Departments of Biochemistry and Medicine and the Graduate Program in Molecular and Systems Pharmacology, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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Cellular redox, maintained by the glutathione (GSH)- and thioredoxin (Trx)-dependent systems, has been implicated in the regulation of a variety of biological processes. The redox state of the GSH system becomes oxidized when cells are induced to differentiate by chemical agents. The aim of this study was to determine the redox state of cellular GSH/glutathione disulfide (GSH/GSSG) and Trx as a consequence of progression from proliferation to contact inhibition and spontaneous differentiation in colon carcinoma (Caco-2) cells. Results showed a significant decrease in GSH concentration, accompanied by a 40-mV oxidation of the cellular GSH/GSSG redox state and a 28-mV oxidation of the extracellular cysteine/cystine redox state in association with confluency and increase in differentiation markers. The redox state of Trx did not change. Thus the two central cellular antioxidant and redox-regulating systems (GSH and Trx) were independently controlled. According to the Nernst equation, a 30-mV oxidation is associated with a 10-fold change in the reduced/oxidized ratio of a redox-sensitive dithiol motif. Therefore, the measured 40-mV oxidation of the cellular GSH/GSSG couple or the 28-mV oxidation of the extracellular cysteine/cystine couple should be sufficient to function in signaling or regulation of differentiation in Caco-2 cells.
oxidation-reduction; cysteine; cystine; intestine; cell cycle
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INTRODUCTION |
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CONSIDERABLE EVIDENCE DEMONSTRATES that redox signaling mechanisms function in cell regulation and growth control. Glutathione (GSH) is the major low molecular weight thiol in cells and plays a central role in controlling cellular thiol/disulfide redox state, which is essential for normal redox signaling (19). A relationship between cellular GSH concentration and proliferation has been demonstrated in several studies, wherein GSH precursors or increasing cellular GSH has been found to enhance cell proliferation (2, 16, 39). Intracellular GSH levels are higher in actively proliferating cells (7, 34) and decrease gradually during serum starvation as cells become quiescent (34). GSH depleting agents such as buthionine sulfoximine inhibit cell growth in cultured cells. This antiproliferative effect is reversed by supplemental GSH or GSH precursors that return intracellular levels of GSH to normal (19, 34). These and other studies have clearly demonstrated a relationship between cellular GSH levels and proliferation, although the mechanisms involved are still unclear.
Apart from the involvement of cellular GSH in proliferation,
changes in the cellular GSH/glutathione disulfide (GSSG) redox state
(Eh) have been implicated in cell cycle responses such as proliferation, differentiation, and apoptosis. Proliferating
cells have Eh values ranging from 260 mV to
230 mV
(19, 22). This pool becomes oxidized (
220 mV to
190
mV) on growth arrest, either due to differentiation (22,
33) or contact inhibition (15). Cells undergoing
apoptosis are further oxidized to between
170 mV and
165 mV
(7, 17). These findings suggest that as cells progress
from proliferation through contact inhibition, differentiation, and
finally apoptosis, there is a natural progression from a more reduced to a more oxidized Eh.
Intestinal epithelium is maintained by continual cell proliferation, with a turnover of the entire epithelial layer every 4-6 days in humans. Studies in which buthionine sulfoximine was used to deplete GSH show that GSH is required for normal intestinal function (25) and that depletion of GSH exaggerates growth suppressive effects of thiol oxidants (28). Decreased GSH concentration, oxidation of the GSH pool, and inhibition of intestinal growth indices occurred with restriction of diet to 25% of ad libitum caloric intake in rats (18). A decrease in GSH concentration in HT29 cells was observed in association with time in culture and decrease in proliferation rate (22). These results indicate that changes in GSH and GSH/GSSG redox are associated with changes in intestinal cell growth.
Thioredoxin (Trx) and Trx reductase system complement the GSH system for maintenance of protein thiol/disulfide redox (14). Trx is a highly conserved, low molecular weight protein with a redox-active disulfide/dithiol in the active site (Cys-Gly-Pro-Cys). This active site is ideally suited to control protein function via the redox state of structural or catalytic thiol groups. Trx functions in ribonucleotide reduction for DNA synthesis and maintenance of c-Fos, c-Jun, p53, NF-kB, and other transcription factors in their reduced, active forms (11, 12, 36). The redox state of Trx has been implicated in control of apoptosis (32, 38) and cell growth (30), and expression of Trx is often increased in cancer cells (3). We have recently developed methods to quantify redox changes of Trx in cells (37), and this allows investigation of whether oxidation of Trx occurs concomitantly with oxidation of GSH during changes in cell growth.
The present study was designed to determine whether the redox states of GSH and Trx are independently controlled during transition of cells from proliferation to differentiation. For this, we selected a cell model (Caco-2) in which progression from growth to contact inhibition and differentiation occurs without added differentiating agents. Caco-2 is a well-described human colon carcinoma cell line that spontaneously differentiates on becoming confluent and becomes morphologically and functionally similar to enterocytes (8). The results show that the cellular Eh became oxidized as the cells progressed from proliferation through contact inhibition and differentiation. However, no change in the redox state of the Trx pool was detected under these conditions. Thus the redox states of the two central antioxidant and redox-regulating systems are regulated independently in this model of intestinal cell progression.
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MATERIALS AND METHODS |
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Chemicals. MEM and FBS were purchased from Life Technologies (Grand Island, NY). Goat anti-human Trx was purchased from American Diagnostica (Greenwich, CT), and horseradish peroxidase-labeled anti-goat IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The cell proliferation ELISA kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Chemiluminescence detection reagents were from Amersham Pharmacia Biotech (Piscataway. NJ) and Pierce (Rockford, IL). Dye reagent was purchased from Bio-Rad (Hercules, CA). HPLC-grade methanol was purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals were purchased from Sigma (St. Louis, MO).
Cell culture. The human colon cancer cell line, Caco-2, was obtained from the American Type Culture Collection (Rockville, MD). Cells were studied between passages 21 and 30. For all experiments, cells were cultured in MEM, supplemented with 2 mM glutamine and 10% fetal bovine serum in 95% air-5% CO2 at 37°C. For GSH/GSSG, cysteine/cystine (Cys/CySS) redox, and Trx measurements, cells were plated in six-well plates at a density of 8 × 104 cells per well. Cells were seeded in 96-well plates at a density of 104 cells per well for measurements of 5-bromo-2-deoxyuridine (BrdU) incorporation.
Cells were harvested and medium was collected on day 1 after plating and subsequently at 3-day intervals (until day 25) for GSH/GSSG and Cys/CySS measurements. To allow all cells to be analyzed together, cells were plated at a density of 8 × 104, on days 25, 22, 19, 16, 13, 10, 7, 4, and 1 before harvesting for Trx and cell cycle analyses. For all experiments, cell medium was changed in 3-day intervals.GSH, GSSG, Cys, and CySS determination.
Cells were treated with ice-cold 5% (wt/vol) perchloric acid,
containing 0.2 M boric acid and 10 µM -glutamyl-glutamate
(internal standard), and precipitated proteins were separated from the
acid-soluble supernatant by centrifugation. The protein pellet was
resuspended in 1 M NaOH, and protein concentrations were measured using
the Bradford method (5) with rabbit
-globulin as the
protein standard (Bio-Rad). Intracellular GSH and GSSG levels were
determined after treatment of the supernatant with iodoacetic acid
followed by dansyl chloride (20). For HPLC analysis, the
derivatized samples were separated as previously described
(20) on a Supercosil LC-NH2 column (5 µm:
4.6 × 25 cm) (Supelco, Bellefunk, PA) with the use of a 2690 HPLC
and autosampler system (Waters, Milford, MA). Detection was obtained by
fluorescence using band-pass filters (305- to 395-nm excitation, 510- to 650-nm emission; Gilson Medical Electronics, Middletown, WI). Thiols
were quantified by integration relative to the internal standard and
expressed as nmol/mg protein. Conversion to molar values was obtained
by using 5 µl cell vol/mg protein (18).
Eh calculation.
The cellular and extracellular Eh values were calculated by
using the appropriate forms of the Nernst equation (in mV) for the
respective GSH/GSSG (22) and Cys/CySS (19)
pools: Eh = 252 + 30 log
([GSSG]/[GSH]2) for pH 7.2 in Caco-2 cells, and
Eh =
250 + 30 log
([CySS]/[Cys]2) for pH 7.4 in culture medium, where
respective concentrations are expressed in molarity.
Enzyme assays.
Alkaline phosphatase (AP) and -glutamyltranspeptidase (
-GT)
activities were determined as markers for differentiation. For AP
activity, cell lysates were assayed using 7 mM
p-nitrophenylphosphate as substrate and
2-amino-2-methylpropan-1-ol as solvent. To determine AP activity, the
product (p-nitrophenol) produced per minute was measured and
normalized for cellular protein (4).
-GT activity was
measured using
-glutamyl-p-nitroanilide as substrate and glycylglycine as the
-glutamyl acceptor (29).
Cell cycle and apoptosis measurements. Cells were trypsinized and fixed with 100% ethanol at room temperature. Fixed cells were incubated with RNase for 30 min and then treated with propidium iodide (100 µg/ml) (26). Flow cytometric analyses were performed using a FACScan flow cytometer using the CellQuest software (Becton-Dickinson, Mountain View, CA).
BrdU incorporation. Caco-2 cells were plated in 96-well plates and cultured as described above. To measure DNA synthesis, cells were incubated in the presence of 100 µM BrdU for 2 h. After labeling, BrdU incorporation into cellular DNA was measured by a colorimetric immunoassay using a commercially available cell proliferation ELISA kit. Absorbance from peroxidase reaction with substrate (tetramethylbenzidine) was measured by a scanning multiwell spectrophotometer at 450 nm with a reference wavelength of 650 nm (Molecular Devices, Sunnyvale, CA). BrdU incorporation for all experiments was expressed as the percentage of day 1.
Redox Western blot analysis. To determine the relative amounts of reduced and oxidized Trx, cellular protein was extracted in guanidine-lysis buffer containing 10 mM iodoacetic acid (10, 38). Purified Trx (containing fully oxidized, partially oxidized, and fully reduced forms of Trx) was used as a standard to identify different redox states of Trx. Samples were incubated for 30 min at 37°C and centrifuged (735 g for 2 min) in G-25 microspin columns (Amersham Pharmacia Biotech) to remove excess iodoacetic acid. Concentration of protein in the eluate was determined by the Bradford method, and equivalent amounts of protein were loaded for each well. Cell lysates were resolved by electrophoresis with a 5% stacking gel and a 15% nondenaturing, nonreducing, resolving gel. Proteins were transferred onto nylon membranes (Amersham Pharmacia Biotech), and the membranes were blocked with 5% nonfat milk for 1 h. The membranes were incubated with primary Trx antibody for 1 h, incubated with horseradish peroxidase-conjugated anti-goat secondary antibody for 1 h, and developed according to the chemiluminescence system. Densitometry was performed for quantification.
Statistical analysis. Tests for statistically significant differences (P < 0.05) were performed by ANOVA, and Students' t-test was used to specifically test for differences between proliferating (days 1-13) and differentiated (days 19-25) cells. Data are given as means ± SE; P values < 0.05 were considered significant.
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RESULTS |
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Protein concentration in Caco-2 cells during culture.
Cellular protein was measured as a function of time in culture and used
as an index of cell growth. Results show that cellular protein content
increased until day 16, at which point there was no further
change (Fig. 1). Visual inspection of the
cells in culture showed that they became confluent by day
10, indicated by a dotted vertical line marked "c" in all
figures. The continuous increase in protein content after this point
indicated that cell division or hypertrophy occurred until day
16. After this time, the cells appeared nonproliferating (see
BrdU incorporation into cellular DNA and Cell cycle
analysis), indicated by a broken vertical line marked
"np" in all figures.
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BrdU incorporation into cellular DNA.
To measure cellular proliferation, BrdU incorporation into DNA was
measured as a function of time in culture. The results (Fig.
2) showed an increase in BrdU
incorporation at day 4, which was maximal at day
10. There was no change from days 10-13, but the level of BrdU incorporation decreased at day 16 and
remained at this level until day 25. BrdU incorporation rate
in cells at days 4-13, the period of
rapid replication, was significantly higher than at later time points
(days 19-25). These data show that
BrdU incorporation occurs under all conditions but that there is an
identifiable decrease in this measure of DNA synthesis at day 16 in association with cessation of the
increase in protein.
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Cell cycle analysis.
Cell cycle analysis was performed to further elucidate Caco-2 growth
characteristics. A significant increase in the percentage of cells in
the G0/G1 stage of the cell cycle (Fig.
3A)
occurred at days 19-25
compared with days 1-13. In contrast,
a significantly greater percentage of cells were in the synthesis (S)
phase of the cell cycle at the earlier time points (Fig.
3B). There was no significant change in percentage of cells
in the G2/M stage (not shown). The percentage of
apoptotic (sub-G0) cells was significantly increased at
later time points (6-8%, days 16-25)
compared with earlier time points (4-5%, <10 days), but only a
small percentage of the cell population was undergoing
apoptosis (Fig. 3C ) (23).
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Differentiation markers.
Cellular differentiation follows growth arrest at the transition
between the first phase (G1) and the S of the cell cycle (35). To determine the time course of differentiation,
activities of two differentiation markers, AP and -GT, were measured
as a function of days in culture. There was no significant change in
the activities of either marker from days 1-16,
but there was a significant increase in both activities on day
19 and subsequent days (Fig. 4).
These results are consistent with the BrdU and cell cycle analysis data
and confirm the expected spontaneous shift from the proliferative to
the differentiated phenotype in these cells in association with the
change from a growing to a stationary population.
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Cellular GSH, GSSG, and GSH/GSSG redox, Eh.
Having defined proliferation and differentiation as days before and
after day 16, respectively, experiments were performed to
determine whether the Eh was different in these growth
phases. Intracellular concentrations of GSH decreased continuously
throughout the experiment despite supply of the same concentration of
CySS in the medium every 3 days. Concentration of GSH before day
16 was significantly higher than the concentration after day
16 (Fig. 5A). In
contrast, no significant changes were observed in GSSG concentration
(Fig. 5A). As a consequence of the change in GSH concentration without a corresponding change in the GSSG concentration, the calculation of the redox state of GSH/GSSG couple showed that the
differentiated cells were 40 mV more oxidized than the dividing cells
(Fig. 5B).
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Extracellular Cys/CySS redox.
Previous studies showed that depletion of Cys/CySS from the culture
medium is sufficient to oxidize the cellular GSH/GSSG pool
(27). To determine whether the marked oxidation of
cellular GSH/GSSG redox associated with differentiation could be due to depletion of extracellular Cys and CySS, cell medium was collected at
each medium change and analyzed for remaining Cys and CySS concentrations. Cys concentration was low and did not change
significantly (Fig. 6A). CySS
concentration was decreased from the original amount in the
culture medium (200 µM) and was significantly lower during maximal
proliferation. After day 16, CySS concentration returned
toward the day 1 level (Fig. 6B). These results
support the interpretation that maximal CySS utilization occurred
during the active growth phase. At days 7 and 10 when cellular BrdU incorporation rate was maximal, Cys/CySS redox was
maximally reduced (Fig. 6B). These results show that
extracellular redox is more reduced in actively proliferating cells
than in differentiated cells, with a change of redox of ~28 mV
between day 7 and days 18-25. Results also indicate that in differentiated cells, the extracellular Cys/CySS
pool was not depleted, and therefore Cys depletion was not a factor
contributing to the oxidation of intracellular GSH redox observed
during this phase.
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Effect of progression from proliferation to differentiation on Trx
redox state.
To determine whether the oxidation state of the redox-active protein
Trx was altered in association with differentiation, cell extracts were
examined using a redox Western blot analysis (37) that
allows separation and quantification of the different redox forms of
Trx. Both reduced and oxidized Trx bands were present in all samples at
all time points. The relative abundance of the oxidized Trx band
compared with the total Trx levels showed no change in the oxidation
state of Trx as a function of time after day 1 (Fig.
7A and B). However,
there was an increase in expression of Trx protein as indicated by the
redox Western blot and confirmed by direct Western blot analysis (Fig.
7A). Thus the results show that, unlike the GSH/GSSG pool,
the Trx pool increases rather than decreases and does not undergo
oxidation in association with Caco-2 differentiation. Results therefore
indicate that the redox states of these two major redox-active
thiol/disulfide pools are independent of each other during the
transition from proliferation to differentiation in Caco-2 cells.
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DISCUSSION |
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Cell proliferation, differentiation, and death are a continuous process in intestinal mucosa homeostasis. Although cancer cell lines, such as Caco-2 and HT29, may not completely replicate normal growth characteristics, they provide useful in vitro models for study of cellular growth control. The GSH/GSSG redox (Eh) has been strongly implicated to be involved in all three stages of cell development. When induced chemically to differentiate, HT29 cells became more oxidized by 60 mV compared with the proliferative cell population (22). In addition, differentiation induced by butyrate in Caco-2 cells was accompanied by apoptosis (23), which results in a further oxidation of GSH/GSSG redox (6, 17). Present data confirm and extend these results, showing that 1) oxidation of the GSH pool occurs as Caco-2 cells spontaneously progress from proliferation to differentiation, 2) oxidation of the extracellular Cys/CySS pool occurs under the same conditions, 3) increased apoptosis (albeit at low levels) is also inherent in this natural progression, and 4) the GSH pool operates independently from the Trx pool under conditions of enterocyte-like differentiation in Caco-2 cells.
Caco-2 cells when grown to confluence under standard culture conditions
cease division and spontaneously differentiate in a manner similar to
normal enterocytes (8). To minimize error in
interpretation of the data, we used different approaches to define this
progression. Analysis of cell cycle and BrdU incorporation rates
demonstrated that cell division was significantly higher before
day 16. In contrast, two differentiation markers (AP,
-GT) were significantly higher at times after day 16.
Thus the results showed that although Caco-2 cells are heterogeneous, a
demarcation between proliferating and differentiated cells could be
obtained by comparing cells before day 16 to those after
day 16.
Measurement of GSH revealed a substantial decrease in the intracellular
GSH concentration in association with decreased cell division and
expression of differentiation markers. These results are consistent
with observations in other cell types that show GSH concentration is
high in actively proliferating fibroblasts and decreases on serum
starvation (34) and GSH concentration in cultured
hepatocytes is high when cells are cultured at low density and
decreases as cell density is increased (24). However, the
factors primarily responsible for determining steady-state GSH
concentrations remain unclear. Measurements of Cys and CySS remaining
in the culture medium show that the decrease in GSH is not due to
depletion of Cys and CySS. A previous study of butyrate-induced differentiation of HT29 cells showed there was no change in expression of either the catalytic or regulatory subunit of glutamate-cysteine ligase despite a sixfold decrease in GSH concentration
(22). Decrease in cellular GSH in differentiated cells
could occur as a consequence of increased export; extracellular GSH
concentrations were low (not shown), but -GT activity increased with
differentiation (Fig. 4) and may have resulted in enhanced rates of GSH degradation.
The redox state of GSH/GSSG was 40 mV more reduced before day 16 than after day 16, indicating that cells became substantially oxidized as they progressed from actively proliferating to nonproliferating differentiated cells. The extent of oxidation is similar to that observed when HT29 cells were induced to differentiate using butyrate (60 mV) (22). Also consistent with this, contact-inhibited fibroblasts were found to be 35 mV more oxidized than proliferating fibroblasts (15). Mechanisms responsible for these redox changes remain undefined. As indicated above, depletion of Cys (27) does not appear to be responsible. However, the concomitant changes in extracellular Cys/CySS redox raise the possibility that functional responses associated with cellular GSH/GSSG redox may be causally related to changes in extracellular redox or cellular Cys/CySS redox, neither of which has been extensively studied.
Although several studies have addressed the GSH system during cell division and differentiation, relatively little information is available concerning the other major thiol/disulfide system, i.e., Trx. Trx functions as a cofactor in synthesis of deoxyribonucleotides for DNA synthesis, and increased Trx is associated with enhanced proliferation in cancer cells (31). Trx contains a conserved dithiol motif that undergoes reversible oxidation in its catalytic function. In the present study, we measured the redox state of Trx based on change in mobility due to reaction of the reduced form with iodoacetic acid (37). Results show an apparent increase in the expression of Trx protein but no change in the redox state of Trx as cells progressed from proliferation to differentiation. Trx reductase was not measured, but the increase in Trx without change in redox may also require an increase in expression of this enzyme to maintain the steady-state redox. In combination with measured oxidation of GSH/GSSG under these conditions, results support the interpretation that the GSH and Trx systems are complementary in function but further show that the redox states of the two systems are independently controlled.
A summary of the present data, placed in the context of existing
knowledge of the cellular GSH/GSSG redox system (see Ref 19), is
provided in Fig. 8. Both in vivo
(18) and other in vitro (22) studies show an
oxidation in GSH/GSSG redox during the transition from proliferating to
nonproliferating, differentiated cells. Present results show that the
redox change is inherently associated with this transition and is not a
function of manipulations, such as the addition of a differentiating
agent or removal of a required growth factor.
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The magnitude of oxidation of GSH/GSSG redox observed in the Caco-2 cells during differentiation is sufficient to contribute to redox-dependent regulation of proteins with redox-sensitive thiols (19) provided that adequate glutaredoxin activity is present to mediate such effects (13). Currently, no information is available on possible changes in expression of glutaredoxin during differentiation. However, the current data show that Trx, which also has a central role in thiol/disulfide regulation, has increased expression but no detectable redox change in association with differentiation. In addition, the results show that the extracellular Cys/CySS redox in these cultured cells also changes in association with the proliferation/differentiation state. Although it is not possible to extrapolate these findings to normal cells or to cell function in vivo, the data show that the two central cellular antioxidant and redox-regulating systems (GSH and Trx) are controlled independently and that cellular and extracellular redox may be dynamically coupled with cellular growth control.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants ES-011195, ES-009047, DK-55850, and RR-00039.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. P. Jones, Dept. of Biochemistry Rollins Research Center, Rm. 4131, Emory University, 1510 Clifton Rd. NE, Atlanta, GA 30322 (E-mail: dpjones{at}emory.edu).
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.
10.1152/ajpgi.00183.2002
Received 14 May 2002; accepted in final form 20 August 2002.
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