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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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The expression of the cell proliferation marker, Id2, after the depletion of the GSH.The cells were plated as usual and after attaching, 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. The protein extracts were obtained at 6 h, 24 h, and 72 h of culture and western blotting was performed as described in materials and methods. At upper panel, western blot analysis of Id2 and β-tubulin in 3T3 fibroblasts at 6, 24, and 72 h of culture is shown. Lower panel shows the relative Id2 to β-tubulin band intensity [Mean±SD (n = 4)] derived from densitometry.
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pone-0006413-g007: The expression of the cell proliferation marker, Id2, after the depletion of the GSH.The cells were plated as usual and after attaching, 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. The protein extracts were obtained at 6 h, 24 h, and 72 h of culture and western blotting was performed as described in materials and methods. At upper panel, western blot analysis of Id2 and β-tubulin in 3T3 fibroblasts at 6, 24, and 72 h of culture is shown. Lower panel shows the relative Id2 to β-tubulin band intensity [Mean±SD (n = 4)] derived from densitometry.

Mentions: In order to find a molecular explanation for the differences in the rate of growth found when glutathione levels were depleted with DEM, we studied the expression of the cell cycle-related protein Id-2 (inhibitor of DNA binding 2). Figure 7 shows that Id-2 expression decreased in the DEM, but not in the BSO treated cells or in the DEM+GSHe treated cells. Thus, depletion of nuclear, but not cytoplasmic glutathione levels is able to decrease Id-2 expression, impairing cell growth.


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 expression of the cell proliferation marker, Id2, after the depletion of the GSH.The cells were plated as usual and after attaching, 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. The protein extracts were obtained at 6 h, 24 h, and 72 h of culture and western blotting was performed as described in materials and methods. At upper panel, western blot analysis of Id2 and β-tubulin in 3T3 fibroblasts at 6, 24, and 72 h of culture is shown. Lower panel shows the relative Id2 to β-tubulin band intensity [Mean±SD (n = 4)] derived from densitometry.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006413-g007: The expression of the cell proliferation marker, Id2, after the depletion of the GSH.The cells were plated as usual and after attaching, 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. The protein extracts were obtained at 6 h, 24 h, and 72 h of culture and western blotting was performed as described in materials and methods. At upper panel, western blot analysis of Id2 and β-tubulin in 3T3 fibroblasts at 6, 24, and 72 h of culture is shown. Lower panel shows the relative Id2 to β-tubulin band intensity [Mean±SD (n = 4)] derived from densitometry.
Mentions: In order to find a molecular explanation for the differences in the rate of growth found when glutathione levels were depleted with DEM, we studied the expression of the cell cycle-related protein Id-2 (inhibitor of DNA binding 2). Figure 7 shows that Id-2 expression decreased in the DEM, but not in the BSO treated cells or in the DEM+GSHe treated cells. Thus, depletion of nuclear, but not cytoplasmic glutathione levels is able to decrease Id-2 expression, impairing cell growth.

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