ABNORMAL DNA-SYNTHESIS IN POLYAMINE DEFICIENT CELLS REVEALED BY BROMODEOXYURIDINE-FLOW CYTOMETRY TECHNIQUE

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 t he thymidine analogue bromodeoxyuridine (BrdUrd) and they were then fi xed directly after the labelling period. In addition, cells were label led for 30 min and they were then allowed to progress in BrdUrd-free m edium during a defined post-labelling time before fixation. An indirec t immunofluorescence technique, using the monoclonal BrdUrd antibody a nd the intercalating stochiometric DNA stain, propidium iodide, was ap plied to enable quantification of cellular BrdUrd and DNA contents, re spectively, by flow cytometry (FCM). By comparing the mean DNA content of BrdUrd-labelled cells to the mean DNA contents of G(1) and G(2) ce lls, a relative measure of the position of the BrdUrd-labelled cells w as 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, w hile their progression through S phase was impaired. DNA histograms of BrdUrd-labelled control cells fixed directly after labelling showed t hat most cells were found in early and late S phase, while DNA histogr ams of BrdUrd-labelled DFMO-treated cells showed that most cells were in early S phase, indicating a delayed progression through S phase. An alysis 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 ind ex was significantly higher in DFMO-treated, growth-inhibited cells th an in early plateau phase control cells indicating an S phase accumula tion in the former cells.