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

Fredlund, Jan O; Johansson, Maria C LU ; Baldetorp, Bo LU and Oredsson, Stina LU (1994) In Cell Proliferation 27(5). p.243-256
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 stochiometric DNA stain, propidium iodide, was applied to enable quantification of cellular BrdUrd and DNA contents, respectively, by flow cytometry (FCM). By comparing... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Cell Proliferation
volume
27
issue
5
pages
243 - 256
publisher
Wiley-Blackwell
external identifiers
  • scopus:0028270237
ISSN
1365-2184
DOI
10.1111/j.1365-2184.1994.tb01422.x
language
English
LU publication?
yes
id
5f70a3ed-5027-4d63-bb06-9fbbb8f06137 (old id 31443)
date added to LUP
2007-06-19 14:32:16
date last changed
2017-01-01 04:21:34
@article{5f70a3ed-5027-4d63-bb06-9fbbb8f06137,
  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 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.},
  author       = {Fredlund, Jan O and Johansson, Maria C and Baldetorp, Bo and Oredsson, Stina},
  issn         = {1365-2184},
  language     = {eng},
  number       = {5},
  pages        = {243--256},
  publisher    = {Wiley-Blackwell},
  series       = {Cell Proliferation},
  title        = {Abnormal DNA synthesis in polyamine deficient cells revealed by bromodeoxyuridine-flow cytometry technique},
  url          = {http://dx.doi.org/10.1111/j.1365-2184.1994.tb01422.x},
  volume       = {27},
  year         = {1994},
}