Flow cytometric BrdUrd‐pulse‐chase study of X‐ray‐induced alterations in cell cycle progression

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd)‐pulse‐chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X‐rays and pulse‐labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd‐free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G 2 , i.e. the fraction of BrdUrd‐negative G 2 cells and the fraction of BrdUrd‐positive cells that had not divided, showed radiation dose‐dependent delays. The magnitude of the effects indicates that the cells irradiated in G 2 and in S are equally delayed. S phase transit of cells irradiated in S or in G 1 did not appear to be affected. There were apparent changes in flow of cells out of G 1 , which could be explained by the delayed entry of G 2 cells into the compartment because of G 2 arrest. Thus, in asynchronous cells the method was able to detect G 2 delay in those cells irradiated in S and G 2 phases and demonstrate the absence of cell‐cycle delays in other phases.