Hirayama, Ryoichi*; Uzawa, Akiko*; Takase, Nobuhiro*; Matsumoto, Yoshitaka*; Noguchi, Miho; Koda, Kana*; Ozaki, Masakuni*; Yamashita, Kei*; Li, H.*; Kase, Yuki*; et al.
Mutation Research; Genetic Toxicology And Environmental Mutagenesis, 756(1-2), p.146 - 151, 2013/08
Nakajima, Nakako*; Brunton, H.*; Watanabe, Ritsuko; Shrikhande, A.*; Hirayama, Ryoichi*; Matsufuji, Naruhiro*; Fujimori, Akira*; Murakami, Takeshi*; Okayasu, Ryuichi*; Jeggo, P.*; et al.
PLOS ONE (Internet), 8(8), p.e70107_1 - e70107_14, 2013/08
Heavy particle irradiation can produce complex DNA double strand breaks (DSBs) within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, can create low linear energy transfer (LET) damage distant from the track. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe ions, H2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered H2AX foci. We also identified simple H2AX foci distant from the track. They are rapidly repaired. Clustered H2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple H2AX foci. However, mitotic entry was observed when 10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.
Hirayama, Ryoichi*; Uzawa, Akiko*; Matsumoto, Yoshitaka*; Noguchi, Miho; Kase, Yuki*; Takase, Nobuhiro*; Ito, Atsushi*; Koike, Sachiko*; Ando, Koichi*; Okayasu, Ryuichi*; et al.
Radiation Protection Dosimetry, 143(2-4), p.508 - 512, 2011/02
We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV/m carbon ions and X-rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X-rays was 1.68, and this value was not changed after 1 h rejoining time (1.40). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X-rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X-rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.
Hamada, Nobuyuki*; Imaoka, Tatsuhiko*; Masunaga, Shinichiro*; Ogata, Toshiyuki*; Okayasu, Ryuichi*; Takahashi, Akihisa*; Kato, Takamitsu*; Kobayashi, Yasuhiko; Onishi, Takeo*; Ono, Koji*; et al.
Journal of Radiation Research, 51(4), p.365 - 383, 2010/07
Noguchi, Miho; Hirayama, Ryoichi*; Druzhinin, S.*; Okayasu, Ryuichi*
Radiation Physics and Chemistry, 78(12), p.1184 - 1187, 2009/12
Herbimycin A (HA), as in Geldanamycin, binds to conserved pockets of heat shock protein 90 (Hsp90) and inhibits its chaperone functions. Hsp90 plays an integral role in cancer cell growth and survival, because it maintains the stability of several key proteins by its chaperone's activity. It is known that some of the proteins associated with radiation responses are functionally stabilized by Hsp90. In this study, we investigated the effect of HA on radiosensitivity in human cancer cells and the mechanism related to the sensitization. In order to gain a mechanistic insight of this sensitization, we examined repair of DNA double strand breaks (DSBs) in irradiated human cancer cells pre-treated with HA, as unrepaired DSBs are thought to be the main cause of radiation-induced cell death. Cellular radiosensitivity was determined by clonogenic assay, and the DSB rejoining kinetics was examined by constant field gel electrophoresis. SQ-5, a lung squamous carcinoma cell line, showed synergistic increase in radiosensitivity when cells were pre-treated with HA. In addition, HA significantly inhibited repair of radiation induced DSBs. These results suggest that the combination of HA and ionizing radiation may be a useful therapeutic strategy for treating certain cancer cells.
Hirayama, Ryoichi*; Matsumoto, Yoshitaka*; Kase, Yuki*; Noguchi, Miho; Ando, Koichi*; Ito, Atsushi*; Okayasu, Ryuichi*; Furusawa, Yoshiya*
Radiation Physics and Chemistry, 78(12), p.1175 - 1178, 2009/12
The contribution of OH radical-mediated indirect action by particle beams under hypoxic irradiation condition was investigated by using a radical scavenger. V79 cells were irradiated with 150 MeV/nucleon helium ions at an LET of 2.2 keV/mm in the presence or absence of DMSO, and their colony survivals were determined. The contribution of indirect action to cell killing under hypoxic condition was estimated to be 52 %. We conclude that OH radical mediated indirect action still has a half in total contribution on cell killing under hypoxic condition.
Hirayama, Ryoichi*; Ito, Atsushi*; Tomita, Masanori*; Tsukada, Teruyo*; Yatagai, Fumio*; Noguchi, Miho; Matsumoto, Yoshitaka*; Kase, Yuki*; Ando, Koichi*; Okayasu, Ryuichi*; et al.
Radiation Research, 171(2), p.212 - 218, 2009/02
The biological effects of radiation originate principally in damages to DNA. DNA damages by X-rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster V79 cells were exposed to high-LET radiations of 20 to 2106 keV/m in the presence or absence of DMSO and their survival was determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET. However, the contribution did not reach zero even at very high LETs and was estimated to be 32% at an LET of 2106 keV/m. Therefore, even though the radiochemically estimated G value of OH radicals was nearly zero at an LET of 1000 keV/m, indirect action by OH radicals contributed to a substantial fraction of the biological effects of high-LET radiations. The RBE determined at a survival level of 10% increased with LET, reaching a maximum value of 2.88 at 200 keV/m, and decreased thereafter. When the RBE was estimated separately for direct action (RBE(D)) and indirect action (RBE(I)); both exhibited an LET dependence similar to that of the RBE, peaking at 200 keV/m. However, the peak value was much higher for RBE(D) (5.99) than RBE(I) (1.89). Thus direct action contributes more to the high RBE of high-LET radiations than indirect action does.
Noguchi, Miho; Hirayama, Ryoichi*; Okayasu, Ryuichi*
no journal, ,
We investigated radiosensitization effect and its mechanism of Hsp90 inhibitor 17-AAG in human tumor cell lines irradiated with high LET carbon ions. Human tumor cell lines, DU145 derived from prostate carcinoma and normal human fibroblasts HFL III were incubated for 24 h in the presence of 17-AAG at concentration of 100nM. The cells were then irradiated with carbon ions (290MeV/nucleon, LET70keV/um) and several biological endpoints were compared. Cellular radiation sensitivity was determined by clonogenic assay and DNA double strand break (DSB) repair kinetics were examined by constant field gel electrophoresis. DU145 cells showed an increase in carbon ions-induced cell death when pre-treated with 17-AAG. The radiosensitivity enhancement ratios measured at a survival rate of 10% were 2.13 for DU145 cells. In contrast to the tumor cell lines, normal human fibroblasts with carbon irradiation showed no radiosensitization with 17-AAG pre-treatment. Our constant field gel electrophoresis studies indicated that 17-AAG had almost no effect on carbon ion-induced DSB repair in DU145 cells. On the other hand, radiation induced Rad51 foci formation showed different kinetics between the carbon ion alone and the combined treatment with 17-AAG and carbon ions in DU145 cells. Our findings suggest that mechanisms other than inhibition of DSB repair could be involved with the radiosensitization by 17-AAG in tumor cells irradiated with carbon ions. However, limited inhibition of homologous recombination by this agent may still be a possibility.
Noguchi, Miho; Yu, D.*; Hirayama, Ryoichi*; Kubota, Nobuo*; Okayasu, Ryuichi*
no journal, ,
The aim of this study is to evaluate the radiosensitization of Hsp90 inhibitor 17-Allylamino-17-demethoxygeldanamycin (17AAG), specifically the effect of 17AAG on the DNA DSB repair machinery. Our constant field gel electrophoresis studies indicated that pretreatment with 17AAG for 24 hours inhibited radiation induced DSB repair in two cancer cell lines (DU145 and SQ-5). The treatment of 17AAG alone leads to the reduction of Rad51 protein expression by western blotting, and the combined treatment with X-irradiation caused a delay in the formation of nuclear Rad51 foci by immuno-staining. These results suggest that 17AAG affects the key protein(s) for HRR, resulting in the radiosensitization of tumor cells. Our data show for the first time that 17AAG is a DNA DSB repair inhibitor, predominantly affecting the homologous recombination pathway.
Noguchi, Miho; Hirayama, Ryoichi*; Druzhinin, S.*; Okayasu, Ryuichi*
no journal, ,
Herbimycin A binds to conserved pockets of heat shock protein 90 (Hsp90) and inhibits its chaperone functions. Hsp90 plays an important role in tumor cell growth and survival though maintaining stability of proteins by its chaperone activity. It is known that some of the proteins associated with radio-resistance are functionally stabilized on Hsp90. In this study, we investigated the effect of herbimycin A on radiation sensitivity in human tumor cells and the mechanism related to the sensitization. For mechanistic insight, we examined repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with herbimycin A. The lung squamous carcinoma cell cine, SQ-5, was used. Cells were treated with 1, 2, and 4 M herbimycin A for 24 h before X-irradiation. SQ-5 cells showed increased radiation sensitivity when pre-treated with herbimycin A. In addition, herbimycin A significantly inhibited repair of radiation induced DSBs. It is possible that one of the proteins associated with DNA DSB repair might be degraded by this drug as previously shown with another Hsp90 inhibitor 17-AAG, a geldanamycin derivative.
Takahashi, Momoko; Noguchi, Miho; Hirakawa, Hirokazu*; Okayasu, Ryuichi*
no journal, ,
Ionizing radiation has been widely used as a tool for tumor treatment. However, there are still difficulties to treat the solid tumor with only irradiation. Several studies have been shown that the combination therapy using both X-irradiation and antitumor drug is more effective to tumor compared with X-irradiation only. Previously Noguchi et al. showed the Hsp90 inhibitor 17AAG induced tumor cell death effectively with X-irradiation. Hsp90 is the enzymes activated by cellular stress and activates several tumor-related genes. To clarify whether or not the combination therapy of carbon ion beam and 17AAG is effective to solid tumors, we tested the growth of solid tumor with the combination of X-irradiation and 17AAG and compared the results from study.
Okayasu, Ryuichi*; Hirakawa, Hirokazu*; Noguchi, Miho; Yu, D.*; Takahashi, Momoko; Hirayama, Ryoichi*; Fujimori, Akira*
no journal, ,
17AAG, an Hsp90 inhibitor was shown to radiosensitize certain human tumor cells exposed to X-rays, while this sensitization was not clearly observed in normal human cells. The mechanism of this was suggested to come from inhibition of DNA double strand break (DSB) repair, particularly impairment of homologous recombination repair (HRR) pathway by this drug (Noguchi et al 2006). Key proteins associated with HRR seem to be affected by this inhibitor. To our surprise, tumor radiosensitization with 17AAG was also observed in cells exposed to high LET carbon ions (70 kev/um). Independently we also found that knockdown of BRCA2, a key HRR protein significantly radiosensitized human tumor cells. These results indicate that there seem to be a radio-sensitization mechanism associated with the combination of HRR inhibition and high LET radiation, and this may occur particularly in S-phase cells. Furthermore, we also used mouse xenograft model to examine the combined effect of 17AAG and high LET carbon irradiation. For this purpose, SQ5 human lung tumor cells were implanted on the leg of nude mice and the tumor growth was observed in the combined treatment as compared with radiation or drug treatment alone. Our preliminary results indicate that tumor growth was more inhibited in the 17AAG and carbon irradiation than carbon or 17AAG treatment alone. These data suggest that an effective tumor control might be obtained by combining an HRR inhibitor with high LET carbon irradiation.
Hirayama, Ryoichi*; Matsumoto, Yoshitaka*; Uzawa, Akiko*; Takase, Nobuhiro*; Tsuruoka, Chizuru*; Wada, Mami*; Noguchi, Miho; Kase, Yuki*; Matsufuji, Naruhiro*; Ito, Atsushi*; et al.
no journal, ,
The contribution of indirect action mediated by OH radicals in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action of radiation without affecting direct action. Exponentially growing Chinese hamster ovary cells under oxic and hypoxic conditions were exposed to X-rays and iron ions having a dose-averaged LET at 200 keV/micrometer in the presence or absence of DMSO, and the cell survival was determined using the colony formation assay. The contributions of indirect action of 76% and 50% were found for X-rays under oxic and hypoxic conditions, respectively. In contrast, the contributions of indirect action for iron ions were estimated to be 42% and 32%. The RBE values were 2.8 for oxic and 5.3 for hypoxic, and the OER values were 2.8 for X-rays and 1.5 for iron ions. When the RBE and OER were estimated separately for direct action (RBE(D) and OER(D)) and indirect action (RBE(I) and OER(I)); the RBE(D) was larger than RBE(I) under both normal and low oxygen concentrations. The OER(D) values for both X-rays and iron ions were lower than that for OER(I). Thus, direct action of radiation gives a remarkably higher RBE and lower OER for cell killing than indirect action. It is possible that particle beams may be highly effective in treating cancer if the particle therapy can only use the portion of direct action out of total radiation actions; this would be exemplified by the usage of radioactive 9C-ion beams and boron neutron capture.