Detection of S-phase overreplication following chronic hypoxia using a monoclonal anti-BrdUrd

We have examined cytokinetic perturbations induced in Chinese hamster V-79 cells in vitro during and following exposure to chronic hypoxia employing simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content. These data indicate hypoxia inhibited G1 progression into S-phase, but did not significantly delay G2M division and progression into G1. Also, upon reaeration after 20 hr in hypoxia, cells originally in G1 exhibited significant kinetic delay. BrdUrd pulse/chase and pulse/fix data indicated DNA replication was reduced, but not completely inhibited during hypoxia. Also, between 6 and 20 hr of chronic hypoxia and following reaeration, a subset of the original S-phase cells overreplicated their DNA, such that these cells had greater than 2C DNA content. This subpopulation was estimated on the average to comprise approximately 20% of the total population (30% of the treated S-phase subpopulation) by 24 hr following reaeration after 20 hr hypoxia. These results are discussed in light of the similarities between overreplication and gene amplification observed under certain conditions with other agents, which like chronic hypoxia, are used to transiently disrupt DNA synthesis.