The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

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

Effect of nuclear GSH depletion by DEM and BSO on cell cycle.

<p>Cells were plated and treated as described previously. The cell cycle was studied by flow cytometry, using the level of the fluorescence of the DNA dye propidium iodide (final concentration, 5 µg/mL) at 630 nm fluorescence emission as a measure of the DNA content per cell, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">materials and methods</a>. The cells were detached, fixed in ethanol and stained along the proliferation curve of the 3T3 fibroblasts. The histograms corresponding to each experimental group at 6 h, 24 h, 48 h, and 5 days of culture (of the same representative experiment) were overlaid and presented at the panel A. The effect of the GSH depletion on the cell proliferation, defined by mean percentages of cells in phases S+M/G2±SD, is presented at the panel B, and the effect of the depletion on the cell death, i.e. the percentage of apoptotic and necrotic cells is shown on the panel C. The results presented are mean±SD of 5–17 different experiments.</p

GSH depletion in 3T3 fibroblasts. The total cellular GSH concentration was assessed.

<p>The cells were plated as described previously (4) and after attaching 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. The total cellular concentration of GSH was determined spectrophotometrically as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a>”. The results are presented as mean±SD of 5–12 different experiments.</p

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.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">material and methods</a>). Right panels shows the densitometry results for the level of glutathyolation and oxidation of nuclear proteins, respectively [Mean±SD (n = 3)].</p

The effect of DEM and BSO treatment on the nuclear and cytoplasmic pool of GSH.

<p>The maximum projection images (as presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) were analysed by area, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a>”. CMFDA fluorescence in nuclear area (defined by Hoechst staining, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g004" target="_blank">fig. 4A</a> and the CMFDA fluorescence of the cytoplasm area (defined by transmission images, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) is presented at <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g004" target="_blank">fig. 4B</a>. 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.</p

Effect of glutathione depletion on Glutathione S-Transferase (GST) activity.

<p>The 3T3 fibroblasts were treated with 100 µM diethylmaleate (DEM) or with 10 µM buthionine sulfoximine (BSO) immediately after attaching. The activity of the GST was determined at 24 h and 6 days of culture, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and methods</a>.</p

The relationship of the nuclear GSH level and the rhythm of cell growth.

<p>The consequences of the GSH depletion by DEM and BSO on the cell growth are shown on the panel B and C, respectively. Control cells are presented at the panel A. The mean CMFDA fluorescence in nuclear area (defined by Hoechst staining, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) was obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">materials and methods</a>, and the profile of the cell growth was characterized by cell number. The results are presented as mean values of at least 4 experiments.</p

The GSH distribution after its depletion with DEM and BSO.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a>, 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.</p