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Funayama, Tomoo; Yokota, Yuichiro; Suzuki, Michiyo; Sakashita, Tetsuya; Kobayashi, Yasuhiko
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 73, 2015/03
Using a collimating heavy-ion microbeam system, we have explored various effects of heavy-ion hit on biological materials. However, there are limitations of the collimating system in the size of the microbeam spot and in the irradiation speed that cannot be overcome in principle. Thus, we started the development of a focusing microbeam system for target-irradiating individual cells more precisely. In this year, we established the protocol for irradiating "actual" cell sample with scanned beam. In the experiment, the HeLa cells were inoculated on a CR-39 film, then place on the sample stage. The microscopic image of cells was analyzed, and the cells were irradiated with scanned neon microbeam. After irradiation, we found the correspondence of the distribution pattern of the ion hit positions and the 
-H2AX foci on cell nuclei, indicating rapid and accurate irradiation of individual cells with the focusing heavy-ion microbeam.
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.
Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Nakano, Takashi*; Kobayashi, Yasuhiko
no journal, ,
Human lung normal fibroblasts WI-38 and human lung cancer cells H1299/wt
were used. Cells were irradiated with carbon-ion broad beams (LET=108 keV/ 
m), then survival rates of bystander cells after co-culture with irradiated cells were measured using colony formation assay. The survival rates of non-irradiated H1299/wt cells co-cultured with 0.13 Gy irradiated WI-38 increased after 6 and 24 h of co-culture. On the other hand, the bystander cells co-cultured with 0.5 Gy irradiated WI-38 showed decreased survival rates. The survival rates of bystander H1299/wt cells showed a tendency to increase by the addition of Carboxy-PTIO to the co-culture medium, when co-cultured with 0.5 Gy irradiated WI-38. From these results, reduction of survival rates is likely to be caused by NO radical as a mediator in bystander effects between lung normal and cancer cells. However, it is suggested that there might be other signals participated in an increase of survival rates.
Funayama, Tomoo; Yokota, Yuichiro; Sakashita, Tetsuya; Suzuki, Michiyo; Ikeda, Hiroko; Kobayashi, Yasuhiko
no journal, ,
In JAEA-Takasaki, there are two microbeam systems for biological study: collimating heavy-ion microbeam system, and focusing heavy-ion microbeam system. Using the collimating microbeam system, we have carried out the 
cultured-cell studies of heavy-ion hit effect and analysis of bystander effects. Moreover, we have promoted the microbeam studies of small individuals, such as 
, silkworm, and medaka fish. In these studies, we carried out not only researches aimed to explore the effects of heavy-ion radiation, but also the researches of physiological or developmental biology by applying microbeam as the tool of the radio-microsurgery. The focusing microbeam system was developed for overcoming the limitations of the collimating system. Using the system, we developed the experimental system to irradiate cells rapidly and accurately with the scanned microbeam spot of high-energy heavy-ions.
Murata, Kazutoshi*; Noda, Shinei*; Oike, Takahiro*; Takahashi, Akihisa*; Yoshida, Yukari*; Suzuki, Yoshiyuki*; Ono, Tatsuya*; Funayama, Tomoo; Kobayashi, Yasuhiko; Takahashi, Takeo*; et al.
no journal, ,
This study aimed to investigate the effect of carbon ion (C-ion) irradiation on cell motility through the ras homolog gene family member (Rho) signaling pathway in the human lung adenocarcinoma cell line A549. Cell motility was assessed by a wound-healing assay, and the formation of cell protrusions was evaluated by F-actin staining. Cell viability was examined by the WST-1 assay. The expression of myosin light chain 2 (MLC2) and the phosphorylation of MLC2 at Ser19 (P MLC2-S19) were analyzed by Western blot. The data suggest that C-ion irradiation increases cell motility in A549 cells via the Rho signaling pathway and that ROCK inhibition reduces that effect.
