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Kitabatake, Satomi*; Ushiroda, Tota*; Hirayama, Ryoichi*; Furusawa, Yoshiya*; Funayama, Tomoo; Yokota, Yuichiro; Okahata, Yoshio*; Ito, Atsushi*
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 86, 2015/03
Biological effects of high-LET radiation could be understood in terms of the ion track structure. Therefore the evaluation of the contribution of both core and penumbra regions to biological effects is an important issue for the study of high-LET effects. In the present study, we developed a protocol to make a uniform DNA sheet with insoluble nature in aqueous solution, and explored the applicability to the detection of 8-OHdG distributions after heavy-ion irradiation. Water-insoluble DNA sheet was irradiated with proton and neon ion beams at JAEA-Takasaki. After irradiation DNA samples were incubated with an 8-OHdG antibody followed by with a second antibody containing a fluorescence probe. The preliminary results indicated that upon ion irradiation randomly distributed dot-like fluorescence was observed, suggesting that these dots may be from incident ions.
Takahashi, Akihisa*; Kubo, Makoto*; Ma, H.*; Nakagawa, Akiko*; Yoshida, Yukari*; Isono, Mayu*; Kanai, Tatsuaki*; Ono, Tatsuya*; Furusawa, Yoshiya*; Funayama, Tomoo; et al.
Radiation Research, 182(3), p.338 - 344, 2014/09
Times Cited Count：57 Percentile：90.66(Biology)To clarify whether high-LET radiation inhibits all repair pathways or specifically one repair pathway, studies were designed to examine the effects of radiation with different LET values on DNA DSB repair and radiosensitivity. Embryonic fibroblasts bearing repair gene KO were exposed to X rays, carbon-, iron-, neon- and argon-ion beams. Cell survival was measured with colony-forming assays. The sensitization enhancement ratio (SER) values were calculated using the 10% survival dose of wild-type cells and repair-deficient cells. Cellular radiosensitivity was listed in descending order: double-KO cells 
NHEJ-KO cells HR-KO cells wild-type cells. Although HR-KO cells had an almost constant SER value, NHEJ-KO cells showed a high-SER value when compared to HR-KO cells, even with increasing LET values. These results suggest that with carbon-ion therapy, targeting NHEJ repair yields higher radiosensitivity than targeting homologous recombination repair.
Funayama, Tomoo; Wada, Seiichi; Kobayashi, Yasuhiko; Watanabe, Hiroshi*
Radiation Research, 163(2), p.241 - 246, 2005/02
Times Cited Count：32 Percentile：64.73(Biology)As the first step for the analysis of the biological effect of heavy charged-particle radiation, we established a method for the irradiation of individual cells with a heavy ion microbeam apparatus at JAERI-Takasaki. CHO-K1 cells attached on the thin film of ion track detector, CR-39, were automatically detected under fluorescent microscope, and irradiated individually with 
Ar
ion (11.5 MeV/u, LET=1260 keV/
m) microbeam. Without killing the irradiated cells, trajectories of irradiated ions were visualized as etch-pits by treatment of CR-39 with alkaline-ethanol solution at 37
C. The exact positions of ion hits were determined by overlaying images of both cells and etch-pits. The cells that were irradiated with argon ions showed a reduced growth in post-irradiate observations. Moreover, a single hit of an argon ion to the cell nucleus resulted in strong growth inhibition. These results tell us that our truthful irradiation method now enables us to start a precise study of the cellular response of high-LET radiation effects on cells.
Kobayashi, Yasuhiko; Funayama, Tomoo; Wada, Seiichi; Furusawa, Yoshiya*; Aoki, Mizuho*; Shao, C.*; Yokota, Yuichiro; Sakashita, Tetsuya; Matsumoto, Yoshitaka*; Kakizaki, Takehiko; et al.
Uchu Seibutsu Kagaku, 18(4), p.235 - 240, 2004/12
no abstracts in English
Kobayashi, Yasuhiko; Funayama, Tomoo; Wada, Seiichi; Sakashita, Tetsuya
Uchu Seibutsu Kagaku, 18(3), p.186 - 187, 2004/11
no abstracts in English
Kobayashi, Yasuhiko; Funayama, Tomoo; Wada, Seiichi*; Taguchi, Mitsumasa; Watanabe, Hiroshi
Radiation Research, 161(1), p.90 - 91, 2004/01
A single cell irradiation system has been developed for targeting cells individually with a precise number of heavy ions to elucidate radiobiological effects of exactly one particle and to investigate the biological effects of low fluence irradiation with HZE particles. Using the heavy ion microbeam apparatus installed at JAERI-Takasaki, mammalian cells were irradiated in the atmosphere with a single or precise numbers of ions, 13.0 MeV/u 20Ne or 11.5 MeV/u 40Ar. The number of ions traversed the cells attached on the ion track detector CR-39 were counted with a plastic scintillator. Immediately after the irradiation, the position and the number of ion tracks traversed the cell was detected with etching of CR-39 from the opposite side of the cell with alkaline-ethanol solution at 37C. The growths of the cells were observed individually up to 60 hours after irradiation.
Funayama, Tomoo; Wada, Seiichi; Kobayashi, Yasuhiko
Radiation Research, 161(1), p.111 - 112, 2004/01
Using the heavy ion microbeam apparatus, mammalian cells were irradiated in the atmosphere with a single or precise numbers of Ar heavy ions (11.5 MeV/u) with a spatial resolution of a few microns. Positional data of the individual cells attached on the ion track detector CR-39 were obtained at the off-line microscope before irradiation, then the targeting and irradiation of the cells were performed semi-automatically at the on-line microscope of the microbeam apparatus according to the obtained data. Immediately after irradiation, the position and the number of ion tracks traversed the cell was detected with etching of CR-39 at 37 C. The growths of the cells were observed individually up to 60 hours after irradiation. The continuous observation of the individual cell growth indicated that single Ar ion traversal of cell nucleolus resulted to complete growth inhibition of the irradiated cells.
Kobayashi, Yasuhiko; Funayama, Tomoo; Wada, Seiichi; Taguchi, Mitsumasa; Watanabe, Hiroshi
Nuclear Instruments and Methods in Physics Research B, 210(1-4), p.308 - 311, 2003/09
A method for detecting the ion hit tracks on the mammalian cultured cells at the irradiation time was established. The cells were attached to the ion track detector CR-39 (100 m thick), then irradiated with 13.0 MeV/u 20Ne or 11.5 MeV/u 40Ar ion beams. Immediately after the irradiation, the cells were refilled with medium, then the CR-39 was etched from the opposite side of the cell with alkaline-ethanol solution at 37C. With the 15 min etching treatment, we obtained the accurate information about the spatial distribution of irradiated ions without significant effect on the cell growth. The continuous observation of the individual cell growth indicated that the growth of ion hit cell was reduced compared with that of non-irradiated one.
Kobayashi, Yasuhiko; Funayama, Tomoo
Isotope News, (590), p.2 - 7, 2003/06
A microbeam can be used for selective irradiation of individual cells, which can be subsequently observed to ascertain what changes occur to that cell and to neighboring un-irradiated cells. Therefore, the use of microbeam allows direct investigation of cell-to-cell communications such as "bystander effects", that is, radiation effects transmitted from irradiated cells to neighboring un-irradiated cells. Furthermore, a microbeam with sufficient spatial resolution will be useful for analyzing the dynamics of intra-cellular process such as apoptosis and the influence of track-structure of energetic heavy ions by means of highly localized irradiation of a part of a nucleus or cytoplasm.
Wada, Seiichi; Natsuhori, Masahiro*; Ito, Nobuhiko*; Funayama, Tomoo; Kobayashi, Yasuhiko
Nuclear Instruments and Methods in Physics Research B, 206, p.553 - 556, 2003/05
Times Cited Count：3 Percentile：27.69(Instruments & Instrumentation)Determining the biological effects of a very low number of charged particles crossing the cell nucleus is interest for estimating the risk due to environmental exposure to charged particles. Especially it is necessary to detect the radiation damage induced by a precise number of charged particles in the individual cells. To compare the number of ions traversing the cell and the DNA damage produced by the hit ions, we applied comet assay. Cells attached on the ion track detector CR-39 were irradiated with 17.3 MeV/u 12C, 15.7 MeV/u, 10.4 MeV/u 20Ne and 6.9 MeV/u 40Ar ion beams at TIARA, JAERI-Takasaki. After irradiation, CR-39 was covered with 1 % agarose. After electrophoresis the CR-39 was taken off from the slide glass. The agarose gel on the CR-39 was stained with ethidium bromide and the opposite side of the CR-39 was etched with KOH-ethanol solution at 37 
. We observed that the ion particles with higher LET value induced the heavier DNA damage, even by the same number of ion-hits within the irradiated cells.
-radiationYokoya, Akinari; Cunniffe, S. M. T.*; Stevens, D. L.*; O'Neill, P.*
Journal of Physical Chemistry B, 107(3), p.832 - 837, 2003/01
Times Cited Count：26 Percentile：56.43(Chemistry, Physical)no abstracts in English
Funayama, Tomoo; Kobayashi, Yasuhiko
Hoshasen Seibutsu Kenkyu, 37(3), p.334 - 347, 2002/09
In JAERI-Takasaki, the heavy ion microbeam system was developed, and utilized to irradiate biological materials. However, the utilization of microbeam system was mainly in the radio-surgery tool for analyzing insect development and plant root differentiation. This was because some beam technological and biological difficulties existed in irradiating cultured cell with high LET ion beam. Recently, we established a efficient and practical way for irradiating mammalian cultured cell with our microbeam apparatus. In this paper, the outlines of our micro beam system and the actual process for cultured cell irradiation will be described.
Kobayashi, Yasuhiko
Hoshasen Seibutsu Kenkyu, 37(1), p.67 - 84, 2002/03
A single cell irradiation system has been developed for targeting cells individually with a precise number of high-LET heavy ions to elucidate radiobiological effects of exactly one particle and to investigate the interaction of damages produced by separate events. Using the heavy ion microbeam apparatus in TIARA, mammalian cells were irradiated in the atmosphere with a single or precise numbers of heavy ions, 13.0 MeV/u 20Ne or 11.5 MeV/u 40Ar, with a spatial resolution of a few microns.
*; *; *; Kobayashi, Yasuhiko; Watanabe, Hiroshi; *; *; *; *
Oncology Reports, 4(4), p.691 - 695, 1997/07
no abstracts in English
Funayama, Tomoo; Yokota, Yuichiro; Sakashita, Tetsuya; Suzuki, Michiyo; Kobayashi, Yasuhiko
no journal, ,
To explore the single ion hit effect of heavy-ion the cells, we established a method to detect focusing beam spot under the microscopy, and carried out an irradiation of cells by moving cells to the position of focusing beam spot one by one. However, according to the speed limitation of the mechanical stage, the throughput of the irradiation is still comparable with that of the collimating system. To improve the throughput, we next carried out a development of a method to irradiate cells with a scanned beam. The HeLa cells were inoculated on a CR-39 film, and irradiated with scanned neon microbeam. After irradiation, the hit positions of the ion were visualized as etched pits, and the cells were stained with the 
-H2AX antibody. We found the correspondence of the distribution pattern of the etch pits, the cell positions and the -H2AX foci. Thus we concluded that the developed method can irradiate cells rapidly and accurately with the focusing heavy-ion microbeam.
Saito, Katsuyo*; Funayama, Tomoo; Kobayashi, Yasuhiko; Murakami, Takashi*
no journal, ,
Malignant melanoma is one of the most common cutaneous malignancies. Epigenetic modifiers, such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors, have emerged recently as promising anticancer agents and has been expected as a sensitizer for other cancer therapeutics including radiotherapy. In addition, the biological effects of the high linear energy transfer (LET) heavy-ion radiation are more pronounced than the low-LET radiation. These accumulating evidences allowed us to investigate whether the use of HDACi could sensitize melanoma cells for the heavy-ion therapy. Treatment of B16F10 melanoma cells with HDACi in combination with heavy-ion radiation provided enhanced anti-tumor effects. These data suggest that combination of HDACi together with heavy-ion therapy may provide improved therapeutic responses in melanoma patients.
