Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 µm2 with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.
Abstract:11 Heuskin A.C., Osseiran A., Tang J. and Costes S.V., Simulating Space Radiation-Induced Breast Tumor 12Incidence Using Automata, Radiat. Res. 13Estimating cancer risk from space radiation has been an ongoing challenge for decades primarily 14 because most epidemiological data showing evidence of cancer risk from ionizing radiation are derived 15 from studies of atomic bomb survivors, where individuals were exposed to acute dose of gamma-rays 16 instead of chronic exposure of high-LET cosmic radiation. In this work, we introduce a formalism using 17 cellular automata to model the long-term effects of ionizing radiation in human breast for different 18 radiation quality. We first validate and tune parameters for an automata-based two stage clonal 19 expansion model which simulates the age dependence of spontaneous breast cancer incidence in 20 unexposed US population. We then test the impact of radiation perturbation in the model by modifying 21 parameters to reflect both targeted and non-targeted effects of ionizing radiation. 22Targeted effects (TE) reflect the immediate impact of radiation on cell's DNA with classic endpoints 23 being gene mutations and cell death. They are well known and are directly derived from experimental 24 data. In contrast, non-targeted effects (NTE) are persistent radiation effects affecting both damaged and 25 undamaged cells, they are non-linear with dose and they are not well characterized in the literature. TE 26 is first introduced in the model and predictions are compared to epidemiologic data of the A-bomb 27 cohort. TE alone is not sufficient to induce enough cancer and genomic instability which last ~100 days 28 post-exposure independently of dose needs to be added to predict accurately the dose dependence of 29 breast cancer induced by gamma-rays. Finally, by integrating experimental RBE for TE and keeping 30 radiation-induced genomic instability constant with dose and LET, the model predicts that RBE for breast 31 cancer induced by cosmic radiation would be maximum at 220 keV/µm. This work is well suited to 32 explore next the impact of chronic low dose exposure, inter-individual variation and more complex 33 space radiation scenarii. 34 3
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