Abstract [Purpose] To identify the mechanisms that control the DNA metabolic response to ionic and nonionic radiation in human cells, we investigated this issue using the experimental methods that we originally developed. Recently, we noted the indication that chaperones, such as Heat shock protein 27 (HSP27) and the 78-kDa glucose-regulated protein (GRP78), are important factors involved in these control mechanisms (Exp. Cell Res., 298, 584-592, 2004, Exp. Cell Res., 305, 244-252, 2005). In the present study, we searched for HSP27-interacted proteins to clarify the roles of HSP27 in cellular UVC response. [Methods] The HSP27 protein purified as a fusion protein with GST (GST-HSP27) was conjugated with NHS-activated Sepharose. Cell lysates from the human UVC-resistant cells were applied to the affinity column, and subjected to SDS-PAGE and mass spectrometrical analyses. Interaction of candidate proteins with HSP27 was examined by immunoprecipitation analysis, and the function of the candidate in UVC resistance was further examined using small-interfering RNA (siRNA). [Results and Conclusion] Annexin II was specifically bound to GST-HSP27-conjugated Sepharose compared with GST-conjugated Sepharose. The interaction between HSP27 and annexin II was confirmed by coimmunoprecipitation using anti-HSP27 and anti-annexin II antibodies. Protein amounts of both HSP27 and annexin II increased after UVC irradiation in nuclear fractions of UVC-resistant cells. Cells transfected with annexin II-specific siRNA were more susceptible to UVC lethality than negative control siRNA-transfected cells. There was no significant difference in cell proliferation rates between siRNA-treated cells and control cells. These findings suggest that annexin II is an HSP27-interacted protein and involved in UVC resistance in human cells, at least, the cells tested here.

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Recent studies have suggested that the signals from cellular membrane damage are also participated in induction of radiation induced apoptosis, and a system assume to be due to is sphingomyelin–ceramide signal transduction pathway. However, its detail is still unclear. Microbeam is able to irradiate a specific area of target cells, so that it is considered to be effective for elucidating mechanisms underlying membrane/cytoplasmic damage induced apoptosis. In this study, we investigated induction of apoptosis by a selective irradiation of membrane and cytoplasm using heavy-ion microbeam. An activation of sphingomyelinase and an increase of intracellular ceramide signal were observed 5 min after irradiation when Chinese hamster ovary cell line, CHO-K1, was exposed to γ rays. Additively, a treatment of sphingomyelinase inhibitor restrained an induction of radiation induced apoptosis. These results indicate that sphingomyelinase is activated by radiation and consequently induces radiation induced apoptosis. Thus we irradiated cytoplasmic region of the cells by Ar ion microbeam (11.2 MeV/u) at TIARA, JAEA-Takasaki. An Apoptotic fraction induced by cytoplasm irradiation was higher than that of control. An intracellular visualization of ceramide revealed that ceramide is localizing near an ion hit region that is visualized by etching of CR-39 base of the microbeam dish. Thus, it was considered that ceramide was generated at the damage site of the cellular membrane. These results indicate that sphingomyelinase is activated by radiation-induced membrane damage and ceramide is playing a role in induction of apoptosis.

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Stem cells are defined as cells that self-renew indefinitely and also give rise to differentiated cells by suitable stimulation. During thelast decade, it has been revealed that almost all tissues containtissue-specific stem cells (ex. blood, muscle, intestine, gland and brain) which continuously generate the residential differentiated cells responsible for tissue functions and homeostasis, or trangently regenerate cells lost by injury or illness. There is increasing evidence that malignant tumours contain high-hierarchy cells that maintain the characteristics of tissue-specific stem cells and are malignant. Malignant gliomas, for example, contain both proliferating cells expressing stem cell markers and differentiating cells expressing either neuronal markers or glial markers. These recent findings support an idea that malignant glioma can be generated from either neural stem cell (NSC) or glial lineage cell. The hypothesis can be viewed as cancer stem cells. The development of NSC culture has been accomplished by in vitro growth from regions of the developing brain or neurogeneic regions of adult. In this study, we investigate radiation response of NSC, using mouse embryonic neurospher. NSC was harvested from corpus striatum in E14.5d ICR mouse embryo. For 10 days culture, enriched NSC was grown as self-adherent complexes of cells, forming clusters known as neurosphere. These cells were identified by using both nestin and CD133 Ab, which are specific-marker for NSC. More over, we defined that the neurosphere stimulated by serum gave rise to neurones, astrocytes and oligodendrocytes. To estimate self-renewal activity, colony forming assay in soft agar was executed. Regenerate capacity of NSC was exhausting during serial sub-culture. Cell from larger size clone had higher proliferate potential. Additionally, X-ray radio-sensitivity of NSC was similar to identical MEF. In the present, we are carrying out to elucidate radio-sensitivity p53 deficient NSC. Finding radiation response in NSC overcome radio-resistance and provide a therapeutic model for malignant brain cancers.

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DNA double-strand break (DSB) is the most dangerous DNA damage because it can result in loss or rearrangement of genetic information, that leads to cell death, carcinogenesis or aging. It has been recognized that DSB is induced by low dose ionizing radiation (IR) as well as by high dose IR. Two major pathways exist in mammalian cells for the repair of DNA DSB: non-homologous DNA-end-joining (NHEJ) repair and homologous recombination (HR). NHEJ, in contrast to HR, rejoins DNA ends with the use of little or no sequence homology, leading to imprecise joining. Therefore, this repair pathway seems to often fail to preserve the full information content at the site of damage. NHEJ pathway is thought to begin with the binding of Ku70 and Ku80. Differences in the timing and destination of translocations of protein are used to provide fine spatial and temporal control of protein-complex formation and function within the cell. Several proteins involved in DNA DSB repair have been found to change their localization within the cell nucleus after exposure to ionizing radiations. The control mechanism for hetrodimerization and/or subcellular localization of Ku70 and Ku80 appear to play a key role in regulating the physiological function of Ku. In this study, we analyzed about the dynamics of Ku70 at DSB sites in mamamalian cells.

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Reactive oxygen species (ROS) are produced in cells as by-products of endogenous oxygen metabolism as well as exogenous factors such as ionizing radiation and chemical agents. ROS oxidize biologically important molecules such as nucleic acids, proteins and lipids. The oxidation might be involved in mutagenesis, carcinogenesis and ageing. Among oxidized base lesions, 8-oxo-7,8-dihydroguanine (8-oxo-G) can pair with cytosine and adenine and consequently induces A:T to C:G and G:C to T:A transversion mutations. Organisms developed elaborate mechanisms to prevent the mutagenic effects of 8-oxo-G. Studies on mutator mutants of Escherichia coli elucidated that three enzymes, encoded by the mutM, mutY, and mutT genes, play important roles in avoiding mutagenesis. MutT hydrolyzes 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and pyrophosphate. In mammalian cells, similar enzymatic activities have been found, suggesting that similar systems are used to avoid 8-oxo-G-related mutagenesis in mammals. MTH1 has bees identified as human(or mouse)homologue. In this study, we searched candidates for a functional homologue of E. coli MutT in Caenorhabditis elegans and Ciona intestinalis from databases and analyzed their structures and functions.

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Nijmegen breakage syndrome (NBS) is a rare autosomal recessive genetic disorder characterized by immunodeficiency and cancer predisposition. The NBS cellular phenotype includes chromosomal instability, hyper-sensitivity to radiation and abnormal checkpoints activation. NBS1 protein, product of the NBS gene, forms a complex with MRE11 and RAD50, and the RAD50/MRE11/NBS1 (R/M/N) complex plays a crucial role in DNA double strands break (DSB) repair. The human NBS1 has three functional regions: N-terminus fork head associated (FHA) and BRCA1 C-terminus (BRCT) domains, two serine residues at 278 and 343 that are phosphorylated by the ATM or ATR kinases, and the C-terminus MRE11 binding domain and ATM interacting domain. We have previously reported that the R/M/N complex with functional NBS1 is essential for homologous recombination (HR) repair pathway and that FHA domain is required for regulation of normal HR function. To investigate whether mutation in FHA domain presents a dominant negative effect on cellular HR activity, we analyzed HR efficiency in normal or mutant NBS1 expressing HeLa cells carrying a SCneo reporter construct. It was found that mutation in FHA domain induces significant reduction of HR frequency, suggesting that regulation of nuclear localization of R/M/N complex might be crucial for HR function.

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Post-replication DNA repair facilitates resumption of DNA synthesis following replication fork stalling at sites of DNA damage. Despite the importance of RAD18 and polymerase h for post-replication repair, the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. In this report, we present evidences that human RAD18 protein preferentially binds to forked DNA structures and that RAD18 and replication protein A bind long single-stranded DNA in a cooperative manner, which are known to be localized at stalled replication forks. The SAP domain of RAD18 is crucial for RAD18 binding to the DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at sites of DNA damage in vivo, and does not guide DNA polymerase h to stalled replication forks. The SAP-domain mutant fails to suppress the UV-sensitivity of Rad18-knockout cells. Furthermore, RAD18 is required for guiding polymerase h (and also polymerase _) to stalled replication sites. These results suggest that RAD18 is recruited to stalled replication forks via interactions with forked DNA or long single-stranded DNA structure, which are required for initiating post-replication repair.

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Translesion DNA synthesis is essential for the maintenance of chromosomal integrity as well as the DNA repair function. It has been suggested that functions of REV genes are required for translesion DNA synthesis, essential for induction of mutations and prevention of cell death caused by ionizing radiation. It has been suggested that functions of the REV genes are required for error-prone post-replication repair, essential for induction of mutations and prevention of cell death caused by ionizing radiation. REV1 is the deoxycytidyl transferase and a member of the Y-family DNA polymerase. The activity is capable of extending a primer terminus by insertion of dCMP opposite a variety of damaged bases. REV3and REV7 encode an error-prone DNA polymerase, pol zeta. Genetic data suggest that those proteins form specialized machinery for translesion DNA synthesis. We have demonstrated that human REV1 forms a stable heterodimer with REV7. Recently, it has been found that REV1 interacts with all of the Y-family DNA polymerases, pol eta, iota and kappa. These results suggested the central role of REV1 in the translesion DNA synthesis. In this report, we focused on the deoxycytidyl transferase activity of REV1, since the novel activity has been maintained throughout eukaryotic evolution, implying a contribution to survival.

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  H2AXs surrounding the DSB region are phosphorylated at Ser139 to form γ-H2AX focus. Therefore, it is assumed that γ-H2AX foci are possible landmarks of DSB regions in the nucleus. However, many aspects about the mechanism of its phosphorylation has not still been elucidated.
  In the present study, we compared the formation of γ-H2AX foci in three cell lines after some DSB evoking stimuli. Apparent γ-H2AX foci were formed in DU145 and BALB-3T3 after the irradiation of proton beams(200MeV, 0.5 - 1.0 Gy) and hyperthermia(44°C, 10min), whereas those in interphase HeLa cells were faint. H2AX in our HeLa cell does not seem to lack the capability of phosphorylation, since chromosomes in mitosis revealed apparent γ-H2AX foci after the irradiation of proton beams. Moreover, replication stall brought to HeLa cells by the administration of hydroxyurea caused strong fog-like H2AX phosphorylation in nuclei, as seen in other 2 cell lines.
  These results suggest that at least 2 different mechanisms are involved in the phosphorylation of H2AX , and the major pathway in response to DSB formation is lost or diminished in our HeLa line at the interphase of the cell cycle. Relationship between H2AX and other factors concerning DSB detection and repair were further examined.

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DNA double-strand breaks (DSB) are serious damage which leads to genomic instability or cell death. Hence, mammalian cells recognize DSBs immediately and repair them when they are generated into genome DNA. Since genome DNA is compacted into chromatin structure with histone protein in higher eukaryotic cells, the chromatin structure might be remodeled to recognize and repair the damaged DNA with specific modifications of histones. The phosphorylation of histone H2AX is one of important modification in response to DNA damage. Histone H2AX is a variant of histone H2A in higher eukaryote and the C-terminus of histone H2AX is phosphorylated in response to generation of DSB. This phosphorylation is indispensable for the recruitment of DNA damage-related proteins to DSB sites. In addition, the amino acid sequence of histone H2AX suggests acetylation, ubiquitylation and sumoylation of histone H2AX and these modification may be included in DNA damage response. Therefore, we investigated the role of histone H2AX in DSB-induced damage response.
When we analyzed the phosphorylation of histone H2AX after exposure to ionizing radiation(IR), two extra-bands were detected by g-H2AX andibody. Histone H2AX is also sumoylated in N-terminus in vitro assay. Moreover, we detected that histone H2AX was acetylated on N-terminus using anti-acetyl histone H2A antibody. Thus, histone H2AX might be modified in several manners following DNA damage. We discuss the role of these modification in IR-induced foci formation and DSB repair.

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Many proteins involved in DNA replication and repair are known to undergo post-translational modification. Ubiquitination is now considered to play important roles for functioning of such proteins in DNA damage responses. PCNA, a protein that functions as sliding clamp for a number of proteins involved in DNA replication and repair, has been shown to be mono-ubiquitinated by the Rad6-Rad18 complex in cells treated with various DNA-damaging agents, and further poly-ubiquitinated by the Rad5-Mms2-Ubc13 complex. Such modifications of PCNA result in activating error-prone pathway by recruiting a DNA polymerase for translesion synthesis to the site of DNA damage or error-free pathway by damage avoidance of unknown mechanism. Recently, we have found that RFC, functioning as a loader of the PCNA clamp onto DNA, also undergoes ubiquitination in cells treated with alkylating agents. This ubiquitination is dependent on the Rad6-Rad18 complex. In addition, some mutant forms with a single amino acid substitution was found to be ubiquitinated without DNA damaging treatment when expressed in human cells. Furthermore, we constructed multiple cell lines expressing the mutant or wild-type protein with a tag and observed that the cell lines expressing the mutant protein are hypersensitive to alkylating agents while no significant difference in normal growth rate was found between the cell lines expressing the mutant and wild-type.

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Recent genetic studies in Caucasian population and European cohorts revealed several gene polymorphisms related to radiation susceptibility in breast cancer patients. This study examined the association of six of these polymorphisms in the genes XRCC1 (Arg399Gln), XRCC3 (Thr241Met), APEX1 (Asp148Glu), ATM (Asp1853Asn), SOD2 (Val16Ala), and TGFb1 (C-509T) with the risk of early adverse skin reactions within 3 months of starting radiotherapy in 399 Japanese patients with breast cancer. The patients were divided into two groups according to the NCI-CTC scoring system for radiation dermatitis: the low-grade; grade <= 1, n = 290, and the high-grade; grade > 1, n = 109. We found an association between the Arg399Gln genotype in XRCC1 and the risk of early adverse skin reactions (P < 0.015). Asp1853Asn in ATM was not polymorphic in the breast cancer patients in our study or in 115 healthy Japanese women. No Japanese patients showed early adverse skin reactions in association with the markers C-509T in TGFb1, Val16Ala in SOD2, Asp148Glu in APEX1, or Thr241Met in XRCC3. In addition, seven polymorphisms in APEX1, 53 in ATM, 3 in TGFb1, and 6 in XRCC1 were selected from the jSNP and dbSNP databases and investigated. None showed an association with the risk of early adverse skin reactions. These findings suggest that radiation susceptibility associated with gene polymorphisms may differ among ethnic groups, and highlighting the importance of analyzing specific genetic factors within individual populations.

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Maintenance of genome integrity requires both DNA repair and DNA-damage-induced cell-cycle arrest. The ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases, which are members of phosphatidyl inositol 3-kinase-like kinase family, play central roles in DNA damage checkpoints. ATM is defective in ataxia-telangiectasia (A-T), a disease characterized by cancer susceptibility and radio sensitivity. ATM is primarily activated by DNA double-strand breaks (DSBs) caused by ionizing radiation (IR) or radiomimetic drugs. ATM exists as inactive dimmers in undamaged cells but dissociates into active monomers after exposure to IR. ATM activation is stimulated by the MRE11-RAD50-NBS1 (MRN) complex. The C-terminus of NBS1, which include the MRE11 binding domain and the ATM binding domain, is required for ATM activation, whereas the N-terminus region is dispensable. ATR is the primary kinase responsible for signaling replication stress. Activation of the ATR-mediated pathway also requires NBS1. However, its precise mechanism is unknown. In this study, we established NBS cell lines expressing various truncated NBS1 proteins and analyzed checkpoint activation.

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Both of known murine thymine glycol (TG)-DNA glycosylase (TGG), mNTH1 and mNEIL1 are bifunctional with apurinic/apyrimidinic (AP) lyase activity. mNTH1 and mNEIL1 are thought to localize mainly in mitochondria and nuclei, respectively. We have reported a novel monofunctional TGG activity in nuclear extract of various mouse organs. We prepared nuclear extract following hypertonic treatment of isolated spleen nuclei. Although the monofunctional activity is quite abundant in spleen, the extract also contains large amount of 3'-OH forming activity like AP endonuclease or polynucleotide kinase (PNK). Majority of the activity was successfully separated by hydroxyapatite column chromatography. Furthermore, the activity was almost completely suppressed in the presence of 10 mM EDTA. When Western blot analysis of hydroxyapatite column chromatography fractions was performed with rabbit anti-mNEIL1 polyclonal antibody, no band was detected in the fractions having monofunctional TGG activity. The result suggests that the novel monofunctional TGG protein other than mNEIL1 or the related proteins existed in normal mouse organs. The activity was subsequently separated with ion-exchange column and analyzed by 2D-PAGE. The analyses of candidate spots were in progress. When Western blot analysis of mouse liver nuclear extract was performed with the anti-mNEIL1 antibody, three bands (about 44, 29 and 17 kDa) were observed. The apparent sizes were corresponded with those of mNEIL1 and the mNEIL1 variants, suggesting that mNEIL1 variant proteins were expressed in normal mouse liver.

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Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, bird-like face, growth retardation, and cancer predisposition. Cells from NBS patients exhibit radiosensitivity, S-phase checkpoint defect, and chromosome instability. NBS1, the gene product mutated in NBS, forms a complex with MRE11 and RAD50 that is involved in the repair of DNA double-strand breaks (DSBs) and DNA damage checkpoints. Although the function of NBS1 has been extensively studied using cell lines derived from NBS patients carrying hypomorphic mutations, the biological function of NBS1 has not fully been elucidated on the basis of NBS cells, which is devoid of NBS1. In order to investigate the NBS1 function more precisely, we tried to generate Nbs1-knockout mice. Consistent with previous reports, Nbs1^-/- mice died at early stages of embryonic development. However, we could establish the Nbs1^-/- embryo fibroblast cell line. In this cell, a truncated Nbs1 protein was still found, while this truncated protein is smaller than that in NBS patient cells. We analyzed the function of truncated NBS1 products in both NBS patient cells and Nbs1-deficient mouse cells.

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Radiation-induced DNA double strand breaks (DNAdsb) play an important role in cell death and may result in cancer if they are not repaired correctly. In mammalian cells, DNAdsb are mainly repaired through non-homologous end joining in which a key function is performed by double-stranded DNA-dependent protein kinase (DNA-PK). In this study, the relationships between radiation sensitivity and DNA-PKcs activity/expression were examined in different human thyroid cancer cell lines. Thyroid cancer cells could be clearly divided into two groups according to their radiation sensitivity. A significant correlation was observed between DNA-PK activity and protein level of DNA-PKcs. A good correlation was found between D₁₀ value and DNA-PK activity. Radioresistant cells had a high level of DNA-PKcs in the nucleus while radiosensitive cells displayed low levels of nuclear DNA-PKcs. Radiation sensitivity correlates with the protein level and activity of DNA-PKcs, suggesting that it may be a new criterion for determining the effectiveness of radiation therapy of thyroid cancers.

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Purpose: ATM, protein responsible for a rare genetic disease with hyper-radiosensitivity, is the one of the earliest repair proteins sensing DNA double-strand breaks and phosphorylates substrate proteins such as Mre11, NBS1, 53BP1, and p53. We examined the interactions between ATM and DNA-PKcs, a typical NHEJ repair protein, after cells exposure to high LET irradiation and the comparison was made with x-rays. Materials and methods: Normal human (HFLIII, MRC5VA), and AT homozygote (AT2KY, AT5BIVA, AT3BIVA) cells were used. ATM kinase specific inhibitor KU55933 was added 10uM for 1hr before irradiation. X rays and high LET radiation (carbon (70keV/um) and iron (200keV/um)) were used. The efficiency of DSB repair (NHEJ) was detected by immunofluorescence technique with DNA-PKcs (pTh2609, pS2056) and ATM (pSer1981) antibodies. Results and discussion: Compared with normal cells, AT cells irradiated with high LET radiation showed significantly higher radiosensitivity. In normal cells, the phosphorylation of DNA-PKcs was clearly detected after high LET irradiation, though the peak of phosphorylation was delayed when compared to X-irradiation. In contrast, in AT cells only a few phosphorylation foci were detected when irradiated with high LET radiation. These data suggest that the phosphorylation of DNA-PKcs with X-ray is mostly ATM-independent, and the phosphorylation of DNA-PKcs with high LET radiation is mostly ATM-dependent probably due to its complex nature of DSB. The analysis of DSB repair proteins is underway using Western blotting. We are also examining the possibility of activation of HRR pathway when NHEJ function is reduced using antibodies such as Rad51.

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Clustered DNA damage induced by a single radiation track is a unique feature of ionizing radiation. Recent in vitro studies have shown that the repair of lesions within clusters may be retarded, but less is known about the processing and the mutagenic effects of such clustered damage in vivo. Using a plasmid-based assay in Escherichia coli, we have investigated the mutagenic potential of bistranded clustered damage sites which consist of 8-oxo-7,8-dihydroguanine (8-oxoG) and dihydrothymine (DHT) at defined separations. We found a significantly higher mutation frequency for the clustered DHT + 8-oxoG lesions than that for either a single 8-oxoG or a single DHT in wild-type and in glycosylase-deficient strains of E. coli. From these results and similarities with the mutability of respective 8-oxoG + AP clusters, it is suggested that removal of 8-oxoG within clustered damage site is retarded, probably reflecting the preferential excision of DHT initially. For a certain fraction of clusters, however, 8-oxoG may be initially removed from the cluster. To gain further insights on the processing of the DHT + 8-oxoG cluster, several potential intermediates after 8-oxoG removal were assessed. DHT + AP or DHT + Gap containing cluster had a relatively low mutation frequency, whereas AP + AP or Gap + AP cluster was found to strongly retard replication. These results led us to suggest that, when either 8-oxoG or DHT is initially excised from a cluster containing 8-oxoG and DHT, the base remaining within the resulting damage will not be further converted to an AP site or to a single strand break in vivo.

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Clustered DNA damage is defined as two or more lesions induced within 1-2 helical turns (10-20bp) of DNA. Ionizing radiation-induced DNA damage, especially DNA double strand breaks (DSB) are considered to be a major factor for radiation induced chromosomal aberration and cell death. Many studies have shown the experimental evidence of DSB induction and its repair efficiency, as well as intracellular signal transduction caused by DSB. It is predicted that the non-DSB clustered damage shows high biological effects. It is, however, technically difficult to directly detect non-DSB clustered damage site as well as its effect in living cells. In this study, we investigated the potential of single strand break (SSB) to influence the mutagenicity of base lesion in Escherichia coli. We used plasmid based assay developed in our laboratory to measure the mutation frequency induced by bistranded clustered damage. As models of clustered damage, we used synthesized oligonucleotides carrying a SSB and 8-oxo-7,8-dihydroguanine (8-oxoG) at a restriction enzyme recognition site. Damaged DNA was transfected into wild-type or glycosylase-deficient strains (fpg, mutY, fpg mutY) of E coli and mutation frequency was assessed by the inability to cut by the restriction enzyme. The mutation frequency of 8-oxoG was enhanced by the SSB situated on opposite strand. When SSB was present in tandem on the same strand of an 8-oxoG, however, these clustered damages showed lower mutation frequency than a single 8-oxoG lesion. These results suggest that SSB opposed to 8-oxoG have an inhibitory effect on repair of 8-oxoG, but that, in the case of 8-oxoG and SSB positioned in tandem, 8-oxoG can be removed, at least partly, by the repair process of SSB. We propose that the mutagenic potential of 8-oxoG depends on whether SSB is located on either strand, same or opposite, to 8-oxoG.

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DNA mismatch repair (MMR) is a conserved process correcting nucleotide mismatches generated during DNA replication. In eukaryotes, the MSH2-MSH6 hetero dimmer initiate the repair of base-base and small insertion or deletion mismatches while the MSH2-MSH3 hetero dimmer recognize larger insertion or deletion mismatch. By disrupting either MSH2 and MSH2 we generated two DT40 cell lines expressing either MSH6 and MSH3 or MSH2-MSH6 hetero dimmer, respectively. We found that MSH2-dificient cells exhibit hypersensitivity to alkylation base damage induced by MMS and to nucleotides damage by UV irradiation or 4NQO. Conversely, MSH3 deficient cells exhibit similar sensitivity as wild type cells to MMS, 4NQO and UV. This MSH2 deficient, hypersensitive phenotype was synergistically increased by concomitant deletion of major BER proteins, either Polbeta or FEN1, or NER protein, XPA. Interestingly, a MSH3/XPA double deficiency produce synergistic effect against UV damage even though MSH3 deficient cells were not show sensitivity to UV irradiation. Taken together, these results suggest that functional links exist between MMR and both base and nucleotide excision repair in vertebrate cells.

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Genomic instability has been commonly observed in various tumor cells and is considered the earliest cellular event in the process of carcinogenesis. Although genomic instability can be induced by ionizing radiation the molecular mechanisms underlying this process are poorly understood. Cell cycle checkpoints play a key role in cell survival following DNA damage. The failures of DNA repair and cell cycle regulation in response to DNA damage are thought to be important factors in the early stages of genomic instability. Several genes associated with DNA damage repair and cell cycle regulation are also related to radiation susceptibility. By a large-scale expression profiling of 15 human cell lines and three mouse strains having varying degrees of susceptibility to ionizing radiation, we identified 200 genes correlated with radiation susceptibility. We constructed an shRNA library of these 200 genes. We screened this library using 96-well format cell proliferation assay based on loss of function analysis, after X-irradiation. We identified 12 genes involved in radiation susceptibility. Eight of the 12 genes have been reported to directly or potentially regulate cell cycle, and biological functions of remaining four genes have not been reported. We then performed cell cycle analysis of cells transfected with shRNA vectors against these four genes following X-irradiation, and found that knockdown cells of one of these 4 genes did not accumulate in G₂/M phase. This result suggests that this gene is associated with the G₂/M checkpoint after DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, and to develop new radiation sensitizing agents for radiotherapy.

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Base excision repair (BER), a predominant pathway to repair small base damages, includes two sub-pathways, short-patch which is dependent on polymerase β (polβ) and long-patch BER which is dependent on PCNA. Recent studies have shown that mouse polβ knockout cells cannot repair the methylated bases by BER in both G1 and S phases. But it is possible that other BER enzymes can be affected in the absence of polβ. In this study, to compare the mode of BER in human cells, we generated a stable human cell line in which polβ expression has been decreased using RNA interference and analyzed the effect of methyl methansulfonate (MMS) in wild type and knockdown cells. We first found the target RNA sequence and constructed the plasmid which expressed shRNA. After transfection to HeLa RC355 cells, we obtained the stable zeocin resistant cells and analyzed siRNA-induced 50% silencing of polβ using Western Blot. The cells were treated for 1h with different concentrations of MMS and examined the survival after 10 days. The number of colonies of knockdown cells was apparently decreased at 1.5mM or higher MMS concentrations. These results suggest the vital role of human polβ in the repair of MMS-induced damages.

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The proteins responsible for radiation sensitive disorders,NBS1, kinase ataxia-telangiectasia-(A-T)-mutated (ATM) and MRE11, interact through the C-terminus of NBS1 in response to the generation of DNA double-strand breaks (DSBs) and are all implicated in checkpoint regulation and DSB repair, such as homologous recombination (HR). We measured the ability of several NBS1 mutant clones and A-T cells to regulate HR repair using the DR-GFP or SCneo systems. ATM deficiency did not reduce the HR repair frequency of an induced DSB, and it was confirmed by .findings that HR frequencies are only slightly affected by deletion of ATM-binding site at the extreme C terminus of NBS1. In contrast, The HR-regulating ability is dramatically reduced by deletion of the MRE11-binding domain at the C-terminus of NBS1 and markedly inhibited by mutations in the FHA/BRCT domains at the N-terminus. This impaired capability in HR is consistent with a failure to observe MRE11 foci formation. Furthermore, normal HR using sister chromatid was completely inhibited by the absence of FHA/BRCT domains. These results suggested that the N- and C-terminal domains of NBS1 are the major regulatory domains for HR pathways, very likely through the recruitment and retention of the MRE11 nuclease to DSB sites in an ATM independent fashion.

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Once DNA double-strand breaks (DSBs) are generated into the genome DNA, the cells arrest the progression of cell cycle immediately and then repair the damaged DNA. ATM kinases plays a central role in these DNA damage response. ATM is the mutated gene in radiation-hypersensitive Ataxia-Telangiectasia syndrome (A-T). AOA (ataxia oculpmotor apraxia) 1-3 are also characterized as human autosomal recessive ataxias as well as A-T. The responsible genes of AOA1 and AOA2 are Apraxin and Senataxin respectively, suggesting that these gene products have roles in DNA damage response. Hence, the mutated gene in AOA3 may also function in DNA damage response. Therefore, we investigated the DNA damage response in lymphoblastoid cell ( ATL2) and fibroblast (PSF) from AOA3 patient. Both ATL2 and PSF cells showed the hypersensitivity to exposure of ionizing radiation (IR) and treatment of camptotesin (CPT), suggesting that AOA3 cells have defects in DNA damage response. While, the autophosphorylation of ATM on Ser1981, which is a key phenomenon for regulation of cell cycle checkpoints, was induced at the normal level in ATL2 cells. However, ATL2 cells showed the aberration in p21 induction, controlled by p53, and ATM-dependent phosphorylation of SMC1. Moreover, PSF cells showed the abnormality of p53 induction and SMC1 phosphorylation. These results suggested that the mutated gene in AOA3 syndrome functions in DNA damage response and is involved in the regulation of p53 and SMC1.

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DNA-protein cross-links (DPCs) are ubiquitous DNA lesions and are produced by ionizing radiation, UV light, aldehydes, and other genotoxic compounds. However, the repair mechanism of DPCs is largely unknown. In the accompanying genetic study, we have shown that DPCs are repaired by nucleotide excision repair (NER) and homologous recombination in E. coli. In the present study, we analyzed the biochemical aspects of DPC repair by NER. Since our genetic study suggested that the repair of DPCs by NER was dependent on the size of cross-linked proteins, we systematically constructed oligonucleotide substrates containing peptides and proteins of various sizes (0.2-44 kDa). DPCs were introduced as cross-links between oxanine in DNA and proteins or peptides. The DPC substrates were incubated with UvrABC nuclease (prokaryotic NER proteins), and products were analyzed by denaturing PAGE. As for other bulky DNA lesions, UvrABC incised the 8th and 5th phosphodiester bonds 5' and 3' to DPC, respectively, yielding a 12mer fragment. However, the incision efficiency decreased with increasing the size of cross-linked proteins. The incision efficiency was marginal for 14-16 kDa proteins, indicating that NER cannot process oversized DPCs. To confirm the in vivo role of NER, E. coli cells were treated with formaldehyde (FA), a DPC-inducing agent, and chromosomal DPCs were analyzed. DPCs were actively removed in wild type cells but poorly in NER-deficient cells.

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Ionizing radiation and UV light induce DNA-protein cross-links (DPCs). In the present study, we analyzed the sensitivity of repair-deficient E. coli mutants to DPC-inducing agents to elucidate the DPC repair pathway. Formaldehyde (FA) and 5-azacytidine (azaC) were used as DPC-inducing agents. FA covalently traps DNA binding proteins of various sizes, and azaC specifically traps the DNA cytosine methylase (Dcm in E coli K12). When cells were treated with FA or azaC, the uvrA mutant exhibited hypersensitivity to only FA, whereas the recA mutant exhibited hypersensitivity to both FA and azaC, indicating that FA-induced DPCs are repaired by both nucleotide excision repair (NER) and homologous recombination (HR), whereas the azaC-induced DPC is processed by HR exclusively. Moreover, the recB but not recF mutant was hypersensitive to FA and azaC, indicating that the HR of DPCs proceeds through the RecBCD pathway and that replication of DPC-containing chromosomes results in the formation of double-strand break ends processed by RecBCD helicase/exonuclease. In vitro and in vivo analysis of DPC repair by the NER system revealed that the excision efficiency for DPCs was dependent on the size of cross-linked proteins, which accounted for the differential involvement of NER in the repair of FA- and azaC-induced DPCs. We are also assessing the sensitivity of other mutants deficient in DNA polymerases, base excision repair, and other repair pathways. These data will also be presented in the meeting.

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Radiation cancer therapy based on the predictive assay might improve the outcome of cancer therapies in patients with normal p53 function. In more advanced cancer therapy, it is crucial to develop therapeutic strategies against mutated p53 cancer cells, and thus the strategy of radiation enhancement regardless of p53 status is currently the focus of intense research. We previously demonstrated that siRNA targeting NBS1 enhanced radiation sensitivity of cancer cells p53-independently. In this report, we showed siRNA targeting XIAP (XIAP-siRNA) enhanced radiation sensitivity and radiation-induced apoptosis more effectively even in mutated p53-transfected non-small cell lung cancer cells (H1299/mp53) than in the wild-type p53-transfected cells (H1299/wtp53). Furthermore, we showed that pifithrin-α, an inhibitor of p53 activation, did not result in efficient radiation sensitization in XIAP -siRNA-treated H1299/wtp53 cells. These results suggest that dysfunction of p53 as a transcription factor is not a cause of radiation sensitization in XIAP-siRNA-treated H1299/mp53 cells.

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Ginseng is one of the most widely recognized herbal drug and reported to have a wide range of therapeutic and pharmacological uses. In the present study oral administration of Panax ginseng extract at 10 mg/kg body weight/day for 4 days before whole body exposure to radiation was found to be effective with the LD_50/30 values, giving a dose reduction factor (DRF) of 1.3223. Whole body gamma radiation (8.0 Gy) exposure to mice significantly elevated the transaminases activity (ALT & AST) and alkaline phosphatase activity in serum. It also decreases the antioxidant potential of cell (GSH, P<0.001; glutathione peroxidase, P<0.05; glutathione-S-transferase, P<0.05; glutathione reductase, P<0.01; catalase, P<0.05 and SOD, P<0.01). Due to decrease in antioxidant potential and generation of reactive oxygen species a highly significant increase (P<0.001) in malondialdehyde (lipid peroxides) formation was also observed in liver. Our results demonstrated that Panax extract (10mg/kg body weight, orally) is effective in protecting the gamma radiation induced pathological alterations in liver. The pre-treatment of Panax ginseng significantly decreases the transaminases activity and alkaline phosphatase activity in serum. It also significantly increases the antioxidant potential of cell and thus decreases the level of lipid peroxides in liver of mice, suggesting its role in protection against radiation induced membrane damage. Pre and post treatment of Panax ginseng with sodium arsenite (4.0mg/kg body weight i.p.) also reduces the arsenic induced cellular damage in liver of Swiss albino mice. It significantly declines the level of lipid peroxides, transaminases and alkaline phosphatase activity by enhancing the antioxidant defenses of liver. From the present study it may be speculated that the Panax ginseng could elevate the antioxidant potential of cell and thus reduces the radiation and arsenic induced hepatic lesions.

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In the present study, we report the radioprotection activity of γ-tocopherol-N,N-dimethylglycine ester (γ-TDMG), a potent post-irradiation protector against bone marrow death. Mice (C3H, 10 weeks, male) were injected with γ-TDMG intraperitoneally before or after X-irradiation. γ-TDMG was suspended in 0.5% methyl cellulose solution. The protection activity was evaluated as the 30-day survival of mice after the irradiation. γ-TDMG showed potent protection activity in both pre- and post-irradiation administration, and post-administration was more effective. The 30-day survival was 98% (n=42). The optimal concentration was found to be 100 mg/kg body weight. When administered at 1 h, 10 h and 24 h post-irradiation, the survival was 86, 75, and 40%, respectively, showing that it was effective even administered at 24 h after irradiation. Subcutaneous administration was also effective. An analog, α-TDMG also showed comparable potent radioprotection, and other analogs, γ- and α-tocotorienol dimethylglycine ester were also effective. γ-TDMG is a single component, stable molecule of new class of radioprotector.

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Intraperitoneal injection of beta-glucan greatly reduces mortality of mice exposed to whole body X-ray radiation and tumor growth in tumor bearing mice. Since the leukocyte and lymphocyte number was increased by a single dose of beta-glucan, the radioprotective effect of beta-glucan is probably mediated at least in part by a hemopoietic action in irradiated mice. In addition, both of the NK and LAK activity increased significantly by repeated dose of beta-glucan. Augmented immunological activity as seen in increased NK and LAK activity by beta-glucan seems to play a role in preventing secondary infections associated with irradiation, and to contribute probably to attenuated tumor growth in tumor-bearing mice through enhanced anti-tumor immunity. From these, beta-glucan is expected to be promising for the treatment of cancer patients receiving radiotherapy.

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The Fibroblast growth factor (FGF) family polypeptides regulate cell proliferation, differentiation, and metabolisms. FGFs need heparan sulfate (HS)/ heparin as a co-factor to activate FGF receptors (FGFRs). Although FGF1 and FGF2 are structurally related, they are different in receptor specificity and heparin dependence; activity of FGF1 is more dependent on exogenous heparin than FGF2. We previously reported systematic construction and expression of various forms of FGF1/FGF2 chimeric proteins in an attempt to address structure-activity relationships as well as creating useful molecules. One of those, designated FGF-C, in which residues 44-86 of FGF1 was substituted with the corresponding region of FGF2, was studied in the present study. The FGF-C was analyzed for its heparin dependence, receptor specificity and protective potential against radiation-induced damage. A cell line that has neither endogenous HS nor FGFR was stably transfected with various types of FGFR, and was used to evaluate their proliferation in response to FGF1 and FGF-C in the presence and absence of exogenous heparin. Not only FGF-C demonstrated FGF1-like receptor specificity and potency in the presence of heparin, its activity in the absence of heparin was more potent than FGF1. When C3H mice were intraperitoneally administered with FGF-C prior to whole body γ-irradiation (10Gy), it stimulated regeneration of small intestine crypts with a potency similar to FGF1. These results suggest clinical usefulness of FGF-C for its protective potential against radiation-induced damage.

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The mechanism of radioprotective effect by DMSO is believed that hydroxyradical are scavenged and DNA strand breaks are suppressed immediately after irradiation. However, this theory is not completely confirmed by experiments. Here, we examined the mechanisms of radioprotective effect by 0.5% DMSO treated CHO cells. First of all, significant reduction of micronuclei formations by X-irradiation was observed in 0.5% DMSO treated cells. Cell killing effect was also suppressed by this DMSO treatment. Interestingly, this suppressive effect of DMSO on micronuclei induction was not observed in Ku80 deficient xrs5 cells and DNA-PKcs deficient Scid cells. These results imply that there is a relationship between radioprotective effects of DMSO and non-homologous end joining pathway (NHEJ). We next examined the kinetics for disappearance of 53BP1 foci after X-irradiation. In the case of 15 min after irradiation, the number of foci were not different between DMSO treated cells and non-treated cells. However, the number of foci at 1 hr and 2 hr after irradiation were reduced in time dependent manner, and faster disappearance of foci was observed in DMSO treated cells. It is suggested that the repair kinetics for DNA double strand breaks are faster in DMSO treated cells. We believe that radioprotective effect by DMSO is not caused by suppression of initial DNA damage level, but caused by effective clearance of DNA double strand breaks through the enhanced NHEJ repair.

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Carbon-ion therapy has been shown to be effective for a treatment of malignant tumors because of a good dose distribution and a high relative biological effectiveness (RBE). We are able to calculate physical processes in a phantom irradiated by heavy-ion beams with the advancement in computer technologies. However, no general theory to calculate biological effect has not been established for heavy-ion beams due to the extremely complexities of chemical and biological processes. A certain model is necessary to predict the biological effects on any irradiation condition for the heavy-ion therapy of high precision.
So far, we found that a combination of the Kiefer-Chatterjee track structure model and the Hawkins' Microdosimetric Kinetic Model (MKM) is very useful in explaining the survival curve of aerobic in-vitro cells for high-energetic ion beams. However, there are actually hypoxic cells in a large tumor, which are typically radiation-resistant. This time, we verified practical effectiveness of the MKM for predicting the biological effect of hypoxic cells for heavy-ion beams when we have known the MKM parameters of the same cells in aerobic condition.
In the result, we could estimate the MKM parameters in the hypoxic condition from the parameters in the aerobic condition for V79 and HSG cells by assuming the oxygen enhancement ratio (OER) of 3 for X-rays and a half radius of aerobic domain which represents a considerable region for radiation effect in the MKM. The reason why the domain in the hypoxic condition became the half radius of the aerobic condition is thought of as loss of production and dispersion of active oxygen (oxygen radical, hydrogen peroxide, ozone etc.) which causes lethal damages in the cell.

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Edaravone, a clinical drug used widely for the treatment of acute cerebral infarction, is reported to scavenge free radicals. In the present study, we investigated the radioprotective effect of edaravone on X-ray-induced apoptosis in MOLT-4 cells. Apoptosis was determined by the dye exclusion test, AnnexinV-PI binding assay, cleavage of caspase, and DNA fragmentation. We found that edaravone significantly suppressed the X-ray-induced apoptosis. The amount of intracellular ROS production was determined by the chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate system. We found that the intracellular ROS production by X-irradiation was completely suppressed by the addition of edaravone. The accumulation and phosphorylation of p53 and the expression of p21^WAF1, a target protein of p53, which were induced by X-irradiation, were also suppressed by adding edaravone. We conclude that the free radical scavenger edaravone suppresses X-ray-induced apoptosis in MOLT-4 cells by inhibiting p53.

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Radio-protective effect of AA-2G (L-ascorbic acid 2-glicoside ) was evaluated in apoptosis induction of mouse spleen after total body 5Gy irradiation. We also investigated the effect of AA-2G for the anti-oxidative enzymes activation (catalase and superoxide dismutase) in plasma and splenocytes following irradiation. The ascorbic acid concentration in mouse plasma increased and maintained a high level during 30-90 min after oral administration of AA-2G. After 1 hour of AA-2G administration at the concentration of 25mg/body, the concentration of the ascorbic acid in plasma was increased 4-10 times higher than the control. Mice were irradiated in plastic tubes by gamma-ray at 5 Gy. The splenocytes were isolated 1-24 hours after total body irradiation and the apoptosis was measured with Elisa kit (Roche). The activation of the anti-oxidative enzymes in plasma and splenocytes was measured by catalase or SOD assay kit (Calbiochem). The treatment of AA-2G reduced the apoptosis induction of mouse splenocytes and increased the activation of catalase in splenocytes following irradiation.

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In the present study, we examined radioprotecition of tocopherol monoglucoside (TMG), a water-soluble vitamin-E derivative. The radioprotection by TMG was evaluated as 30-day survival of C3H/HeSlc 10-week-old male mice after X-ray irradiation. TMG (650 mg/Kg BW) was administered subcutaneously to mice immediately after 7.0 Gy (0.5 Gy/min) whole body X-irradiation. The survival of TMG-injected mice after irradiation was 80.0 %, while that of control mice was only 26.7 %. We also evaluated the prevention by TMG of radiation-induced tumors of rat mammary. When dams at day 21 of lactation were exposed to X-rays at a total dose of 1.5 Gy (0.3 Gy/min) and then treated with diethylstilbestrol as a tumor promoter, the incidence of mammary tumors (81.8 %) was increased compared to that of the non-irradiated control (0 %). The administration of TMG (600 mg/ Kg BW i.p.) after the irradiation significantly reduced the incidence (59.0 %). TMG is an effective agent for protection of bone marrow death of mice even when administered subcutaneously, and for prevention of radiation-induced mammary tumors of rats.

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[Purpose]Generally, we can confirm the effect of radioprotection agent (cysteine, cysteanime, amifostine, etc) by administering it before irradiation. There were some reports that mutation rate fall down by administering the ascorbic acid which absorb long lived radicals after irradiation. Therefore, in our study, we are aimed at studying cell death and the radioprotection effect of the ascorbic acid for the DNA double-strand break.
[Result] With administration of ascorbic acid after irradiation, we confirmed that DNA damages were decreased in G0-PCC assay and gamma-H2AX assay. We also observed radioprotection effect in cell survival.
[Discussion] It is important that a radioprotection effect appears even if we administer the ascorbic acid not only before irradiation but also after irradiation. This effect may be whether ascorbic acid scavenges long lived radicals or adding ascorbic acid may promote PLDR. However, the detailed mechanism is not yet clear. Further development can expect the protection of the normal tissues at radiation therapy, an emergency measure at the time of the emergency radiation exposure and a protection of the astronaut based on this study. Ascorbic acid has a few side effects and it is generally a material recognized well. In addition, we are carrying out further research of ascorbic acid with heavy charged particles.

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Although high-dose radiation exposed to whole body results in gastrointestinal injury and finally leads to death, a real effective radioprotector has not yet been established. Ursodeoxycholic acid (UDCA), a major component of bear bile, has been widely used to treat pain, inflammation, and hepatobiliary diseases. Recently, several potential mechanisms of the anti-apoptotic action of UDCA have been proposed, including inhibition of mitochondrial membrane depolarization and enhancement of methionine adenosyltransferase activity. In the present study, we investigated whether UDCA can protect rat intestinal epithelial IEC-6 cells from high-dose irradiation. IEC-6 cells were cultured in DMEM with 5% FCS and were treated with 50, 100, and 200 microM UDCA alone or in combination with inhibitors of MEK and p38 MAPK or PI3K, and irradiated with gamma rays at a dose of 20 Gy. Analyses using Hoechst staining showed that UDCA suppressed radiation-induced apoptosis of the cells in a dose-dependent manner. UDCA in the presence of an inhibitor of MEK further decreased the number of apoptotic cells, whereas PI3K inhibitor attenuated the anti-apoptotic effects of UDCA. Western blot analyses showed that treatment with UDCA inhibited phosphorylation of ERK, enhanced phosphorylation of Akt, but did not affect p38 MAPK phosphorylation in irradiated IEC-6 cells. UDCA also increased expression of Bcl-2 and Bcl-xL and suppressed caspase 9 activation, but did not change expression of Bax and cytochrome c. In conclusion, UDCA protects intestinal cells from irradiation-induced apoptosis in vitro through inhibition of the MEK/ERK pathway, activation of the PI3K/Akt pathway, and via inhibition of caspase/mitochondria pathways.

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Purpose: It has been reported that heparan sulfate (HS) participates in almost every stage of leukocyte transmigration through the blood-vessel wall in inflammatory responses. However, it is not known whether HS administration could prevent radiation-induced intestinal injury. In this study, we investigated the effect of HS on local (slit) radiation-induced intestinal injury. Methods: ICR mice, 8-10 weeks old, 25-31 g body weight, were used. Mice were anesthetized and irradiated to the slit width of 7.5 mm or 10 mm abdominal region (X-ray; a single dose of 30 Gy, 150 kV, 5 mA, 1.0 mm Al + 0.2 mm Cu filters, 0.75 Gy/min). Mice were intraperitoneally administered with HS at a dose of 1 μg/g body weight at 1 day before irradiation and 5 days to 10 days daily after irradiation. Mice were weighed daily after irradiation, and the survival was recorded. At 13 day or 30 day after irradiation, the intestine of mice was obtained to assay histological change by Haematoxylin-eosin staining. Results: The body weight of mice treated with HS increased significantly compared with the untreated control on day 8 after irradiation with slit width of 7.5 mm. In addition, the survival of mice treated with HS was significantly higher than that of the untreated control mice on day 13 after irradiation with slit width of 10 mm. In the histological observation, the radiated control mice showed ulceration and granulation tissue formation in the intestinal mucosa. The regenerating intestinal crypts were also less. In contrast, the mice treated with HS exhibited a lot of regenerating crypts and healing stage of the ulceration. Conclusions: These results suggest that HS may play a role in prevention against radiation-induced intestinal injury. Further experiments will be needed to study the protective mechanism of HS.

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Several FGFs are able to reduce and improve radiation-induced tissue damage, but there are no reports about whether FGF12 plays a role in radiation sensitivity. In this study, we demonstrated for the first time that FGF12 was expressed in the human leukemic mast cell line HMC-1. The overexpression of FGF12 in HMC-1 cells decreased the ionizing radiation-induced apoptosis, and siRNA-mediated repression of FGF12 expression augmented the apoptosis in HMC-1 cells. In contrast, the mitogen-activated protein kinase (MAPK) scaffold protein islet brain 2 (IB2), which was reported to bind to FGF12, was also expressed in HMC-1 cells, and suppressed radiation-induced apoptosis by itself; however IB2 did not interfere with the anti-apoptotic effect of FGF12. The FGF12-IB2 complex was expected to be involved in the p38 MAPK signaling pathway; however, SB203580 (an inhibitor of p38 MAPK) did not suppress the anti-apoptotic effect of overexpression of FGF12. Instead, FGF12 strongly suppressed the marked augmentation of apoptosis induced by inhibition of the MEK/ERK pathway with PD98059. In addition, the expression of Fgf12 transcripts was detected in murine cultured mast cells, which were derived from bone marrow or fetal skin. These findings suggest that FGF12 suppresses the radiation-induced apoptosis in mast cells independently of IB2.

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Fibroblast growth factors (FGFs) have such important roles in numerous biological events such as angiogenesis, wound repair and so on, that they may also be able to protect intestine against radiation injuries. FGF receptor 2 IIIb (FGFR2b; KGFR) is a high-affinity receptor for FGF1, FGF7 and FGF10, which can be expressed only by epithelial cells. In this study, we demonstrated that total body irradiation of γ-ray induced the expression of KGFR transcripts in jejunum of Balb/c mice, although these transcripts were not detected until 16 hours after irradiation. In contrast, other FGFRs, which could react with FGF1, but not FGF7 and FGF10, were expressed in spite of irradiation. Intraperitoneal administration of FGF1, FGF7 or FGF10 at 24 hours before irradiation increased the crypt number at 3.5 days after irradiation. Especially, FGF1 induced the more crypt survival than FGF7 or FGF10. The number of apoptotic cells in crypts of jejunum was also lower in FGF1-treated mice than that in other FGF-treated mice at 16 hr after irradiation. In addition, FGF1 increased the LD_50/6 of C3H mice. Moreover, the administration of each FGF at 24 hr after irradiation also increased the crypt number; however it did not show any significant difference in the level of increase among the FGF-treated mice. These findings suggest that FGF1 protects jejunum against the radiation-induced injuries more effectively than FGF7 and FGF10, because FGF1 could react with all other FGFRs except KGFR at an early phase of radiation exposure.

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Polaprezinc, an anti-gastric ulcer drug, is a chelate compound consisting of zinc and L-carnosine. Polaprezinc has been shown to prevent gastric mucosal injury. The mechanisms of anti ulcer effects of polaprezinc have been thought of its antioxidative actions, cytoprotective effects in gastric mucosa and stimulation of wound healing. The effect of polaprezinc on ionizing radiation-induced apoptosis was studied in the jejunal epithelial crypt cells of rats. Seven- to 8 week-old Wistar rats, which were treated 100 mg/kg of polaprezinc as polaprezinc group or 2 % carboxymethylcellulose sodium (CMC) as control group, were exposed to whole body X-ray irradiation at 2 Gy. The jejunum was resected at 1, 2, 4 and 6 hours after irradiation and immersed in neutral-buffered formalin. After fixation, the jejunum was cut longitudinally and processed for embedding in paraffin blocks. The number of apoptosis was counted in each crypt in H&E-stained sections. Immunopositive cells for TUNEL and active caspase-3 were also counted. Accumulation of p53 and the expression of p21 in jejunum after irradiation was examined by Western blot analysis. Polaprezinc treatment given prior to radiation resulted in the significantly reduction in number of apoptotic jejunal crypt cells, and decreases in p53 accumulation and p21 expression. Polaprezinc has protective effect against ionizing radiation induced apoptosis in jejunal crypt cells by way of p53 pathway.

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We reviewed an anticancer effect and radiation protection of various agaricus. We reviewed antioxidation, SOD activity, immunisation activity to review mechanism. We transplanted an SCC -7 cancer cell using C3H and performed 6Gy/ three times division local irradiation. We administered it every day, and the medication method of various agaricus measured it. We used an SOD activity detection kit as an antioxidation experiment and tested it. We used it and analyzed CD4/CD8 by flow cytometry in T lymphocyte of C57BL mouse.In addition, we examined IL2, IL12. With control group, protection of Japanese product agaricus and T lymphocyte of a Brazilian product agaricus treated group was found in comparison with irradiation group. In addition, a rise of humoral immunity is thought about from IL2, activity of IL12. We compared it with control group, and, about antioxidation action, activity of SOD was recognized glucan treated group. In addition, tumor growth of various agaricus treated groups was inhibited. Various agaricus lowers activity of dehydrase, and it is thought that we disturbed metabolism of tumor cells while, on the other hand, putting up activity of a catalase. It is thought that we restrain fissiparity of a cancer cell by immunisation activity by glucan when various agaricus causes cell protein and a fall of a division index.

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Exploration of new mitigators, which could be effective if administered after accidental irradiation exposure before the appearance of overt biological consequences, is useful to improve the prognosis of victims as well as to develop more effective and safe clinical therapy. As the cell killing effect plays an important role in the postnatal death (bone marrow death) induced by a high dose fetal exposures (6.5Gy) in the very late fetal period of gestation, in this study combined post-irradiation application of anti-apoptosis reagents was tested as a strategy aiming at reduction of the postnatal mortality.
Pregnant ICR mice were exposed to whole body X-irradiations on E18 at a dose of 6.5Gy and a dose rate of 1.8Gy/min, which could result in about 40% of death in the neonates before weaning. Sodium orthovanadate (Na₃VO₄, VD) at a dose of 15 mg/kg, or its combination with a pan-caspase inhibitor (Z-VAD) at a dose of 1 mg/mouse was intraperitoneally administrated to the pregnant mice 5 minutes after the irradiation. The dams were left for delivery and the neonates were monitored for postnatal survival and development (body weight). The hemogram of peripheral blood and bone marrow of femora in the young offspring (7 weeks old mice) were analyzed respectively with a blood cell differential automatic analyzer and the micronucleus test. Administration of VD alone significantly reduced the detrimental effect on postnatal survival and development, and combined application (VD+Z-VAD) showed a more statistically remarkable efficacy for some endpoints. These findings indicate that administration of combined anti-apoptosis agents would have a great possibility for clinical application in the future as a therapeutic treatment for high dose radiation exposure.
This work was supported in part by a grant for a project of the Radiation Emergency Medical Preparedness by the NIRS.

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Cerebellum is a vital organ responsible for the motor coordination and recently it has been reported to be involved in cognitive function. Reactive oxygen species are implicated in neurodegeneration and cognitive disorders due to higher vulnerability of neuronal tissues. Therefore, present study aimed at investigating the role of melatonin against high-LET ⁵⁶Fe particle irradiation induced oxidative damage and apoptosis in mice cerebellum. Radiation induced oxidative damage was examined using a neuronal-specific terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), quantitative histopathology, DNA damage (comet assay), carbonyl content and 4-HAE + MDA (4-hydroxyalkenal + malondialdehyde) status of cerebellum. Radiation exposure augmented the number of TUNEL positive cell, DNA migration in the comet tail and carbonyl and 4-HAE + MDA level of cerebellum. Melatonin pretreatment significantly inhibited the oxidative damage to bimolecules as well as cerebellar apoptosis. Melatonin treated irradiated group showed higher counts of intact Purkinje cell as compared to vehicle treated irradiated mice. In addition, radiation induced augmentation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and decline of total antioxidant capacity (TAC) in serum were also ameliorated by melatonin pretreatment. Present results represent the potential evidence supporting the antioxidant and neuroprotective function of melatonin.

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Purpose Radiation response is complex especially when the effect is assessed in a tissue which naturally accommodates different types of cells. In this work we examined whether interactions between epithelial and mesenchymal cells can modulate the extent of radiation-induced DNA damage in one or both type of cells. Materials and Methods Human primary thyrocytes (PT, normal epithelial cells) and normal diploid fibroblasts (BJ, mesenchymal cells) after homologous or heterologous conditioned medium transfer were irradiated with a single acute dose of γ-rays. Radiation-induced DNA damage was evaluated in terms of the number γ-H2AX nuclear foci per cell or by Comet assay. Results Reciprocal conditioned medium transfer prior to irradiation resulted in the reduction of DNA damage in both types of cells indicating the involvement of paracrine soluble factors. The DNA-protected state of cells was achieved within minutes after heterologous conditioned medium transfer. The diminished DNA damage was not detected after reciprocal conditioned medium transfer between BJ and epithelial cancer cell lines. Conclusions The results imply the existence of paracrine soluble factor-mediated reciprocal interactions between normal epithelial and mesenchymal cells which protect DNA from radiation-associated genotoxic stress.

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[Purpose] It is expected that novel 2-nitroimidazole acetamide (TX-1877) derivatives which were designed and synthesized by H.Hori and colleagues have the activities of radiosensitization, tumor growth control, suppression of metastasis and immunopotentiation. In the present study it was investigated the hypoxic cell radiosensitization effect of the derivatives.
[Materials and Methods] Chinese hamster V79 cells were grown in log phase in alpha-MEM supplemented with 10% FBS, trypsinized and suspended in the medium supplemented with a sensitizer (1mM). Cell suspension of 0.5 ml in a glass tube was flushed with mixed gas (95% N₂+5% CO₂) for 1 hour at room temperature and then irradiated with 6MV X-rays. After irradiation cells were plated and incubated on dishes for colony formation assay. TX-2068 (addition of sugar group into TX-1877), TX-2243,TX-2244,TX-2245, TX-2246 (additon of Ac) and etanidazole were used as the sensitizer.
[Results] Oxygen enhancement ratio of hypoxic cells after irradiation was 2.7 based on 10% survival dose (D₁₀). Enhancement ratio (ER) (ratio of D₁₀) of etanidazole was 1.3. ERs of TX-2068 and TX-2244 were also similar (ER=1.3-1.4) to that of etanidazole. It is suggested that TX-2068 and TX-2244 are useful as the hypoxic cell radiosensitizer.

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[Purpose] Glioblastoma is one of the most common tumor types resistant to radiation and anti-tumor drugs. Arsenite was reported to show synergistic radiosensitization effect in vitro and in vivo. But, the mechanism of radiosensitization is not wellÅ@understood. Thus, we decided to study the mechanism of anti-tumor effect of arsenite by itself at first. [Materials and Methods] U87MG, glioblastoma cell line, was used. Cell growth was measured by counting number of cells at various time points. Cell death was measured by trypan-blue staining. [Results] Growth of U87MG was significantlly decreased by arsenite alone, and insignificant number of cell death was also observed. The morphology of U87MG cells exposed to arsenite revealed senescence like shapes and also these cells are positive for SA-É¿-galactosidase as known senescence marker. Our result indicates that arsenite alone induced senescence-like growth arrest in U87MG cells. Expression patterns of senescence associated proteins are being analyzed by Western blotting and the data will be presented.

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Radiation therapy is one of the main methods of cancer treatment currently available to improve prognosis, but its effectiveness depends on the radiation sensitivity in tumor. Therefore, it is very important to establish modalities to increase radiation sensitivity. One of such ways is to inhibit DNA repair mechanisms, because cells with mutations in DNA repair genes, including MRE11 and NBS1, are highly sensitive to ionizing radiation. MRE11 and NBS1 form MRN complex, which has a central role in a response to radiation-induced DNA damage. Therefore, inhibition of these protein interactions could inhibit the function of the MRN complex and affect the radiation sensitivity. In addition, biochemical studies have demonstrated DNA binding activity and nuclease activity of MRN complex, suggesting increased radiosensitivity by inhibition of these activities. We will report on some approaches to inhibit the MRN complex and evaluate their usefulness in radiosensitization.

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Sulforaphane (SFN), an isothiocyanate present in broccoli, is one of the most promising chemopreventive agents. It inhibits cell proliferation and induces apoptosis in different tumor cell lines, suggesting that SFN might be useful as chemotherapeutic agent for cancer treatment. In this study, we investigated the radiosensitizing effect of SFN in two human pancreatic cancer cell lines, MIA PaCA-2 and PANC-1. Radiation survival curves obtained showed that SFN radiosensitized both cell lines, but potentiated X-ray sensitivity more in MIAPaCa-2 than PANC-1 cells. In addition, SFN radiosensitized MIA PaCA-2 cells to apoptosis, but not PANC-1. The results obtained indicates that SFN-mediated radiosensitization by inducing apoptosis in MIA PaCa-2 cells is not regulated by Bcl-2 and Bax. A combined treatment of SFN and X-rays resulted in a decrease in the level of XIAP in both cells. MIA PaCa-2 cells treated with SFN and X-rays exhibited a marked increase in protein level of Apaf-1, which is essential for activation of caspase-9. These results indicate that Apaf-1 is an important role in SFN-induced radiosensization by inducing apoptosis. Thus, SFN appears to be a promising radiosensitizing agent for use against human carcinomas.

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