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Journal Articles

LET dependency of human normal dermal cells survival in carbon ion irradiation

Yoshida, Yukari*; Mizohata, Kensuke*; Matsumura, Akihiko*; Isono, Mayu*; Yako, Tomoko*; Nakano, Takashi*; Funayama, Tomoo; Kobayashi, Yasuhiko; Kanai, Tatsuaki*

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 81, 2015/03

In the clinical application of carbon-ion (C-ion) radiation therapy in Japan, different RBE values of carbons have been used for clinical and biological endpoints. The biological RBE (bRBE) was estimated by a method that is based on the linear-quadratic (LQ) model, and was defined ${it in vitro}$ at the 10% surviving fraction of human salivary gland (HSG) tumor cells. However, many of biological parameters, that is, type of tissues, different sort of cells, oxygenation levels, and all, could affect radiosensitivity. Thus, normal human dermal fibroblasts (NHDF) cells were exposed to C-ion beams at Gunma University (10-80 keV/micrometer) and TIARA (108 and 158 keV/micrometer). The surviving fractions were analyzed with colony formation assays. The experimental RBE (eRBE) values were estimated from the radiation dose survival curve fitted by LQ model, and defined ${it in vitro}$.

Journal Articles

Nonhomologous end-joining repair plays a more important role than homologous recombination repair in defining radiosensitivity after exposure to high-LET radiation

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:51 Percentile:90.51(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.

Journal Articles

Exploration of "over kill effect" of high-LET Ar- and Fe-ions by evaluating the fraction of non-hit cell and interphase death

Mehnati, P.*; Morimoto, Shigeko*; Yatagai, Fumio*; Furusawa, Yoshiya*; Kobayashi, Yasuhiko; Wada, Seiichi; Kanai, Tatsuaki*; Hanaoka, Fumio*; Sasaki, Hiroshi*

Journal of Radiation Research, 46(3), p.343 - 350, 2005/09

 Times Cited Count:30 Percentile:63.93(Biology)

no abstracts in English

Oral presentation

Study on bystander effects induced by carbon-ion beams between normal fibroblasts and lung cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this study is to detect bystander effect between normal fibroblasts and lung cancer cells with different p53 status and to elucidate its mechanisms. In the study, we used human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wtp53 line that can produce normal p53 proteins in their DNA damage response. We irradiated cells with carbon-ion broad beams (18.3 MeV/u, LET = 108 keV/$$mu$$m; Dose = 0.5 Gy) or $$gamma$$-rays (Dose = 0.5 Gy), then calculated survival rates of bystander cells after 6 or 24 hours cocultute of irradiated and non-irradiated cells. When we cocultured irradiated H1299/wtp53 cells with non-irradiated WI-38 cells, it was found that survival rates of WI-38 cells were not decreased at all. Consequently, it was suggested that bystander factors were not released from irradiated H1299/wtp53 cells.

Oral presentation

Study on bystander cell-killing effects induced by carbon-ion beams between normal fibroblasts and lung cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

In this study, we investigated radiation-induced bystander effects between normal fibroblasts and cancer cells to elucidate the responses between normal tissues and tumor in heavy-ion radiotherapy. In our experiments, human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wtp53 line which is genetically modified to produce normal p53 proteins in their DNA damage response were used. Cells were irradiated with carbon-ion broad beams (LET = 108 keV/$$mu$$m, Dose = 0.5 Gy) or Co-60 $$gamma$$-rays (LET = 0.2 keV/$$mu$$m, Dose = 0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells were calculated using colony formation assay. It was consequently found that survival rates of non-irradiated WI-38 cells increased when the cells were co-cultured with irradiated H1299/wtp53 cells. From this result, we conclude that some signals are released from irradiated H1299/wtp53 cells to promote cell adhesion and growth of bystander cells.

Oral presentation

Analysis of radiation-induced bystander effects between lung normal and cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this study is to detect radiation-induced bystander effects between lung normal and cancer cells using co-culture system. In our experiments, human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wt${it p53}$ line which is genetically modified to produce normal p53 proteins in their DNA damage response were used. Cells were irradiated with carbon-ion broad beams (LET = 108 keV/$$mu$$m, Dose = 0.5 Gy) or $$^{60}$$Co-$$gamma$$-rays (LET = 0.2 keV/$$mu$$m, Dose = 0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells were calculated using colony formation assay. When we co-cultured irradiated H1299/wt${it p53}$ with non-irradiated WI-38, it was found that survival rates of WI-38 increased from 10% to 20% after 24-hours co-culture. Consequently, it was suggested that some factors were released from irradiated H1299/wt${it p53}$ to promote cell growth and adhesion of bystander cells.

Oral presentation

Comprehensive detection of radiation-induced bystander effects using co-culture system

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this research is to detect comprehensively what kind of influence radiation-induced bystander effects have, between lung normal and cancer cells using co-culture system. In our experiments, human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wt${it p53}$ line were used. Cells were irradiated with carbon-ion broad beams (WI-38: 0.13 Gy, H1299/wt${it p53}$: 0.5 Gy) or $$^{60}$$Co $$gamma$$-rays (0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells was calculated using colony formation assay. When we co-cultured irradiated H1299/wt${it p53}$ with non-irradiated WI-38, it was found that survival rates of WI-38 increased about 10% after 24-hours co-culture. On the other hand, when we co-cultured irradiated WI-38 with non-irradiated WI-38, it was found survival rates of WI-38 decreased about 15% after 6-hours and 24-hours co-culture. Consequently, when the kinds of irradiated cells differed, it found out that heavy-ion induced bystander responses change a lot, and it was thought that these results are important to upgrade heavy-ion radiotherapy.

Oral presentation

Construction of experimental systems to analyze bystander effects induced by heavy-ion microbeam between different type cells

Ikeda, Hiroko; Funayama, Tomoo; Yokota, Yuichiro; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

We have so far analyzed bystander effects between lung cancer cells H1299/wt${it p53}$ and lung normal cells WI-38, using clonogenic assay to judge reproductive cell death. Consequently, when the kinds of irradiated cells differed, it found out that heavy-ion induced bystander responses change a lot. In this research, we decided to use the heavy-ion microbeam system at JAEA-Takasaki to clarify inner cellular and/or intercellular molecular mechanisms concerned with the bystander effects. This equipment is an effective tool and it enabled to elucidate mechanisms which were not made clearly in our previous study using broad beam system. Although there are many reports of bystander effects between the same type cells using microbeam, since there is still no report of bystander effects between different type cells using heavy-ion microbeam, the establishment of experimental system is needed. Now, we are constructing experimental systems to irradiate a part of cells which carried out mixed culture in the same plate using microbeam, and we will report the acquired findings and future development.

Oral presentation

Time-dependent change of heavy-ion induced bystander response in different cells or same cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this research is to comprehend a characteristic phenomenon by comparing bystander responses between different cells and same cells. In our experiments, human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wt${it p53}$ line were used. Cells were irradiated with carbon-ion broad beams (WI-38:0.13 Gy, H1299/wt${it p53}$:0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells was calculated using colony formation assay. When we co-cultured irradiated WI-38 with non-irradiated WI-38, it was found that survival rates of WI-38 decreased about 15% after 6-hours and 24-hours co-culture. On the other hand, when we co-cultured irradiated H1299/wt${it p53}$ with non-irradiated WI-38, it was found survival rates of WI-38 increased about 10% after 24-hours co-culture. Consequently, when the kinds of irradiated cells differed, it found out that heavy-ion induced bystander responses change a lot.

Oral presentation

Comprehensive detection of radiation - induced bystander effects; Are bystander effects negative or positive ?

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this research is to detect comprehensively what kind of influence radiation-induced bystander effects have, between lung normal cells WI-38 and lung cancer cells H1299/wt${it p53}$ using co-culture system. In our experiments, cells were irradiated with carbon-ion broad beams (WI-38:0.13 Gy, H1299/wt${it p53}$:0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells was calculated using colony formation assay. Consequently, it was thought that survival rates of non-irradiated cell were increased by co-culture between different cells. So we used the heavy-ion microbeam system at JAEA-Takasaki to clarify molecular mechanisms concerned with the bystander effects. Now, we are constructing experimental systems to irradiate a part of cells which carried out mixed culture in the same plate using microbeam, and we will report the acquired findings.

Oral presentation

Estimation of heavy-ion-induced bystander effects between lung normal and cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this research is to elucidate comprehensively what kind of influence heavy-ion-induced bystander effects have in cancer therapy. In our experiments, human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wt${it p53}$ line were used. Cells were irradiated with carbon-ion broad beams (WI-38: 0.13 Gy, H1299/wt${it p53}$: 0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells was calculated using colony formation assay. When we co-cultured irradiated WI-38 with non-irradiated WI-38, it was found survival rates of WI-38 decreased about 10%-15% after 6-hours and 24-hours co-culture. On the other hand, when we co-cultured irradiated H1299/wt${it p53}$with non-irradiated WI-38, it was found that survival rates of WI-38 increased about 10% after 24-hours co-culture. Consequently, it was thought that survival rates of non-irradiated cells were increased by co-culture between different type cells.

Oral presentation

Comprehensive detection of radiation-induced bystander effects using lung normal and cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this research is to detect comprehensively what kind of influence radiation-induced bystander effects have, between lung normal cells WI-38 and lung cancer cells H1299/wt${it p53}$ using co-culture system. In our experiments, cells were irradiated with carbon-ion broad beams (WI-38: 0.13 Gy, H1299/wt${it p53}$: 0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells was calculated using colony formation assay. Consequently, it was thought that survival rates of non-irradiated cell were increased by co-culture between different cells. So we used the heavy-ion microbeam system at JAEA-Takasaki to clarify molecular mechanisms concerned with the bystander effects. Now, we are constructing experimental systems to irradiate a part of cells which carried out mixed culture in the same plate using microbeam, and we will report the acquired findings.

Oral presentation

Establishment of experimental systems for analyzing bystander effects induced by heavy-ion irradiation between normal and cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this study is to make clear the bystander effect between cancer cells and normal cells and to establish a new approach using microbeam for elucidating its molecular mechanism. When irradiated cells and non-irradiated cells were co-cultured without physical contact, it was found that clonogenic abilities of non-irradiated bystander cells increased between different type cells but decrease between same type cells. This suggests that bystander responses were greatly different between the same type cells and different type cells. However, we could detect only the medium-mediated bystander response in the co-culture experiment. Since there is another pathway that induces bystander response via gap junction intercellular communications, we planned to irradiate the mixed culture of different type cells using the heavy-ion microbeam system for elucidating its molecular mechanism. Now, we are constructing the new experimental approach and will report the latest findings.

Oral presentation

Heavy-ion induced bystander effects between different type cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kobayashi, Yasuhiko; Kanai, Tatsuaki*

no journal, , 

In this study, we used human lung normal fibroblast cell line WI-38, and human lung cancer cell line H1299/wtp53. To detect only the medium-mediated bystander effects, we adopted cell co-culture systems. The result indicated that the clonogenic abilities of the non-irradiated cells were increased when the cells irradiated with carbon ions transmitted bystander signals via medium to the different type of the cells. In contrast, the clonogenic abilities were decreased when irradiated cells were co-cultured with the same type of non-irradiated cells. From the result, it is suggested that there is a large difference in heavy-ion induced bystander responses via medium between the same type cells and different type cells. Now, we start the establishment of experimental systems for irradiating a part of confluent cells of mixed culture in the same dish using microbeam.

Oral presentation

Analysis of carbon-ion induced bystander effects between different type cells and same type cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Kobayashi, Yasuhiko

no journal, , 

The purpose of this research is to compare bystander responses between different type cells and same type cells, and is to clarify their influence on heavy-ion radiotherapy. In our experiments, human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wt${it p53}$ line were used. Cells were irradiated with carbon-ion broad beams (WI-38:0.13 Gy, H1299/wt${it p53}$:0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells was calculated using colony formation assay. Consequently, it is suggested that there is a large difference in heavy-ion induced bystander responses via medium between the different type cells and the same type cells.

Oral presentation

Analysis of dose-response in bystander effects between human lung normal and cancer cells

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${it p53}$ were used. Cells were irradiated with carbon-ion broad beams (LET=108 keV/ $$mu$$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${it p53}$ 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${it p53}$ 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.

Oral presentation

LET dependency of relative biological effectiveness in normal human dermal fibroblasts irradiated with carbon ion beam

Mizohata, Kensuke*; Yoshida, Yukari*; Matsumura, Akihiko*; Isono, Mayu*; Yako, Tomoko*; Ando, Koichi*; Funayama, Tomoo; Ono, Tatsuya*; Nakano, Takashi*; Kanai, Tatsuaki*

no journal, , 

Clinical RBE (cRBE), which was used to decide a clinical dose of carbon ion radiotherapy, is calculated by multiply experimental RBE (eRBE) by scaling factor. A value of eRBE was measured by colony formation assay of HSG cells in past, and adopted to the therapy of all patients and organ. However, there are question whether it was proper to use conventional eRBE in a different organization, different cell type. Therefore, we irradiated X-rays or a carbon line to NHDF cell and performed a colony assay to determine RBE of different cell type. The value of RBE was calculated from the cell survival rate fit in LQ model. As a result, the value of RBE was increased in LET-dependent manner, and the RBE obtained from the NHDF cell exhibited higher value than that of HSG cells. Thus, we concluded that it might be necessary to change scaling factor when evaluate different organ.

Oral presentation

Analysis of medium-mediated bystander effects induced by carbon-ion between human lung normal and cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kobayashi, Yasuhiko; Kanai, Tatsuaki*

no journal, , 

The purpose of this research is to analyze carbon-ion induced bystander effects via medium, and is to understand a characteristic phenomenon between different type cells. In this research, human lung normal fibroblasts WI-38 and human lung cancer cells H1299/wt${it p53}$ were used. Cells were irradiated with carbon-ion broad beams (LET=108 keV/$$mu$$m, Dose=0.13, 0.5 Gy), then survival rates of bystander cells after 6- or 24-hours co-culture with irradiated cells were measured using colony formation assay. When dose was changed, we found that bystander responses are also different between irradiated WI-38 and non-irradiated H1299/wt${it p53}$.

Oral presentation

Analysis of medium-mediated bystander effects induced by carbon-ion irradiation between human lung normal and cancer cells

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Nakano, Takashi*; Kobayashi, Yasuhiko

no journal, , 

In this study, human lung normal fibroblasts WI-38 and human lung cancer cells H1299/wt${it p53}$ were used. Cells were irradiated with carbon-ion broad beams, then survival rates of bystander cells after co-culture with irradiated cells were measured using colony assay. The survival rates of non-irradiated bystander cancer cells co-cultured with 0.13 Gy irradiated normal cells increased after 6-hours co-culture. On the other hand, the bystander cells co-cultured with 0.5 Gy irradiated normal cells showed decreased survival rates. These results indicated that the bystander responses of the cancer cells changes according to the irradiation dose on the normal cells. In addition, the survival rates of bystander cancer 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 normal cells. From these results, reduction of survival rates is likely to be caused by NO radical as a mediator in bystander effects.

Oral presentation

Patterning irradiation for contact co-culture of different type cells using heavy-ion microbeam

Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Kanai, Tatsuaki*; Nakano, Takashi*; Kobayashi, Yasuhiko

no journal, , 

There have been some reports on bystander effects induced by proton microbeam in contact co-cultured different type cells, but there are few reports using heavy-ion microbeam. So, we have established a new contact co-culture system between human lung normal fibroblast cell line WI-38 and human lung cancer cell line H1299/wt${it p53}$ in the same dish. We have also adapted patterning irradiation systems which automatically irradiated to cancer cells (or normal cells) in a certain range by making use of the target cell irradiation technique at JAEA-Takasaki. Thereby, we were able to successively irradiate to 250 sites of confluent cancer area (lengthwise: 5 mm) so as not to overlap the irradiated range, using carbon-ion microbeam collimated by aperture of $$Phi$$20 $$mu$$m. Now, we are analyzing DNA damage and repair of patterning irradiation samples by evaluating focus numbers of immunostained 53BP1 and $$gamma$$-H2AX. The details of the method and findings will be reported in the talk.

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