Departments of 1Animal Sciences and 2Veterinary Pathobiology, 3Division of Nutritional Sciences, and 4Institute for Genomic Biology, University of Illinois, Urbana, Illinois
Submitted 7 April 2005 ; accepted in final form 12 June 2005
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ABSTRACT |
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cell proliferation; contact inhibition; glutathione
Increased concentrations of the tripeptide GSH or reduction of the redox state of the GSSG/2GSH couple measured as an increase in the GSH-to-GSSG ratio or a decrease in the calculated reduction potential derived from the Nernst equation have been associated with cell proliferation (4, 11, 25, 50, 51). A parallel decrease in reactive oxygen species (ROS) production has also been associated with cell proliferation (31, 53). Furthermore, a decrease in the GSH concentration ([GSH])-to-GSSG concentration ([GSSG]) ratio, and the consequent increase in the reduction potential, as well as an increase in ROS production have been associated with decreased cell growth (10, 42, 50, 53).
Conversely, a mitogenic effect of ROS has been proposed (9). Several studies have shown that high ROS concentrations are needed for proliferation (33, 48). In fact, a decrease in ROS and an increase in intracellular low-molecular-weight thiols accompany contact inhibition of cell growth (38, 46). NO-mediated signaling is also involved in both induction (14, 59) and inhibition (2, 16) of cell proliferation as well as the induction of genotoxicity, mitochondrial damage, and apoptosis (30). These differential effects seem to be concentration dependent (13).
The NAD+/NADH and NADP+/NADPH couples have received less attention regarding their role in cell proliferation (7, 36). NAD works as a carrier, taking electrons from catabolic reactions and redirecting them into the oxidative phosphorylation chain, enabling cells to maximize energy production from catabolic metabolism (34). The NAD+ concentration ([NAD+])-to-NADH concentration ([NADH]) ratio plays an important role in regulating intracellular redox and is often considered a readout of the metabolic state (32). Structurally related reduced NADPH is a major source of electrons for reductive biosynthesis and is also used as an electron donor to maintain the overall reduced thiol state in the cell (50). In recent years, there has been increasing recognition that both [NAD+]-to-[NADH] and [NADPH]-to-[NADP+] ratios and concentrations fluctuate with the physiological state of the cell and affect the ability of certain transcription factors to bind DNA (49, 58).
Overall, there is an apparent contradiction concerning the magnitude and direction of the redox changes experienced by the cell during proliferation and contact inhibition of growth. Depending on how the redox state of the cell is defined (i.e., which redox active species or couple is quantified) and on when it is measured, proliferating cells may appear more reduced or oxidized. The aim of this study was to measure redox and metabolic changes associated with proliferative stages through contact inhibition of growth by using nontransformed intestinal epithelial cells as a model system. The redox state of the GSSG/2GSH and NAD(P)+/NAD(P)H couples, relative concentrations of ROS and NO, and the ATP concentration ([ATP])-to-ADP concentration ([ADP]) ratio were measured to establish the magnitude and the temporal relationship of the oxidative and reductive events in an attempt to clarify the previously cited contradictions.
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MATERIALS AND METHODS |
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Cell culture and treatment. The rat intestinal crypt cell line IEC-6 (47) was obtained from the American Type Culture Collection (Manassas, VA), and maintained in DMEM supplemented with glucose (25 mM), and dispensable amino acids (each 0.1 mM), sodium bicarbonate (44 mM), penicillin (40,000 U/l) and streptomycin (40,000 µg/l), HEPES (15 mM), 10% FBS, Fungizone (250 µg/ml), and insulin (0.1 U/ml). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and serially passaged in 75-cm2 tissue culture flasks (Corning, Corning, NY). Cells were seeded at a density of 1.5 x 105 cells in six-well cell culture plates (area 9.5 cm2; Corning) and grown for the periods indicated for each experiment. For all experiments, cells were used between passages 17 and 27, and cell medium was replaced every 3 days.
Growth curve. IEC-6 cells were seeded at a density of 1.5 x 105 cells per well (6-well plate), cultured, and harvested every day for up to 12 days. After harvesting, the cells were stained with Trypan blue, and live cells were counted with a hemocytometer.
Cell cycle analysis. Cells were seeded in a six-well plate as described above. Each day, cells were washed twice (first with PBS and then with trypsin) and then lifted with trypsin and mixed with 10% FBS in PBS. The cells were centrifuged, fixed with ice-cold ethanol (70%), and kept at 20°C for subsequent analysis. Before the analysis, cells were washed twice in PBS and treated for 30 min with propidium iodide (50 µg/ml) and RNase (100 µg/ml; Qiagen, Valencia, CA). Flow cytometric analysis was performed with an EPICS XL-MCL flow cytometer controlled by SYSTEM II software (Beckman Coulter, Miami, FL). The data were analyzed with Summit V3.1 software (Cytomation, Fort Collins, CO).
Cell volume. Cell volume was estimated with a Beckman Coulter counter (Beckman). A cell volume distribution was performed. A mean cell volume of 1.413 pl (SD 0.507), calculated for early confluent cells, was used to calculate metabolite concentrations.
Cellular DNA quantification. Total DNA concentrations from crude cell homogenates were determined by fluorometry with bisbenzimidazole (Hoechst 33258; Molecular Probes, Eugene, OR) as described previously (28).
GSH and GSSG determination. [GSH] and [GSSG] were determined as described previously (41) with minor modifications. Briefly, cells were washed twice with HBSS, lifted with a cell scraper (Fisher) into HBSS, and lysed by repeated freeze-thaw cycles. The sample was divided for the determination of two pools, A and B. Pool A consisted of reduced and oxidized GSH, whereas pool B included only the oxidized GSH. For pool A, the sample was mixed with DTT (50 mM) and Tris (100 mM; pH 8.5). For pool B, the sample was mixed with NEM (4 mM), and after 2 min of incubation, Tris (pH 8.5) and DTT (100 mM) were added. In both cases, samples were incubated for 30 min and then 5% SSA was added to precipitate the proteins. The samples were then centrifuged and derivatized with OPA solution (50 mg OPA in a 1:10 methanol-0.4 M potassium borate buffer, pH 9.9) for 5 min at room temperature, and sodium phosphate buffer (pH 7.0) was added. Samples were separated on a reverse-phase column (Atlantis, Waters, Milford, MA) at 30°C coupled to the same type of guard column in a Waters 2695 HPLC system with autosampler system (Waters). Compounds were eluted with 7.5% (vol/vol) methanol in sodium phosphate buffer (10 mM, pH 7.0) and detected with a Hitachi F-1080 fluorescence detector with excitation at 340 nm and emission at 420 nm. Peak areas were integrated with Millennium 3.2 software (Waters). Reduced GSH values were determined by differences between pools A and B. [GSH] and [GSSG] were expressed per cell, using 6.1 pg DNA/cell (19) and 1.413 pl as cell volume. The reduction potential for the GSSG/2GSH couple was calculated by using the Nernst equation at 37°C and pH 7.2 [half-cell reduction potential (Ehc) = 252 mV (61.5/2)*log([GSH]2/[GSSG])].
Western blot analysis.
IEC-6 cells were seeded at a density of 1.5 x 105 cells in six-well plates on consecutive days. After 2, 3, and 4 days, equal protein concentrations (15 µg) were size separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with a rabbit polyclonal anti--glutamylcysteine synthetase (
-GCS) heavy subunit (
-GCSH) primary antibody (Neomarkers, Fremont, CA) and horseradish peroxidase-conjugated anti-rabbit IgG (Sigma) as a secondary antibody. Bands were detected with an enhanced chemiluminescence system (ECL, Amersham Biosciences, Piscataway, NJ). Immunoblots were scanned by optical densitometry (GS-710 Calibrated Imaging Densitometer, Bio-Rad, Hercules, CA) to quantify the relative level of protein expression between treatments, using the peak density option in Diversity Database 2.2.0 software (Bio-Rad).
Quantification of ROS and NO. ROS activity was detected by flow cytometric analysis using the fluorescein-labeled dye dichlorodihydrofluorescein diacetate (H2DCF; Molecular Probes). Cells were treated with 10 µM H2DCF for 30 min at 37°C, washed with PBS, and dispersed with trypsin, and endogenous peroxides were measured with a MoFlo MLS high-speed flow cytometry instrument (Cytomation). NO production was detected by flow cytometric analysis using the fluorescein-labeled dye 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate (Molecular Probes). Cells were treated with 5 µM H2DCF for 45 min at 37°C, washed with PBS, and incubated in PBS for 15 min. The cells were then dispersed with trypsin, and NO was measured with the MoFlo MLS high-speed flow cytometry instrument. Data were analyzed with Summit V3.1 software. Ten thousand events were recorded for each analysis. Results were calculated as the mean fluorescence intensity of treated relative to control cells. Results of different experiments were normalized with Immuno-Bright calibration beads (Coulter Source, Marietta, GA).
NAD(P)(H), ATP, and ADP. [NADPH]-to-[NADP+], [NAD+]-to-[NADH], and [ADP]-to-[ADP] ratios and concentrations were determined according to the method described by Lazzarino et al. (29) with modifications. Briefly, cells were washed with PBS, nitrogen-saturated precipitation solution [acetonitrile-10 mM KH2PO4 (3:1) pH 7.4; 4°C] was added, and the cells were collected and centrifuged at 16,000 g for 4 min at 4°C. Ice-cold chloroform was added to the supernatant, which was mixed and centrifuged at 16,000 g for 4 min at 4°C. Chloroform extraction was repeated twice, keeping the aqueous phase. Samples were filtered and transferred to an autosampler vial or stored at 80°C until HPLC analysis. Separation was performed on a Waters 2695 separations module at 25.526°C on a reverse-phase column (Kromasil C18) coupled to a guard column (Waters Symmetry C18), using tetrabutylammonium hydroxide as an ion-pairing reagent. Detection was performed with a Waters 2487 dual absorbance detector at 260 nm [for ATP, ADP, NAD(P)] serially connected to a Hitachi F-1080 fluorescence detector with excitation at 360 nm and emission at 465 nm [for NAD(P)H]. Peak areas were integrated with Millennium 3.2 software. The intracellular Ehc values were calculated by the Nernst equation for each NADPH-to-NADP+ ratio [Ehc = 321.15 mV (61.5/2)*log(NADPH/NADP+)] and NADH-to-NAD+ ratio [Ehc = 322.15 mV (61.5/2)*log(NADH/NAD+)] at 37°C and pH 7.2.
Statistics.
Results are expressed as means ± SE. A minimum of two independent experiments with triplicate samples per time point were performed per analysis. Statistical analysis was performed with SAS (version 8.02; SAS Institute, Cary, NC). For comparison between days, a general linear model procedure was used [Variable = Day + error () and Variable = Day + (Day)2 +
], and a Tukey's W procedure was used to separate different populations (SAS 8.02). Comparisons between the different stages were performed with contrasts. Other comparisons were performed with paired Student's t-tests (SAS 8.02). Statistical significance was set at P < 0.05.
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RESULTS |
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Intracellular [GSH] and [GSSG] and calculation of redox state of the GSH/GSSG couple.
[GSH] increased from day 1 to day 3 and then successively decreased until day 14. [GSH] on day 3 were significantly higher and [GSH] on days 914 were significantly lower than the others (Fig. 2A). [GSSG] decreased from day 1 to day 3 and then increased again from day 11 to day 14. [GSSG] on days 1, 2, 13, and 14 were significantly higher compared with the other days. From day 1 to day 3, the increase in [GSH] was accompanied by a decrease in GSSG, but the changes in [GSSG] were not of sufficient magnitude to account for the total changes in GSH. A decrease in GSH and the opposite increase in GSSG were also observed during the last 3 days (Fig. 2A). The GSSG/2GSH couple became more reduced (35 mV) from day 1 to day 3, when cells were actively proliferating, and then became more oxidized (+45 mV) toward the end of the study period (Fig. 2B). The GSSG/2GSH couple was more oxidized after day 9 compared with the previous days (P < 0.01). The range of reduction potential of the GSSG/2GSH couple varied from 171.6 ± 8.6 mV, when cell growth was arrested by contact inhibition, to 216.8 ± 2.8 mV, when they were actively proliferating on day 3, just before confluence. To determine the extent to which observed changes in [GSH] resulted from an increase in de novo synthesis, expression of the catalytic subunit of -GCS (
-GCSH), the enzyme involved in the rate-limiting step of GSH synthesis (20), was measured. Changes in the amount of
-GCSH preceded changes in the synthesis of GSH (Fig. 3). Expression of
-GCSH was significantly higher on day 2, when GSH concentrations were increasing, lower on day 3, when GSH values peaked, and significantly lower on day 4, when [GSH] started to decrease (Fig. 3).
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DISCUSSION |
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We observed an increase in GSH concentrations at the beginning of the logarithmic growth phase and a subsequent decrease simultaneous with a decrease in proliferation due to contact inhibition. Consistent with previous studies, the data confirm that [GSH] are greater during the exponential growth phase and then decrease as cells become confluent and begin to differentiate (4, 42, 51). The +45-mV oxidation of the Ehc of the GSSG/2GSH couple observed between cells actively proliferating on day 3 and contact-inhibited cells at day 12 is similar to published data (5, 22, 42). A change of this magnitude in the redox state of GSH is sufficient to regulate the activity of proteins with redox-sensitive thiols, assuming that sufficient glutaredoxin activity is present to mediate such effects (23, 37, 50). The present data indicate that changes in the GSH pool may be generated in part by shuttling GSH equivalents to and from the GSSG pool. Specifically, [GSSG] and [GSH] fluctuated in opposite directions at the beginning and end of the study. However, the changes in [GSSG] were not of a sufficient magnitude to explain the observed changes in GSH alone. Temporal changes were also observed in expression of -GCSH protein, which is the catalytic subunit of
-GCS, the rate-limiting enzyme in the de novo synthesis of GSH (20). Together these data indicate that both major pathways in GSH synthesis, de novo production and regeneration of GSH from GSSG, underlie [GSH] changes during cell proliferation. The decrease in
-GCSH expression coincided with a decrease in ROS concentrations and an increase in [GSH] during the same period. This is consistent with previous observations that
-GCSH synthesis is induced by an increase in ROS (52) and a decrease in GSH (8).
The present study indicates a role for ROS in both proliferation and contact inhibition. These data are consistent with an oxidative event being necessary to initiate proliferation (38, 43) and with previous observations that ROS production is greater in sparse than in confluent cells (45). As suggested by Schafer and Buettner (50), an oxidative stimulus similar to that observed in this study may be responsible for the subsequent increase in [GSH]. This effect may be mediated, at least in part, by activation of -GCS transcription through a 5' antioxidant response element (40). To the contrary, the decrease in proliferation due to contact inhibition was also accompanied by an increase in ROS concentrations, denoting a potential dual effect for ROS in cell proliferation as well as in contact inhibition. The extent of the oxidative stimulus may be responsible for this differential role (15, 50).
NO-mediated signaling is involved in cell proliferation, growth inhibition, and apoptosis (14, 16, 39). In this study, relative [NO] were high at the beginning of the culture period, decreased when the cells reached confluence, and then remained constant, suggesting a role of NO in cell proliferation. Relative [NO] were similar between confluent but proliferating cells compared with contact-inhibited cells, indicating that in contrast to ROS, NO is apparently not involved in signaling related to growth arrest. This observation seems to contradict other findings that NO is associated with the cessation of cell proliferation (16, 57). NO is involved in the regulation of GSH (27) and superoxide concentrations (44). Moreover, high ROS concentrations are responsible for modulating NO bioactivity during cell proliferation (3). Although we observed a temporal relationship among ROS, NO, and GSH, additional studies are needed to determine the extent of cross-regulation among these redox molecules in the context of cell growth regulation.
[NAD(P)H]-to-[NAD(P)+] ratios were high in proliferating cells and low in G1-arrested cells (36). In agreement, in the present study, the [NADPH]-to-[NADP+] ratio was greatest at the beginning of the culture and then decreased, reaching a minimum at confluence and remaining low through contact inhibition. The decrease in stage 1 may reflect a flux of electrons from NAD(P)H to GSSG to generate more GSH or the use of NADPH electrons for biosynthetic pathways as well as for ROS and NO production. The NAD+/NADH couple became more oxidized during stage 1 but was reduced during stage 3, when cell proliferation decreased by contact inhibition, contradicting previous observations of an oxidation of NADH during contact inhibition in fibroblasts (7). These couples are capable of acting as sensors of the redox or energetic state of the cell and thereby directly regulate cellular processes (32, 49, 58). The observed changes in the [NADPH]-to-[NADP+] ratio (from 4.3 ± 0.56 to 0.1 ± 0.007) and in the [NAD+]-to-[NADH] ratio (from 2.98 ± 1.07 to 20.5 ± 2.82) are similar to changes shown to affect the binding properties of transcription factors that regulate expression of the light-dark cycle proteins (49) and may thus be involved in the regulation of cell proliferation and contact inhibition. Interestingly, when cells were contact inhibited, the NAD+/NADH couple was more reduced, the GSSG/2GSH couple was more oxidized, and the [ATP]-to-[ADP] ratio reached its highest value, consistent with the mechanism proposed by Jones (23), in which GSH redox state is controlled by metabolism. In this mechanism, electrons derived from GSH are used to reduce NAD+ to NADH. This creates an electron flux from NADPH to NADH and eventually to the mitochondrial electron transport chain, generating more ATP in cases when the NAD+/NADH couple is oxidized, such as nutrient deprivation.
As summarized in Fig. 7, changes in the redox state of distinct couples are associated with the processes of cell proliferation and growth arrest by contact inhibition. However, the direction of these changes varied among the different couples, indicating that it is not possible to describe the redox state of the cell by examining just one couple. It is more precise to consider changes in the redox state of a specific couple or changes in the concentration of a redox-active species associated with a particular cellular process. In other words, it is not possible to state that the cell is more reduced (or oxidized) when it is actively dividing, but it is possible to indicate that the GSH couple is more reduced in proliferating relative to contact-inhibited cells. Schafer and Buettner (50) defined the redox environment as "the summation of the products of the reduction potential and the reducing capacity of the linked couples present in an organelle, cell or tissue." They pointed out that because the GSSG/2GSH couple provides a relatively large pool of reducing equivalents, its reduction potential effectively determines the intracellular redox environment and correlates with biological stages (proliferation, confluence, differentiation, apoptosis, and necrosis). Although our data generally agree, reliance on this definition alone may overlook an opposite change in another couple present in lower concentrations that may be critical for a particular regulatory mechanism. For example, the present data demonstrate that in stage 1 of culture, the GSSG/2GSH couple became more reduced while the NAD(P)+/NAD(P)H couples became more oxidized. Furthermore, organelles such as mitochondria, nuclei, and endoplasmic reticulum have differential pools of redox-active species compared with the cytoplasm (6, 56). These independent pools add spatial complexity to the system, providing cells additional regulatory options, perhaps according to organelle-specific processes. In addition to spatial separation, different couples may exhibit dependent or independent changes in redox status, depending on whether they are linked enzymatically.
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GRANTS |
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ACKNOWLEDGMENTS |
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Kajorn Kitiphongspattana is currently a Postdoctoral Scholar in the Department of Medicine at the University of Chicago.
Present address of K. Ishii-Schrade: Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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