Impairment of DNA replication within one cell cycle after seeding of cells in the presence of a polyamine-biosynthesis inhibitor

Chinese hamster ovary (CHO) cells in the plateau phase were seeded in the absence or presence of 5 mM 2-difluoromethylornithine (F(2)MeOrn), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. The thymidine analogue bromodeoxyuridine (BrdUrd, 5 mu M) was added to the culture medium 30 min before sampling of the cells, which occurred 1-17 h after seeding. Using flow cytometry, coupled with an indirect immunofluorescence technique, which utilized monoclonal BrdUrd and secondary fluorescein-isothiocyanate-conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. To determine if there was a perturbation in the progression of cells through the S phase, the distribution of BrdUrd-labelled cells in the S phase was evaluated in two ways: (a) by calculating the mean DNA content of BrdUrd-labelled cells in relation to the mean DNA contents of G(1) and G(2) cells (relative movement(zero)) and (b) by studying DNA histograms of BrdUrd-labelled cells. By using both evaluation methods, we show that DNA replication was impaired during the first cell cycle that was initiated after seeding CHO cells in the presence of F(2)MeOrn. The cells appeared to enter the S phase normally but were then delayed in their progression through this phase. The impairment of F(2)MeOrn treatment on DNA replication was apparent at 9 h after seeding, a time point at which the putrescine pool was depleted, the spermidine pool was approximately halved, and the spermine pool was unaffected, when compared to corresponding pools of control cells. When cells were seeded in the presence of F(2)MeOrn and putrescine, the effect on DNA replication was prevented. The rates of incorporation of [H-3]uridine and [H-3]leucine into RNA and protein, respectively, were the same in control and in F(2)MeOrn-treated cells for at least up to 11 h after seeding.