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Yokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Sakashita, Tetsuya; Suzuki, Michiyo; Kobayashi, Yasuhiko
JAEA-Review 2015-022, JAEA Takasaki Annual Report 2014, P. 67, 2016/02
The role of nitric oxide (NO) in bystander effect was investigated. Human fibroblasts were irradiated with 
-rays (LET: 0.2 keV/
m) or carbon-ion beam (108 keV/m), and then, co-cultured with the non-irradiated cells. After 24 h culture, the survival rates of non-irradiated cells and the concentrations of nitrate, an oxide of NO, in the medium were measured. The survival rates of non-irradiated cells decreased in dose-dependent and radiation quality-independent manners. Negative relationships between survival rates and nitrite concentrations existed, indicating the amounts of produced NO are an important determinant of bystander effects. Next, a reagent producing two molecules of NO in a half-life of 100 min was added in the culture medium. After incubation of 24 h the survival rates of treated cells did not decrease, suggesting NO produced intracellularly has an important role to lead the bystander effect but is not the signal molecule for intercellular communication.
Yokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Kobayashi, Yasuhiko
Isotope News, (741), p.21 - 25, 2016/01
Our article published on the International Journal of Radiation Biology (2015) was reviewed. We investigated the dependence of the bystander cell-killing effect on radiation dose and quality, and related molecular mechanisms. Human fibroblasts were irradiated with -rays or carbon ions and co-cultured with non-irradiated cells. Survival rates of non-irradiated cells decreased and nitrite concentrations in co-culture medium increased with dose. Their dose responses were similar between -rays and carbon ions. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of non-irradiated cells. Negative relationships were observed between survival rates and nitrite concentrations. From these results, it was concluded that the bystander cell-killing effect mediated by NO radicals depends on irradiation doses, but not on radiation quality. NO radical production appears to be an important determinant of bystander effects.
Hattori, Yuya; Yokoya, Akinari; Watanabe, Ritsuko
BMC Systems Biology (Internet), 9, p.90_1 - 90_22, 2015/12
Times Cited Count：17 Percentile：66.49(Mathematical & Computational Biology)The radiation-induced bystander effect is a biological response observed in non-irradiated cells surrounding an irradiated cell, which is known to be caused by two intercellular signaling pathways. However, the behavior of the signals is largely unknown. To investigate the role of these signaling pathways, we developed a mathematical model to describe the cellular response to direct irradiation and the bystander effect, with a particular focus on cell-cycle modification. The analysis of model dynamics revealed that bystander effect on cell cycle modification was different between low-dose irradiation and high-dose irradiation. We demonstrated that signaling through both pathways induced cell cycle modification via the bystander effect. By simulating various special and temporal conditions of irradiation and cell characteristics, our model will be a powerful tool for the analysis of the bystander effect.
Tomita, Masanori*; Matsumoto, Hideki*; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke*; Maeda, Munetoshi*; Kobayashi, Yasuhiko
Life Sciences in Space Research, 6, p.36 - 43, 2015/07
A radiation-induced bystander response is generally known as a cellular response induced in unirradiated cell by receiving bystander signaling factors released from directly irradiated cells of a cell population. Bystander responses induced by high-LET heavy ions at low fluence are an important problem concerning the health of astronauts in the space environment. Here we set out NO-mediated bystander signal transductions induced by high-LET heavy-ion microbeam irradiation in normal human fibroblasts. Our findings suggest that Akt- and NF-
B-dependent signaling pathway involving COX-2 plays an important role in the NO-mediated high-LET heavy-ion-induced bystander responses. Additionally, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells, which are distinguish form directly irradiated cells.
Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko*; Ikeda, Hiroko; Kobayashi, Yasuhiko
International Journal of Radiation Biology, 91(5), p.383 - 388, 2015/05
Times Cited Count：11 Percentile：64.63(Biology)We investigated the dependence of the bystander cell-killing effect on radiation dose and quality, and related molecular mechanisms. Human fibroblasts were irradiated with -rays or carbon ions and co-cultured with non-irradiated cells. Survival rates of non-irradiated cells decreased and nitrite concentrations in culture medium increased with increasing doses. Their dose responses were similar between -rays and carbon ions. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of non-irradiated cells. Negative relationships were observed between survival rates and nitrite concentrations. From these results, it was concluded that the bystander cell-killing effect mediated by NO radicals in human fibroblasts depends on irradiation doses, but not on radiation quality. NO radical production appears to be an important determinant of -ray- and carbon-ion-induced bystander effects.
Yokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Sakashita, Tetsuya; Suzuki, Michiyo; Kobayashi, Yasuhiko
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 75, 2015/03
We investigated the bystander effect induced by -rays or carbon ions and analyzed the role of nitric oxide (NO) in the effect. Normal human fibroblasts were used. Cells inoculated on a porous membrane were irradiated with varying doses of -rays or carbon ions. Irradiated cells were then non-contact co-cultured with non-irradiated cells for 24 h. After co-culture, the survival rates of non-irradiated bystander cells co-cultured with irradiated cells decreased with increasing dose and bottomed out at 0.5 Gy or higher doses. This indicates that the bystander effect is dependent on irradiation dose but independent of radiation quality. Next, a specific NO scavenger c-PTIO was added to the culture medium during irradiation and co-culture. This treatment prevented the reduction in survival rates of bystander cells, clearly indicating that NO has an important role in the bystander effect.
Matsumoto, Hideki*; Tomita, Masanori*; Otsuka, Kensuke*; Hatashita, Masanori*; Maeda, Munetoshi*; Funayama, Tomoo; Yokota, Yuichiro; Suzuki, Michiyo; Sakashita, Tetsuya; Ikeda, Hiroko; et al.
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 76, 2015/03
The objective of this project is to elucidate molecular mechanisms for the induction of radioadaptive response through radiation-induced bystander responses induced by irradiation with heavy ion microbeams in JAEA. We found that the adaptive response was induced by Ar (520 MeV 
Ar
) microbeam-irradiation of a limited number of cells, followed by the broad beam-irradiation and that the adaptive response was almost completely suppressed by the addition of carboxy-PTIO, as a nitric oxide (NO) scavenger. In addition, we found several genes induced specifically and preferentially when radioadaptive response could be induced. We confirmed that 
expression was specifically induced only when radioadaptive response could be induced. Our findings strongly suggested that radioadaptive response can be induced by NO-mediated bystander responses evoked by irradiation with heavy ion microbeams.
Tomita, Masanori*; Matsumoto, Hideki*; Otsuka, Kensuke*; Funayama, Tomoo; Yokota, Yuichiro; Suzuki, Michiyo; Sakashita, Tetsuya; Kobayashi, Yasuhiko
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 77, 2015/03
Radiation-induced bystander responses are defined as responses in cells that have not been directly targeted by radiation but are in the neighborhood of cells that have been directly exposed. In this study, we aim to clarify a role of bystander response to sustain the homeostasis of damaged tissue using heavy-ion microbeams. We established the heavy-ion microbeam irradiation method to a 3D cultured human epidermis. Using this method, a viable cell rate of the 3D cultured human epidermis irradiated with 260 MeV 
Ne-ion microbeams or broadbeams was analyzed by the MTT method.
locus in normal human fibroblasts induced by C-, Ne- and Ar-ion microbeamsSuzuki, Masao*; Funayama, Tomoo; Yokota, Yuichiro; Muto, Yasuko*; Suzuki, Michiyo; Ikeda, Hiroko; Hattori, Yuya; Kobayashi, Yasuhiko
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 78, 2015/03
We have been studying the radiation-quality dependent bystander cellular effects, such as cell killing, mutation induction and chromosomal damage, using heavy-ion microbeams with different ion species. This year we focused on the ion-species dependent bystander mutagenic effect on locus in normal human fibroblasts. The confluent culture were irradiated using a 256 (16
16)-cross-stripe method using C, Ne and Ar microbeam. Gene mutation on locus was detected with 6-thioguanine resistant clones. The mutation frequency in cells irradiated with C-ion microbeams was 6 times higher than that of non-irradiated control cells and of the sample treated with specific inhibitor of gap-junction cell-to-cell communication. On the other hand, no enhanced mutation frequencies were observed in cells irradiated with either Ne- or Ar-ion microbeams. There is clear evidence that the bystander mutagenic effect via gap-junction communication depends on radiation quality.
Wada, Seiichi*; Ando, Tatsuhiko*; Watanabe, Aya*; Kakizaki, Takehiko*; Natsuhori, Masahiro*; Funayama, Tomoo; Sakashita, Tetsuya; Yokota, Yuichiro; Kobayashi, Yasuhiko
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 79, 2015/03
So far, we clarified that X-ray irradiation induced cell killing by bystander effect mediated-secreted factor. This phenomenon was related with sphingomyelinase (SMase). In this study we analyzed mechanism of secreted SMase from irradiated cells after irradiation. SMase was detected in the culture medium after irradiation by SDS-PAGE. Then, SMase was detected in the exosome of culture medium, but not out of exosome after irradiation. This result indicates that SMase was secreted as exosome from the irradiated cells.
Autsavapromporn, N.*; Plante, I.*; Liu, C.*; Konishi, Teruaki*; Usami, Noriko*; Funayama, Tomoo; Azzam, E.*; Murakami, Takeshi*; Suzuki, Masao*
International Journal of Radiation Biology, 91(1), p.62 - 70, 2015/01
Times Cited Count：31 Percentile：93.49(Biology)Radiation-induced bystander effects have important implications in radiotherapy. Their persistence in normal cells may contribute to risk of health hazards, including cancer. This study investigates the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of harmful effects in progeny of bystander cells. Confluent human skin fibroblasts were exposed to microbeam radiations with different linear energy transfer (LET) by which 0.036
0.4% of the cells were directly targeted by radiation. Following 20 population doublings, the cells were harvested and assayed for micronucleus formation, gene mutation and protein oxidation. The results showed that expression of stressful effects in the progeny of bystander cells is dependent on LET.
Suzuki, Masao*; Autsavapromporn, N.*; Usami, Noriko*; Funayama, Tomoo; Plante, I.*; Yokota, Yuichiro; Muto, Yasuko*; Suzuki, Michiyo; Ikeda, Hiroko; Hattori, Yuya; et al.
Journal of Radiation Research, 55(Suppl.1), P. i54, 2014/03
Kobayashi, Yasuhiko; Funayama, Tomoo; Wada, Seiichi; Sakashita, Tetsuya; Kakizaki, Takehiko; Hamada, Nobuyuki*; Yokota, Yuichiro; Furusawa, Yoshiya*
KEK Proceedings 2005-5, p.6 - 8, 2005/10
no abstracts in English
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*; 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 37
C. 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.
Yokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Kobayashi, Yasuhiko
no journal, ,
To get the whole picture of bystander effects is necessary to better understand the effect of low-dose irradiation. Normal human fibroblasts were irradiated with carbon ions or rays. After irradiation, irradiated and non-irradiated cells were co-cultured in the upper and lower parts of porous membrane, respectively. The survival rates of bystander cells decreased with dose and bottomed out at around 80%. In addition, the survival rates of bystander cells were not different between carbon ions and rays at the same doses. These indicate that the bystander effect depends in part on irradiation dose but not on radiation quality. Treatment of a specific scavenger of nitric oxides suppressed the reduction of survival rates of bystander cells. There were also negative relationships between the survival rates of bystander cells and the nitrite concentrations of the medium, suggesting that the amounts of nitric oxide released are an important factor related to the bystander effect.
Hattori, Yuya; Yokoya, Akinari; Watanabe, Ritsuko
no journal, ,
no abstracts in English
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.
Kaminaga, Kiichi; Kanari, Yukiko; Sakamoto, Yuka; Narita, Ayumi; Usami, Noriko*; Kobayashi, Katsumi*; Noguchi, Miho; Yokoya, Akinari
no journal, ,
no abstracts in English
