FLOW CYTOMETRIC BRDURD-PULSE-CHASE STUDY OF X-RAY-INDUCED ALTERATIONSIN CELL-CYCLE PROGRESSION

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd )-pulse-chase method detects perturbed cell kinetics we applied it to measure eel cycle progression delays following exposure to ionizing ra diation, Since this method will allow both the use of asynchronous cel l populations and the determination of the alterations in cell cycle p rogression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. E xponentially growing Chinese hamster ovary (CHO) K1 cells were irradia ted with graded doses of X-rays and pulse-labelled with BrdUrd immedia tely thereafter. Cells were subcultured in a BrdUrd-free medium for va rious time intervals and prepared for flow cytometric analysis. Of fiv e flow cytometric parameters examined, only those that involved cell t ransit through G(2), i.e. the fraction of BrdUrd-negative G(2) cells a nd the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays, The magnitude of the effects indicate s that the cells irradiated in G(2) and in S are equally delayed. S ph ase transit of cells irradiated in S or in G(1) did not appear to be a ffected. There were apparent changes in flow of cells out of G(1), whi ch could be explained by the delayed entry of G(2) cells into the comp artment 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 phas es.