DNA damage and repair kinetics after microbeam radiation therapy emulation in living cells using monoenergetic synchrotron X-ray microbeams

A novel synchrotron-based approach, known as microbeam radiation therapy (MRT), currently shows considerable promise in increased tumour control and reduced normal tissue damage compared with conventional radiotherapy. Different microbeam widths and separations were investigated using a controlled cell culture system and monoenergetic (5.35 keV) synchrotron X-rays in order to gain further insight into the underlying cellular response to MRT. DNA damage and repair was measured using fluorescent antibodies against phosphorylated histone H2AX, which also allowed us to verify the exact location of the microbeam path. Beam dimensions that reproduced promising MRT strategies were used to identify useful methods to study the underpinnings of MRT. These studies include the investigation of different spatial configurations on bystander effects. H2AX foci number were robustly induced in directly hit cells and considerable DNA double-strand break repair occurred by 12 h post-10 Gy irradiation; however, many cells had some H2AX foci at the 12 h time point. H2AX foci at later time points did not directly correspond with the targeted regions suggesting cell movement or bystander effects as a potential mechanism for MRT effectiveness. Partial irradiation of single nuclei was also investigated and in most cases H2AX foci were not observed outside the field of irradiation within 1 h after irradiation indicating very little chromatin movement in this time frame. These studies contribute to the understanding of the fundamental radiation biology relating to the MRT response, a potential new therapy for cancer patients.