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野口 実穂; 横谷 明徳; 神長 輝一; 藤井 健太郎; 鈴木 啓司*; 宇佐美 徳子*
no journal, ,
In this study, to clarify activation of autophagy on energy deposition in the cell nucleus or cytoplasm by the photoelectric effect, we investigated change of autophagic activity in human fibroblast cells irradiated with soft X-ray microbeam (5.35keV).Using a synchrotron X-ray microbeam, we irradiated 25 to 61 cells by targeting nuclei with square X-ray microbeam (10 m 10 m). For irradiating cytoplasm, 60 m 60 m square X-ray microbeam was used with a metal mask of 10m 10 m central area not to irradiate the cell nucleus. Induction of autophagy was measured using the fluorescent probe, Cyto-ID Green, which stains specifically autophagic vacuoles. Irradiated cells were treated with the dye 15 min before observation. The cells observed by a fluorescent microscope were quantified as mean values of the fluorescent intensity per cell. We observed autophagic fluorescence in nucleus- or cytoplasm-irradiated cells at 1 day to 4 days after irradiation. Some of these cells showed highly localized strong fluorescence. Such localized fluorescence was rarely observed when irradiated with a wide beam from a conventional higher energy X-ray machine (150 kVp). A lot of nucleic irradiated cells, rather than cytoplasm-irradiated cell, showed cell death especially at 4 days after irradiation. When irradiated with the wide beam X-rays, on the other hand, the cells sustained irreversible growth arrest, and maintained their viability. These results indicate that the soft X-ray microbeam exposure is a powerful probe to provide us an aspect of autophagic activation by photoelectric (Auger) effect in a particular part of cells.
神長 輝一; 嘉成 由紀子*; 坂本 由佳*; 野口 実穂; 成田 あゆみ*; 藤井 健太郎; 宇佐美 徳子*; 小林 克己*; 鈴木 啓司*; 横谷 明徳
no journal, ,
We performed selective exposure to HeLa-Fucci cells of a specific cell cycle using synchrotron X-ray microbeam. The results suggested that, not only the irradiated, but also non-irradiated cells surrounding the exposed cells also undergo cell cycle arrest as a consequence of a novel "bystander" effect. When irradiated to spheroids of the HeLa-Fucci cells as a model of 3D cellular tissues, we have succeeded to track the cell cycle modulations in the spheroid using a confocal microscopy. Further we performed sub-cellular irradiation to target a cell nucleus or cytoplasm to look at the effect on mitochondria in human fibroblast cells stained with a specific chemical. Even when the nucleus was irradiated, the potential of mitochondria was enhanced for 12 h. It is inferred that not only DNA damage repair but also certain responses to cellular damage might need excess production of ATP. These clearly show that the live cell imaging appears to be a promising method for single cell tracking.