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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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Related in: MedlinePlus

The pattern of overall S-glutathionylated and oxidized nuclear proteins induced by the two GSH depleting agents at 6 h, 24 h and 6 days in culture.A.- Glutathionylated proteins: equal amount of nuclear extracts were loaded and separated by a 12% SDS gel under non-reducing conditions. S-glutathionylated proteins were detected by Western blot using anti-glutathione monoclonal antibody. B.- Oxidized proteins: equal amounts of nuclear extracts were derivatized and separated by electrophoresis in an acrilamide gel. Western blotting and subsequent immunodetection of carbonylated proteins were performed according to the protocol recommended by the manufacturer (see material and methods). Right panels shows the densitometry results for the level of glutathyolation and oxidation of nuclear proteins, respectively [Mean±SD (n = 3)].
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pone-0006413-g008: The pattern of overall S-glutathionylated and oxidized nuclear proteins induced by the two GSH depleting agents at 6 h, 24 h and 6 days in culture.A.- Glutathionylated proteins: equal amount of nuclear extracts were loaded and separated by a 12% SDS gel under non-reducing conditions. S-glutathionylated proteins were detected by Western blot using anti-glutathione monoclonal antibody. B.- Oxidized proteins: equal amounts of nuclear extracts were derivatized and separated by electrophoresis in an acrilamide gel. Western blotting and subsequent immunodetection of carbonylated proteins were performed according to the protocol recommended by the manufacturer (see material and methods). Right panels shows the densitometry results for the level of glutathyolation and oxidation of nuclear proteins, respectively [Mean±SD (n = 3)].

Mentions: In order to study the changes that take part during the short period when GSH is depleted that is not reversed thereafter, we addressed a kinetic assessment of oxidized and glutathionylated nuclear proteins in DEM treated cells versus control. As presented in figure 8 A at 6 and 24 h in culture the fibroblasts showed a sustained decrease in the pattern of nuclear glutathionylated proteins. However, in those cells incubated with DEM that were in culture for 6 days glutathionylation of nuclear proteins increased to values even higher than controls or BSO treated cells. In BSO treated cells the expression of nuclear glutathionylated proteins was similar to 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)

The pattern of overall S-glutathionylated and oxidized nuclear proteins induced by the two GSH depleting agents at 6 h, 24 h and 6 days in culture.A.- Glutathionylated proteins: equal amount of nuclear extracts were loaded and separated by a 12% SDS gel under non-reducing conditions. S-glutathionylated proteins were detected by Western blot using anti-glutathione monoclonal antibody. B.- Oxidized proteins: equal amounts of nuclear extracts were derivatized and separated by electrophoresis in an acrilamide gel. Western blotting and subsequent immunodetection of carbonylated proteins were performed according to the protocol recommended by the manufacturer (see material and methods). Right panels shows the densitometry results for the level of glutathyolation and oxidation of nuclear proteins, respectively [Mean±SD (n = 3)].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006413-g008: The pattern of overall S-glutathionylated and oxidized nuclear proteins induced by the two GSH depleting agents at 6 h, 24 h and 6 days in culture.A.- Glutathionylated proteins: equal amount of nuclear extracts were loaded and separated by a 12% SDS gel under non-reducing conditions. S-glutathionylated proteins were detected by Western blot using anti-glutathione monoclonal antibody. B.- Oxidized proteins: equal amounts of nuclear extracts were derivatized and separated by electrophoresis in an acrilamide gel. Western blotting and subsequent immunodetection of carbonylated proteins were performed according to the protocol recommended by the manufacturer (see material and methods). Right panels shows the densitometry results for the level of glutathyolation and oxidation of nuclear proteins, respectively [Mean±SD (n = 3)].
Mentions: In order to study the changes that take part during the short period when GSH is depleted that is not reversed thereafter, we addressed a kinetic assessment of oxidized and glutathionylated nuclear proteins in DEM treated cells versus control. As presented in figure 8 A at 6 and 24 h in culture the fibroblasts showed a sustained decrease in the pattern of nuclear glutathionylated proteins. However, in those cells incubated with DEM that were in culture for 6 days glutathionylation of nuclear proteins increased to values even higher than controls or BSO treated cells. In BSO treated cells the expression of nuclear glutathionylated proteins was similar to 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