@article{oai:repo.qst.go.jp:00078564,
 author = {Konishi, Teruaki and Kobayashi, Alisa and Ohsawa, Daisuke and Autsavapormporn, Narongchai and Ahbrizal Tengku Ahmad, Tengku and Liu, Cuihua and Oikawa, Masakazu and Wang, Jun and Konishi, Teruaki and Kobayashi, Alisa and Ohsawa, Daisuke and Liu, Cuihua and Oikawa, Masakazu},
 journal = {平成３０年度　共用施設（PASTA＆SPICE、NASBEE、X/γ線照射装置）成果報告書},
 month = {Dec},
 note = {Objective: Direct exposure of the nucleus to radiation, is the primary cause of various radio-biological effects. However, the cytoplasm is equally exposed to radiation during treatments that result in activation of intra-cellular response. Thus, the present study is aimed at investigating 1) whether cytoplasmic irradiation affect DSB repair, when the cytoplasm and nucleus is irradiated sequentially, and 2) whether the cytoplasmic irradiation alone is sufficient to induce DNA double strand breaks (DSB) in the nucleus.

Material and Methods: To distinguish the radiobiological effects between nuclear and cytoplasmic irradiation, all the experiments were conducted using the SPICE-NIRS microbeam (SPICE),[1] that can target precisely the nucleus (N) and/or (C) with desired number of 3.4 MeV protons. We examined the kinetics of DSB repair in WI-38 normal human fibroblast cells that were irradiated by microbeam targeted to the N, C, or N+C. Cells were fixed at 1, 4, 8, 16, and 24 hours post-irradiation. Subsequently, they were immuno-stained with antibodies against γ-H2AX, a DSB marker, and imaged using SPICE-Offline microscope system to quantify the residual DSB in each nucleus. 

Results: Microbeam irradiation induced significant γ-H2AX, directly proportional to the number of protons delivered per N. In the C-targeted cells, γ-H2AX levels did not increase significantly, compared to controls, 1-hour post irradiation. However, 4 hours post-irradiation, γ-H2AX levels were significantly increased in C -targeted cells, compared to non-irradiated controls, and the increase was proportional to the number of protons delivered. Cells irradiated with 500 protons per N, showed lowered residual γ-H2AX levels in N+C cells additionally irradiated with 500 or 1000 protons targeted to the C, 16 hours and 24 hours post-irradiation, respectively. 

Conclusion: Our results suggest that cytoplasmic damage triggers enhanced repair of DSBs that are induced upon nuclear irradiation. 

Acknowledgments
Authors would like to thank the staffs of Electrostatic Accelerator section, NIRS for technical assistance on the operation of SPICE‑NIRS microbeam. This work was supported by grants from the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant‑in‑Aid for Challenging Exploratory Research (16K15586) of Japan, and the International Open Laboratory program of NIRS. 
This work was published in Konishi T et al., Journal of Radiation and Cancer Research, 2018. [2]

Reference
1.Konishi, T., Oikawa, M., Suya, N., Ishikawa, T., Maeda, T., Kobayashi, A., Shiomi, N., Kodama, K., Hamano, T., Homma-Takeda, S. et al. SPICE-NIRS microbeam: a focused vertical system for proton irradiation of a single cell for radiobiological research. J Radiat Res. 54(4), 736-47 (2013).
2.Konishi, T., Kobayashi, A., Tengku Ahmad, T. and Wang, J. Enhanced DNA double strand break repair triggered by microbeam irradiation induced cytoplasmic damage. Journal of Radiation and Cancer Research. 9(4), 183-189 (2018).},
 pages = {25--27},
 title = {Studies on radiation induced defensive intra and inter-cellular response using SPICE-NIRS microbeam (S16-IOL01:2019)},
 year = {2019}
}