S phase synchrony in monolayer CHO cultures

Abstract

Parameters are described for reproducible S phase synchrony of Chinese hamster ovary cells growing in monolayer, adapting a method described by Tobey & Crissman [1] for CHO cells growing in suspension culture. Cells are collected at the G1/S boundary in hydroxyurea after reversal of an early G1 block induced by isoleucine deprivation. The entire population enters the S period within 60 min after removal of hydroxyurea and proceeds through the S period with minimal decay of synchrony, as evidenced by autoradiographic and rate studies on [ 3H]TdR uptake. In addition, a method is described for obtaining cells synchronized during two successive S periods. The presence of hydroxyurea during G1 does not measurably affect the rate of uptake of [ 3H]uridine or [ 3H]leucine into TCA-insoluble material; however, cultures released from the hydroxyurea block at 10 h incorporate slightly more [ 3H]uridine (but not [ 3H]leucine) in the next 6 h than cultures maintained in hydroxyurea over this interval. Delaying entry into S with hydroxyurea for as long as 15 h does not significantly change the initial rate or duration of DNA synthesis upon removal of hydroxyurea, arguing against the build-up of substances responsible for initiation of replicons. Furthermore, if DNA synthesis is delayed with hydroxyurea in one cell cycle, a constant minimal interval of 15 h elapses before the population enters into the next S phase, suggesting that the timing of the S period is coupled to the timing of the previous S.