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The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

Markovic J, Mora NJ, Broseta AM, Gimeno A, de-la-Concepción N, Viña J, Pallardó FV - PLoS ONE (2009)

Bottom Line: Both agents decreased total cellular glutathione although depletion by BSO was more sustained.Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain.

ABSTRACT

Background: Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.

Principal findings: We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.

Conclusions: Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

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Comparison of the effect of GSH depletion by DEM and BSO on cell growth.Cells were plated and after attaching, treated with 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe. At 6 h, 24 h, 48 h, 72 h, and 5 days of culture cells were detached by trypsinization and counted. The proliferation curves are created on the basis of the mean±SD of 6–16 different experiments.
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pone-0006413-g001: Comparison of the effect of GSH depletion by DEM and BSO on cell growth.Cells were plated and after attaching, treated with 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe. At 6 h, 24 h, 48 h, 72 h, and 5 days of culture cells were detached by trypsinization and counted. The proliferation curves are created on the basis of the mean±SD of 6–16 different experiments.

Mentions: Control 3T3 fibroblasts grew slowly during the first 6 hours in culture and then started to grow faster during 48–72 hours. By day 5 of culture cells reached its confluence limit and stopped growing (see figure 1). Fibroblasts incubated with 10 µM BSO exhibited a rate of growth similar to that of controls, even growing for a longer time than untreated cells (day 5 of culture). However cells treated with DEM 100 µM showed a very low growing profile. Five days after plating, less than 1.5×106 cells were present in the culture dish (250,000 cells were plated) compared with more than 2×106 in the untreated group. When DEM-treated cells were co-incubated with 1 mM glutathione ethyl ester (GSHe), to replenish GSH levels, cells grew at a similar rate as controls.


The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

Markovic J, Mora NJ, Broseta AM, Gimeno A, de-la-Concepción N, Viña J, Pallardó FV - PLoS ONE (2009)

Comparison of the effect of GSH depletion by DEM and BSO on cell growth.Cells were plated and after attaching, treated with 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe. At 6 h, 24 h, 48 h, 72 h, and 5 days of culture cells were detached by trypsinization and counted. The proliferation curves are created on the basis of the mean±SD of 6–16 different experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712766&req=5

pone-0006413-g001: Comparison of the effect of GSH depletion by DEM and BSO on cell growth.Cells were plated and after attaching, treated with 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe. At 6 h, 24 h, 48 h, 72 h, and 5 days of culture cells were detached by trypsinization and counted. The proliferation curves are created on the basis of the mean±SD of 6–16 different experiments.
Mentions: Control 3T3 fibroblasts grew slowly during the first 6 hours in culture and then started to grow faster during 48–72 hours. By day 5 of culture cells reached its confluence limit and stopped growing (see figure 1). Fibroblasts incubated with 10 µM BSO exhibited a rate of growth similar to that of controls, even growing for a longer time than untreated cells (day 5 of culture). However cells treated with DEM 100 µM showed a very low growing profile. Five days after plating, less than 1.5×106 cells were present in the culture dish (250,000 cells were plated) compared with more than 2×106 in the untreated group. When DEM-treated cells were co-incubated with 1 mM glutathione ethyl ester (GSHe), to replenish GSH levels, cells grew at a similar rate as controls.

Bottom Line: Both agents decreased total cellular glutathione although depletion by BSO was more sustained.Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain.

ABSTRACT

Background: Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.

Principal findings: We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.

Conclusions: Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

Show MeSH
Related in: MedlinePlus