Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays

Noguchi, Miho; Kanari, Yukiko; Yokoya, Akinari; Narita, Ayumi; Fujii, Kentaro

Radiation Protection Dosimetry, 166(1-4), p.101 - 103, 2015/09

https://doi.org/10.1093/rpd/ncv157

Real-time observation of irradiated HeLa-cell modified by fluorescent ubiquitination-based cell-cycle indicator using synchrotron X-ray microbeam

Narita, Ayumi; Kaminaga, Kiichi; Yokoya, Akinari; Noguchi, Miho; Kobayashi, Katsumi*; Usami, Noriko*; Fujii, Kentaro

Radiation Protection Dosimetry, 166(1-4), p.192 - 196, 2015/09

https://doi.org/10.1093/rpd/ncv156

For the knowledge about irradiation effects of mammalian cells depending on the cell cycle, most of them had been analyzed by statistical approches. Our purpose is to establish the method for real-time observation of irradiated cells under a microscope. Fluorescent ubiquitination-based cell cycle indicator (FUCCI) human cancer (HeLa) cells (red indicates G1; green, S/G2) were exposed to a synchrotron X-ray microbeam. Cells in either G1 or S/G2 were selectively irradiated according to cell color observed in the same microscopic field in a culture dish. Time-lapse micrographs of the irradiated cells were acquired for 24 h after irradiation. The cell cycle was strongly arrested by irradiation at S/G2 and never progressed to G1. In contrast, cells irradiated at G1 progress to S/G2 with a similar time course as non-irradiated control cells. These results show single FUCCI cell exposure and live cell imaging are powerful methods for studying radiation effects on the cell cycle.

Visualization of cell-cycle modification by ionizing irradiation in single HeLa cells using fluorescent ubiquitination-based cell-cycle indicator

Kaminaga, Kiichi; Sakamoto, Yuka; Kanari, Yukiko; Noguchi, Miho; Yokoya, Akinari

Journal of Radiation Research, 55(Suppl.1), p.i127 - i128, 2014/03

https://doi.org/10.1093/jrr/rrt159

Significance of DNA Polymerase I in processing of clustered DNA damage

Shikazono, Naoya; Akamatsu, Ken; Takahashi, Momoko*; Noguchi, Miho; Urushibara, Ayumi; O'Neill, P.*; Yokoya, Akinari

Mutation Research; Fundamental and Molecular Mechanisms of Mutagenesis, 749(1-2), p.9 - 15, 2013/09

https://doi.org/10.1016/j.mrfmmm.2013.07.010

We examined the biological consequences of bi-stranded clustered damage sites, consisting of a combination of DNA lesions using a bacterial plasmid-based assay. The transformation efficiencies were significantly lower for the bi-stranded clustered GAP/AP lesions than for either a single GAP or a single AP site. When the two lesions were separated by 10-20 bp, the transformation efficiencies were comparable with those of the single lesions. This recovery of transformation efficiency for separated lesions requires DNA polymerase I (Pol I) activity. Analogously, the mutation frequency was enhanced in a bi-stranded cluster containing a GAP and an 8-oxoG, and Pol I was found to play an important role in minimising mutations induced as a result of clustered lesions. These results indicate that the biological consequences of clustered DNA damage strongly depend on Pol I activity.

The Mutagenic potential of 8-oxoG/single strand break-containing clusters depends on their relative positions

Noguchi, Miho; Urushibara, Ayumi; Yokoya, Akinari; O'Neill, P.*; Shikazono, Naoya

Mutation Research; Fundamental and Molecular Mechanisms of Mutagenesis, 732(1-2), p.34 - 42, 2012/04

https://doi.org/10.1016/j.mrfmmm.2011.12.009

The effect of a single strand break associated with base lesion(s) in vivo remains largely unknown. In the present study we determined the mutagenicities of two- and three-lesion clustered damage sites containing a 1-nucleotide gap (GAP) and 8-oxo-7,8-dihydroguanine(s) (8-oxoG(s)). The mutation frequencies (MFs) of bi-stranded two-lesion clusters (GAP/8-oxoG), especially in mutY-deficient strains, were high and were similar to those for bi-stranded clusters with 8-oxoG and base lesions/AP sites, suggesting that the GAP is processed with an efficiency similar to the efficiency of processing a base lesion or an AP site within a cluster. The MFs of tandem two-lesion clusters comprised of a GAP and an 8-oxoG were comparable to or less than the MF of a single 8-oxoG. The mutagenic potential of three-lesion clusters, which were comprised of a tandem lesion (a GAP and an 8-oxoG) and an opposing single 8-oxoG, was higher than that of a single 8-oxoG, but was no more than that of a bi-stranded 8-oxoGs. We suggest that incorporation of a nucleotide opposite 8-oxoG is less mutagenic when a GAP is present in a cluster than when a GAP is absent. Our observations indicate that the repair of a GAP is retarded by an opposing 8-oxoG, but not by a tandem 8-oxoG, and that the extent of GAP repair determines the biological consequences.

A Model for analysis of the yield and the level of clustering of radiation-induced DNA-strand breaks in hydrated plasmids

Shikazono, Naoya; Yokoya, Akinari; Urushibara, Ayumi; Noguchi, Miho; Fujii, Kentaro

Radiation Protection Dosimetry, 143(2-4), p.181 - 185, 2011/02

https://doi.org/10.1093/rpd/ncq528

Enhanced radiation-induced cell killing by Herbimycin A pre-treatment

Noguchi, Miho; Hirayama, Ryoichi*; Druzhinin, S.*; Okayasu, Ryuichi*

Radiation Physics and Chemistry, 78(12), p.1184 - 1187, 2009/12

https://doi.org/10.1016/j.radphyschem.2009.07.008

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.

Contributions of direct and indirect actions in cell killing by high-LET radiations

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

https://doi.org/10.1667/RR1490.1

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.

17-allylamino-17-demethoxygeldanamycin enhances the cytotoxicity of tumor cells irradiated with carbon ions

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.

The Mutation frequency of 8-oxo-7,8-dihydroguanine when single strand break is closely present on the same or the opposite strand

Noguchi, Miho; Urushibara, Ayumi*; Yokoya, Akinari; Shikazono, Naoya

no journal, , 

Ionizing radiation induces clustered DNA damage which is defined as two or more lesions induced within 1-2 helical turns (10-20bp) of DNA by a single radiation track. Recent in vitro studies predict 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 lesions in Escherichia coli. We used plasmid based assay 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. Clustered damage containing 8-oxoG and SSB located on opposite strand raised mutation frequencies. However, clustered damage containing 8-oxoG and SSB positioned in tandem gave lower mutation frequency than single 8-oxoG lesion. We propose that the mutagenic potential of 8-oxoG depends on whether SSB is located on either strand, same or opposite, to 8-oxoG.