Abnormal DNA synthesis in polyamine deficient cells revealed by bromodeoxyuridine—flow cytometry technique

Abstract

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.

Ordered Cell Cycle Phase Perturbations in Chinese Hamster Ovary Cells Treated with an S‐Adenosylmethionine Decarboxylase Inhibitor

“…For cells in culture, 50% inhibitory concentrations were found to be 0.1 -3 pM for L1210 cells (Regenass et al, 1994) and 0.1 -0.5 pM for hormon-dependent and independent breast cancer cells (Manni et al, 1995). In our studies using CHO cells, we have used 20 pM CGP 48664 to obtain growth inhibition equivalent to that obtained after treatment with 5 mM F,MeOm (Fredlund et al, 1994;Fredlund, 1996) since we wished to compare the cell cycle kinetic effects obtained with the two inhibitors. The cell number was 50% lower in cultures treated with 20 pM CGP 48664 compared to control cells at 2 days after seeding and then the treated cells ceased to proliferate.…”

“…For the BrdUrd labelling experiments, a number of replicate cultures, consisting of 0.5X lo6 plateau-phase cells seeded into fresh medium in the absence or presence of 20 pM CGP 48664 in 65-cm2 petri dishes, were set up. Before choosing a CGP 48664 concentration of 20 pM for the present experiments, we performed growth curve experiments using a concentration range of 0.5-20 pM: 20 pM CGP 48664 produced similar growth inhibition as that obtained when treating CHO cells with 5 mM F,MeOrn (Fredlund et al, 1994;Fredlund, 1996). During the 3-day experimental period, no effects on mitochondria1 morphology were observed as judged by vital staining with rhodamine 123 (Chen, 1988) (not shown).…”

“…The method used was originally described by Dolbeare et al (1983) and has been modified by Schutte et al (1987) and Van Erp et al (1988). We have previously described the staining procedure in detail (Fredlund et al, 1994).…”

Impairment of DNA Replication within One Cell Cycle after Seeding of Cells in the Presence of a Polyamine‐Biosynthesis Inhibitor

“…The method was originally described by Dolbeare et al (1983) and it has been modified by Schutte et al (1987) and Van Erp et al (1988). We have described the staining procedure in detail in Fredlund et al (1994) and in the present study it was used with only a minor modification. Before DNA denaturation, cells were incubated in freshly made pepsin/ HCl solution (0.2 mg pepsin/ml in 0.1 M HCI) for 30 min at 37°C in a shaking water bath, to isolate nuclei.…”

“…However, we have recently presented evidence indicating that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in culture in the presence of a polyamine biosynthesis inhibitor (Fredlund et al, 1994). The present study was designed to more specifically evaluate when DNA replication was affected in relation to polyamine depletion after seeding plateau phase CHO cells in the absence or presence of the polyamine synthesis inhibitor 2-difluoromethylornithine (F,MeOrn).…”