Advanced

Proliferation of cultured fibroblasts is inhibited by L-iduronate-containing glycosaminoglycans

Westergren-Thorsson, G LU ; Onnervik, Per-Ola; Fransson, L A LU and Malmström, A LU (1991) In Journal of Cellular Physiology 147(3). p.30-523
Abstract

We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in... (More)

We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1-100 micrograms/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%-50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates, with moderately high sulfate and L-iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate and L-iduronate contents. The antiproliferative effect of exogenous glycans appeared after a lag period of 3-4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth-promoting effects in densely seeded cultures.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cell Count, Cell Division, Cell Nucleus, Cells, Cultured, Dermatan Sulfate, Fibroblasts, Gentian Violet, Glycosaminoglycans, Heparin, Heparitin Sulfate, Humans, Iduronic Acid, Lung, Journal Article, Research Support, Non-U.S. Gov't
in
Journal of Cellular Physiology
volume
147
issue
3
pages
30 - 523
publisher
John Wiley & Sons
external identifiers
  • scopus:0025817877
ISSN
0021-9541
DOI
10.1002/jcp.1041470319
language
English
LU publication?
yes
id
9fbdea51-8a32-48dc-81f3-495c8e427c02
date added to LUP
2017-06-27 14:22:01
date last changed
2017-07-09 05:02:34
@article{9fbdea51-8a32-48dc-81f3-495c8e427c02,
  abstract     = {<p>We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1-100 micrograms/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%-50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates, with moderately high sulfate and L-iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate and L-iduronate contents. The antiproliferative effect of exogenous glycans appeared after a lag period of 3-4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth-promoting effects in densely seeded cultures.</p>},
  author       = {Westergren-Thorsson, G and Onnervik, Per-Ola and Fransson, L A and Malmström, A},
  issn         = {0021-9541},
  keyword      = {Cell Count,Cell Division,Cell Nucleus,Cells, Cultured,Dermatan Sulfate,Fibroblasts,Gentian Violet,Glycosaminoglycans,Heparin,Heparitin Sulfate,Humans,Iduronic Acid,Lung,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {3},
  pages        = {30--523},
  publisher    = {John Wiley & Sons},
  series       = {Journal of Cellular Physiology},
  title        = {Proliferation of cultured fibroblasts is inhibited by L-iduronate-containing glycosaminoglycans},
  url          = {http://dx.doi.org/10.1002/jcp.1041470319},
  volume       = {147},
  year         = {1991},
}