Chinese hamster ovary cells were seeded in the absence or presence of the polyamine synthesis inhibitor 2-difluoromethylornithine (DFMO). At 1-4 days after seeding, the cells were labelled for 15-120 min with the thymidine analogue bromodeoxyuridine (BrdUrd) and they were then fixed directly after the labelling period. In addition, cells were labelled for 30 min and they were then allowed to progress in BrdUrd-free medium during a defined post-labelling time before fixation. An indirect immunofluorescence technique, using the monoclonal BrdUrd antibody and the intercalating stoichiometric DNA stain, propidium iodide, was applied to enable quantification of cellular BrdUrd and DNA contents, respectively, by flow cytometry (FCM). By comparing the mean DNA content of BrdUrd-labelled cells to the mean DNA contents of G1 and G2 cells, a relative measure of the position of the BrdUrd-labelled cells was obtained (relative movement). Relative movement data, obtained from control and DFMO-treated cells fixed directly after BrdUrd labelling, indicated that DFMO-treated cells entered S phase at a normal rate, while their progression through S phase was impaired. DNA histograms of BrdUrd-labelled control cells fixed directly after labelling showed that most cells were found in early and late S phase, while DNA histograms of BrdUrd-labelled DFMO-treated cells showed that most cells were in early S phase, indicating a delayed progression through S phase. Analysis of relative movement of cells that were allowed to progress in BrdUrd-free medium after labelling showed that DFMO treatment resulted in a significant lengthening of the DNA synthesis time. Labelling index was significantly higher in DFMO-treated, growth-inhibited cells than in early plateau phase control cells indicating an S phase accumulation in the former cells.
The polyamines putrescine, spermidine, and spermine are needed for normal cell cycle progression and polyamine-depleted cells cease to proliferate. We have investigated cell cycle perturbations in Chinese hamster ovary cells seeded in the presence of 4-amidinoindan-I-one 2'-amidinohydrazone (CGP 48664), a potent inhibitor of S-adenosylmethionine decarboxylase, an enzyme which is essential for the synthesis of spermidine and spermine. At 9 h and at 1, 2, and 3 days after seeding, cells were labelled with the thymidine analog bromodeoxyuridine (BrdUrd) for 30 min, after which the BrdUrd-containing medium was removed and the cells were allowed to progress through the cell cycle in BrdUrd-free medium before sampling (post-labelling time). Using flow cytometry, coupled with an indirect immunofluorenscence technique, utilizing monoclonal anti-BrdUrd and secondary fluorescein-isothiocyanate-conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. By investigating the movement of BrdUrd-nonlabelled G , cells into S phase during the post-labelling time, a measure of the G,/S transition was obtained. The time of appearence in G , of BrdUrd-labelled cells which had divided was used to monitor the length of the G,+M phase. A measure of the S phase length (DNA synthesis time) was obtained by monitoring the progression of BrdUrd-labelled cells through S phase. CGP 48664-induced spermine depletion significantly increased the length of the S phase already 9 h after seeding cells in the presence of the inhibitor. No effects 011 the G,/S transition or on the length of the G,+M phase were observed until 2 days after seeding.
We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G1/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine- flow cytometric method to more specifically study the G1/S transition, the S phase length, and the progression of cells from S phase through G2+M and into G1, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G1/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G1 phase occurred later than the effect on S phase progression. These results imply that the G2+M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.
Chinese hamster ovary (CHO) cells in the plateau phase were seeded in the absence or presence of S mM 2-difluoromethylornithine (F,MeOm), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. The thymidine analogue bromodeoxyuridine (BrdUrd, 5 pM) 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, and G, cells (relative movement,,,,) and (b) by studying DNA histograms of BrdUrdlabelled 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,MeOrn. The cells appeared to enter the S phase normally but were then delayed in their progression through this phase. The impairment of F,MeOm 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,MeOm and putrescine, the effect on DNA replication was prevented. The rates of incorporation of ['Hluridine and ['H]leucine into RNA and protein, respectively, were the same in control and in F,MeOrn-treated cells for at least up to 11 h after seeding.
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