Enhanced DNA double-strand break repair by sequential microbeam irradiation of the nucleus and cytoplasm.

Direct hits to the nucleus by radiation are known as the primary cause of various radio-biological effects. However, the cells will have equal chances of being exposed at the cytoplasm, and results in damages that may activate various signaling pathways that mediates inter-cellular response. To distinguish the radiobiological effect between nucleus and cytoplasm irradiation, microbeam irradiation system is the most advantageous technology. In this study, all the experiments were conducted on the SPICE-NIRS proton microbeam irradiation system (SPICE). SPICE is a highly advanced microbeam irradiation system, which provides 3.4 MeV proton beam with a size of 2 micrometer in diameter size that can target the nucleus and/or cytoplasm precisely with desired number of protons 1,2. The feature of SPICE enabled not only the cellular response of targeted cells but also the studies of radio-bystander effect studies of in vitro 3-5 as well as in vivo 6,7.We examined the velocity of DNA double-strand break (DSB) repair in microbeam-irradiated WI-38 normal human fibroblast cells that were microbeam targeted in the nucleus, cytoplasm, or both. Cells were fixed at various time points between one to twenty-four hours post-irradiation. Subsequently, they were immunostained using antibodies against γ-H2AX, which is used as marker for DSB, to quantify the residual DSB per nucleus in images obtained using a microscope.Microbeam irradiation induced significant γ-H2AX activity in proportion to the number of protons delivered per nucleus. In nucleus-targeted cells, γ-H2AX levels did not increase significantly in comparison with non-irradiated controls. However, at four hours post-irradiation, γ-H2AX levels were significantly higher than in the controls, and the increase was proportional to the number of protons delivered. For the cells irradiated with 500 protons per nucleus, we found less residual γ-H2AX with additional 200 and 500 proton deliveries to the cytoplasm at 16 hours and 24 hours post-irradiation, respectively. Taken together, cytoplasmic damage enhances repair of DSB induced by irradiation of the nucleus. Further study is needed to identify the type of cytoplasmic damage and mechanism of intracellular signaling responsible for the enhanced cellular response to DSB. Current status of SPICE-NIRS microbeam will be also described in the presentation.\nReferences 1.Konishi, T., Oikawa, M., Suya, N.,et al., SPICE-NIRS microbeam: a focused vertical system for proton irradiation of a single cell for radiobiological research, J. Radiat. Res., 2013, 54, 736-747.2.Konishi, T., Ishikawa, T., Iso, H., Yasuda, N., Oikawa, M., Higuchi, Y., Kato, T., Hafer, K., Kodama, K., Hamano, T., Suya, N., and Imaseki, H. Biological studies using mammalian cell lines and the current status of the microbeam irradiation system, SPICE, Nucl. Instrum. Meth. B. 2009, 267, 2171-2175.3.Kobayashi A, Autsavapromporn N, Tengku Ahmad TAF, et al., Bystander WI-38 cells modulate DNA double-strand break repair in microbeam-targeted A549 cells through gap junction intercellular communication. Radiat. Prot. Dosim., (in press)4.Kobayashi, A., Tengku Ahmad, TAF., Autsavapromporn, N., et al., Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bi-directional signaling, Mutat. Res., 2017, 803-805, 1-8.5.Autsavapromporn, N., Plante, I., Liu, C., et al., Genetic changes in progeny of bystander human fibroblasts after microbeam irradiation with X-rays, protons or carbon ions: the relevance to cancer risk, Int. J. Radiat. Biol., 2015, 91, 62-70.6.Choi, V. W., Yum, E. H., Konishi, T.,et al., Triphasic low-dose response in zebrafish embryos irradiated by microbeam protons, J. Radiat. Res. 2012, 53, 475-481.7.Choi, V. W., Konishi, T., Oikawa, M., et al., Adaptive response in zebrafish embryos induced using microbeam protons as priming dose and X-ray photons as challenging dose, J. Radiat. Res., 2010, 51, 657-664.The 7th Asian Pacific Symposium on Radiation Chemistr