A method for the cell-cycle-specific analysis of radiation-induced chromosome aberrations and breaks.

The classical G ₂ -assay is widely used to assess cell-radiosensitivity and cancer phenotype: Cells are exposed to low doses of ionizing-radiation (IR) and collected for cytogenetic- analysis ˜1.5 h later. In this way, chromosome-damage is measured in cells irradiated in G ₂ -phase, without retrieving information regarding kinetics of chromosome-break-repair. Modification of the assay to include analysis at multiple time-points after IR, has enabled kinetic-analysis of chromatid-break-repair and assessment of damage in a larger proportion of G ₂ -phase cells. This modification, however, increases the probability that at later time points not only cells irradiated in G ₂ -phase, but also cells irradiated in S-phase will reach metaphase. However, the response of cells irradiated in G ₂ -phase can be mechanistically different from that of cells irradiated in S-phase. Therefore, indiscriminate analysis may confound the interpretation of experiments designed to elucidate mechanisms of chromosome-break-repair and the contributions of the different DSB-repair-pathways in this response. Here we report an EdU based modification of the assay that enables S- and G ₂ -phase specific analysis of chromatid break repair. Our results show that the majority of metaphases captured during the first 2 h after IR originate from cells irradiated in G ₂ -phase (EdU ^- metaphases) in both rodent and human cells. Metaphases originating from cells irradiated in S-phase (EdU ⁺ metaphases) start appearing at 2 h and 4 h after IR in rodent and human cells, respectively. The kinetics of chromatid-break-repair are similar in cells irradiated in G ₂ - and S-phase of the cell-cycle, both in rodent and human cells. The protocol is applicable to classical-cytogenetic experiments and allows the cell-cycle specific analysis of chromosomal-aberrations. Finally, the protocol can be applied to the kinetic analysis of chromosome-breaks in prematurely-condensed-chromosomes of G ₂ -phase cells. In summary, the developed protocol provides means to enhance the analysis of IR-induced-cytogenetic-damage by providing information on the cell-cycle phase where DNA damage is inflicted.
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