high-LET charged particles using microbeam irradiation systems. On the other hand, it has not yet fully recognized bystander response to low-LET photons. Here, it has shown that bystander cell killing induced by synchrotron radiation (SR) X-ray microbeam irradiation was mainly mediated by nitric oxide (NO). Cell nucleus of confluent normal human lung fibroblast WI-38 cell was irradiated with a 5.35 keV monochromatic SR X-ray microbeam cut in 5 μmx5 μm square. All of the cells on dish were harvested and plated 24 h after irradiation. Surviving fractions were determined by colony formation assay. Surviving fractions were decreased between 0.25-1.5 Gy and were 0.85 at 1.4 Gy in the case of 5 cells in the center of dish were irradiated. Above 2.0 Gy, surviving fractions were recovered approximately 1.0, suggesting that the induction of bystander cell killing may need surviving activities in targeted cells, because the dose resulting in 37% cell survival was about 2.0 Gy. Bystander cell killing was significantly suppressed by pretreatment with aminoguanidine (an inhibitor of inducible NO synthase) or carboxy-PTIO (a scavenger of NO), but not by DMSO (a scavenger of reactive oxygen species) or lindane (an inhibitor of gap junction). These results suggest that NO is the chief initiator/mediator of bystander cell killing induced by SR X-ray microbeam irradiation.

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Our studies using X-ray microplanar beams generated with synchrotron radiation clearly showed that the recovery from reproductive cell death was quite different between normal and tumor cells. One possible mechanism might be the bystander effect between irradiated and unirradiated cells by X-ray microplanar beams, however it is still unknown not only biological effect itself but also its mechanism. This year we focused on the relationship between p53-gene status and bystander response to explain the phenomenon. Two p53-wild type normal human cells, one p53-wild type tumor cell line and two p53-mutated type tumor cell lines were irradiated with 25µm-thin (200µm beam space) X-ray microplanar beams generated with the SPring-8 at Japan Synchrotron Radiation Research Institute. The results showed that the recovery from reproductive cell death was occurred in the p53-wild type cells after 12h of post-irradiation incubation, while no recovery was observed in the p53-mutated type cells. Furthermore, the recovery observed in the p53-wild type cells was suppressed to the similar level of the reproductive cell death observed in the p53-mutated type cells, when using a specific inhibitor of gap-junction mediated cell-cell communications together. Our present results suggest that p53-mediated cellular responses play an important role of repair process between irradiated and unirradiated cells with X-ray microplanar beams.

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Purpose:
The purpose is the development of the microbeam system using a laser-plasma x-ray and is to verify DNA double strand breaks induced by the laser-plasma x-ray irradiation.
Materials and Methods:
A tabletop Ti:Sapphire laser was used to generate the Kα x-ray pulse from a copper (Cu) target. The laser energy, the pulse width, the shot rate, laser spot size on the target surface, the intensity were approximately 150mJ, 70fs, 10Hz, 30μm, and 3x10¹⁷W/cm², respectively. The number of Cu- Kα photon and pulse duration were approximately 3x10¹⁰ photons/4πsr/pulse and 1ps, respectively. The x-ray was focused on human lung adenocarcinoma cell line (A549) by using the polycapillary x-ray lens. The measurement of the x-ray spot size and the dose estimation were confirmed with Gafchromic film EBT. In order to detect the DNA double strand breaks (DSB) induced by laser-plasma x-ray irradiation, the immunofluorescence staining was performed with anti γ-H2AX antibody and anti phosphorylated ATM antibody.
Results and Discussion:
The absorption dose of laser-plasma x-ray was 0.12mGy per laser pulse. Result from immuno fluorescence staining after the laser-plasma x-ray irradiation, γ-H2AX and phosphorylated ATM focus formation in the nucleus was detected. The focus positive cells existed range from 600 to 900μm in diameter, this length almost corresponded with the x-ray spot size. These results show that the laser-plasma x-ray can induce DSB in the cancer cells and apply to radiobiological study. The laser-plasma x-ray have attractive features such as monochromaticity, high brightness, focusing ability, and ultra-short duration compared with conventional x-ray. Near future, we will start to research the radiation response to the ultra intense and short pulse x-ray.

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At STS in Kazakhstan, about 460 nuclear tests were performed between 1949 and 1989. Residents around STS were affected with chronic and repeated long-term exposure to low level mixed (external and internal) radiation. REA has been promoting ‘Study on Health Effects of Radiation in Residents in and around STS’ since 2001, with cooperation of NNC, and CSPRE. Data were collected from Archives, Citizen Registration Office, etc. As of the end of March, 2009, personal data (date of birth, sex, race, etc.) were collected for 131,700 persons, including 51,900 of exposed population. Among this population, number of persons whose residential history between 1949 and 1963 was obtained and thereby whose dose can be calculated, was 20,400. Out of the latter, vital status became clear for 16,800 persons (alive, 7,500; dead, 9,300). Control population was not used in statistical analysis at present, because it has not been long after study of this population had been initiated. Among causes of death in exposed population, circulatory disorders were 42%, followed by neoplasms (21%), mainly of esophagus and stomach. Individual radiation doses were calculated with use of calculating formula advocated by Ministry of Health, Russian Federation. Relationship between radiation dose and ICD-10-coded cause of death was analyzed using data of exposed population. Positive trend between effective dose and mortality from diseases of the circulatory system and ischemic heart diseases was suggested by the statistical analyses. (This study is a part of projects supported by Special Accounts from MEXT, Japan.)

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The Atomic Bomb Survivor Database at Nagasaki University has been storing medical information on Nagasaki atomic bomb survivors. As of March 2009, the mean age of atomic bomb survivors was 75.4 years old (73.2 years old in males and 76.7 years old in females). We assessed health conditions of atomic bomb survivors using trends in their mortality by the distance from hypocenter.
As of April 1, 1970, the number of atomic bomb survivors registered was 76,806, among whom 65,273 were directly exposed. The number of deaths observed during 1970-2004 in directly exposed survivors was 26,159. Death from cancer was observed in 6,744, cerebrovascular diseases in 4,351, and heart diseases in 4,400. We divided observation period into 7 periods: 1970-74, 1975-79, 1980-84, 1985-89, 1990-94, 1995-99 and 2000-04. We also categorized the distance from hypocenter as 0-1.9 km, 2.0-2.9 km, and 3.0 km or over. We calculated age-adjusted mortality rates using Japanese standard population constructed from 1985 national census.
The mortality from all causes showed a tendency to decrease with year in both sexes for all distance groups; in males, the mortality after 1980 showed a tendency to be slightly higher in those exposed within 1.9 km compared to the other groups, and in females, similar tendency was observed during 1970-79 and after 1990.
The mortality from cancer showed in males a tendency to be higher after 1985 in those exposed within 1.9 km compared to the other groups, while in females, although higher until 1979 in those exposed within 1.9 km compared to other groups, no difference among distance groups was observed after 1979.

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Recently it has been revealed by the large scale pool analyses of lung cancer case-controls studies that a level of 100 Bq/m3 in door radon did elevate a risk for lung cancer significantly. The levels of in door radon in Japan were estimated to be below the half level of world mean. However, there is a concern that the levels might elevate along with the increase of energy-saving houses with higher air tightness and lower ventilation rate. In the study, (1) we will measure indoor radon at 3900 homes in Japan in order to obtain the population-weighted mean of in door radon in Japan, and (2) we will estimate the attributable fraction of radon for lung cancer among smokers and non-smokers in Japan utilizing a mathematic model by EPA.
A passive radon-thoron discriminative detector (RadoSys) was set at either living or bed room for 6 months. In the present report, we will discuss about 2800 measurements done from Oct. 2007 ~Aug. 2009. At present, we have 2122 measurements covering 45 prefectures excluding Hiroshima and Nara. The distribution of indoor radon was compatible with log-normal distribution and those distributions in spring-summer and autumn-winter seasons could be adjusted by multiplying correction factors. Here we report data of 2122 houses: arithmetic mean (after correction) and SD were 15.2± 17.4 Bq/m³, geometric mean and geometric SD were 11.5 ×÷ 2.0 Bq/m³, lowest and highest values were 0.1 and 332 Bq/m³, respectively. Judging from mean and SD of log-normal transformed data, the probability of in door radon more than 100 Bq/m³ is about 0.1%, but in high radon areas such as Iwate and Okinawa, such a probability will be several percent. In these areas more intensive survey must be conducted.

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The Life Span Study (LSS) of A-bomb survivors has provided the important source of risk estimates of radiation exposure in humans, and played a valuable role in establishing radiation protection system.
Since it is known that the presence of random errors in the individual radiation dose estimates for the A-bomb survivors causes underestimation of radiation effects in dose-response analyses, the method which adjusts measurement errors in dose estimates, known as regression calibration (RC), is applied in most of recent analyses using LSS data. The main reason for applying RC method is its wide applicability, and this applicability enables a great many studies using LSS data to conduct analyses in the same way.
Though it is expected that the RC method removes almost of bias in risk estimates caused by measurement errors in the individual radiation dose, the performance of RC method in LSS settings has not evaluated enough.
Therefore, we conducted a simulation study to quantify the performance of RC method. We assumed that there were two types of measurement errors inherent in the radiation dose estimates, which were classical and Berkson errors. We generated the data in a similar setting to the LSS data, and assumed Poisson regression models, and analyzed in three ways, using no adjusting method, using RC method, and using true covariate.

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There is an increasing concern on effects of radiation and/or radionuclides on non-human biota. In order to elucidate the radiation effects on the non-human biota and the environment, it is essential to develop a new method to assess and estimate the radiation dose. However, the intake and metabolism of radionuclides in the body of non-human biota is little known. In this study, we describe a new method to predict the endodermal transfer (percutaneous uptake) ratios of radionuclides with modification of filter paper tests on the Acute Toxicity Tests according to OECD/NEA.
To investigate the percutaneous uptake of three radionuclides, ¹⁰⁹Cd, ¹³⁴Cs and ⁶⁰Co in earthworms (Eisenia fetida, hereafter earthworm), they were exposed to the radionuclide solution soaked in filter paper.Filter paper exposure used 50 mL plastic vials lined with Advantec No.1 filter paper contaminated with 1 mL solutions of ¹⁰⁹Cd or mixture of ¹³⁴Cs and ⁶⁰Co for individual worm exposure.Each vial was kept horizontal at 20 ^oC under darkness for 3 days, and the radioactivities in the worms were measured every day using a gamma spectrometer with a high purity germanium detector. Prior to the measurement, individual earthworm was washed and blot-drying to remove the radionuclides on the surface of the body.
Percutaneous uptake ratios (the activities in an earthworm to the activities added to a vial) of every radionuclide were increased with time; that of ¹³⁴Cs was the greatest, 10%, compared with those of ¹⁰⁹Cd and ⁶⁰Co, which were approximately 5%. The mode of uptake ratios with time was different between ¹³⁴Cs and the others; the uptake mode of the former had approached almost plateau at the end of the tests, and those of the latter were linear. The radioactivities in the water and paper used for removing the radionuclides from the surface of earthworms' body were less than 10% of those in earthworms.
In general, we concluded that this new method presented here is a useful tool to estimate the uptake of radionuclides in earthworm easily. The method showed that ¹³⁴Cs was absorbed into earthworms rapidly compared with ¹⁰⁹Cd and ⁶⁰Co.

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Cardiovascular diseases are the most common birth defects and adult diseases requiring medical and therapeutic intervention. In order to collect basic data for prevention of, as well as safety and therapeutic applications in, diseases related to environmental stresses, the biologically unique effects of chemical agents and radiation exposure need to be evaluated. The focus of this study is the abnormal development of the arterial pole of the heart and pharyngeal arch following maternal stress exposure to a chemical and ionizing radiation.
Pregnant rodents were given either chemicals or ionizing radiation, and we report on the relationships between each exposure group and subsequently observed embryonic lethality as well as external and visceral defects, especially craniofacial and cardiovascular, on day 18 of gestation. We observed a high incidence of teratogenesis, especially cardiovascular, pharyngeal and facial anomalies, in both treated groups. These results indicate sensitivity to chemicals and ionizing radiation exposures in fetuses leading to DNA damage, embryonic lethality, dysfunction of neural crest cells and pharynx development, which form neurocristopathy syndrome and neurocristopathy-induced cardiovascular and pharyngeal syndrome, respectively. The most common cardiovascular defects involved septation and alignment of the arterial pole of the heart. Particularly, craniofacial and cardiac outflow tract defects, including tetralogy of Fallot, atrioventricular septal defects and aortic arch anomalies noted following exposure, were quite similar to those found in studies of avian neural crest ablation model and to human syndromes.
We have previously studied teratogenesis and cardiovascular anomalies following similar maternal environmental stress exposure. It has shown that cardiovascular system diseases and tumorigenesis are some of the most common causes of sufferings in Hiroshima atomic bomb survivors and in their ultimate mortality. Studies were done on the role of neural crest cells in the structural development of the arterial pole by using several different animal models of human heart defects. Affected neural crest and neural crest cells that migrate to the pharynx and cardiovascular system and interfere with various developmental processes in the heart and pharynx are essential for abnormal craniofacial and cardiovascular development, as such cells disrupt signaling gene and transcription factors in the pharynx and cardiovascular system. This animal model is expected to contribute in investigating the mechanism of such dysfunction in neural crest and heart field in human syndromes and diseases, as well as their relative biological effectiveness.

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It has been suggested that the susceptibility of DNA damage to repair strongly depends on the track structure of the radiation represented by a linear-energy transfer (LET) value. In order to clarify the characteristics of complex DNA damage induced by high LET radiation, the yields of single- and double-strand breaks (SSB and DSB), base lesions and clustered damage induced in closed-circular plasmid DNA (pUC18) were measured after exposing to various kinds of radiation (He, Ne and C ions; 2 to 900 keV/μm) obtained from JAEA-TIARA and NIRS-HIMAC. In order to focus on the effect of direct energy deposition from radiation track, we prepared hydrated DNA (35 water molecules per nucleotide). Base excision repair enzymes, EndoIII and Fpg, were used to detect oxidative base lesions. The obtained results show that 1) the yield of prompt SSBs did not depend significantly on the LET of the helium ions, 2) whereas the yield of DSBs increased with increasing LET, 3) The yields of isolated nucleobase lesions and clustered damage sites visualized by additionally induced DSB by enzymatic treatment decreased drastically with increasing LET, and 4) very few enzyme-sensitive sites were induced above 100 keV/μm. 5) C and Ne ions induce less base lesions than He ions when compared in the same LET region. These results indicate that the yield of cluster of nucleobase lesions, which are less readily processed by the base excision repair proteins, depends not only on LET but also ion species irradiated.

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DNA double strand breaks (DSB) is thought to be the most critical damage for cell death. However, a considerable discrepancy is often observed between LET dependence of the frequency of cell death and that of the DSB yield. Also, several studies on the DSB yields as a function of LET have reported different tendency in the DSB-LET relationships. To obtain insight of the relationship between DSB and cell death, it is necessary to clarify the actual LET dependence of DSB.
In this study, event-by-event Monte Carlo track structure simulation technique was used to evaluate the LET-DSB relationship. Both of direct and indirect actions to DNA molecules can be considered in the simulation of strand break induction by irradiation of several types of ions with wide energy range. The strand breaks in diluted solution, cell mimetic condition and humid sample mimetic condition were calculated. The simulation results show the DSB yields increase with the increase of LET for all tested conditions. This result supports the idea that the increase of energy deposition density induces the increase of biological effect as a result of the increase of dense and severity of DNA damage. Although, our result is not consistent with some recent studies reporting the decrease or consistency of DSB yields with the increase of LET. To explain the cause of such discrepancy, some points in experimental detection system of DSB and the problems in calculation of absorbed dose (Gy) for particle beams will be discussed.

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DNA double-strand breaks (DSBs) represent the most serious threat among the cellular effects of ionizing radiation. XRCC4 and Artemis play key roles in non-homologous end-joining (NHEJ), the predominant repair pathway of DSBs, in multicellular eukaryotes. XRCC4, a component of DNA ligase IV complex, is an essential factor for activation and stabilization of the ligase itself in the process of NHEJ. On the other hand, Artemis may not be required for whole NHEJ system, but rather functions in processing of a subset of complex DNA ends prior to ligation. To elucidate roles of these proteins on risks related to radiation-induced events in human, we established XRCC4 and Artemis gene deficient cells in human colon carcinoma cell line HCT116 by gene targeting. In this current study, we compared sensitivities of the mutant human cells, XRCC4^-/- and Artemis^-/-, to various DNA damaging agents by clonogenic survival assay. XRCC4^-/- cells exhibited serious sensitivities to X-rays, etoposide and 5-fluorodeoxyuridine, and modest sensitivities to camptothecin, methyl methanesulfonate, cisplatin, mitomycin C, hydroxyurea and aphidicolin as compared to parental HCT116 cells. Consistent with the above-mentioned functions of these proteins in NHEJ, sensitivities of Artemis^-/- cells to most of those DNA damaging agents were equivalent to or lower than those of XRCC4^-/- cells. Nevertheless, Artemis^-/- cells were more sensitive to DNA cross-linking agents, mitomycin C or cisplatin, than XRCC4^-/- cells. By contrast, Artemis^-/- cells were significantly resistant to hydroxyurea than parental cells. These results suggest that Artemis also function in some DNA damage response pathways other than NHEJ.

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Ionizing radiation (IR) induces chromosomal double-strand breaks (DSBs) repaired by two pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), in mammalian cells. Cutting sections of DSBs induced by IR show structurally complex, not easy to connect by DNA ligase. Recently, we reported that repair of DSB introduced connectable I-SceI site, which is not real DSB induced by IR. Therefore, this connectable I-SceI repair model was not appropriate for real DSB repair. In this study, to clarify the relative contribution of each repair pathway for such complex DSBs, we developed a system to investigate the DSBs that occur in an endogenous single-copy human gene in human lymphoblastoid TK6 cells. Two DSBs, inversely arranged I-SceI restriction sites, were introduced at the either side of exon 5 of the TK gene (chromosome 17), and we named this cell line as TSCE206. We assessed for DSB repair, un-connectable I-SceI site repair, by transfection with the I-SceI expression vector in TSCE206. It was shown that the mutation frequency of TSCE206 expressed I-SceI was about 2-fold higher than TSCE105, which was introduced connectable I-SceI site. Molecular analysis showed the deletion size of TSCE206 mutants was larger than TSCE105 mutants. Furthermore, the mutants repaired by HR, which was rarely type in TSCE105, were observed in TSCE206. Thus, the repair pathway by NHEJ required making of large deletion to connect un-connectable DSB, which was radiation-like-double strand break. Additionally, we suggested that HR repair pathway might function in the situation, which was not easy to repair by NHEJ.

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We have previously shown that a radiation sensitive medaka mutant ric1 strain has the defects in repair of DSBs, induction of apoptosis and regulation of cell cycle after γ-rays irradiation [Aizawa et al., 2004; Hidaka et al., The 49th Annual Meeting of JRRS], suggesting that the responsible gene, ric1, is involved with the initial steps of DSB repair. It is well known that DSBs are repaired either by homologous recombination (HR) or by non-homologous end-joining (NHEJ) mechanisms. Then we elucidated each repair ability of ric1 cell lines by using HR assay and EJ assay, which can detect HR or NHEJ repair frequency by using reporter constructs and a rare-cutting I-SceI endnuclease. DR-GFP construct for HR assay and End-Joining construct for EJ assay, each containing one or two I-SceI site, express green fluorescent protein when the each construct repair by HR or NHEJ after DNA digestion by I-SceI. We found that the HR repair frequency was significantly lower in ric1 cells than in wild-type cells. We are currently analyzing NHEJ repair frequency, effect of ATM inhibitor and the change in quantity of gamma-H2AX after gamma-rays irradiation. These results will reveal ric1 function. The data will also be presented in the meeting.

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It is well known that high LET heavy particle radiation produces complex DNA lesions which are difficult to repair. Although many researches focused on complex DNA double strand breaks, our study presents complex DNA single strand breaks and base damages and homologous recombination repair. When cells were irradiated after G1-synchronization, heavy ions more efficiently inactivate cells and produce chromatid aberrations than x-ray does. In homologous recombination deficient cells, we observed significantly higher chromatid type aberrations after high LET exposure. This results suggested that DNA lesions formed in G1-phase were converted into DNA double strand breaks during DNA replication and they are complex single strand breaks or base damages produced by high LET radiation.

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Clustered DNA damage, defined as two or more lesions within one to two helical turns of DNA by a single radiation track, is a unique feature of ionizing radiation. We are currently focusing on the biological effects of non-dsb type of clustered damage in vivo, whose effect has remained largely unknown. We have chosen an experimental approach that utilizes synthetic DNA damage as a model of radiation induced clustered damage. One of the advantages of this strategy is that the effect of clustered damage with defined configurations could be examined in detail. Using a bacterial plasmid-based assay, we have investigated the biological consequences of bistranded clustered damage sites which are comprised of combinations of base lesions, apurinic/apyrimidinic (AP) sites, and single strand breaks (SSBs). Plamids were ligated with oligonucleotides with clustered lesions. Following transformation of the ligated plasmids into wild type strain of Escherichia coli, clustered damage-induced mutations were characterized. Although the transformation efficiencies were low, the major types of mutation induced by the AP + AP or AP + SSB cluster were G insertion opposite AP and a 1-bp deletion at the base pair with AP. On the other hand, mutations were occasionally found at base pairs where lesions had not been placed with clusters such as dihydrothymine (DHT) + 7,8-dihydro-8-oxoguanine (8-oxoG), and DHT + SSB. From these results, we suggest that, in a bistranded base cluster, 1) either base lesion is remained unrepaired and 2) some mutations arise not at but very close to the base pair where base lesions were originally placed.

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ATM-dependent foci formation of checkpoint factors is important for the induction of DNA damage response. Previously, we developed the index to quantify the signals sufficient for initiating the G2/M checkpoint, which is the Sum Of Integrated Density (SOID). The SOID is calculated based on the intensity of the fluorescence for histone H2AX phosphorylation. It integrates foci number, foci area and density, reflecting the molecular numbers of checkpoint factors. In the present study, we determined the SOID value in cells that overcame the G2 checkpoint, and compared the SOID value in those, in which G2/M checkpoint was inactivated by ATM inhibitor KU55933 (KU). We also analyzed chromosomal aberrations to estimate the type of damage which was detected as phosphorylated H2AX foci.
Normal human diploid cells were exposed to low dose of X-irradiation. We found that mitotic cells disappeared two hours after 0.4 Gy of X-rays, whereas no such G2 arrest was observed with 0.1 Gy. Analysis of phosphorylated H2AX foci in the G1 cells, which progressed after irradiation of 0.4 Gy, showed SOID of less than 5000. We also examined chromosomal aberrations in 0.4 Gy-irradiated cells. The average number of chromosomal aberrations in irradiated cells was 2, but it increased to 5 with KU treatment. Most of the chromosomal aberrations were gaps and breaks.
These results indicated that there is a threshold in the DNA damage signal essential for the execution of G2/M checkpoint. For 0.4 Gy of X-rays, it is about 5000 SOID, which corresponds to 3~4 of chromosomal aberrations.

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Anticancer agents induce DNA lesions such as DNA-protein crosslinks (DPCs), interstrand and intrastrand crosslinks (ICLs), double strand breaks (DSBs), and base modifications to various degrees. DPCs, ICLs, and DSBs block DNA replication and hence exert strong lethal effects on cells. However, it has not been fully elucidated to what extent individual DNA lesions contribute to cell death upon treatment with anticancer agents. To clarify this, we analyzed the relationship between the cell-killing efficacies of anticancer agents and the yields of DPCs, ICLs, and DSBs formed in treated cells. HeLa cells were incubated with different types of anticancer agents including alkylating agents [mitomycin C (MMC) and melphalan (L-PAM)], platinum compounds [cisplatin (cis-Pt) and oxaliplatin (L-OHP)], a DNA methylase inhibitor [5-aza-2'-cytidine (azadC)], and topoisomerase inhibitors [camptothecin (CPT) and etoposide (VP-16)]. The lethal effects of anticancer agents were analyzed by colony formation. The doses that gave 10% cell survival were L-PAM (4.23 μM), L-OHP (1.5 μM), azadC (1.35 μM), cis-Pt (0.68 μM), VP-16 (0.43 μM), MMC (0.025 μM), and CPT (0.0076 μM). To quantify DNA damage, cells were treated with these doses of anticancer agents, and genomic DNA was isolated and purified by CsCl density gradient ultracentrifugation. We established a Western blotting method for highly sensitive detection of DPCs and are currently analyzing DPCs. We will also quantify ICLs and DSBs produced by individual anticancer agents. The results will be presented in the meeting.

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Cells from patient with Ataxia Telangiectasia are highly sensitive to ionizing radiation. This enhanced radiosensitivity attributes to the defective repair of double-strand DNA breaks (DNAdsb) or the misrepair. DNAdsb are repaired by multiple pathways including homologous recombinatioal repair, Ku-mediated nonhomologous end joining and 53BP1-mediate d nonhomologous end joining. Although ATM is generally considered to contribute to homologous recombination, there are contradictory reports if ATM is involved in nonhomologous end joining or ATM does not contribute to homologous recombination of I-SceI induced DNAdsb. Thus, association of ATM with DNA repair pathways still remains unclear. In this study using a selective ATM inhibitor and RNAi knockdown of 53BP1 in Ku70 defective cells, we examined the expression of repair proteins and the phosphorylation by western blot.
Intriguingly, 53BP1 knockdown cells showed the higher clonogenic survival than that used to be in the parental Ku70 defective cells, presumably due to the increased homologous recombination. The increased radiosensitivity was suppressed after treatment with ATM inhibitors, perhaps due to the decreased DNA repair capacity. We are currently investigating the roles of ATM expression in this enhanced cell survivals.

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Clustered DNA damage is defined as two or more lesions generated within 1-2 helical turns (10-20bp) of DNA. Ionizing radiation has a characteristic to deposit energy to local area on DNA and to induce clustered DNA damage. Theoretical studies and experimental data suggest an increased complexity and severity of clustered DNA damage with increasing LET of ionizing radiation. It is proposed that ionizing radiation produces clustered DNA damage including single strand break (SSB) and double strand break (DSB) with several oxidative base lesions. It is postulated that clustered DNA damage is incompletely or incorrectly repaired, and is strongly related to biological effects, such as cell death and mutation.
We measured mutation frequencies induced by two types of clustered damage carrying a SSB and an 8-oxo-7,8-dihydroguanine (8-oxoG) each on the complementary strands or both on the same DNA strand in E. coli. We found that the clustered damage with 8-oxoG and SSB each placed on complementary strands had a higher mutation frequency than single 8-oxoG, while the clustered damage with 8-oxoG and SSB positioned on the same strand had a slightly lower mutation frequency than single 8-oxoG either in wild type or in glycosylase (Fpg and/or MutY) deficient mutants. These results suggest that SSB opposed to 8-oxoG retards the excision of 8-oxoG by formamidopyrimidine DNA glycosylase (Fpg). However, processing of the tandem lesion of SSB and 8-oxoG remains to be elucidated. In this study, using in vitro enzyme assay, we investigated the processing of clustered damage containing 8-oxoG and SSB on the same DNA strand.

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Purpose: XRCC4, in association with DNA ligase IV and XLF, is necessary for the ligation of the two DNA ends, as the final step of DNA double-strand break repair through non-homologous end-joining. It is shown that XRCC4 is phosphorylated in vitro and in vivo by DNA-PK, which is considered the molecular sensor of DNA double-strand breaks. We have so far identified four phosphorylation sites, in addition to two identified by others. Among four phosphorylation sites, two were phosphorylated in cellulo, i.e., in murine leukemia M10-derived XRCC4 expressing cell lines. On the other hand, we have been unable to detect the phosphorylation of other two sites. Considering a possible difference in the manner of phosphorylation between human and rodent cells, we examined phosphorylation status of XRCC4 in human cells.
Methods: We collected XRCC4 protein from 0.5L culture of human leukemia MOLT-4 cells, either left unirradiated or harvested 30min after 20Gy ⁶⁰Co gamma-irradiation), by immunoaffinity column chromatography. The phosphorylation status was analyzed by Western blotting using phosphorylation-specific antibodies corresponding to respective sites.
Results and discussion: We could detect the phosphorylation of all of four phosphorylation sites. The phosphorylation was enhanced after irradiation. This observation indicated that the manner of phosphorylation of XRCC4 in response to DNA damage is considerably different between human and murine cell lines. We will also report a new, rapid assay system to evaluate XRCC4 function in terms of its ability to sustain proliferative capacity after irradiation.

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