Abstract
An X-ray microbeam irradiation system using synchrotron radiation has been developed at the Photon Factory, KEK, Japan, and is now being fully operated for various biological experiments. The minimum beamsize by the slit is 5-micrometer-square, and the size of the microbeam can be changed quickly and arbitrarily by the high-precision slit installed in the system. This specification might be ideal to investigate localization of the damage induction and subsequent cellular responses in cells irradiated with subcellular-sized beam.
To visualize the DNA damage induction, the microbeam-irradiated cells were immuno-stained by phosphorylated histone H2AX (gamma-H2AX) antibody. Most DNA double strand breaks were observed in localized area in cell nuclei, the size of which was almost the same as the beam size.
GFP-tagged protein enables us to visualize cellular responses in living cells. We constructed Chinese hamster ovary (CHO) cells having GFP-tagged Rad51, one of the protein related homologous recombination. Within 1 hour after irradiation, significant number of foci of GFP-Rad51 could be observed at the microbeam-irradiated site in the majority of the irradiated cells, and remained at least 5 hours after irradiation. The foci were appeared in the restricted area in the cell nuclei, which was corresponded to the damage induced area visualized with gamma-H2AX. In some of the cells, the foci could not appeared at all even after 12 hours, and the percentage of the population was almost same as the estimated population of G1-phase cells at the irradiated time.
