Glutathione Is Recruited into the Nucleus in Early Phases of Cell Proliferation

Abstract

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S؉G 2 /M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G 0 /G 1 ) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 ؎ 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G 0 /G 1 phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and performs many physiological functions (1). We have reported that cellular glutathione decreases in apoptosis (2).Although the role of nuclear GSH in the synthesis of DNA (3) and in protection against oxidative damage or ionizing radiation (4) is well established, little is known about the concentration of GSH in the nucleus and its regulation. This is due to two main factors. The first is methodological: it is impossible to determine the nuclear concentration of GSH using standard cell fractionation and analytical approaches (for a review see Söderdahl et al. (5). In view of this problem, we used confocal microscopy.The second factor is that most, if not all, of the reports share the common view of nuclear GSH distribution in a static situation. Cells are usually studied under steady state conditions i.e. when they are confluent (G 0 /G 1 phase of the cell cycle). The nucleus changes dramatically during the different phases of the cell cycle. Thus, studies addressed to determining the nuclear GSH distribution must take cell cycle physiology into account. To our knowledge there is a lack of information about the cellular distribution of glutathione during the different phases of the cell cycle and the possible correlation between cellular growth and nuclear GSH levels. We report here that GSH concentrates in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and it redistributes uniformly between nucleus and cytoplasm when cells reach confluence. Nuclear Bcl-2 may be responsible for this change, as its expression changes in parallel with glutathione levels in nuclei. EXPERIMENTAL PROCEDURES Cell Culture3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (25 units/ml penicillin, 25 g/ml streptomycin, and 0.3 g/ml amphotericin B) in 5% CO 2 in air at 37°C in 25 or 75 cm 2 f...

Regulatory T Cells Interfere with Glutathione Metabolism in Dendritic Cells and T Cells

“…Previous studies have shown that GSH concentrates in the nucleus during early stages of cell proliferation but later redistributes approximately equally between the nuclear and cytoplasmic compartments (24). We imaged changes in GSH in localization during T cell activation and found that GSH co-localized with nuclear DNA during the early stages of T cell activation (Fig.…”

“…For quantitative analysis of the GSH fluorescence intensity, the nuclear perimeter was defined by the area of Hoechst staining, and the cell perimeter was defined from the bright field image of the cell seen by light microscopy as described previously (24). The cytoplasmic volume was the difference in the whole cell volume and the nuclear volume.…”

Glutathione – linking cell proliferation to oxidative stress

“…Data such as that shown in Figure 4 demonstrates that the nuclear GSH pool is in equilibrium with that of the cytosol. However, studies on animal and plant cells have demonstrated GSH is compartmentalized in the nucleus during the cell cycle (5)(6)(7)(8)107).…”

“…Like other small molecules, GSH diffuses freely between the cytosol and nucleus through the nuclear pore complex (4). It is rather surprising therefore that the nuclear GSH pool is much more resistant to depletion than the cytosolic pool, a property that is particularly important during cell proliferation (5)(6)(7)(8). Although relatively little is known about the nuclear thioredoxins (TRX) and glutaredoxins (GRXs) or their functions in plants (9) it is probable that these redox proteins participate in the plethora of thiol-dependent redox regulation mechanisms and post-translational modifications that are required for plant growth and development, particularly through functions in the nuclei.The many important roles of glutathione in plants have been well documented in recent reviews (1,2,10) and hence the following discussion will focus on how GSH functions as a regulator of plant development, with a particular focus on the nuclear GSH and the regulation of mitosis.…”

Defects in a New Class of Sulfate/Anion Transporter Link Sulfur Acclimation Responses to Intracellular Glutathione Levels and Cell Cycle Control

“…Our finding that glutathione overaccumulates in smt15-1 but not in sac mutants is a possible clue for understanding the cell cycle defects in this mutant. Glutathione levels and subcellular localization have been linked to cell cycle control in both plants and animals (Markovic et al, 2007;Pallardó et al, 2009;Pellny et al, 2009;Diaz Vivancos et al, 2010a, 2010b, although its specific impact on cell cycle-related processes is not completely understood. Changing the redox state of cell cycle regulators through glutathionylation has also been shown to influence the cell cycle (Chiu and Dawes, 2012).…”