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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 GSH distribution after its depletion with DEM and BSO.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. At 24 h after plating the cells were stained as described in Materials and Methods and observed by confocal microscopy in the chamber provided with 5% CO2 and at 37°C. Images were taken by light microscopy (transmission) and by confocal microscopy, as described in Materials and Methods, to capture blue fluorescence of nuclei (Hoechst-nuclei), green fluorescence that marks GSH (CMFDA-GSH) and red fluorescence of dead cells (PI-dead cells) (results not shown). Z series of at least 8 planes were obtained and maximum projection images were created and analysed. The representative experiment (of five) is presented.
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pone-0006413-g003: The GSH distribution after its depletion with DEM and BSO.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. At 24 h after plating the cells were stained as described in Materials and Methods and observed by confocal microscopy in the chamber provided with 5% CO2 and at 37°C. Images were taken by light microscopy (transmission) and by confocal microscopy, as described in Materials and Methods, to capture blue fluorescence of nuclei (Hoechst-nuclei), green fluorescence that marks GSH (CMFDA-GSH) and red fluorescence of dead cells (PI-dead cells) (results not shown). Z series of at least 8 planes were obtained and maximum projection images were created and analysed. The representative experiment (of five) is presented.

Mentions: Figure 3 depicts untreated fibroblasts during 24 hours in culture showing that GSH (CMFDA staining-green fluorescence) co-localizes with DNA (Hoechst staining-blue fluorescence). However, cells incubated with 100 µM DEM showed a homogeneous GSH distribution within the cells. Surprisingly, inhibition of GSH synthesis by BSO was unable to change the distribution of cellular GSH, showing a cellular glutathione pattern different from the one shown in the DEM-treated cells. Indeed, GSH was high in the nucleus and low in the cytoplasm, like in the control group. Thus, each GSH-depleting agent induces different responses in the distribution of cellular GSH when fibroblasts were proliferating. Co-incubating the cells with 100 µM DEM and 1 mM GSHe showed similar glutathione distribution to untreated cells.


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 GSH distribution after its depletion with DEM and BSO.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. At 24 h after plating the cells were stained as described in Materials and Methods and observed by confocal microscopy in the chamber provided with 5% CO2 and at 37°C. Images were taken by light microscopy (transmission) and by confocal microscopy, as described in Materials and Methods, to capture blue fluorescence of nuclei (Hoechst-nuclei), green fluorescence that marks GSH (CMFDA-GSH) and red fluorescence of dead cells (PI-dead cells) (results not shown). Z series of at least 8 planes were obtained and maximum projection images were created and analysed. The representative experiment (of five) is presented.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006413-g003: The GSH distribution after its depletion with DEM and BSO.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. At 24 h after plating the cells were stained as described in Materials and Methods and observed by confocal microscopy in the chamber provided with 5% CO2 and at 37°C. Images were taken by light microscopy (transmission) and by confocal microscopy, as described in Materials and Methods, to capture blue fluorescence of nuclei (Hoechst-nuclei), green fluorescence that marks GSH (CMFDA-GSH) and red fluorescence of dead cells (PI-dead cells) (results not shown). Z series of at least 8 planes were obtained and maximum projection images were created and analysed. The representative experiment (of five) is presented.
Mentions: Figure 3 depicts untreated fibroblasts during 24 hours in culture showing that GSH (CMFDA staining-green fluorescence) co-localizes with DNA (Hoechst staining-blue fluorescence). However, cells incubated with 100 µM DEM showed a homogeneous GSH distribution within the cells. Surprisingly, inhibition of GSH synthesis by BSO was unable to change the distribution of cellular GSH, showing a cellular glutathione pattern different from the one shown in the DEM-treated cells. Indeed, GSH was high in the nucleus and low in the cytoplasm, like in the control group. Thus, each GSH-depleting agent induces different responses in the distribution of cellular GSH when fibroblasts were proliferating. Co-incubating the cells with 100 µM DEM and 1 mM GSHe showed similar glutathione distribution to untreated cells.

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