Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both hind legs of Balb/cA nude Mice. The mice then received nicotinamide injection or carbogen gas (95% O2, 5% CO2) inhalation with or without mild temperature hyperthermia (MTH, 40 centigrade, 60 min). After each treatment, the mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors. SAS/mp53 tumors showed significantly larger values in the size of not only the HF but also the diffusion-limited chronically HF than SAS/neo tumors. MTH could efficiently release the chronically HF, irrespective of p53 status. These supported the fact that, in gamma-ray irradiation and cisplatin treatment, the enhancement in combination with MTH was more remarkable in SAS/mp53 cells than SAS/neo cells. [J Radiat Res 44:407 (2003)]

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We studied the cell cycle progression and the induction of cyclinB1 and p21 in cultured human fibroblasts after irradiation with heavy ion beams at HIMAC of NIRS. Asynchronous human normal fibroblasts (NB1RGB) in monolayer were irradiated with X-rays, carbon ion beam (75 keV/um), Si ion beam (250 keV/um), and Fe ions (200-440 keV/um) at room temperature. Cell cycle distribution and the protein induction were measured by flow cytometory. After irradiation, both G₁ and G₂ block were observed independently on ions. From 30 to 50 percent of NB1RGB cells have accumulated at G₂/M phase. Percentage of cell number at G₂/M phase increased with dose up to 2 Gy. CyclinB1 was expressed only in G₂/M phase cells. The percentage of cells in which cyclin B1 was expressed increased with incubation time after irradiation up to 8 h. No increment of cyclin B1 expression was observed from 2 Gy to 10 Gy. P21 was expressed both in G₁ and G₂/M cells after 3 h and longer incubation after irradiation. [J Radiat Res 44:407 (2003)]

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Oxidative DNA damages are mainly generated by reactive oxygen species produced during cellular metabolism as well as exposure to radiation. The spectrum of DNA damage includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine is an important mutagenic lesion. The mutgenic base is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. Until now there is no study with Ciona intestinalis . In this study, we identified and characterized Ciona intestinalis cDNAs encoding the 8-oxoguanine DNA glycosylase genes. The reasons why we choose Ciona intestinails are that firstly, it's genome is the smallest of any experimentally manipulable chordate. Secondly, Ciona intestinails has a short life cycle only 6~8 weeks. Thirdly, this configuration is the most simplified and primitive chordate body plan. We found that the cDNA library from Ciona intestinails contains homologs of the human Ogg1 gene. [J Radiat Res 44:408 (2003)]

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Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing DNA double-strand breaks (DSBs). XRCC4 is NHEJ protein employed in DNA DSBs repair and in V(D)J recombination. To address the role of XRCC4 in human cells, we used gene targeting in human somatic tissue culture, human HCT116 colon cancer cells, to functionally inactivate the XRCC4 locus. Human XRCC4 heterozygous cell lines displayed haploinsufficient with reduction in cell proliferation but not hypersensitivity to ionizing radiation. A second round of gene targeting generated homozygously null XRCC4 cell lines. These cells showed a growth defect and hypersensitivity to ionizing radiation. [J Radiat Res 44:408 (2003)]

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Purpose: To determine the change in radiosensitivity of Escherichia coli by overexpression of various DNA glycosylases, which may provide an evidence of in vivo response against radiation-induced clustered DNA damage in Escherichia coli. Materials and methods: Quantitative survival assay was performed in alpha-ray, gamma-ray, X-ray and H₂O₂ survival experiments. Results and Conclusion: The mutant defective in MutM, Nth, Nei DNA glycosylases (mutMnthnei) was less sensitive to gamma-rays and X-rays than the wild-type strain. Overexpression of MutM and human Ogg1 resulted in enhanced sensitivity to such low LET radiation. The enhancement of radiation sensitivity occurred in a dose-dependent fashion. On the contrary, the mutMnthnei mutant was more sensitive to hydrogen peroxide than the wild-type strain. Clustered DNA damage may be transformed into double strand breaks by DNA glycosylases that excise closely opposing lesions, or lesions closed to opposing strand breaks. The level of the increase in radiosensitivity depends on the substrate specificity of DNA glycosylases. [J Radiat Res 44:408 (2003)]

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Oxidative DNA base lesions produced by reactive oxygen species exert genotoxic and cytotoxic effects on living organisms. 5-Formyluracil (fU) is a major oxidative thymine lesion and induces transition mutations if not repaired. In the previous study, we partially purified and characterized rat fU-DNA glycosylase (FDG). The analysis of the FDG activity to various base lesions suggested that FDG was a rat homologue of SMUG1. In this study, we have performed immunological analysis of FDG using hSMUG1 antibodies. The FDG fraction from rat liver exhibited a positive Western band with a molecular mass of 30 kDa. The fU-releasing activity in rat and human cell free extracts were effectively neutralized by the antibodies. Futhermore the antibodies also neutralized the activities for 5-hydroxyuracil and 5-hydroxymethyluracil but not for thymine glycol in the cell free extracts. These data confirm that mammalian FDG is SMUG1. We are currently studying the substrate recognition mechanism of hSMUG1. [J Radiat Res 44:408 (2003)]

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DNA-dependent protein kinase (DNA-PK) is composed of Ku70, Ku86 and catalytic subunit (DNA-PKcs) and acts as a sensor of DNA double-strand breaks (DSBs) during non-homologous end-joining (NHEJ). Here we demonstrated that DNA-PKcs formed nuclear foci rapidly after exposure to ionizing radiation. DNA-PKcs foci were observed just after 5 Gy of X-irradiation. Wortmannin inhibited XRCC4 phosphorylation but not DNA-PKcs foci formation. On the other hand, both DNA-PKcs foci formation and XRCC4 phosphorylation were reduced by Ku86 siRNA. These results suggest that DNA-PKcs foci formation requires Ku proteins and precedes its activation. DNA-PKcs did not response to DNA replication arrest, while NBS1 and histone H2AX, which are essential for homologous recombination (HR), were phosphorylated and formed foci. Also, X-ray-induced DNA-PKcs foci did not colocalize with phosphorylated H2AX. These results further suggest that DNA-PK selectively recognize DSBs repaired by NHEJ independent of HR-related proteins. [J Radiat Res 44:409 (2003)]

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Radiosensitization together with abrogated cell cycle checkpoints are most pronounced actions of caffeine among its many diversified activities. Caffeine has recently been considered to overcome the G2/M and S checkpoint responses through its inhibition of phosphorylation of ATM /ATR kinase substrates including Chlk1, Chk2 and P53. We have found that homozygouly Np95-inactivated embryonic stem (ES) cells are more sensitive to X-rays, UV-light, MNNG, and HU than ES wild type cells, mimicking the phenotype of ATRkd human fibroblasts or Chk1-/- cells. To explore the relationship between ATR and NP95 we examined the effect of Np95 status on the caffeine action in ES cells. Two mM caffeine in the post irradiation medium enhanced the cytotoxicity of UVC in Np95 +/+ cells, but not in Np95 -/- cells, while synergism was more pronounced in Np95 -/- cells than Np95 +/+ cells following the treatment with X-rays. [J Radiat Res 44:409 (2003)]

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Base excision repair (BER) is one of the pathways to avoid mutations in living cells. Damaged bases are excised by DNA glycosylases and then AP lyases nick DNA, followed by repair synthesis and rejoining by DNA polymerases and DNA ligases. Increased frequency of mutations in BER-defective mutants in E. coli and S. cerevisiae indicates that BER plays an important role in the cells. However, the effects of BER deficiency in multicellular organisms are yet unknown. In human, defect of nucleotide excision repair results in serious hereditary diseases, while no desease has been found to relate to BER deficiency. So we investigated the influence of BER deficiency in multicellular organsim using C. elegans. Recently, we identified the uracil DNA glycosylase homolog gene (Ceung) in C. elegans, and clarified the glysocylase activity of purified recombinant CeUNG. In this study, we examined the phenotypic properties of C. elegans defective in UNG activity using RNAi. [J Radiat Res 44:409 (2003)]

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After X-irradiation of cells, p53-binding protein 1 (53BP1) binds to chromatin at sites of DNA double-strand breaks in a phosphorylation-dependent manner. The Tudor plus Myb domain, the minimal region of 53BP1 for chromatin binding, binds directly to both double-stranded and single-stranded DNA, and stimulate end-joining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro, suggesting 53BP1's role in the repair of DNA double-strand breaks. In the last meeting, we showed that a colon cancer cell line SW48 is deficient in 53BP1 expression, and that expression of 53BP1 in SW48 cells significantly reduce the number of cells containing X-ray-induced chromosomal aberrations. To determine whether 53BP1-p53 interaction is necessary for stimulation of repair by 53BP1 for X-ray-induced chromosomal aberrations, we established a SW48 cell line that expressed mutant 53BP1 that did not bind to p53. The importance of interaction between 53BP1 and p53 in the repair of DNA double-strand breaks will be discussed. [J Radiat Res 44:409 (2003)]

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Recently we present the evidence for novel roles of histone acetylases. The TIP60 histone acetylase is purified as a multimeric protein complex. This complex has a RuvB homologue which has a role in DNA recombinational repair in E.coli. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence , suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis. We present here that how the histone acetylase TIP60-containing complex is involved in DNA damaged repair. Recently, we purified the TIP60 complex after DNA damage and revealed that TIP60 complex come to have a g-H2AX after DNA damage. Interestingly we also cleared that histone acetylase of TIP60 complex is required for the phosphorylation of H2AX. [J Radiat Res 44:410 (2003)]

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DNA replication stalls at lesions such as pyrimidine dimers in a template strand. DNA polymerases from hyperthermophilic archaea recognize the presence of template uracil and stall DNA synthesis. To analyze the recognition mechanism, the binding mode of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA was examined by gel-shift assay and atomic force microscopy (AFM). Pol B1 tightly and specifically bound to uracil-DNA and retarded the mobility of DNA on agarose gel. When primer/template DNA was incubated with Pol B1 plus dNTPs, the presence of template uracil significantly inhibited the formation of double-stranded DNA. In the AFM image, single-stranded and double-stranded DNAs were observed as sphere and linear forms, respectively. Omission of dNTPs and primer from the reaction completely blocked the formation of double-stranded DNA and inhibited the formation of intermediates where Pol B1 appeared to bind to uracil-DNA. These results suggest that Pol B1 more efficiently recognizes template uracil by processive sliding on template DNA rather by random diffusion-mediated mechanism. [J Radiat Res 44:410 (2003)]

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When cells were exposed to ionizing irradiation, the cells were arrested and do not enter in mitosis. Otherwise, cells carrying unrepaired or misrepaired DNA damages might be lethal or prone to cancer. Recently accumulating evidences have been supported that DNA-PK and ATM play an important role in this mechanism, but it is still unclear how these proteins works cooperate. Using the ATM mutated cells AT5BISV and LY 294002, a specific inhibitor of DNA-PK, we investigated the role of DNA-PK and ATM in radiohypersensitivity and control of cell cycle check point following irradiation. Colony Surviving assay showed that 50 uM of LY294002 made AT5 cells 2 order hypersensitive to irradiation. AT5 cells in G2/M phase were accumulated of 60% of total, but more than 10% cells were escaped from G2 and reached in mitosis. The mitotic chromosomes of AT5 were quite badly damaged, whilst those of control cells were almost repaired. These findings suggested that DNA-PK and ATM cooperatively control DNA repair machinery and cell cycle check point regulation, particularly G2-M transition control. [J Radiat Res 44:410 (2003)]

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Werner syndrome (WS) is a disorder with multiple features of premature aging. The responsible WRN gene encodes a protein possessing DNA helicase and exonuclease. To examine the differential function of helicase and exonuclease of WRN protein, we investigated cell cycle progression after treatment with hydroxyurea (HU) in a WS cell line (WS780) and a human cell line (293delta231) defected in a WRN exonuclease activity. The results indicated that WS780 cells were highly sensitive to HU treatment, showing a more delayed recovery from the DNA replication (S) arrest than a control GM638 cell line. In contrast, the WRN exonuclease defective cell line (293delta231) showed no difference in the recovery from the S arrest as compared with a control 293 cell line. The present results suggest that WRN protein play a role in the recovery from the S arrest although WRN exonuclease is not involved in this process. [J Radiat Res 44:410 (2003)]

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Fanconi anemia (FA) is an autosomal recessive disorder characterized by skin pigmentation, high incidence of cancer, and a diverse variety of congenital malformations. The cells from FA patients display chromosome instability, and hypersensitivity to DNA cross-linking agents, such as mitomycin C. At least eight genes, FANCA to FANCG, are responsible for FA and, among them, FANCD1 revealed to be identical with breast cancer associated gene BRCA2. It have been reported that BRCA2 functions in homologous recombination repair by interacting with Rad51. In fact, CAPAN-1 cells expressing truncated BRCA2 protein show failure of nuclear localization of Rad51 in response to DNA damage and are sensitive to radiation. We report here that several FA-D1 cells show normal nuclear localization of Rad51, while they are radiation sensitive. By using DR-GFP system, we are assaying the ability of homologous recombination in FA-D1 cells. [J Radiat Res 44:411 (2003)]

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It has been already reported that the repair of radiation induced DNA double strand breaks(DNA-dsbs) is inhibited with antimony potassium tartrate(APT), but whether APT induces DNA-dsbs or not is not well known. The effect of APT on DNA was studied in Chinese hamster ovary(CHO) cells and glutathione(GSH)-deficient CHO. CHO cells were treated with APT for 2h. Then DNA-dsb was induced by gamma-irradiation at a dose of 40Gy and the degree of DNA-dsb was determined by gel electrophoresis immediately after irradiation or following 30-min repair period. DNA-dsbs was induced by exposure to 0.8 and 0.02mM APT in normal and GSH-deficient cells, respectively. DNA-dsb repair was completely inhibited by exposure to 0.4 and 0.02mM APT in normal and GSH-deficient cells, respectively. GSH is a radical scavenger and DNA-dsbs was induced by lower concentration of APT in GSH-deficient cells. These results show that antimony is a radical inducer and toxicity of antimony is, in part, derived from DNA-dsbs. [J Radiat Res 44:411 (2003)]

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DNA double-strand breaks (DSBs) induced by ionizing radiation represent the most serious damage in cells. Immediately after the formation of DSB, histone H2AX is phosphorylated at the site of DSB. The phosphorylated H2AX (γH2AX) foci can be observed with an immunohistochemical staining. In this research, we examined that γH2AX serves to an index for existence of DSBs which are unrejoined for a long time participating in the radiation susceptibility. 2Gy X-rays were irradiated cells with varying radiosensitivity and the change in the number of foci were observed with time. In all cell lines, foci formation was seen immediately after irradiation, and the number of foci reached a peak at 30 to 60 minutes. At 12 hours, many foci were remained in sensitive cells but having almost disappeared in resistant cells. That is, compared with resistant cells, it was suggested that the rejoining activity of DSBs in sensitive cells is descended. Our result has suggested that the quantity of residual γH2AX serves as an index of radiation susceptibility. [J Radiat Res 44:411 (2003)]

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Reactive oxygen species are continuously generated in living cells. They interact with DNA, which produce a wide variety of base damage and cause mutations. Bacteria and eukaryotes have evolved DNA repair systems to maintain the genetic stability and to prevent mutations. Many enzymes are involved in the base excision repair pathways in E. coli and eukaryotes. For instance, MutT dephosphorylates 8-oxo-dGTP and prevents the incorporation of 8-oxodGTP produced in nucleotide pool. 8-oxoG mispairs with A and causes G:C to T:A transversions. E. coli Nth has glycosylase activity that recognizes and excises thymine glycol (Tg). Tg blocks DNA replication in vitro. The phenotypes of DNA repair defective mutant are well studied in E. coli and S. cerevisiae, while little in multicellular organisms. In humans, the defect of nucleotide excision repair results in serious hereditary diseases. In order to investigate the effect of DNA repair deficiency in multicellular organisms, we first identified the homologs of E. coli MutT and Nth in C. elegans. [J Radiat Res 44:411 (2003)]

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8-oxoguanine (8-oxoG) is an abundant and critical base modification. It is excised from the DNA by MutM in E. coli and Ogg1 in S. cerevisiae and mammalian cells. However, any structural and functional homologs of them are not yet identified in Shizosaccharomyces pombe. Recently, Nth-Spo, an ortholog of E. coli Nth, was cloned from the S. pombe. It removes oxidative pyrimidines from the DNA. E. coli Nth and Nei remove 8-oxoG from the 8-oxoG:G mispairs in DNA. We examined whether the Nth-Spo functions as a DNA glycosylase/AP lyase for 8-oxoG in DNA. The Nth-Spo was expressed in E. coli and partially purified using glutathione affinity column chromatography. The DNA glycosylase activity was assayed for 8-oxoG:A, 8-oxoG:G or 8-oxoG:C containing duplex oligonucleotides. The Nth-Spo efficiently removed 8-oxoG from 8-oxoG:A and 8-oxoG:G pairs from DNA. The activity to remove 8-oxoG from 8-oxoG:A and 8-oxoG:G mispairs was much higher than that from 8-oxoG:C. Furthermore, we report about the effects of Nth-Spo expression in mutMmutY and nthnei mutants of E. coli. [J Radiat Res 44:412 (2003)]

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It has been suggested that Deinococcus radiodurans possesses a DNA damage response mechanism. However, little is known about the molecular basis for the control of the inducible proteins. We found that, like in E. coli, RecA is the sole protein required for LexA cleavage. We also found that the co-protease activity rather than recombination activity of RecA contributes to the high proficient DNA repair in D. radiodurans. Interestingly, it has been shown that the D. radiodurans genome encodes different two LexA homologues (LexA and LexA2). We analyzed the function of these genes by generating gene disruption strains. The lexA2 disruptant strain exhibited much higher resistance than the wild-type, suggesting that DNA repair genes are down-regulated by LexA2. Furthermore, to gain insight into contributions of LexA and LexA2 in the DNA damage response mechanism, we examined the gene dosage effects of lexA and lexA2. [J Radiat Res 44:412 (2003)]

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Ionizing radiation induces DNA double-strand breaks, which are the major cause of the detrimental effects of radiation. They activate a checkpoint protein, ATM, and it phosphorylates a variety of proteins to exert its functions. Histone H2AX, a member of the histone H2A family, is phosphorylated immediately after irradiation, and phosphorylated H2AX forms discrete foci. Because the number of foci corresponds to the number of DNA double strand breaks, it has generally been thought that the phosphorylation of H2AX is caused by DNA double strand breaks. Present study reveals phosphorylated histone H2AX foci on mitotic cells irradiated with X-rays. As expected, phosphorylated H2AX foci were found at the ends of chromosome fragments in metaphase cells. In addition, the foci were detected on chromosomal bridges between two sister nuclei in anaphase. Furthermore, we found duplicated phosphorylated H2AX foci exactly at the same sites on both sister chromatids 20 hours after irradiation. These results indicate that a change in higher-order chromatin structure, caused by DNA double-strand breaks, is involved in the phosphorylation of histone H2AX. [J Radiat Res 44:412 (2003)]

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In mammalian cells there are two major pathways for spontaneously arising and ionizing radiation (IR) induced DNA double strand breaks: homologous recombination (HR) and non-homologous end-joining (NHEJ). While a deficiency in any step of either pathway could render cells hypersensitive to IR, the resultant effect on mutations with allelic losses has not been fully understood. We measured spontaneous and X-ray induced mutagenesis with LOH in DNA ligase IV deficient SX10 cells and its parental SR-1 cells both heterozygously inactivated at the aprt locus. In SX10 cells, spontaneous mutation frequency was clearly higher than in the parental SR-1 cells. The mutation frequencies were not significantly elevated after X-irradiation in SX10, whereas a dose dependent increase was noted for SR-1. [J Radiat Res 44:412 (2003)]

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Ku, a heterodimer of Ku70 and Ku80, plays a key role in multiple nuclear processes, e.g. DNA repair, chromosome maintenance, and transcription regulation. We generated cell lines expressing the human Ku80 tagged with the green fluorescent protein color variants. The tagged Ku80 complements a deficiency in Ku-deficient mammalian cells. Therefore, these cells should prove useful in the analysis of the function of Ku in living cells. We also established stable cell lines expressing Ku80-mutants to which mutations were introduced by the site-directed mutagenesis technique. [J Radiat Res 44:412-413 (2003)]

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UV-B easily produces cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts. They inhibit cellular and physiological activity and impair genetic stability. Responses of plants to UV-B varies among species and cultivars. Cucumber and spinach are sensitive to UV-B. Hypersensitivity of a rice cultivar to UV-B has been reported to be due to reduced photoreactivation (PR). We have previously reported a nucleotide sequence of a gene for CPD photolyase in spinach. The gene complemented E. coli deficient in PR. We here further investigated a gene for spinach CPD photolyase. E. coli deficient in PR was transformed with the cloned gene and the content of CPD was determined by ELISA mehtod. The extent of repair of CPD was much higher in E. coli treated with visible light than untreated control. The gene has been reported to highly express in flower in Arabidopsis. However in spinch the expression in leaf was much higher than other organs, root, stem and flower. [J Radiat Res 44:413 (2003)]

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We have found a novel monofunctional thymine glycol-DNA glycosylase in the mouse organs. The activity was purified using the extract prepared from isolated nuclei of mouse liver by repeated high salt extraction. By the use of the extraction buffer containing 0.42M NaCl, most of lower molecular weight proteins including mNTH1 and major APendonuclease, could be separated from the target activity which was extracted more slowly. The specific activity in this extracted fraction was about 300 times higher than that previously observed in nuclear extract. This extracted fraction was further purified using UNO-S1 and UNO-Q1 column (BIO-RAD). The optimum pH and salt condition of the target activity were 100mM KCl and pH 8.0, respectively. The activity was resistant to 0.58 mM EDTA. An apparent single band was observed on SDS-PAGE of active UNO-Q1 fractions. The estimated molecular weight (about 45,200Da) agree with one of candidate proteins observed in previous purification using the extract from isolated nuclei by sonication. [J Radiat Res 44:413 (2003)]

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Deinococcus radiodurans is well known to exhibit remarkable radioresistance that is supposed to be due to efficient capability to repair DNA double-strand breaks through homologous recombination. In Escherichia coli, two distinct pathways are known for recombination repair (RecBCD and RecFOR). In the case of D. radiodurans, because of the absence of recB and recC genes, it is expected that the RecFOR pathway is important for DNA homologous recombination. For the last few years, we have investigated on the D. radiodurans recR gene. In this meeting, we will report some results obtained by the analysis of recF-disrupted mutant. [J Radiat Res 44:413 (2003)]

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Ionizing radiation frequently causes oxidative DNA damage in cells. It has been suggested that functions of the REV1 and REV7 genes are induction of mutations and prevention of cell death caused by ionizing radiation. However, in higher eucaryotes, functions of homologues are poorly understood and appear somewhat different from the yeast case. It has been suggested that human REV1 interacts with human REV7, this being specific to higher eucaryotes. Here we show that purified human REV1 and REV7 proteins form a heterodimer in solution, which is stable through intensive purification steps. Results from biochemical analysis of the transferase reactions of the REV1-REV7 complex demonstrated, in contrast to the case of yeast Rev3 whose polymerase activity is stimulated by assembly with yeast Rev7, that human REV7 did not influence the stability, substrate specificity or kinetic parameters of the transferase reactions of REV1 protein. A possible molecular role of the REV7 subunit may be to help assembly of the REV1 protein to a large complex containing REV3 and/or other DNA polymerases in higher eucaryotes. [J Radiat Res 44:413 (2003)]

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Using Bacillus subtilis sacB gene expression system, mutation spectrum analysis of the radioresistant bacterium Deinococcus radiodurans was performed. The sacB encodes levansucrase that confers sucrose sensitivity when the gene is expressed in cytoplasm. Mutations in sacB, thereby, allow cells to grow in a medium containing sucrose. After acute exposure to gamma-rays, the sacB mutation frequency was readily increased. Whereas mutation spectrum after UV irradiation was similar to that of non-irradiated control, mutation spectrum after gamma-irradiation showed characteristic features; +1 frameshift at adenine cluster, and increased transposition frequency of an insertion sequence. These results suggest that D. radiodurans possesses mutagenesis mechanism that is activated through radiation-induced DNA damage. [J Radiat Res 44:414 (2003)]

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Radiation-induced mutations were studied in vivo with a transgenic mouse called HITEC, which carries the rpsL gene as a reporter for mutations. Although we had previously investigated the increase in mutation frequency caused by ~5 Gy of gamma-rays in mouse liver, no obvious increase in mutation frequency was observed due to a high background mutation frequency. We therefore performed a partial hepatectomy to amplify the mutation frequency. HITEC mice were irradiated with 5 Gy of gamma rays before hepatectomy and the remnant liver was resected after hepatectomy. The mutation frequency was then analyzed. In mice in which hepatectomy was done 2 days after irradiation and the remnant liver was resected 10 days after hepatectomy, the mutation frequency was approximately two-fold higher. In mice in which hepatectomy was done 10 days after irradiation and the remnant liver was resected 2 or 10 days after hepatectomy, the mutation frequency was two to three times greater. These results indicate that partial hepatectomy increases the frequency of radiation-induced mutations. [J Radiat Res 44:414 (2003)]

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It has been known that ultraviolet light (UV) A, B and C damage the cells and lead mutation and/or death by generating reactive oxygen species (ROS) in the cell and/or forming photoproducts such as pyrimidine dimer on DNA. In order to elucidate the ratio of repairable damage induced by each wave length, the sensitivity of Escherichia coli mutants of DNA repair and of ROS scavenging enzymes to UV-A, B and C was investigated. Three UV lamps illuminating each wave length were used. DNA repair mutants used were WP2(wild), WP2uvrA(uvrA), CM571(recA) and WP100(uvrArecA) and ROS scavenging enzyme lacking mutants used were DSH7(wild), DSH19(katEG), DSH56(sodAB) and DSH76(katEGsodAB). As the results, it is suggested that UV-A, B and C induce photoproducts including pyrimidine dimer on DNA which are repairable and that UV-A and B generate ROS in the cells but UV-C does not. [J Radiat Res 44:414 (2003)]

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GRP78 has been reported to be a molecular chaperon whose expression is increased in response to endoplasmic reticulum stress such as glucose starvation and anoxia. In this annual meeting of the last year, we suggested that GRP78 is involved in the resistance of human cells to far-ultraviolet light (UVC) lethality. In the present study, we investigated whether GRP78 regulates the susceptibility of human cells to UVC mutagenicity and the mechanisms underlying the GRP78-invloved UVC resistance. We established human cells, in which the expression levels of GRP78 proteins were decreased by transfection of antisense cDNA of GRP78. The sensitivity of the cells to UVC mutagenicity was examined by ouabain-resistance test. The removal kinetics of the UVC-damaged DNA were also analyzed by ELISA assay. The transfected cells showed higher sensitivities to UVC mutagenicity and lower removal ability of the damaged DNA than did the vector control cells. These findings suggest that GRP78 is involved in the UVC resistance of human cells possibly via DNA repair process. [J Radiat Res 44:414 (2003)]

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Mouse lymph node cell (M10) is highly sensitive against X-ray irradiation. Growth of this cell depended on the UV-rays irradiation. The cell growth was repressed in proportion to the irradiation time. Dynamic changes of 597 genes after the UV-B (310 nm) irradiation were investigated on DNA array membranes (Atlas #7741-1,CLONTECH) using P-33 labeling probe. Radioactivities obtained from imaging analyzer (BAS1500, Fuji film co. ltd.) were analyzed using software of ArrayGauge. The expression of mRNA was further analyzed by EX-ARRAY program. Seven housekeeping genes were used as standards. Irradiations for 10-second, 20-second and 30-second were done, and changes in the gene expression after the cultivation for 2 h were compared with cells without irradiation. As for a group of activated genes, UV-rays reached more than 80 kinds in 597 genes for 10-second irradiation, while 110 kinds as for a group of repression genes. Activated genes decreased due to the irradiation time, and it became clear that repression genes increased in proportion to the irradiation time. [J Radiat Res 44:415 (2003)]

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The cataract is promoted by UV irradiation. It is well known that the main causes of UV induced cataract are reactive oxygen species which are generated by UV irradiation. Metallothionein regulates detoxification from heavy metals, and free radical scavenging in living tissues. In this study, we analyzed the expression and induction of Metallothionein-I (MT-I) by UV irradiation using mouse lens epithelial cell line (alphaTN 4-1). AlphaTN 4-1 was irradiated by UV with various doses, and then the cells were incubated for several hours. After incubation each cells were collected and the expression of MT-I mRNA was analyzed by the quantitative RT-PCR method. The relative amounts of MT-I mRNA were normalized against GAPDH mRNA. MT-I was constantly expressed in alphaTN 4-1 and induced by UV irradiation. The amounts of MT-I mRNA changed with increasing dose of UV irradiation. These results suggest that MT-I is activated by UV irradiation and can function as a radical scavenger in lens. [J Radiat Res 44:415 (2003)]

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Telomeres at terminal regions of linear chromosomes in eukaryotes play important roles not only in maintaining the chromosome integrity but also in the exercise of normal cellular functions. Here we studied how UVB irradiation affects telomere and telomerase in cultured murine cells. A TRF method and a TRAP assay measured the telomere length and telomerase activity, respectively. Immediately after UVB irradiation no change in telomere length was shown, but an apparent shortening of telomere was observed markedly in normal BALB cells compared with SCID cells 24 h after UVB irradiation. Telomere activity was strikingly enhanced in a dose dependent fashion 1 h after UVB irradiation: up to 20 times in SCID cells and 10 times in BALB cells as high as non-irradiated cells. Nine-fold high activity was still held in SCID cells 24 h after UVB irradiation. These results suggest that UVB-induced high telomerase expression may repair UVB-damaged telomeres. It is also suggested that DNA-PKcs, deficient in SCID cells, may be involved in the repair process of UVB-damaged telomeres. [J Radiat Res 44:415 (2003)]

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In an attempt to understand the biological effect of physiological and chronic low-dose rate UV-B irradiation, cultured human cells were irradiated with low-dose rate UV-B in situ using a CO₂ incubator equipped with a UV-B tube. The dose-dependent formation of cyclobutane pyrimidine dimers and (6-4) photoproducts in normal human fibroblastic cells, XP 2OS cells, and human keratinocytes (HaCaT) were observed during the chronic irradiation. Synthesis of DNA and RNA in the cells were substantially reduced during the chronic exposure associated with the accumulation of G₁/S phase cells, indicating that DNA replication and transcription were immediately blocked. The intracellular oxidative level elevated continuously with increasing irradiation time. Furthermore, all of the signaling molecules examined, ERK, JNK, p38, and p53, were kept phosphorylated during the irradiation. These observations suggest that DNA damage-dependent S-phase block and p53 phosphorylation, as well as continuous generation of reactive oxygen species followed by MAPK phosphorylation, were coincidentally taking place during the chronic exposure. [J Radiat Res 44:415 (2003)]

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We previously showed that cyclobutane pyrimidine dimer (CPD) photolyase was deficient in UVB-sensitive rice, and that the CPD photolyase deficiency might result from a mutation of CPD photolyase. In this study, we carried out the genetic analysis of correlation between UVB-sensitivity and mutation of CPD photolyase using F2 plants generated by reciprocally crossing UVB-resistant Sasanishiki (Japonica rice cultivar) and UVB-sensitive Surjamkhi (Indica rice cultivar), and F3 lines generated by self-fertilizing of F2 plants. The CPD photolyase activity in Surjamkhi was significantly lower than that in Sasanishiki. There were two variations in the deduced amino acid sequence of CPD photolyase in these two cultivars. We investigated the UVB sensitivity in F2 plants and the genotype of CPD photolyase in F3 lines. Consequently, we found that strong correlation exists between the UVB-sensitivity and mutation of CPD photolyase in UV-sensitive and -resistant rice cultivars. [J Radiat Res 44:416 (2003)]

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Japanese rice (Oryza sativa L.) cultivars vary widely in their sensitivity to UVB radiation. We previously showed that cyclobutane pyrimidine dimer (CPD)-photorepair activity in UVB-sensitive Norin 1 was lower than in its close relative UVB-resistant Sasanishiki, and that the CPD-photorepair deficiency in Norin 1 resulted from a functionally altered photolyase. There was a variation between the deduced amino acid sequences of Norin 1 and Sasanishiki CPD photolyases. These results suggested that the difference in the UVB-sensitivity between those two cultivars might be due to the structural alteration of CPD photolyase. In this study, we investigated the deduced amino acid sequences of photolyases, CPD-photolyase activities in vitro and the UVB-sensitivities of close relatives and progenitors of Sasanishiki and Norin1. Two genotypes of CPD photolyase were found. One is the "Sasanishiki type" and the other was "Norin 1 type". We discuss the relationship among the difference in the UVB-sensitivities, CPD photolyase activities and genotypes of CPD photolyase. [J Radiat Res 44:416 (2003)]

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Rice cultivars (Oryza sativa L.) vary widely in their sensitivity to UVB. We found that the cyclobutane pyrimidine dimer (CPD) photorepair activities in UVB-sensitive cultivars were lower than these in UVB-resistant cultivars in several rice cultivars and a possibility that these. These differences in the photorepair activities maight correlate with the differences in the deduced amino acid sequences of CPD photolyase. Wild rices are thought to be progenitors of rice cultivars, and to have many variations in CPD photolyase genes. In this study, we investigated the variations in UVB-sensitivities,deduced amino acid sequences of CPD photolyases and the activities of CPD photolyases in several wild rice plants (O.rufipogon, O.meridionalis) originated from various geography, Asia and Australia. We found that there were variations in deduced amino acid sequences of CPD photolyases in wild rices having different UVB-sensitivities. [J Radiat Res 44:416 (2003)]

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Although the skin carcinogenicity of UVB and UVA has been established, those of solar UV, which consists of UVA and longer wavelength part of UVB, are only deduced from these UVB and UVA data and have never been directly evaluated. We studied mutation induction kinetics in the mouse skin directly exposed to sunlight. Using biological dosimetry systems, we show here poorer skin protection against genotoxic effect of solar UVB than those of UVA and full wavelength-range UVB, revealing that the longer UVB included in sunlight is more effective to induce skin mutations than the shorter UVB. Practically, at the same skin erythematogenic doses, sunlight would induce more mutations than UVB. [J Radiat Res 44:416 (2003)]

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Radiation induced acute changes of mouse hematopoietic functions and its recovery were studied in different LET of heavy ions, carbon (13, 30 keV/mm), neon (32, 55 keV/mm), silicon (56 keV/mm), argon (85 keV/mm), iron (200 keV/mm). After whole body irradiations up to 3 Gy, the subsequent hematopoietic parameters were followed by the blood analysis for 8 days. All hematological functions, spleen weight, granulocyte-monocyte colony forming unit (GM-CFU) in bone marrow and spleen indicated dose dependent reduction within 2 days after the irradiation and followed by a slow recovery. GM-CFU in spleen showed the highest sensitivity to all particles, showing a large reduction down to less than 1% of the control at 3 Gy irradiation. Of the particle exposed Si (55 keV/mm) induced the largest reduction and the slowest recovery, whereas Fe (200 keV/mm) indicated the least reduction and the fastest recovery. The results suggested a strong LET dependency of the hematopoietic progenitor cells which may have a RBE maxima less than 100 keV/mm. [J Radiat Res 44:417 (2003)]

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In the measurement of cosmic rays on board aeroplanes, it is essentially important to provide suitable devices. A portable Si ionization chamber fulfill the demand because it is cheap, compact and easy to handle. Another type of candidate, a wristwatch type detector with a GM tube, appeared very recently. Its performances are rather poor than the Si type, especially for short distance flights, but are satisfactory when it flies over long distances like Narita-Paris. Correlation coefficient between the GM- and Si-detector is as good as 0.999 in such long distance flights. The shortest flight for safe usage of the GM-detector would be at least 4,000km, which is like Narita-Bangkok. [J Radiat Res 44:417 (2003)]

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Bacterial spores can survive in extreme environments and potential agents of interplanetary dispersal. In the preflight project of EXPOSE, various microorganisms are exposed to stresses to be encountered during the flight, and we are studying the survival and mutagenesis of Bacillus subtilis spores. In the first simulation experiment, dry spores are exposed to high vacuum (about 10⁻⁴ Pa) for 100 minutes or 7 days and UV radiation. Induction of rifampicin-resistant mutants was observed with UV and longtime exposure to vacuum. We have analyzed sequence changes in rpoB gene of the mutants, and found that about a half of those obtained from vacuum exposure carried tandem-double base substitutions, the majority of which belonged to a single allele (r201) of CA to TT change. This demonstrates that the exposure to high vacuum causes forced dehydration in the vicinity of DNA, resulting in unique base-substitution mutations. [J Radiat Res 44:417 (2003)]

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It is afraid that disruption of the ozone layer by flon would still continue more fifty years in spite of international control of its production. One% decrease of the ozone layer produces 2% increase of UV arrival on earth. UV irradiation in sunlight is the most important factor of skin cancer. This study intended to estimate biological effects of UVA, UVB and UVC irradiation on cell DNA with single cell electrophoresis assay (comet assay). Hepatic cells isolated from ICR mouse were applied to special microscope slides for comet assay and electrophoresis was performed under alkaline electrophoresis buffer after UV irradiation in sunlight. The relations between intensity of individual UV irradiation and DNA damage were investigated. [J Radiat Res 44:417 (2003)]

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Recent studies from our laboratory have shown that He ions induced chromatin breaks in unirradiated bystander cells. In this study, we examine the potential contribution of irradiated medium to the bystander effect. We can plate cells in either side or both sides of double mylar dishes. Since He ions can only traverse a very limited distance, cells plated on the other side of a medium-filled mylar dish (target cells) would have no chance of being hit by He ions. After one side (with or without cells) was irradiated by the He ions, the target cells were collected for cytogenetic assay using the PCC technique. Our results show that the number of aberrations in cell irradiated group is slightly higher than those in medium irradiated group. Furthermore, when transfer the medium of cell irradiated group, chromatin fragments were produced in the target cells added to the medium, while no damage was observed added to medium irradiated group. These results clearly show that some species induced chromosomal damage exists in medium of cell irradiated group. [J Radiat Res 44:417-418 (2003)]

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We investigated the p53 signal pathway by hypergravity in human glioblastoma cell line, A172. Hypergravity (20g) induced the accumulation and the phosphorylation of p53 at 3h. But hypergravity did not significantly induced Waf-1 or Bax. Then, we examined the expression of genes by using Human Oligo Chip 30K (Hitachi Software Engineering Co., Ltd.). This DNA chip contained oligo DNA from 30,000 genes. Any of p53 inducible genes did not induced, but some genes concerned with cell signaling pathway or cytoskeleton of the cell induced by hypergravity (20g, 6h). We considered that the p53 signal pathway by hypergravity is different from other p53 signal pathway like DNA damage signal. DNA chip revealed that cells induced many gene expressions to adapt the environment of hypergravity. [J Radiat Res 44:418 (2003)]

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We examined the effects of extremely low frequency magnetic fields (ELFMFs) and/or X-rays on mutations using pTN89 plasmids. The plasmids were subjected to sham exposure or exposed to an ELFMF (5 mT), with or without X-ray irradiation (10 Gy). Increased mutation frequency was not detected following exposure to a magnetic field alone, or after sham exposure. Sequence analysis of the supF mutant plasmids revealed that base substitutions were dominant on exposure to X-rays alone and X-rays plus an ELFMF. Deletions were detected in only the combined treatments with X-rays and an ELFMF, but not with X-rays alone. From these results, we could not detect direct effects of ELFMFs on DNA, but exposure to ELFMFs immediately before or after X-ray irradiation may enhance the mutation frequency. [J Radiat Res 44:418 (2003)]

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Mutagenicity of complex exposure to static magnetic field(MF) and 50Hz MF was investigated. We developed a exposure system that can generate a 5T static MF and a 1mT 50Hz MF simultaneously with temperature control. To investigate the mutagenicity, bacterial mutation assay (Ames test) using Salmonella typhimurium TA98 and TA100, and also yeast mutation assay using Saccharomyces cerevisiae XD83 were performed. It was found that gene conversion/recombination frequency in S. cerevisiae XD83 was slightly increased by exposure to a complex MF while point mutation frequency in S. cerevisiae and S. typhimurium was not affected. This result is consistent with previous studies for static MFs, however we observed smaller mutagenicity in this study than one by a 5T static MF alone. This suggests that complex exposure of 50Hz MF with static MF might cause weak protective effects. In future study, different exposure condition will be examined for estimation of the effect of complex MF exposure that was generated in our environment. [J Radiat Res 44:418 (2003)]

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We investigated the effect of 10 and 6 tesla (T) static magnetic fields (SMFs) on the expression of proto-oncogenes using western blot hybridization methods. 10 T is the highest magnetic flux density and 6 T has the strongest magnetic field gradient (41.7 T/m) in our SMF exposure system. We used the human promyelocytic HL-60 cells. HL-60 cells were exposed to the SMFs for 1, 2, 4, 8, 24, 36, 48, and 72 hours. The cells exposed to either 6 T or 10 T SMF for periods of 1 - 48 hours did not exhibit remarkable differences in levels of c-MYC and c-FOS expression, as compared to sham-exposed cells. On the contrary, c-JUN protein expression increased in HL-60 cells following exposure to 6 T SMF for 24, 36, 48, and 72 hours. These results suggest that 10 T SMF is unlikely to alter the expression of proto-oncogenes. However, the exposure to strong magnetic field gradient might induce the c-JUN expression. [J Radiat Res 44:418-419 (2003)]

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To assess the possibility that extremely low frequency (ELF) magnetic fields obstructed the damage repair process, we examined the gene conversion frequency and cell cycle kinetics in DNA repair proficient strain and in nucleotide excision repair (NER) deficient strain of diploid yeast Saccharomyces cerevisiae. Yeast cells were irradiated with UV followed by exposure to magnetic fields up to 30mT. After exposure, the colony-forming ability was scored as revertants in which gene conversion had restored the functional allele of ARG4. Cell cycle analysis was performed using flow cytometry. A gene conversion rate was increased by the combined exposure in DNA repair proficient cells. The UV-induced G1 arrest was inhibited by exposure to ELF magnetic field in both strains. These results suggest that exposure to high density ELF magnetic fields decreased the efficiency of NER by suppressing G1 arrest, which turned into the enhancement of the UV-induced gene conversion. [J Radiat Res 44:419 (2003)]

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Mechanism underlying ultrasound-mediated transfection (USMT) is possibly explained with interaction between effects of cavitation and resposes of plasma membrane. We focused on the plasma membrane to see if altering it could enhance USMT. PC-3 and T24 from urological cancers were used as target cells and the luciferase gene was used as a reporter. Levovist, a contrast agent, was used to facilitate cavitation. Lidocaine, a local anesthetic, or heat was applied for membrane modification. For in vitro transfection, cells in a tube were sonicated and for in vivo transfection, cells in the rat bladder were transabdominally sonicated with 1 MHz ultrasound. Sonicated cells were incubated and subjected to lucifearase assay. Luciferase expression in transfected cells increased with increasing lidocaine concentration and increased with rising temperature. Obtained data suggested different mechanisms in the enhancements by lidocaine and heat. These results indicated membrane modification significantly enhanced USMT, suggesting this could be useful for the ultrasound-mediated gene therapy. [J Radiat Res 44:419 (2003)]

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