Influence of ionizing radiation on proliferation of murine cells and cytotoxicity of EdU revealed by dual pulse and chase analysis

Kurzfassung

Astronauts are constantly exposed to different irradiation doses, resulting in distinct short- and long-term consequences for the organism. Impaired cell cycle progression and decreased clonogenic survival can be caused by elevated radiation levels. To fully understand the effects of different radiation types and doses, a constant development of new methods is indispensable. In order to determine the current cell cycle phase the DNA content can be visualized by DNA intercalating agents and for the analysis of cellular proliferation thymidine analogs can be incorporated into newly synthesized DNA. To measure the effects of irradiation on cellular proliferation and on cell cycle progression in one experiment, a new approach is used by combining two methods. The new assay is called dual pulse labeling. Two different thymidine analogs were used in combination to allow labeling at two different time points, followed by the staining of the DNA. Impaired proliferation of MEF NEMO KO cells was observed after X-irradiation. Irradiation with silicon ions lead to an increased number of MEF in the G2/M phase of cell cycle. Furthermore, cytotoxic side effects were determined for the application of one thymidine analog (5-Ethynyl-2´-deoxyuridine) in mouse embryonic fibroblasts. The results of this study facilitate a new possibility to assess the consequences of radiation on the cell cycle progression and cellular proliferation in one experiment, using fluorescence microscopy and flow cytometry as a read out. Furthermore, the results show that the new thymidine analog EdU can be useful in many scientific questions, but it is inevitable to check for the suitability of the used cell line and to keep the time frame of the experiment restricted.