The major risks of radiation exposure have been shown experimentally with mice to approach zero risk with decreasing low dose-rate irradiation. This demonstrates the critical contribution of DNA repair. However, DNA repair is not perfect. There must be defense mechanisms other than DNA repair. In order to investigate the in vivo role of p53 in surveillance against mutation, and particularly to address the effect of dose rate and the significance of p53-dependent apoptosis, we have used wild-type p53(+/+) and null p53(-/-) mice to determine the mutant frequency at the TCR locus following exposure to gamma-rays. After acute gamma-irradiation with 3 Gy (@ 61 Gy/h), the TCR mutation frequency was higher than the control level. However, when an equal dose of 3 Gy was given but at a lower dose-rate of 0.07 Gy/h, the frequency of TCR mutants did not increase at all for p53(+/+) mice, whereas the same dose of 3 Gy remained mutagenic for p53(-/-) mice, which are unable to carry out p53-dependent apoptosis. This can explain why there is no detectable increase in mutations in p53(+/+) mice after protected irradiation with 3 Gy because, if a fraction of apoptosis-sensitive cells retain unrepaired DNA damage after gamma-irradiation, almost all of them deleted by p53-dependent apoptosis. These results indicate that the p53 gene is indispensable for a threshold effect in the risk of radiation at low doses or dose-rates.

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Radiation therapy for thyroid papillary cancer (PTC) rarely induces anaplastic transformation, which shows aggressive behavior. Ret gene rearrangement is a well known molecular alteration observed in PTC and associated with thyroid tumorigenesis. Segmental jumping translocation (SJT) is defined as unbalanced translocations involving a donor chromosome arm or chromosome segment fused to several different recipient chromosomes, suggesting chromosomal instability, and mainly reported in treatment-related leukemia characterized by multiple copies of the ABL oncogenes. While, only a few studies have reported the SJT in solid cancer. In this study, we found Ret SJT in anaplastic thyroid cancer (ATC) induced after radiation therapy for PTC by fluorescence in situ hybridization (FISH). Formalin-fixed paraffin-embedded thyroid tissues from a 44-yo male was used for analyses. He was operated under the diagnosis of PTC and treated by internal radiation with 131I. The tumor recurred at 57-yo and histologically diagnosed as ATC. The FISH analysis demonstrated nuclei showing several Ret signals and two chromosome 10 signals in ATC but not in PTC, suggesting SJT of Ret gene in ATC. The frequency of SJT-positive cells was approximately 30%. PTC showed a wild type Ret signals and neither Ret/PTC1 nor PTC3 by RT-PCR. SJT of Ret may be associated with anaplastic transformation of thyroid cancer after radiation therapy, indicating chromosomal instability.

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Somatic rearrangements of the tyrosine kinase receptor RET have known to involve radiation-associated papillary thyroid carcinoma. Major types of rearrangements are RET/PTC 1, 2 and 3 that is more than 90% of PTC with RET rearrangements. We investigated the induction of RET rearrangements in human normal thyroid cell by gamma radiation. Human normal thyroid cells were exposed 0, 1, 3, 5, and 10Gy gamma radiation and harvested 2 to 9 days after irradiation. The induction of RET/PTC1, 2 and 3 were analyzed by reverse transcription-polymerase chain reaction and sequencing. No RET/PTC induction observed in cells harvested 2days. RET/PTC1 and RET/PTC3 detected 5 to 9 days after irradiation. RET/PTC1 was more prevalent than RET/PTC3 after each dose and RET/PTC1 was generated dose-dependently by gamma radiation. But, RET/PTC2 was not generated. This study demonstrated a dose-dependent induction of RET/PTC1 in human normal thyroid cells by gamma radiation. Theses results suggested an immediate association between RET rearrangements and gamma radiation exposure in human normal thyroid cells.

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Thyroid gland is an organ particularly vulnerable to radiation as known from previous studies that investigated relationship between thyroid neoplastic disorders and radiation exposure in man. In particular, there is an evidence of increased risk of papillary thyroid cancer (PTC) in the individuals exposed to ionizing radiation especially if exposure had taken place at the young age. In spite of extensive investigations, specific molecular events underlying radiation-related carcinogenesis are not fully understood. Mutation patterns of the oncogenes implicated in thyroid carcinogenesis (ret/PTC and TRK rearrangements, BRAF, RAS, Gsa) or TP53 tumor suppressor gene have not proved a distinctive molecular signature that may unambiguously be associated with radiation-induced thyroid tumors.An alternative approach to elucidation of the basics of thyroid cancer development in radiation victims may be the identification of individual genetic characteristics of the patients with radiation-associated PTCs. In our work, we have profiled a set of SNPs in several genes whose products are involved in the DNA damage pathway, TP53, ATM and HDM2. As a result, several SNPs displaying a significant association with radiation-related or sporadic adult or childhood PTC have been detected. These findings demonstrate that molecular epidemiology studies hold a promise in the problem of detection of the genetic traits potentially contributing to or modifying the susceptibility to radiation-induced thyroid carcinogenesis in humans.

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We previously reported that the relative risk of MDS among Hiroshima A bomb survivors was 2.53 at 1 Sv between 1985 and 1999. In order to clarify the molecular mechanism of MDS genesis including radiation-associated MDS, we studied the point mutations of AML1/RUNX1. AML1 point mutations were frequently detected in both sporadic and secondary MDS/AML(RAEB, RAEBt and MDS derived AML), while rarely detected in RA and RARS. The frequency was 17% (15/88) in sporadic MDS/AML and 50% (11/22) in secondary MDS/AML. Each 7 and 8 of 15 mutations in sporadic MDS/AML were localized in N-terminal (including Runt homology domain) and C-terminal region of AML1, respectively, whereas 10 of the 11 mutations in secondary MDS/AML were in N-terminal region. The majority of the AML1 mutants lost the trans-activation potential. In AML1-mutated MDS/AML cases, cytogenetically normal karyotype, -7/7q-, +8 and -5/5q- were observed in 42%, 29%, 23% and 0%, respectively. The additional mutations were detected in 38 % of these cases, including RAS pathway genes, c-kit, FLT-3 and p53. These results suggested that AML1 point mutations, in cooperation with additional genetic changes, play a major role in the development of MDS/AML, especially in secondary MDS/AML.

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PCS syndrome is the human mitotic-spindle checkpoint disorder, characterized by mosaic variegated aneuploidy (MVA) and premature chromatid separation (PCS). The clinical manifestations include severe microcephaly, Dandy-Walker anomaly, and development of Wilms tumor.Recently, Rahman et al. reported that MVA syndrome is caused by biallelic mutations in BUB1B. In this study, we describe BUB1B mutation in seven patients with PCS syndrome.We identified monoallelic BUB1B mutations in all seven patients; a splice site mutation, a single base deletion, a missense mutation, and a nonsense mutations in one family each. No mutation was found in the second alleles in the seven families, but western blot and haprotype analysis of BubR1 indicated a modest decrease of their transcripts. These results suggest that the patients were compound heterozygotes with the null mutation and the hypomorphic mutation of the BUB1B gene, and that >50% decrease in BubR1 expression is involved in the PCS syndrome.

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Purpose: To identify haplotypes that associated with the risk of adverse skin reactions following radiotherapy in a Japanese breast cancer population, we conducted a case-control association analysis and haplotype inference using unphased genotype data. Methods: Using DNA samples collected from 184 Japanese breast cancer patients who qualified for breast-conserving surgery and radiotherapy, we genotyped 905 SNPs from 127 candidate genes for radiation susceptibility. The adverse skin reactions were clinically graded according to the National Cancer Institute Common Toxicity Criteria. We first conducted a case-control analysis exploring the associations between all SNPs and adverse skin reactions by using logistic regression analysis. Second, Linkage disequilibrium mapping and haplotype-tagging SNPs selection were performed. Finally, haplotype frequencies of each selected locus were inferred. Genetic effects of the inferred haplotypes were analyzed in the same statistical way for the SNPs. Results: Twelve candidate loci involving 24 SNPs showed statistically significant association with the risk of adverse skin reactions. Six of 12 loci generated 42 haplotypes and 4 haplotypes of them associated with the risk of adverse skin reactions. This haplotype-based association analysis revealed that there were the adverse skin reaction-risk or -protective haplotypes. Conclusion: These findings suggested that genetic background is one of the factors that associated with the risk for adverse skin reactions following radiotherapy.

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Bistranded clustered damage generated by ionizing radiation (IR), which contains two or more closely opposed base lesions and/or single-strand breaks (SSBs), is assumed to result in error-prone repair or double-strand breaks (DSBs), and hence leads to adverse biological effects. Although the formation and repair mechanisms of direct DSBs by IR, a special form of clustered damage, have been studied extensively, those of clustered base damage (CBD) containing base lesions or base lesions+SSB are less well understood. In the present study, we have quantified the yield of CBD and assessed the repair efficiency of CBD by base excision repair enzyme.
pDEL19 plasmid DNA was irradiated in aqueous solution by gamma-rays, carbon and iron ion beams, treated with Endo III or Fpg, and subjected to agarose gel electrophoresis. The yields of isolated (base damage and SSBs) and clustered (DSBs and CBD) damage were determined from conformational changes of plasmid DNA. The fraction of clustered damage was 2-3% of total damage and independent of the type of radiation. The ratio of DSBs and CBD was 1:1, indicating that CBD accounts for half of clustered damage.
hOGG1 is a major human DNA glycosylase for oxidative purine lesions generated by IR and oxidizing agents. The damage-excising activity of hOGG1 for CBD containing two closely opposed 8-oxoG lesions was analyzed. hOGG1 excised 8-oxoG and induced DSBs even if two 8-oxoG lesions are less than 5 nucleotides apart. However, the efficiency of DSB formation varied dramatically depending on the configuration of 8-oxoG lesions.

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The high biological effectiveness of accelerated high LET heavy ions could be caused predominantly by the induction of complex clustered DNA damages (CCDs), leading to non-repairable DNA double-strand breaks (DSBs). NBS1 is a causative gene for Nijmegen breakage syndrome (NBS), and is essential for DSB repair by homologous recombination (HR) and cell cycle checkpoint. We have shown that NBS1 recognizes the induction of CCDs, but cannot restore cell survival and abnormal G2 checkpoint after exposure to high LET heavy ions. NBS1 recruited to the vicinity of DSBs induced along the track of heavy ions and formed indissoluble large foci through the dynamic movement in the cell nucleus even 16 h after exposure. On the other hand, X-ray-induced discrete NBS1 foci were almost dispersed within 8 h after irradiation. Abnormal G2 checkpoint and failed Chk2 phosphorylation were observed in the cells derived from NBS patients after both X-ray and heavy ion irradiation. In NBS cells expressing full length NBS1, these defects could be complemented after X-irradiation but not after exposure to heavy ions. In addition, sensitivity to X-rays of NBS cells was restored in NBS cells expressing NBS1. However, the sensitivity to heavy ions at high LET of NBS cell expressing NBS1 was not significantly different from that of NBS cells. These results suggest that heavy ion-induced CCDs are recognized by NBS1 but are difficult to repair by HR. NBS1 regulates G2 checkpoint through Chk2 phosphorylation to avoid abnormal cell cycle progression in response to the severity of DNA damages.

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To investigate a role of 53BP1 in repair of DNA double-strand breaks(DSBs) in vertebrate cells, we established, using hyperrecombinogenic chicken B cell line DT40, a 53BP1 knockout cell line and double knockout cell lines deficient in 53BP1 and either Ku, DNA ligase IV (Lig IV), ATM or Artemis. 53BP1-/- cells showed intact intra-S and G2/M phase checkpoint for DNA DSBs. We carried out an epistasis analysis, using colony formation assay in G1 synchronized cells, and revealed that there are at least three sub-pathways in non-homologous end-joining (NHEJ), 1) the core NHEJ which is dependent on Ku/DNA-PK, 2) Artemis dependent pathway which includes ATM, and 3) 53BP1-dependent pathway. These three pathways lead to Lig IV in the final ligation step. ATM deficiency showed only a marginal effect on the colony formation, and Artemis was modulated by DNA-PK. In contrast to core NHEJ and Artemis dependent pathway, 53BP1 dependent pathway was resistant to PI-3 kinase inhibitor Wortmannin, and showed high contribution to DSB repair only in G1, but not in early S phase.

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It has been generally accepted that LDRE results from the SLD repair. As the dose rate is lowered and the treatment time protracted, more and more SLD can be repaired during the exposure. Recently we found that SLD recovery is due to DSB repair mediated by HR. To study the molecular mechanism of LDRE, we analyzed the knock-out mutants KU70^-/-, RAD54^-/-, and KU70^-/-/RAD54^-/- of the chicken B-cell line, DT40. Rad54 participates in the HR repair of DSBs in S and G2 phase, while Ku proteins are involved in NHEJ and work in whole cell cycle. Survival enhancement by LDR irradiation was observed in parent DT40 and RAD54^-/- cells but not in NHEJ deficient KU70^-/- and KU70^-/-/RAD54^-/- cells. In the LDRE, NHEJ pathway was more important than HR pathway. This suggests that LDRE are not directly attributable to the SLD repair because the SLD repair results from the HR pathway of DSBs in S and G2 phase. Under continuous LDR irradiation, dividing NHEJ-deficient cells will be irradiated and killed in G1 phase. NHEJ pathway plays an important role in LDRE. We studied further the effect of low dose-rate irradiation using the NHEJ deficient KU70^-/- and KU70^-/-/RAD54^-/- cells.

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Recent studies indicate that IR-induced nuclear foci of phosphorylated ATM (S1981), 53BP1, and NBS1 are involved in the induction of DNA-damage checkpoint. Although most of the initial foci disappear in a few hours after irradiation, there are foci remaining long after irradiation. To examine the relationship between residual foci and G1 arrest, we analysed dimensional dynamics of the remaining foci of phosphorylated ATM, 53BP1, and NBS1 proteins. Twenty-four hours after 1 Gy of X-rays, phosphorylated ATM foci were remained in 63% of irradiated normal human diploid cells, and 32% of the foci-positive cells have enlarged foci, whose sizes were more than 1 µm in diameter. When synchronized cells at G0 were irradiated with 1 Gy of X-rays, and were released, these enlarged foci were exclusively detected in cells that did not proceed to the S phase. Interestingly, enlarged foci of phosphorylated ATM showed 100% colocalization with large foci of 53BP1 and NBS1. Furthermore, cells with 53BP1/NBS1 large foci were not increased in 1 Gy-irradiated A-T cells (4.5% in control, 3.6% at 24h after IR), whereas they were significantly increased in 1 Gy-irradiated normal human cells (4.5% in control, 26% at 24h after IR). These results indicate that growing of 53BP1 and NBS1 foci size, which depends on enlarged phosphorylated ATM foci remained several hours after irradiation, might play a critical role in G1 checkpoint induction.

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Phosphorylation of p53 by ATM and ATR is important for its stabilization and activation after DNA damage. Caffeine is known to inhibit ATM and ATR kinase activities, whereas wortmannin represses that of ATM. Both reagents dramatically abolished the suppression of DNA synthesis in sperm-irradiated zygotes, whereas they had no effect on the unirradiated zygotes. These suggest that S checkpoint in mouse zygotes is mainly controlled by the ATM pathway. Microinjection p53 mutant, which lacks the DNA binding capacity, interfered with the suppression of DNA synthesis in sperm-irradiated zygotes. Inhibiting transcription with α-amanitin, an inhibitor of RNA polymerase II, had no effect on the suppression of DNA synthesis in sperm-irradiated mouse zygotes. The above data suggest that p53 suppresses DNA synthesis in a transactivation-independent manner. DNA binding of p53 might be important in S checkpoint of sperm-irradiated mouse zygotes.To investigate the role of p53 in sperm-irradiated mouse embryo, they were immunostained with Rad51 which is required for recombination repair. Rad51 was stained in mouse zygotes irradiated directly but not un-irradiated. Surprisingly, Rad 51 was also stained in male and female pronuclei of sperm-irradiated mouse zygotes. m5s cells were transiently transfected with GFP-p53 wt or GFP-p53 R273H. DNA binding domain of p53 associates with Rad 51 in vivo. These suggest that p53 could recruit Rad51 to replication fork for repair.

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p53 tumor suppressor has been documented to be involved in maintaining stability of the genome through either cell cycle arrest or apoptosis. Recently, the regulation of DNA repair by p53 tumor suppressor has been an emerging important topic in the field of radiation research and oncology, largely distinct and separable from more-studied cell cycle arrest and apoptosis response regulated by p53 (JBC 2005). The crucial role of p53 and its downstream gene Gadd45 in nucleotide excision repair (NER) of DNA damage induced by UV or industrial mutagens has been shown (MCB 2000), but much of its detailed mechanism remains to be elucidated. Moreover, p53 and its downstream gene DNA polymerase beta also participate in base excision repair (BER) of DNA damage induced by free radicals or alkylating mutagens (Cancer Research 2003, Oncogene 2002b), still at an early stage of investigation. Indeed, we demonstrated the relevance of redox factor (ref1) in the mechanism of p53 activation by the reduction of p53 protein under the treatment of selenomethionine (SeMet) (PNAS 2002, Oncogene 2002a). In addition, we found that SeMet also protects cells exposed to ionizing radiation (IR) as well as to UV. Furthermore, transcription activity of p53 was decreased in ref-1 mutant cells under SeMet, suggesting that the redox status of p53 might have protective effects against IR exposue. In conclusion, p53-mediated DNA repair under redox signaling modulation would be an emerging field in radiation research for the cancer prevention and therapy.

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While p53 is known to play a key role in apoptosis induced by ionizing radiation (IR), p53 mutation was reported in more than 50% of tested cancer cells. Many of these cancers, despite the loss of p53 function, are still responsive to radiotherapy, suggesting a p53-independent action of IR. To explore its underlying mechanism, human U937 leukemic cells which have a p53-null mutation were irradiated with -rays at 7 Gy. This treatment resulted in a cell death, which was accompanied by an elevation in cellular ROS levels. The cell death was consistently reversed by the exogenous application of catalase, suggesting that ROS act as death mediators. IR also induced the mitochondrial permeability transition (MPT), and inhibitors for either the MPT or mitochondrial respiratory chain attenuated the IR-induced ROS production. This suggests that the ROS were induced in mitochondria in a MPT-dependent manner. To determine a signaling component upstream of the mitochondrial events, this study focused on JNK because the lethal dose of IR efficiently activated JNK. It was also found that a JNK-specific inhibitor SP600125 suppresses the ability of IR to induce MPT, ROS, and cell death. Overall, the JNK-dependent mitochondrial ROS production appears to be critical for the p53-independent action of IR.

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We have analyzed delayed mutation as part of cellular response to genotoxic damage and found that this occurs at the pink-eyed unstable locus in the somatic cells of F1 mice born to irradiated sperm. However, a whole body mouse system and tissue culture cells are too complex to elucidate the molecular mechanism of delayed mutation.We have now examined delayed mutation in fission yeast. We have constructed three yeast strains which carry tandem duplications differing in length at the ura4 gene. These loci revert to the wild type (WT) through homologous recombination (HR). The frequency of spontaneous HR was linearly related to the length of the repeat. One of the strains carrying a 200 bp repeat unit was used and the frequency of the reversion was found to be dependent on the dose of X-ray to the cells (25-500 Gy). In addition, higher mutation frequencies persisted for 6-10 cell generations after irradiation where damage repair was thought to have been completed.These results suggest that untargeted mutation and delayed mutation is induced in fission yeast by damage to the genome.

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We devised a human HT1080 cell line carrying a tandem duplication in a part of HPRT gene (HPRT-dup cells) to address whether the repeat sequence instability (detected by reversion due to loss of the duplication by intrachromosomal recombination) is implicated in forward mutations at unique sequences in ordinary genes in radiation- induced genomic instability. Previously, we found that there were no absolute correlations in the instability between the reversion mutation from HPRT-dup to HPRT+ and the subsequent forward mutation from HPRT+ to HPRT-. In the present study, we found that many of the clones with the repeat-sequence instability showed 2-5 fold increases in production of reactive oxygen species (ROS) and substantial increase in the number of gamma-H2AX foci. Further, the repeat-sequence instability was not suppressed by fusion of the unstable cells with parental cells (instability-negative), indicating that the nature of the instability is genetically dominant. The results suggest that production of cytosolic diffusible factor(s) that causes persistent intracellular inflammation is responsible for the maintenance of the instability.

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Ionizing radiation induces DNA double-strand breaks. Recently, it was clarified that frequency of mutation and chromosomal abnormality are significantly elevated among progeny of surviving cells. It is thought that these delayed effects are caused by induction of genomic instability in surviving cells. However mechanisms of maintenance of genomic instability and expression of delayed effects are not made clear. From current results of our research, we expect that origin of genomic instability is a large deletion of genome. To clarify this speculation, we isolated HPRT mutated-clones from X-ray irradiated normal human diploid cells. We analyzed presence of each exson of HPRT gene and size of deletion by using STS markers. Then we analyzed relationship between mutation patterns of HPRT gene and frequency of chromosomal abnormality. These results suggest that delayed chromosomal abnormality is induced by a large deletion of more than 0.5Mb containing HPRT gene. As we expected it, delayed chromosomal abnormality originated from large deletion. In addition, we found that dicentric chromosome without fragment is dominant in delayed chromosomal abnormality. In this paper, we present the character of delayed chromosomal abnormality in survived-clones by using WCP FISH analysis.

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C3H/He mice develop acute myeloid leukemia (AML) after whole-body irradiation, and typical chromosome 2 deletions are found in the leukemic cells. Since the cells bearing AML-type deletions expand in the bone marrow around a year after irradiation, the delayed chromosomal aberrations could be responsible for their formation. To test this hypothesis, we have cytogenetically examined primitive hematopoietic cells of irradiated animals. Ten-week male C3H/He mice were exposed to 3 Gy x-rays and sacrificed after 1, 90 and 300 days after irradiation. Bone marrow cells collected from each animal were divided into two batches. One batch was cultured in methylcellulose medium, and metaphase spreads were prepared from each growing colony. The other batch was sorted to obtain Lin⁺ and Lin^-Sca1⁺ cells, which were analyzed with FISH for the AML-type deletions. Non-clonal aberrations in colonies in methylcellulose medium were mostly chromatid breaks, and there was no case of expansion to constitute a subpopulation in the colony. The cells from non-exposed mice also carried a significant number of non-clonal aberrations, which implies the assay itself has a perturbing effect. FISH analysis of the sorted cells detected AML-type deletions in Lin^-Sca1⁺ subpopulation at one day after irradiation, and the frequency of such cells did not decline after 90 days. While the experiments are still going on, there has been no supportive evidence for involvement of delayed chromosomal aberrations in radiation-induced AML in mice.

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We performed a study on Belarusian "liquidators" (clean-up workers), exploring whether increases in the frequencies of germline mutations at microsatellite loci could be found in their progeny. Many liquidators fathered children during the clean-up period and after the work had been terminated. The numbers of families studied were 64 (liquidators) and 66 (controls). A total of 72 loci (31 autosomal, 1 X- linked and 40 Y-linked) were used. DNA was isolated from peripheral blood lymphocytes and the microsatellite loci were amplified by the PCR. At the Y-linked loci, the mutation rates (number of mutations among the total number of loci for the individuals included) are 2.9x10^-3(4/1392) and 2.1x10^-3(3/1458) in the children of exposed and control parents, respectively. This difference is not statistically significant. At the autosomal loci, the corresponding estimates are 5.9 x 10^-3 (11/1862;exposed group) and 8.5x10^-3(18/2108;control). Again, the difference is not significant. The possibility that the Belarusian population might have been unexpectedly exposed to some chemical contaminants in the environment appears unlikely in view of the finding that the spontaneous mutation rates at the same set of loci in several non-Belarusian populations were similar to those in Belarus. The estimated mean radiation dose to the liquidators was small, being about 39 mSv, and this might be one reason why no increases in mutation rates due to radiation could be found.

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The major DNA damage induced by solar UV is cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP) formed between adjacent pyrimidines. These damage plays significant roles in cell killing, mutation and neoplastic transformation, and is repaired by nucleotide excision repair (NER), the only pathway in human. NER needs more than 30 different proteins for the process including damage recognition, removal of the oligonucleotides containing the damage, gap-filling DNA synthesis, and ligation. Defects in NER are associated with autosomal diseases, xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy (TTD). TTD is characterized by brittle hair with reduced sulfur content, ichthyosis, and impaired mental and physical development. The photosensitive form of TTD results from mutations in the same XPD gene as the cancer-prone XP group D (XP-D). Nevertheless, no increase in skin cancers appears in patients with TTD. In this study, we asked whether the cancer-free phenotype in TTD is related to its repair defect. We found that all three TTD cell strains are deficient in the repair of both CPD and 6-4PP. UV sensitivity correlated well with the severity of repair defects. Moreover, mutations of the XPD gene affected both the recruitment of the TFIIH complex to DNA damage sites and the TFIIH expression. The present results indicate that there is no major difference in the repair defect between TTD and XP-D and that the cancer-free phenotype is unrelated to a DNA repair defect.

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UV causes damages in cellular membrane and cytoplasm via generation of reactive oxygen species (ROS) as well as damages in DNA by forming photoproducts. Investigations in this decade clearly demonstrated that two distinct UV responsive intracellular signaling pathways are present, i.e., DNA damage-dependent and -independent pathways. Our previous study has shown that activation of JNK and apoptosis in UVC-irradiated cell population were reduced in hypoxic condition whereas activation of ERK was not affected. Instead, ERK activation was reduced by an inhibitor of receptor tyrosine kinases (RTK). Therefore, contribution of the balance between JNK and ERK, which was regulated by ROS and RTK, respectively, to cell death was suggested. The role of DNA damage-independent pathway is further relevant in cellular responses to UVB. When cells were irradiated with UVB at a dose that forms equivalent amount of DNA photoproducts by UVC, generation of ROS, cell death and mitochondrial cytochrome C release were higher in UVB-irradiated cells. Furthermore, phosphorylation of p53 at ser392 was markedly observed in UVB-irradiated cells that were inhibited by a p38 inhibitor and by an antioxidant. These results indicated the significance of ROS-p38 pathway in UVB irradiated cells, which may act as a protective mechanism against tumorigenesis by eliminating damaged cells. The combinations of acute stress responses as shown above and chronic responses, probably via production of soluble secondary factors, should be examined to better understand UV stress responses in our daily life.

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Ultraviolet light (UV) is categorized according to their wavelength ranges into UVC (<280 nm), UVB (280-320 nm), and UVA (320-400 nm). UVA is subcategorized into UVA1 (340-400 nm) and UVA2 (320-340 nm). Although UV is generally known to induce C -> T mutation at dipyrimidine sites in DNA, wavelength differences could modify the situation, especially for mammals. Vertebrate genome is known to be methylated globally at some specific cytosine residues, and this methylation can enhance formation of one of the UV-specific DNA damage, cyclobutane pyrimidine dimers (CPD) induced, not by UVC, but by UVB or solar UV (>290 nm), which results in hotspot mutations at dipyrimidine sites with a methylated cytosine in mammalian genes, evidenced by mutations in the p53 gene found in human skin cancer.
We found previously, using lacZ-transgenic mice, that mutation induction in UVB-exposed skin epidermis was suppressed at a higher dose range and saturated to a leveled mutant frequency (Ikehata and Ono, Mutat. Res., 508:41-47, 2002). This epidermal phenomenon, called mutation induction suppression (MIS), has a wavelength dependence. The leveled mutant frequency in MIS varies by UV wavelength.

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Chronic UV exposure induce various photocarcinogenesis. Thus far, studies on UV-induced carcinogenesis have focused on UVB-induced DNA mutations. Direct absorption of UVC or UVB leads to formation of cyclobutane-type pyrimidine dimers and pyrimidine pyrimidone (6-4) photoproducts at adjacent pyrimidines. Previous studies suggested that pyrimidine photoproducts are the major classes of UV-induced DNA lesions involved in the cytotoxicity and mutagenesis. Pyrimidine photoproducts induce preferentially transition-type base changes especially at dipyrimidine sites, sometimes called as UV-signature mutations. However, recent reports indicate the involvement of many other factors. Firstly, UVB induces not only pyrimidine photoproducts but also DNA lesions modified by Reactive oxygen species (ROS). Several reports pointed out that types of mutations that are not theoretically caused by pyrimidine photoproducts are frequently observed in the human skin cancers of sun-exposed areas and UVB-induced murine skin cancers. Secondly, recent studies have shown that not only UVB but also UVA is involved in UV-induced carcinogenesis based on animal experiments. UVA induce indirect DNA damage via ROS and lipid peroxidation. ROS have been associated not only with initiation, but promotion and progression in the multistage carcinogenesis model. Recently it has been reported that ogg1 knockout mice is susceptible to photocarcinogenesis. Thirdly, biological responses induced by UV such as inflammatory and immunological responses are also closely involved in photocarcinogenesis.

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Ultraviolet-B (UVB) radiation can damage plant, decreasing growth and productivity. In our over 10 year's field experiments in a northeast of Japan, elevated UVB radiation markedly influenced growth and grain development such as grain size, total nitrogen content, and grain of storage protein composition in rice. We found that the sensitivity to elevated UVB radiation in rice varies widely among cultivars. Such a difference in UVB sensitivity in rice was controlled by at least three major recessive genes. Our previous data also indicated that the photorepair ability of cyclobutane pyrimidine dimers (CPDs) are significantly higher in UV-resistant rice cultivars than in UV-sensitive cultivars, and that the difference in the CPD photorepair ability resulted from a structure/function alteration of photolyase due to the alteration of one or two amino acids in CPD photolyase. Furthermore, a linkage analysis in populations derived from UV-hypersensitive indica cultivar Surjamkhi and UV-resistant japonica cultivar Sasanishiki showed that UVB sensitivity is a quantitative inherited trait and that the CPD photolyase locus is tightly linked with a quantitative trait locus (QTL) that explains a major portion of the genetic variation for this trait. These results suggest that spontaneously occurring mutations in the CPD photolyase gene in rice cause different degrees of sensitivity to UVB. In this meeting, we discuss current status and perspective of UVB and plant research.

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Many recent studies have been conducted on UV-induced DNA damage and its repair in higher plants. DNA repair systems in plants are divided broadly into two categories, photoreactivation catalyzed by two kinds of photolyase activities and light-independent repair pathways such as excision repair system and other means. In addition, there are mechanisms to protect against DNA damage by means of excluding UV-B by leaf waxes and absorbing UV-B by accumulating phenylpropanoid compounds in epidermal and subepidermal cells. However, the whole picture of UV-B resistance in a plant is hardly drawn. For the understanding, induction of UV-B mutants and their analyses are still indispensable. We attempted to produce novel UV-B mutants of Arabidopsis by using ion beams as a mutagen. Because ion beams have high-linear energy transfer (LET) compared with other forms of radiation and gives a superior ability to create mutations. Here, we present the isolation and characterization of new UV-B resistant and sensitive mutants to understand UV-B resistant mechanisms.

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EAOH (Early-onset ataxia with ocular motor apraxia and hypoalbuminemia)/AOA is characterized as a unique disorder. Patients of EOAH have mutations in the APTX gene which encodes Aprataxin. Since Aprataxin has been reported to associate with Xrcc1, PARP-1 and p53, it is supposed to be involved in the repir of DNA single strand breaks.We have established immortal cell line of the cells obtained from a EAOH patient by in introducing the hTERT gene. This EAOH cell was more sensitive to hydrogen peroxide than normal cells. We then analyzed amount of single strand breaks by using the Comet assay and found increased number of single strand breaks in EAOH cells after treatment with hydrogen peroxide. We then analyzed effect of low-dose-rate radiation on this EAOH cells. EAOH cells were more sensitive to low-dose-rate radiation than normal cells and dose dependent amount of single strand breaks were observed after low-dose-rate radiation.

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Ionizing radiation causes DNA double strand breaks (DSBs), which are the cause of detrimental effects of radiation. DNA DSBs are recognized by ataxia-telangiectasia mutated (ATM) protein, and ATM activated through auto-phosphorylation at serine 1981 (S1981) transduces DNA damage signals via phosphorylation of the down-stream effectors. Recently, the effects of low dose ionizing radiation have been received much scientific attention. To understand low dose effects it is indispensable to analyze radiation response in individual cells. In the present study, we examined the induction of DNA damage checkpoint signal at a single cell level using anti-S1981 phosphorylated ATM antibody. Normal human diploid cells are irradiated with various doses of X-rays, and phosphorylated ATM foci were detected by immunofluorescent assay. We found liner dose-dependent induction of phosphorylated ATM foci with doses between 10 mGy and 1 Gy. Interestingly, the mean size of the initial foci (approx. 0.1 micron) changed to 1.0 micron 30 minutes after X-irradiation. Similar foci growth was observed in the foci of phosphorylated histone H2AX at serine 139, 53BP1, and NBS1. Furthermore, we confirmed that the mean size of the phosphorylated ATM foci induced by 50 mGy of X-rays was comparable to that induced by 1 Gy, indicating that phosphorylation-dependent amplification of the signal is involved in DNA damage checkpoint. These results indicate that even a single DSB could be detectable efficiently though signal amplification.

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Although the public today could be exposed to X-rays as high as 1 cGy due to diagnostic procedures, the biological effects of this low dose range have not been established. We searched through more than 23,000 transcripts in normal human fibroblasts using a novel comprehensive expression analysis method (HiCEP). More than two hundred genes were upregulated transiently by 1 cGy of X-rays during the one hour period after irradiation. Within ten genes with increased expression of more than 2 fold in the exposed cells, three encoded proteins which share a known function associated with tumor progression. Our results indicate that human cells respond to doses of radiation as low as 1 cGy, and mechanisms alternative to those involved in moderate/high dose studies have to be considered in understanding the biological effects of diagnostic level radiation.

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Our microbeam irradiation system can deliver a desired number of X-ray photons to an arbitrarily defined target region. We compared the cellular responses of microbeam irradiation with those of broad-field (broadbeam) irradiation.
In broadbeam experiments, 1×10⁵ V79 cells were seeded in a 35 mm dish. The entire plate was irradiated uniformly with 5.35 keV X-rays. Cells were collected by trypsinization and survival fractions were measured with the usual colony assay method. On the other hand, in microbeam experiments, 2×10³ V79 cells were seeded in a specially designed dish for microbeam experiments (Φ 34 mm). The targeted cell nuclei were irradiated with a 10 μm square 5.35 keV X-ray beam. Using the criterion that colonies including more than 30 cells are surviving after 60 hours of incubation, we determined the survival fractions of irradiated cells.
Low-dose hypersensitivity was clearly observed in the dose-survival curves obtained in microbeam experiments. In contrast, the low-dose hypersensitivity was less remarkable in the broadbeam experiments. In broadbeam experiments, the entire cytoplasm, as well as nucleus, was irradiated uniformly with X-rays, while most of the cytoplasm was not irradiated in microbeam experiments. In both experiments, energy deposition in the nuclei was almost equal. These results suggest that the energy deposition in cytoplasm might reduce the cell killing effect in low dose region. We are further analyzing this hypothesis considering the differences in experimental procedures.

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We reported previously that glial cells cultured from young rats indicated the adaptive response to low dose irradiation and that the adaptive response was decreased with aging. In order to investigate its mechanisms, we searched the variant expressions of proteins caused by low dose irradiation in glial cells by using proteomics.
Glial cells were cultured from variously aged Wistar rats. After low dose irradiation, proteins were extracted and subjected to 2D-PAGE. Alterations in the relative intensity of protein spots appearing on proteome profiles were analyzed using PDQuest software (Bio-Rad) and identifications of protein spots were performed using MALDI-TOF MS analysis.
We detected a protein spot, of which expression was increased 3 hr and decreased 24 hr after low dose irradiation, in young cells. The expression of the protein spot was not changed by low dose irradiation in cells from aged rats. From the fact that the adaptive response was demonstrated in young cells and with 3h-interval between low dose pre-irradiation and the challenging irradiation, it is likely that this protein is involved in radiation adaptive response in rat glial cells.

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Last year we have reported that when immature spermatocytes with active DNA repair function were irradiated with low dose, low dose-rate X-rays, mutation frequency became lower than control. Here we present the results of further studies with high and/or low doses and dose-rates. A high dose (10Gy) irradiation at a high dose-rate (500mGy/min) made the mutation frequency significantly higher than control. A 10Gy irradiation at a low dose-rate (50mGy/min) made the mutation frequency higher than control but lower than the high dose-rate experiment. The dose-rate effect was evident. With a low dose (200mGy) irradiation at a high dose-rate, the mutation was not significantly more frequent than control. A 200mGy irradiation at a 50mGy dose-rate resulted in a significant decrease in the mutation frequency. These results suggest that the dose response relationship is non-linear for a low dose-rate irradiation. A possible mechanism of the existence of the threshold is that the DNA repair function is activated by the low dose-rate irradiation and it repaired not only radiation induced damage but also spontaneous one. The background mutation frequency in this experiment was 10 to the 6th/gene which corresponds to that induced by a 4.2Gy irradiation. If a considerable fraction of the spontaneous damage is repaired without error, the background mutation frequency should be reduced, which compensates the increase in the induced mutation and thus forms a practical threshold. Some results using a repair defective strain will also be presented.

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It is extremely important to investigate the genetic effects of the chromosome aberrations caused by radiation exposure at low-dose or low-dose rate from the view points of influences on human health. Here, we used LOH (Loss of Heterozygosity) analysis system in human lymphoblastoid cell TK6, which has been established as a sensitive detection methodology. After TK6 cells were exposed to a low dose-rate gamma rays (1.2 mGy/hr, total 30 mGy), the frequency of mutations induced in thymidine kinase (TK) gene locus, selected as early mutants (EMs), was about 2.8 times as high as that of unirradiated cells. The rate of LOH mutants among EMs was shown to be increased by radiation exposure (0/3→5/7), and most of the LOH mutants were hemi-type observed near the TK locus. On the other hand, the frequency of lately induced TK mutants (LMs) decreased to about 60% of non-irradiation level. Most of obtained LMs showed LOH regardless of the irradiation. In addition, the hemi-type events were detected only after the radiation exposure. It can be suggested that the repair of DNA double double strand breaks by non-homologous end-joining is responsible for the induction of hemi-type LOHs. Moreover, there were almost no differences in the total mutation frequency (EM + LM) between irradiated and unirradiated samples, reflecting a difficulty in the detection of mutation in the low-dose range. These preliminary results are now under the confirmation.

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It is well known that irradiation cause many effects to organismes, like mutation, cell death or tumor. The DSB is major DNA damage by irradiation and, thus this type of damage should be involved in these effects. In order to elucidate the detail effect of DSBs in mammalian cells, we are constructing varions systems to: 1) visualize the DSB repair by introducing fluorescence marker near to the DSB site, 2) isolate proteins which associate to the DSBs site through or after the repair, 3) follow expression pattern of a gene near to the DSB site after the repair. The current data would be presented.

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We have previously reported that the oxygen effect for induction of DNA-DSB was bigger (∼ 6) than that for cell killing level. The large oxygen effects for DNA-DSB decreased to the cell killing level (∼ 3) by DNA repair in a short time. The mechanism of oxygen effect decreased by DNA repair was further examined and here reported. Two chinese hamster cell lines, i.e., wild-type CHO and a Ku80 mutant CHO (xrs6) cells were exposed to 200 kVp X-rays at ice temperature under oxic or hypoxic conditions. Static-field gel electrophoresis (SFGE) was used to determine radiation-induced DSB. Oxygen enhancement ratio (OER) of DSB for CHO cells after X-ray irradiation was 6.3 without repair, and decreased to 3.4 when a repair time of 15 minutes was observed. However, the decrease of OER value was not observed for wild-type CHO cells pretreated with 20 µM wortmannin for 1 hour or xrs6 cells.
We conclude that the radiation-induced DSB by the NHEJ repair system under oxic condition is more efficiently repaired than that under hypoxic condition.

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Ionizing radiation and reactive oxygen species produce various types of oxidative damage to DNA, which cause mutations in cells. Bacteria and eukaryotes have DNA repair systems to prevent mutations. Oxidative base damages are principally repaired by base excision repair (BER) mechanisms. However, there are no indications that oxidative damages are directly reversed on the DNA strand. In this study, we examined whether oxidative damages are directly reversed on the DNA strand. We used pUB3 plasmid irradiated with gamma-rays and subsequently treated with a reducig agent, dithiolthreitol (DTT). E. coli cells were transformed with the pUB3. As a result, the transformation efficiency of pUB3 treated with DTT was higher than that of pUB3 without DTT treatment. We are currently investigating the mutation spectra of the pUB3.

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The Analysis of double strand break repair by chromatin immunoprecipitation in mammalian cellsMikio Shimada, Reiko Ohba and Kenshi KomatsuDepartment of Genome Repair Dynamics, Radiation Biology Center, Kyoto-University, Kyoto 606-8501, JapanRestriction enzyme, similar to ionizing radiation, induces DNA double strand breaks (DSBs) in living cells. I-Sce1 endonuclease recognizes highly specific 18-bp sequence, which is not present in mammalian cells. Since DR-GFP repoter gene contains the I-Sce1 recognition sequence, DSBs can be generated at a specific site of the reporter gene-integrated chromosome by introduction of I-Sce1 endonuclease. Althoug DSBs are rejoined by both homologous recombination (HR) and non homologous end joining (NHEJ), the latter is dominant in mammalian cells. Here we report Chromatin-immunoprecipitation (ChIP) analysis to investigate the repair process from I-Sce1-induced DSBs in mammalian cells. When Ku70 null mouse cell line, NHEJ deficient, was analyzed by ChIP, phosphorylation of H2AX was detected at distances of ~ 1kbp and ~ 4kbp from the I-Sce1- induced DSB. This ChIP analysis totally depends on rejoining rate of DSBs, since this phosphorylation was not detected in mKu70-complemented cell lines. ChIP assay has been successfully applied for analysis of the interaction between chromosome and DNA repair factors in yeast. Present results suggest that ChIP is also a useful tool for analysis of mammalian repair process when DSBs are generated by I-Sce1.

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Base Excision Repair (BER), a predominant pathway to repair small base damages, includes two sub-pathways: a short-patch and a long-patch BER. Since it has been suggested that long-patch BER is dependent on PCNA, it is possible that this pathway is more effective in S phase than in other phases. In this study, to compare the mode of BER in G1 and S phase cells, we first detected the number of MMS-induced AP sites in G1 and S phase cells. We synchronized HeLa RC355 cells in M phase by nocodazole. After releasing the synchronized cells, we examined the number of apparent MMS-induced AP sites in G1 and S phase cells using the ARP method. We found no significant difference between the number of AP sites in both phases, though slightly more AP sites were observed in G1. When compared the total number of methylated bases, we found the number in G1 phase was twice as much as that in S phase cells at 20mM MMS (p<0.05). We have directly analyzed intracellular AP sites using the FARP-1 method. We obtained the similar results as described above. These results suggest that the FARP-1 method is useful to monitor the intracellular BER process. Further analysis is in progress.

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DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand breaks (DSBs) repair, and it consists of DNA-PKcs, Ku70, and Ku86. It has been shown that the promoter regions of these three genes have Sp1 binding sites, and the expression levels are correlated with that of Sp1.The purpose of this study is to clarify the contribution of Sp1 to radiosensitivity of cells through the transcriptional regulation of DSBs-repair genes. We investigated whether Sp1 affects the expression level of DNA-PKcs, Ku70, Ku86, and XRCC4. In addition, we examined the DSBs repair activity in Sp1-down-regulated cells. A human transformed kidney cell line, 293T cells were transfected with siRNA vector targeting Sp1 (siSp1) or the control vector (siC). The vector-transfected cells were selected with G418 sulfate. After seven days selection, protein levels of DNA-PKcs, Ku70, Ku86, and XRCC4 were evaluated by western blotting. The DSBs repair kinetics were determined by pulsed-field gel electrophoresis (PFGE) after 80 Gy X-irradiation. The protein level of Sp1 in siSp1-transfected cells was 31% compared with siC-transfected cells. DNA-PKcs, Ku70, and Ku86 were down-regulated to 6%, 3%, and 53% of the control level, respectively. The protein level of XRCC4 was not changed. These results indicate Sp1-dependent transcriptional regulation of DNA-PK. The PFGE study revealed the apparently lower efficiency of DSBs repair in siSp1-transfected cells than siC-transfected cells. This study showed that Sp1 regulates the transcription of DNA-PK and possibly determines the cellular radiosensitivity.

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Ionizing radiation produces various molecular damages on DNA through the energy deposition to matter. The activities of the repair enzymes could be modified with the chemical structure of DNA damages. We have investigated the radiation effects to DNA using ‘in situ’ spectroscopic methods; mass analysis of the products induced by photo-desorption, spectroscopic analysis for X-ray absorption near edge structure, and the measurement of electron paramagnetic resonance to detected DNA radicals. These analyses enable us to open discussion the ion or radical reaction during irradiation and the reaction pathway to the final product of the DNA molecule. On the other hand, we used not only synchrotron soft X-rays, but also hard X-rays (150kVp) and heavy ions (He²⁺ (50MeV) or C⁶⁺ (320MeV)) as radiation sources. Then we try to clarify the molecular changes by measuring X-ray absorption near edge structure (XANES) irradiated DNA molecules. The K-shell electrons of the element of DNA molecules can be ionized or excited by the soft X-rays. Therefore we can analyze functional groups appeared in DNA molecules irradiated. We used DNA or related molecules (deoxyribose, nucleobases and nucleotides), plasmid DNA (pUC18) and calf thymus DNA as irradiated samples. We will discuss the relationship between the DNA damage and radiation quality in respect of repairbility of damages.

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Oxidative nucleotide lesions as well as oxidative DNA lesions appear to contribute to mutagenesis induced by ionizing radiation. We have studied roles of Y-family DNA polymerases, which are involved in translesional synthesis, in mutations induced by oxidative nucleotide lesions. In this study, we compared mutational property of 8-hydroxy-dGTP (8-OH-dGTP) during DNA replication by cellular extracts with and without DNA polymerase (pol) eta. Plasmid DNA containing the SV40 ori and the supF gene, the unmodified dNTPs, the SV40 large T antigen, and extracts of XP-V cells lacking DNA pol eta were incubated with and without 8-OH-dGTP. Replicated DNA was then transfected into an indicator E. coli strain KS40/pOF105, and the supF mutant frequency was measured. Extracts of HeLa cells were used as controls. Although the supF mutant frequency was increased by addition of 8-OH-dGTP, the frequency of mutation induced by 8-OH-dGTP was similar for XP-V and HeLa cell extracts. Thus, DNA pol eta is not major DNA pol which promotes incorporation of 8-OH-dGTP.

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Ionizing radiation and reactive oxygen species produce various types of DNA damage in living cells. Most of oxidative DNA damage are primarily repaired by base excision repair (BER) system. DNA glycosylases excise damaged bases, releasing AP sites in the DNA. Then AP endonucleases or AP lyases cleave DNA at the AP site, followed by repair synthesis and rejoining by DNA polymerases and DNA ligase. Most of all species so far examined have BER system. Therefore, BER plays an important role in maintenance of genome integrity. Endonuclease III (Nth) is a bifunctional glycosylase which excises oxidized pirimidines such as thymine glycol, and incises the DNA at the resulting AP site. DNA glycosylases are conserved in many organisms, indicating that this enzyme plays a critical role in removing oxidative damage from DNA. In this study, we sub-cloned C. elegans nth homolog gene. Neither OGG1 nor Nei homolog has been identified in C. elegans for oxdative base damage. It is likely that Nth homolog is a major glycosylase for 8-oxoguanine as well as thymine glycol in C. elegans.

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DNA in living cells is highly protected from radiation-induced damage, and the major part of this protection is provided by nuclear proteins. We examined whether the local protective effects of the nuclear proteins modulate the distribution of strand breaks. DNA single-strand breaks with 5'-phosphates in specific genomic sequences in X-ray-irradiated human cells were detected by ligation-mediated PCR and visualized on sequencing gels. We observed protection from strand cleavage at some of the transcription factor-binding sites in promoters. The regions occupied by positioned nucleosomes showed repeated protection patterns with a periodicity of about 10 bases, reflecting the wrapping of DNA helixes around histone cores. Unexpectedly, sites of modest enhancement of cleavage were found near the transcription start site of the c-FOS gene. Thus, the distribution of DNA single-strand breaks in irradiated cells is not uniform, and seems to be dependent on chromatin structure.

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Detection of DNA strand breaks by radiation was carried out using real-time PCR amplification. Gamma irradiation to pBR322 plasmid DNA solutions was done by 137Cs, gammacell 40, as dose of 0Gy, 30Gy, 60Gy, 100Gy, 150Gy, 200Gy and 250Gy. 1003bp, 749bp, 505bp and 242bp fragments were amplified by combined primer sets for the plasmid solution including Tris-HCl as a radical scavenger. Higher amplification values such as over 200% to the non-irradiated solution (100%) were observed and could be depending on rate of closed circular shape (CC) and open circular shape (OC) in the original solution of pBR322 used in each experiment. One suitable explanation that the CC is difficult to be amplified by PCR and after nicking by radicals, the reaction is progressed, was fit to the simulation pattern. Therefore, we carried out the PCR amplification for 13 irradiation points between 0~150Gy and the evident peak was observed at 30Gy. The results of the simulation were not completely fit to the PCR data. The explanation that all CC DNA could not be amplified might be not clear. We also tried to analyze whether the plasmid structure would be depending on the sequences or not. We will present the PCR data for the pBR322 exchanged with other similar length sequences.

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Radiation-induced DNA double strand breaks (DSBs) are recognised by ATM and activated ATM then phsophorylates and activates downstream factors such as histone H2AX. In irradiated interphase cells, phosphorylated histone H2AX foci, which appear immediately after irradiation, colocalise with proteins involved in DNA damage repair. Therefore they are thought to represent DSBs and have a role in DNA damage repair. However, we have found that phospho-H2AX foci are formed at sites of DSB as well as at sites of chromatin aberrations without DSBs. This study aimed to elucidate a possible new role of radiation-induced phosphorylation of H2AX. First, behaviors of phospho-H2AX, phospho-ATM and 53BP1 in irradiated mitotic-phase cells were analysed. In response to X-ray irradiation, phospho-H2AX formed foci in all mitotic-phase cells while phospho-ATM and 53BP1 did not. When these cells successfully divided and progressed to G1-phase, the three proteins formed foci and they colocalised. The results obtained differed from the fact that the proteins colocalise in irradiated interphase cells. This suggests a possible new role for phosphorylated H2AX foci. Since DSBs are lethal in mitotic cells, the phospho-H2AX foci observed in surviving G1 cells are thought to represent the sites of chromatin aberrations without DSBs. Therefore phosphorylation of histone H2AX might have a role in recruiting mediators such as phosphorylated ATM and 53BP1 to the sites of chromatin aberrations not accompanying DSBs.

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DNA damages induced by ionizing radiation are characterized as base modifications, DNA strand breaks and cross-linkings. Most of the DNA damages mentioned above are induced by hydroxyl radicals. Hydroxyl radicals are thought to attack mainly DNA bases but about one fifth of it attacks the DNA backbone to produce the scissions of deoxyribose-phosphate bonds. DNA double strand break may cause genome instability, cell death and carcinogenesis. Studies of the mechanisms of DNA double strand break are one of the most important subjects to clarify the DNA damage induced by ionizing radiation.Recently we could control DNA structures and make compact folded DAN and stretching unfolded DNA by addition of polyamine compounds. In this experiment, we used T4 DNA and made two structures, folded and unfolded DNA by adding spermidine. DNA solutions having different DNA structures were irradiated by cobalt-60 gamma radiation, and DNA double strands breaks were estimated by fluorescence microscopic measurement.For the estimation of DNA double strand breaks, we used two different techniques. One is the direct measurement of DNA length absorbed on the glass and another is an indirect method to estimate DNA length by observation of hydrodynamic radius moving in water by Brawnian movement.In our presentation, we will mention the technical details of our experiments, and relationships between the higher-order structure of DNA molecules and DNA damage by ionizing radiation.

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Werner syndrome (WS) is an autosomal recessive disorder with premature aging. The gene defective in WS, WRN, is located on chromosome 8p. Because the WS gene product (WRN) interacts with several proteins involed in DNA repair in vitro, it has been hypothesized that WRN is involved in DNA repair. However, we found that cells derived from WS patient are not particularly sensitive to ionizing radiation (IR) compared with the control cells in vivo. This result suggests that WRN is not directly involved in DNA double-strand break repair, or that cell cycle-dependent radiation sensitivity of WS cells could be masked in the former assay. In the present study, we investigated whether the cell cycle-specific DNA repair in WS cells is defective or not. Two cell lines used are BJ1-hTERT (hTERT-immortalized normal human fibloblasts) and WS3RGB-T (hTERT-immortalized WS fibroblasts). For the detection of DNA damage, we examined phosphorylated-ATM foci in 1 Gy-irradiated cells. Each cell cycle phases are visualized by cell cycle specific marker, and cells in S and G2 phases were stained with anti-RPA antibody and anti-phosphorylated-hitone H3 antibody, respectively. Without irradiation, the numbers of the foci per cell is slightly higher in WS cells than in the control cells. Immediately after irradiation, both cell lines showed the similar numbers of foci in S and G2 phases, and the foci disappeared similarly thereafter. These results confirm that WRN is not directly involved in DNA double strand repair in both S and G2 phases.

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Several recent studies have suggested that radiation-induced genomic instability involved in radiation carcinogenesis. However the mechanisms of radiation-induced genomic instability have not been clarified. The generation and repair of radiation-induced initial DNA damages in cells have been recently examined by enumeration of phosphorylated histone H2AX (γ-H2AX) foci. This result showed that initial DNA damage induced by a certain amount of dose might not be able to be restored and, there is a possibility remaining for a long time. Although the biological meaning of remains of this initial DNA damage has not been clarified yet, we thought that remaining of initial DNA damage may relate to the induction of the radiation carcinogenesis. In this study, we investigated that time course analysis of radiation -induced initial DNA damage based on γ-H2AX and ATM focus formation in primary human fibroblasts.

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Rad51C is a member of Rad51 paralogs conserved in vertebrates and plays a role in homologous recombination repair. The functional analysis of Rad51 paralogs has been exerted mainly in knocked out DT40 cells, mutated CHO cells and/or knockout mice. Hypersensitivities to radiation and inter-strand cross-linking agents, a reduction in homologous recombination and increased chromosomal aberrations have been reported. In addition, recent reports have shown that Rad51C is required for Holliday junction resolution and Rad51 paralogs are required for telomere maintenance. These findings suggest that Rad51 paralogs play crucial roles in DNA repair and maintenance of chromosomal integrity. We generated the human colon cancer cell line HCT116 harboring Rad51C mutation by gene targeting. They showed hypersensitivities to radiation and inter-strand cross-linking agents, and increases in chromosomal aberrations and aneploidy as observed in DT40 and CHO mumant cells. Furthermore, the cells showed slow growth, which was caused by decreased activities of cyclin E and cyclin A. An increase in p21 and phosphorylation of p53 at serine 15 were also observed. As the increase in p21 was cancelled by the addition of caffeine, G1 checkpoint appeared to be activated by the ATM/ATR-p53-p21 pathway. Furthermore, the ectopic expression of cdk2 reversed increased aneuploidy. Taken together, Rad51C dysfunction leads to activation of G1 checkpoint with reduced cdk2 activity, which may cause aneuploidy.

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Thorotrast was used as a radiological contrast medium during World War II. Since intravascularly administrated Thorotrast was deposited mainly in the liver and the surrounding tissues were exposed to alpha-particles emitted from Thorotrast. We are performing analysis of loss of heterozygosity (LOH) in Thorotrast-induced liver tumors among which relative risk of angiosarcoma (AS) is thought to be characteristically prominent in radiation-induced tumors. LOH frequency was higher in Thorotrast AS than in Thorotrast ICC. This suggests that direct effect of radiation contributes to the induction of AS more than ICC. In order to prove the hypothesis, we assessed common deletion (CD) of mitochondrial DNA which represents weak repair system of DNA in mitochondria. We found that CD frequency of non-tumor part was higher in Thorotrast AS and non-Thorotrast HCC compared with tumor part. In contrast, CD frequency in tumor part was higher in Thorotrast-induced intrahepatic cholangiocarcinoma and cholangiolocarcinoma compared with non-tumor part. These may result from the fact that the remodeling of non-tumor part in Thorotrast AS and non-Thorotast HCC is more dynamic and mitotic activity is less compared with their counterpart, respectively. Furthermore we found 2 novel deletions near to CD. We will discuss the meaning of these newly found deletions in relation to radiation exposures.

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