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.

Visualisation of cell cycle modifications by X-ray irradiation of single HeLa cells using fluorescent ubiquitination-based cell cycle indicators

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

Radiation Protection Dosimetry, 166(1-4), p.91 - 94, 2015/09

https://doi.org/10.1093/rpd/ncv168

Three-dimensional culture of HeLa-FUCCI cells for study of bystander cell cycle effect of high LET particles

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

Journal of Radiation Research, 55(Suppl.1), p.i120 - i121, 2014/03

https://doi.org/10.1093/jrr/rrt166

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.

Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X-rays

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

https://doi.org/10.1093/rpd/ncq478

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.

Recent progress in the energy recovery linac project in Japan

Sakanaka, Shogo*; Akemoto, Mitsuo*; Aoto, Tomohiro*; Arakawa, Dai*; Asaoka, Seiji*; Enomoto, Atsushi*; Fukuda, Shigeki*; Furukawa, Kazuro*; Furuya, Takaaki*; Haga, Kaiichi*; et al.

Proceedings of 1st International Particle Accelerator Conference (IPAC '10) (Internet), p.2338 - 2340, 2010/05

http://accelconf.web.cern.ch/AccelConf/IPAC10/papers/tupe091.pdf

Future synchrotron light source using a 5-GeV energy recovery linac (ERL) is under proposal by our Japanese collaboration team, and we are conducting R&D efforts for that. We are developing high-brightness DC photocathode guns, two types of cryomodules for both injector and main superconducting (SC) linacs, and 1.3 GHz high CW-power RF sources. We are also constructing the Compact ERL (cERL) for demonstrating the recirculation of low-emittance, high-current beams using above-mentioned critical technologies.

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.

The Yield, processing, and biological consequences of clustered DNA damage induced by ionizing radiation

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

Journal of Radiation Research, 50(1), p.27 - 36, 2009/01

https://doi.org/10.1269/jrr.08086

After living cells are exposed to ionizing radiation, a variety of chemical modifications of DNA are induced either directly by ionization of DNA or indirectly through interactions with water-derived radicals. Clustered DNA damage, which is defined as two or more of such lesions within one to two helical turns of DNA induced by a single radiation track, is considered to be a unique feature of ionizing radiation. A double strand break (DSB) is a type of clustered DNA damage. Formation and repair of DSBs have been studied in great detail over the years as they have been linked to important biological endpoints. Although non-DSB clustered DNA damage has received less attention, there is growing evidence of its biological significance. This review focuses on the current understanding of (1) the yield of non-DSB clustered damage induced by ionizing radiation (2) the processing, and (3) biological consequences of non-DSB clustered DNA damage.

LET dependence of the yield of DNA strand breaks, base lesions and clustered damage sites induced in fully hydrated plasmid DNA films by ion particles

Yokoya, Akinari; Ushigome, Takeshi*; Tauchi, Hiroshi*; Suzuki, Masao*; Tsuruoka, Chizuru*; Noguchi, Miho; Fujii, Kentaro; Shikazono, Naoya; Watanabe, Ritsuko

no journal, , 

In order to clarify the characteristics of DNA damage induced by high LET radiation, the yields of strand breaks and base lesions induced in closed-circular plasmid DNA (pUC18) were measured after exposing to various kinds of radiation (He, Ne and C ions; 2 to 900 keV/m). Base excision repair enzymes were used to detect oxidative base lesions. The obtained results show that (1) the yield of prompt SSBs does not depend significantly on the LET, (2) whereas the yield of DSBs increases with increasing LET, (3) The yields of isolated nucleobase lesions detected by enzymes decrease drastically with increasing LET and (4) C and Ne ions induce less base lesions than He ions when compared in the same LET region. These results indicate that the yield of cluster of nucleobase lesions, which are less readily processed by the base excision repair proteins, depends not only LET but also ion species irradiated.