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

Author:
  • Jan O Fredlund
  • Maria C Johansson
  • Bo Baldetorp
  • Stina Oredsson
Publishing year: 1994
Language: English
Pages: 243-256
Publication/Series: Cell Proliferation
Volume: 27
Issue: 5
Document type: Journal article
Publisher: Wiley-Blackwell

Abstract english

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 stochiometric 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 G(1) and G(2) 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.

Keywords

  • Cancer and Oncology

Other

Published
  • ISSN: 1365-2184
Stina Oredsson
E-mail: stina [dot] oredsson [at] biol [dot] lu [dot] se

Professor

Functional zoology

+46 46 222 94 97

B-C208

4

Principal investigator

LUCC - Lund University Cancer Centre

Research group

Animal Physiology

Projects

Cell proliferation

Doctoral students and postdocs

PhD Students, main supervisor

Wendy Soria Sotillo

PhD Students, assistant supervisor

Atena Malakpour Permlid