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

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+G2/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G0/G1) 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 G0/G1 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. 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+G2/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G0/G1) 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 G0/G1 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 (1Viña J. Glutathione: Metabolism and Physiological Function. CRC Press, Boston1990Google Scholar). We have reported that cellular glutathione decreases in apoptosis (2Esteve J.M. Mompó J. García de la Asunción J. Sastre J. Boix J. Viña J.R. Viña J. Pallardó F.V. FASEB J. 1999; 13: 1055-1064Crossref PubMed Scopus (164) Google Scholar). Although the role of nuclear GSH in the synthesis of DNA (3Reichard P. Thelander L. Annu. Rev. Biochem. 1979; 48: 133-158Crossref PubMed Scopus (915) Google Scholar) and in protection against oxidative damage or ionizing radiation (4Biaglow J.E. Varnes M.E. Clark E.P. Epp E.R. Radiat. Res. 1983; 95: 437-455Crossref PubMed Scopus (212) Google Scholar) 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. (5Söderdahl T. Enoksson M. Lundberg M. Holmgren A. Ottersen O.P. Orrenius S. Bolcsfoldi G. Cotgreave I.A. FASEB J. 2003; 17: 124-126Crossref PubMed Scopus (88) Google Scholar). 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 (G0/G1 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. 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (25 units/ml penicillin, 25 μg/ml streptomycin, and 0.3 μg/ml amphotericin B) in 5% CO2 in air at 37 °C in 25 or 75 cm2 flasks. Cultured fibroblasts were washed with phosphate-buffered saline, and acid GSH extracts were obtained in 6% perchloric acid containing 1 mm EDTA. GSH was measured spectrophotometrically using the glutathione S-transferase assay (6Brigelius R. Muckel C. Akerboom T.P. Sies H. Biochem. Pharmacol. 1983; 32: 2529-2534Crossref PubMed Scopus (344) Google Scholar). Determination of reduced (GSH) and oxidized glutathione (GSSG) was carried out using the high-performance liquid chromatography method with UV-visible detection, which we developed to measure GSSG in the presence of a large excess of GSH (7Asensi M. Sastre J. Pallardo F.V. Garcia de la Asuncion J. Estrela J.M. Vina J. Anal. Biochem. 1994; 217: 323-328Crossref PubMed Scopus (164) Google Scholar). The essence of this method consists of minimizing GSH oxidation, which otherwise would result in a large increase in GSSG. Analyses were performed using an Epics Elite cell sorter (Coulter Electronics, Miami). Fluorochromes were excited with an argon laser tuned at 488 nm. Forward angle and right angle light scattering were measured. Samples were acquired for 15,000 individual cells. Cell cycle phases were determined using the fluorescent DNA dye propidium iodide (final concentration, 5 μg/ml) at 630 nm fluorescence emission (8Borras C. Esteve J.M. Vina J.R. Sastre J. Vina J. Pallardo F.V. J. Biol. Chem. 2004; 279: 34332-34335Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Confocal images were acquired using a Leica TCS-SP2 confocal laser scanning unit equipped with argon and helium-neon laser beams and attached to a Leica DM1RB inverted microscope. 3T3 fibroblasts were maintained in culture as described previously (8Borras C. Esteve J.M. Vina J.R. Sastre J. Vina J. Pallardo F.V. J. Biol. Chem. 2004; 279: 34332-34335Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and plated in 2-cm2 Lab-Tek II chambered cover glass (Nunc) for 5 days, 72 h, 48 h, 24 h, 12 h, and 6 h before the experiment so that cells in all phases of the cell cycle were dyed and analyzed on the same day. Triple staining was performed as follows: 2 μg/ml propidium iodide (PI) 4The abbreviations used are: PI, propidium iodide; CMFDA, 5-chloromethylfluorescein diacetate; TBS, Tris-buffered saline; TBST, TBS with Tween 20; FCCP, p-trifluoromethoxycarbonyl cyanide phenylhydrazone; GCS, γ-glutamylcysteine synthetase. (Sigma) to identify dead cells, 2 μg/ml Hoechst (Sigma) to localize nuclei, and 5 μm CellTracker green 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes) to detect GSH (specificity 95%) (9Hedley D.W. Chow S. Cytometry. 1994; 15: 349-358Crossref PubMed Scopus (223) Google Scholar). When indicated, mitochondria were stained with MitoTracker Red 580 (Fig. 6). Cells were first stained with 5 μm CMFDA in cell culture medium for 30 min at 37 °C and 5% CO2. After washing with prewarmed cell culture medium, cells were left to rest for 30 min at 37 °C and 5% CO2 in cell culture medium or, alternatively, were loaded with 250 nm MitoTracker Red 580 (Molecular Probes). In the last 5 min of incubation, 2 μg/ml Hoechst and 2 μg/ml PI were added. After incubation with the fluorochromes, staining solution was replaced with fresh prewarmed cell culture medium, and cells were analyzed. Cell washing procedures did not change glutathione distribution (results not shown). The excitation wavelengths for fluorochromes were 488 nm for CMFDA, 543 nm for IP, 364 nm for Hoechst, and 543 nm for MitoTracker Red 580. The emission apertures for fluorescence detection were 510–540 nm for CMFDA, 585–715 nm for PI, 380–485 for Hoechst, and 575–650 nm for MitoTracker Red. The Z-section series obtained beginning from the nuclear apex and progressing down in 1 ± 0.2-μm increments (at least 10 planes) were converted to maximum projection images to avoid subjectivity in the choice of plane to be analyzed. The distributions of green CMFDA fluorescence (GSH levels) and blue Hoechst fluorescence (nuclei localization) were analyzed by profile and by area as follows. Fluorescence Profile Analysis—A cross-section line of 200 ± 20 μm was drawn through a cell field to compare the intensity and distribution of Hoechst (DNA) and CMFDA (GSH) fluorescence. The graphs obtained from the quantification were overlaid using the following color code: blue for nuclei localization (Hoechst fluorescence) and green for GSH distribution (CMFDA fluorescence). Fluorescence Area Analysis—Perimeters were drawn around the nucleus (according to the area marked with Hoechst) and around the entire cell excluding nucleus area (according to transmission image obtained by light microscopy). The nucleus/cytoplasm ratio for GSH in every cell analyzed was established by dividing the mean of green CMFDA fluorescence of nuclear area by the mean of CMFDA fluorescence in cytoplasmic area. We calculated the nucleus/cytoplasm ratio (n/c) using CMFDA fluorescence in three separate experiments (100 cells/condition). Aliquots of cell lysates (40 μg) were immediately boiled for 10 min to inactivate proteases and phosphatases, electrophoresed in SDS-10 or 12.5% polyacrylamide gels, and electroblotted (Bio-Rad) onto Immobilon-P nylon membranes (Invitrogen). Protein content was determined by a modified Lowry method (10Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7141) Google Scholar). Membranes were blocked with 0.05g/ml skimmed milk in TBS-0.2% Tween 20 (TBST), washed three times at room temperature, and incubated with primary antibodies against Id2 (1:1000) and p107 (1:1000) (both from Santa Cruz Biotechnology) in TBST with 0.05 g/ml bovine serum albumin overnight at 4 °C. Thereafter, the blots were washed again with TBST and further incubated for 1 h with a secondary horseradish peroxidase-linked anti-rabbit IgG antibody (1:2000) (Cell Signaling Technologies). After washing with TBST as above, blots were developed by using the LumiGLOl® reagent as specified by the manufacturer (Cell Signaling Technologies). 3T3 cells were subjected to nuclear protein extraction for the determination of protein oxidation and protein glutathiolation following the instructions for the nuclear extract kit (Active Motif North America). The efficiency of nuclear extraction was determined by measuring the relative activity of a cytosolic enzyme, lactate dehydrogenase, in the nuclei and in whole cell. Nuclear lactate dehydrogenase activity was less than 1.2% of the total cellular activity. To measure the level of nuclear protein oxidation, samples were lysed and derivatized, and Western blotting was performed according to the recommendation of the manufacturer. The level of nuclear protein oxidation was determined by the Oxy Blot protein oxidation detection kit (Chemicon International), which detects carbonylated proteins. Nuclear lysates were obtained in the absence of reductive agents. Western blotting procedure was performed as described previously, and the membrane was probed against anti-glutathione antibody (1:1000; Virogen) at 4 °C overnight. Nuclear lysates were obtained, and Western blotting procedure was performed as described previously. Membrane was probed against anti-bcl2-antibody (1:1000; rabbit polyclonal IgG, Santa Cruz Biotechnology) or, alternatively, against anti-GCS antibody (1:750; NeoMarkers, rabbit polyclonal antibody). Autoradiographic signals were assessed using a Fujifilm scanning densitometer (Fujifilm LAS-1000 plus). Cells were incubated for 30 min to 2 h with freshly prepared 20 μm p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP; Sigma), loaded with CMFDA, PI, and Hoechst, and analyzed by confocal microscopy as described above. Results are expressed as mean ± S.D. The statistical was performed using the least with of The is for all of in which is at the level of We assessed the changes in nuclear GSH in different phases of the cell cycle. after 3T3 fibroblasts to (Fig. at 24 and 48 h after the of cells at the phase and 72 h or cell growth and 6 after when the cells reached most of the cells were in the G0/G1 GSH concentration in whole cells was maximal at 24 h after (Fig. when a of cells was or for when the cell reached confluence and most cells were GSH concentration was at a level We determined the ratio in cells during the cell cycle and that it not change In view of we studied nuclear distribution of GSH have a role in the progression of the cell cycle during the early phases of cell the cell GSH distribution in 3T3 after cells be on the a and The blue staining of the nuclei with Hoechst is in and green staining is in of glutathione staining (Fig. that the fluorescence is located in the nucleus. We a line the cell The of cells that the line a quantification of the fluorescence cell. that the nuclei blue staining in the with the green glutathione staining in the at the beginning of the 12 and 24 h of cells to the glutathione (CMFDA green fluorescence was maximal at 24 of the fluorescence (Fig. and distribution and intensity a maximum correlation of fluorescence at 24 When cells were confluent after 72 h or 5 in correlation the of green and blue fluorescence be The increase culture in total cellular staining for CMFDA with the of glutathione concentration determined spectrophotometrically green (GSH) fluorescence decreased at 72 h and 5 in culture when cell culture reached growth and as a the cell be between the nucleus and and there was a green (CMFDA) on the Thus, when cells to GSH was located mainly in the cell nucleus. when cell growth GSH distribution was the cell. The of the cell to concentrate GSH in the nucleus was measuring the which was calculated after measuring the ratio in cells by in The in 4 a distribution the maximal ratio ± was early after Although a ratio for GSH was maintained during cell growth, as the culture a steady in the nucleus/cytoplasm ratio was the cells reached confluence and most of them were in the G0/G1 phase, the ratio for GSH was and be between the nucleus and the To that most cells at 24 and 48 h in culture were in the phase of the cell cycle, we studied Id2 and p107 expression during cell growth. are when cell division is The in 5 Id2 and p107 expression at 24 and 48 h in that the cells at 24 and 48 h in of Id2 and p107 at 72 h of culture or was GSH was not responsible for the of CMFDA fluorescence. To this we incubated fibroblasts with Hoechst to DNA CMFDA for GSH and MitoTracker for staining 6). A of images obtained the from the cell apex is in 6 experiment of The nuclear is as is the presence of GSH in the nucleus. distribution was To determine glutathione concentrates in the nucleus an we incubated the cells with FCCP, a The in that glutathione concentrates in the nucleus by an the glutathione distribution was in and cells. possible for the reported nuclear increase in GSH during the early phases of cell cycle would be the nuclear GSH We studied the presence of the for GSH synthesis and GSH synthetase. a lack of expression of the FASEB J. 1999; 13: PubMed Scopus Google Scholar) in nuclear samples from whole cells a marked Thus, under our the nuclear synthesis of GSH the nuclear presence of an protein that reported to increase nuclear glutathione D.W. S. S. A. 95: PubMed Scopus Google Scholar). result to a correlation between the expression of Bcl-2 during the cell cycle and the distribution of cellular When cells are dividing and 24 h of the presence of nuclear Bcl-2 is it decreases h and 5 of when cell growth nuclear GSH is during the early phases of cell cycle and when cells are nuclear oxidized are to be higher during cell cycle than at 6 h after This is the as is in GSH is by the in the it be converted into GSSG or and not to the cytoplasm as Western of nuclear extracts the in the glutathiolation of nuclear (Fig. it was higher at 6 h after when nuclear reached its when cells reached confluence and distribution was glutathiolation of nuclear We reported previously that GSH activity in cells in culture (8Borras C. Esteve J.M. Vina J.R. Sastre J. Vina J. Pallardo F.V. J. Biol. Chem. 2004; 279: 34332-34335Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). 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In we have that cells a reduced nuclear by glutathione to the nucleus/cytoplasm ratio is after plating. Thus, glutathione to the nucleus before exponential cell growth. When cells reach at and 5 after GSH distribution is in of the cell nucleus and The phase of the cell cycle must be into when cellular nuclear GSH levels. our that the cell nucleus changes in its oxidative and the role of nuclear glutathione in the physiology of the cell cycle.