@misc{oai:repo.qst.go.jp:00062895,
 author = {Hamada, Nobuyuki and Iwakawa, Mayumi and Imadome, Kaori and Funayama, Tomoo and Sakashita, Tetsuya and Sora, Sakura and NI, Meinan and Imai, Takashi and Kobayashi, Yasuhiko and 浜田 信行 and 岩川 眞由美 and 今留 香織 and 坂下 哲哉 and 今井 高志},
 month = {Nov},
 note = {INTRODUCTION
  Biological effectiveness varies with the linear energy transfer (LET) of ionizing radiation. During cancer therapy or manned space explorations, humans are exposed to high-LET heavy ions, which inactivate irradiated cells more effectively than low-LET photons like X-rays and gamma-rays1. Over the past 16 years, convincing experimental evidence has been presented demonstrating that radiation induces biological effects in non-irradiated bystander cells having received signals from directly irradiated cells2; however, little information is available heretofore concerning the bystander response to heavy ions. Taking into consideration that less irradiated cells coexist with more non-irradiated counterparts in a population exposed to a lower dose of higher-LET heavy ions, the effects arising not merely in irradiated cells but in their bystander cells should be characterized to comprehend the mechanism of heavy-ion action. Here, we set out to investigate the potential impact of heavy ions on the bystander effect, and compare the response of irradiated and bystander cells in confluent human fibroblasts.
\nMATERIALS AND METHODS
  Confluent density-inhibited cultures of AG01522D normal human fibroblasts3,4 were used, and irradiation was conducted at the TIARA facility of JAEA. To see the bystander effect, only 0.0003% of the cells were targeted with microbeams of carbon ions (18.3 MeV/u, 103 keV/µm)5, so that the vast majority of the cells could be considered as non-irradiated bystander cells. The cells were also irradiated with conventional broadbeams of carbon ions (18.3 MeV/u, 108 keV/µm) to see the effects in irradiated cells6-8. Cell survival was determined with the clonogenic assay, apoptosis with the TUNEL staining, the levels of serine 15-phosphorylated p53 proteins with western blotting, and gene expression profiles with the microarray analysis.
\nRESULTS AND DISCUSSION
  Bystander cells showed nearly 10% reductions in the survival9. Whereas apoptotic frequency in irradiated cells increased with time up to 72 h postirradiation, its frequency in bystander cells was elevated twofold at 24 h but declined at 48 h9, suggesting that bystander cells exhibit transient commitment to apoptosis. Whilst the levels of phosphorylated p53 proteins in irradiated cells escalated and remained unchanged at 2 and 6 h, its levels in bystander cells rose twofold at 6 h but not at 2h9, indicating that bystander cells undergo delayed p53 phosphorylation. More than half of the genes whose expression changed at 2 and 6 h in bystander cells were downregulated, and most of the genes upregulated in irradiated cells were downregulated in bystander cells10, suggesting different expression profiles between irradiated and bystander cells. Pathway analysis revealed serial activation of G protein/PI-3 kinase pathway in bystander cells, but NF-kappaB and p21Waf1 pathways in irradiated cells10. Interleukin genes were upregulated in irradiated cells while its receptor gene was upregulated in bystander cells10, indicating the signal transmission from irradiated to bystander cells.
  The bystander effect for the survival and apoptosis was observed similarly irrespective of whether the cells were targeted with microbeams of carbon ions or two types of neon ions (17.5 MeV/u, 294 keV/µm; 13.0 MeV/u, 375 keV/µm)11. With respect to the medium-mediated bystander effect of X-rays (5 keV/µm) and neon ions (13.0 MeV/u, 437 keV/µm) for chromosome aberrations, there was a difference in the types of aberrations but little difference in total yields12. It seems thence likely that the bystander effect is independent of LET, but that its underpinning causes differ with the radiation quality.
\nCONCLUSION
  The bystander effect of heavy ions was manifested as inactivated clonogenic potential, a transient apoptotic response, delayed p53 phosphorylation, and alterations in gene expression at a genome-wide level. Our findings highlight distinct response of irradiated and bystander cells. Such response of normal fibroblasts could be a defensive mechanism that would avert further expansion of aberrant cells, thus maintaining genome integrity and cellular homeostasis.
\nACKNOWLEDGEMENTS
This work was supported in part by a Grant-in-aid for the 21st Century Center of Excellence (COE) Program for Biomedical Research Using Accelerator Technology from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by the Budget of Nuclear Research of the MEXT based on the screening and counseling by the Atomic Energy Commission of Japan.
\nREFERENCES
1) Hamada N, J Radiat Res 50: in press, 2009 (doi: 10.1269/jrr.08070).
2) Hamada N, et al., J Radiat Res 48: 87-95, 2007.
3) Hamada N, et al., Radiat Res 166: 24-30, 2006.
4) Hamada N, et al., Mutat Res 637: 190-196, 2008.
5) Funayama T, Hamada N, et al., IEEE Trans Plasma Sci 36: 1432-1440, 2008.
6) Hamada N, et al., Cancer Lett 268: 76-81, 2008.
7) Hamada N, et al., Radiother Oncol 89: 227-230, 2008.
8) Hamada N, et al., Radiother Oncol 89: 231-236, 2008.
9) Hamada N, et al., Mutat Res 639: 35-44, 2008.
10) Iwakawa M, Hamada N, et al., Mutat Res 642: 57-67, 2008.
11) Hamada N, et al., Biol Sci Space 22: 46-53, 2008.
12) Kanasugi Y, Hamada N, et al., Int J Radiat Biol 83: 73-80, 2007., 8th International Workshop on Microbeam Probes of Cellular Radiation Response},
 title = {Distinct Response of Irradiated Normal Human Fibroblasts and Their Bystander Cells to Heavy-Ion Radiation in Confluent Cultures},
 year = {2008}
}