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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 effect of DEM and BSO treatment on the nuclear and cytoplasmic pool of GSH.The maximum projection images (as presented in the fig. 3) were analysed by area, as described in “Materials and Methods”. CMFDA fluorescence in nuclear area (defined by Hoechst staining, see fig. 3) is presented in fig. 4A and the CMFDA fluorescence of the cytoplasm area (defined by transmission images, see fig. 3) is presented at fig. 4B. The results are mean values of at least 4 different experiments (50–100 cells per experiment). The level of CMFDA fluorescence in the nuclear, cytoplasmic and mitochondrial area after treatment with BSO and DEM at 24 h of culture is presented at the panel C. The analysis of nuclear and cytoplasmic area was performed as described. Mitochondrial area was considered to be marked by perinuclear green fluorescence, as demonstrated previously (4). The results are presented as mean of 3–5 different experiments.
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pone-0006413-g004: The effect of DEM and BSO treatment on the nuclear and cytoplasmic pool of GSH.The maximum projection images (as presented in the fig. 3) were analysed by area, as described in “Materials and Methods”. CMFDA fluorescence in nuclear area (defined by Hoechst staining, see fig. 3) is presented in fig. 4A and the CMFDA fluorescence of the cytoplasm area (defined by transmission images, see fig. 3) is presented at fig. 4B. The results are mean values of at least 4 different experiments (50–100 cells per experiment). The level of CMFDA fluorescence in the nuclear, cytoplasmic and mitochondrial area after treatment with BSO and DEM at 24 h of culture is presented at the panel C. The analysis of nuclear and cytoplasmic area was performed as described. Mitochondrial area was considered to be marked by perinuclear green fluorescence, as demonstrated previously (4). The results are presented as mean of 3–5 different experiments.

Mentions: Quantification of the fluorescence emission in the nuclear and in the cytoplasmic area is shown in Figure 4. Panel A shows nuclear CMFDA fluorescence at different time points in untreated, DEM, DEM+GSHe and BSO treated 3T3 fibroblasts. Clearly DEM treated fibroblasts at 6, 24 and 48 hours show lower fluorescence intensity than controls. In those cells incubated with BSO, CMFDA staining was similar or even higher than in control. Replenishment of GSH with glutathione monethyl ester showed nuclear CMFDA distribution similar than untreated cells. Thus, DEM but not BSO is able to maintain low nuclear GSH levels at 6, 24 and 48 hours of incubation.


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 effect of DEM and BSO treatment on the nuclear and cytoplasmic pool of GSH.The maximum projection images (as presented in the fig. 3) were analysed by area, as described in “Materials and Methods”. CMFDA fluorescence in nuclear area (defined by Hoechst staining, see fig. 3) is presented in fig. 4A and the CMFDA fluorescence of the cytoplasm area (defined by transmission images, see fig. 3) is presented at fig. 4B. The results are mean values of at least 4 different experiments (50–100 cells per experiment). The level of CMFDA fluorescence in the nuclear, cytoplasmic and mitochondrial area after treatment with BSO and DEM at 24 h of culture is presented at the panel C. The analysis of nuclear and cytoplasmic area was performed as described. Mitochondrial area was considered to be marked by perinuclear green fluorescence, as demonstrated previously (4). The results are presented as mean of 3–5 different experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006413-g004: The effect of DEM and BSO treatment on the nuclear and cytoplasmic pool of GSH.The maximum projection images (as presented in the fig. 3) were analysed by area, as described in “Materials and Methods”. CMFDA fluorescence in nuclear area (defined by Hoechst staining, see fig. 3) is presented in fig. 4A and the CMFDA fluorescence of the cytoplasm area (defined by transmission images, see fig. 3) is presented at fig. 4B. The results are mean values of at least 4 different experiments (50–100 cells per experiment). The level of CMFDA fluorescence in the nuclear, cytoplasmic and mitochondrial area after treatment with BSO and DEM at 24 h of culture is presented at the panel C. The analysis of nuclear and cytoplasmic area was performed as described. Mitochondrial area was considered to be marked by perinuclear green fluorescence, as demonstrated previously (4). The results are presented as mean of 3–5 different experiments.
Mentions: Quantification of the fluorescence emission in the nuclear and in the cytoplasmic area is shown in Figure 4. Panel A shows nuclear CMFDA fluorescence at different time points in untreated, DEM, DEM+GSHe and BSO treated 3T3 fibroblasts. Clearly DEM treated fibroblasts at 6, 24 and 48 hours show lower fluorescence intensity than controls. In those cells incubated with BSO, CMFDA staining was similar or even higher than in control. Replenishment of GSH with glutathione monethyl ester showed nuclear CMFDA distribution similar than untreated cells. Thus, DEM but not BSO is able to maintain low nuclear GSH levels at 6, 24 and 48 hours of incubation.

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