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Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA; and
* Department of Internal Medicine, Saga Medical School, Saga, Japan
1Correspondence: Department of Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA. E-mail: taw{at}lsuhsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cellular redox • GSH:GSSG ratio • cell cycle arrest • intestinal cell turnover • EGF
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the ability of certain transcriptional factors to
bind to their cognate DNA (2)
, signal transduction
(3
, 4)
, cell necrosis (5)
, cell apoptosis
(5
, 6)
, and cell proliferation (7
, 8)
.
Oxidative stress is an important pathophysiological stimulus that
affects the cellular redox status. However, besides inducing redox
shift, oxidative stress can directly affect cell functions such as
enzyme activities, cell signaling, and cellular responses
(9)
. Often the effects of redox changes overlap with those
of oxidative stress. For that reason, it is sometimes unclear whether
cellular responses elicited by oxidative stress are mediated by
reactive oxygen species (ROS) directly or by ROS-induced redox
imbalance.
Glutathione (L-
-glutamyl-L-cysteinylglycine) is a ubiquitous
intracellular thiol present in all tissues. Its reduced form (GSH) and
oxidized form (glutathione disulfide, GSSG) constitute the major thiol
redox system in cells, and the redox status of GSH and GSSG is of
crucial importance for cellular function (1)
. Glutathione
has a role in signal transduction (3
, 4
, 10)
, gene
expression (11)
, modulation of protein function (3
, 12)
, necrosis (5)
, and apoptosis (13
, 6)
. It is also associated with cell proliferation
(14
15
16
17
18
19)
and affects growth factor functions (20
, 21)
. However, the role of thiol-disulfide balance in cell
proliferation is unresolved.
In the current study, we used human colon cancer CaCo-2 cells to test the hypothesis that cell proliferation is responsive to the cellular GSH/GSSG status. The specific objectives were to determine 1) whether the induction of mild GSH/GSSG imbalance affects cell proliferation independent of ROS and 2) redox-induced cell cycle change, and 3) whether redox change underlies the stimulatory effect of EGF on cell proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture and treatment
The human colon cancer cell line CaCo-2 was obtained from the
American Type Culture Collection (Rockville, MD). Cells were maintained
in 75 cm2 culture flasks in DMEM supplemented
with 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml
penicillin, and 10 mg/ml gentamicin. The cell cultures were incubated
in humidified atmosphere with 5% CO2/95% air at
37°C. The culture medium was changed every 2 days.
For experimentation, CaCo-2 cells were seeded in 6- or 24-well plates and cultured to 60% confluence in DMEM with serum. Before incubation with redox agents, cell culture media were replaced with fresh serum containing DMEM. In some experiments, incubations were performed in serum-free DMEM. Whenever present, reagents were added to cell cultures at the following final concentrations: diamide, 30 µM; BCNU, 30 µM; NAC, 5 mM; EGF, 100 ng/ml. BCNU and NAC were administered 30 min before diamide treatment. Some cultures were pretreated with 5 mM BSO for 12 h to lower the baseline intracellular glutathione concentration before treatment with diamide or diamide plus BCNU. In addition, BSO was also present throughout the incubation period to prevent the resynthesis of glutathione.
GSH and GSSG determination
Intracellular GSH and GSSG levels were determined by the
high-performance liquid chromatography method of Reed et al.
(22)
. Cells were treated with ice-cold 5% trichloroacetic
acid (TCA), followed by centrifugation to remove TCA-insoluble
proteins. The acid supernatant was derivatized with 6 mM iodoacetic
acid and 1% 2, 4-dinitrofluorobenzene to yield the S-carboxylmethyl
and 2, 4-dinitrophenyl derivatives of GSH and GSSG. Separation of GSH
and GSSG derivatives was achieved on a 25 cm x 4.6 mm x 10
µm Lichrosorb NH2 column.
[3H-] thymidine incorporation
[3H-] Thymidine (1 µCi) was added to each well
3 h before collecting cells. At each time point indicated, labeled
monolayer was washed twice with phosphate-buffered saline (PBS) and the
cells were detached with trypsin-EDTA. Cells were precipitated with 5%
TCA at 4°C. The precipitate was washed twice with 95% ethanol at
4°C, followed by drying at room temperature, and solubilized with 1 N
NaOH. Aliquots of dissolved precipitate were mixed with Poly-fluor
(Packard, Meriden, CT) and the cell-associated radioactivities were
determined using a liquid scintillation counter.
Ornithine decarboxylase (ODC) activity assay
At each time point, the cells were detached with trypsin-EDTA.
After washing twice with PBS, cells were collected by centrifugation.
ODC activity was assayed by a radiometric technique (23)
.
Briefly, cells were resuspended in 500 µl of 0.1 M Tris buffer (pH
7.4) containing 1 mM EDTA, 50 µM pyridoxal 5-phosphate, and 5 mM
dithiothreitol and homogenized. After centrifugation at 12,000 rpm for
30 min, 200 µl aliquot of the supernatant was incubated in stoppered
vials in the presence of 3.5 nmol of L-[1-14C]
ornithine (50 mCi/mmol) for 1 h at 37°C. The
14CO2 liberated by the
decarboxylation of ornithine was trapped on filter paper impregnated
with 20 µl of 2 N NaOH, which was suspended above the reaction
mixture. The reaction was stopped by the addition of 0.3 ml of 10%
TCA. The filter paper was placed in Poly-fluor and radioactivities of
the 14CO2 were measured
using a liquid scintillation counter. Results are expressed as
picomoles CO2 per milligram protein per hour.
Cell cycle analysis by flow cytometry
At the end of each period, the cells were trypsinized, washed
with cold PBS, and fixed in 70% ethanol for 30 min at 4°C. Fixed
cells were washed with PBS and incubated in 1 mg/ml DNAase-free RNAase
for 40 min at 37°C. The RNAase was preheated to 100°C for 15 min to
inactivate DNAase. Propidium iodide (PI, 50 µg/ml) was added and
samples were incubated at 4°C for 30 min in the dark. Flow cytometric
analysis was performed using a FACS Vantage flow cytometer (Becton
Dickinson, San Jose, CA). In each experiment, a minimum of 5 x
105 cells per sample was analyzed. Data were
analyzed using Cell Quest software (Becton Dickinson, San Jose, CA) and
gated on pulse-processed PI signals to exclude doublets and large
aggregates, using a multiparameter gate strategy.
Determination of cellular oxidant production
Oxidant formation was measured using the oxidant-sensitive
nonfluorescent probe DHR. Previous studies have used DHR to detect
reactive oxygen species in cells (24)
. Because the
intracellular oxidation of DHR is mediated by biological oxidants like
peroxynitrite and a variety of secondary hydrogen peroxide-dependent
intracellular reactions that includes
H2O2-cytochrome
c and H2O2-Fe
(2-)
(24
, 25)
, detection of increased
oxidation of this probe is a useful marker of a change in general
cellular oxidant production. In addition, the fluorometric
determination of rhodamine from DHR oxidation has been used to
determine the interactions between superoxide and nitric oxide
(26)
. Intracellularly, DHR is oxidized by two electrons
that results in the formation of rhodamine 123, which possesses high
molar absorptivity and is fluorescent. Since excitation of rhodamine at
500 nm results in light emission at 536 nm, measurement of the amount
of rhodamine at these excitation/emission wavelengths provides a
reasonable quantification of oxidant production.
DHR was prepared as a 25 mM stock solution in nitrogen-purged dimethyl formamide (DMF) and stored in the dark at -20°C. On the day of each experiment, stock DHR was diluted afresh with DMF and added to cells at a final concentration of 5 µM. Cells were then exposed to diamide or diamide plus BCNU for 6 or 24 h. At each time, cells were washed twice with PBS. The cells were harvested using a scraper in 2 ml of PBS and sonicated using a Braun-Sonic sonicator (B. Braun Biotech Int., Allentown, PA). Rhodamine123 accumulation was quantified using an AMINCO Bowman Series 2 luminescence spectrophotometer (Thermo Spectronic, Rochester, NY) at excitation and emission wavelengths of 500 nm and 536 nm, respectively. Results were expressed as relative fluorescence unit/mg protein.
Cell count and viability
Cells were counted using a hemocytometer in the presence of
0.5% trypan blue. The changes in cell number after treatments with
redox agents are presented as fold change above the starting cell
number. Cell viability was determined as the percentage of cells that
excluded trypan blue.
Protein assay
Protein was measured using Bio-Rad Protein Assay kit (Bio-Rad
Laboratories, Hercules, CA) according to the manufacturer’s protocol.
Statistical analysis
Results are expressed as mean ± SE. Data were
analyzed using a one-way ANOVA with Bonferroni corrections for multiple
comparisons. P values of < 0.05 were considered
statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; BCNU is an inhibitor of glutathione reductase that
blocks the conversion of GSSG to GSH (28)
. Figure 1
illustrates the kinetics of intracellular GSH, GSSG, and the
GSH:GSSG ratio in cells exposed to 30 µM diamide or 30 µM diamide
plus 30 µM BCNU. Treatment of cells with 30 µM diamide alone did
not cause a significant change in intracellular GSH, GSSG, or the
GSH:GSSG ratio, indicating that this concentration of diamide was
insufficient to affect the overall cellular glutathione status under
steady state conditions. In addition, cellular GSH/GSSG status was
unchanged in cells treated with 30 µM BCNU alone (data not shown). On
the other hand, 30 µM diamide plus 30 µM BCNU decreased GSH and
elevated GSSG levels within 30 min, resulting in a marked decrease in
the GSH:GSSG ratio. This redox imbalance caused by diamide plus BCNU
returned to baseline levels within 3 h. The intracellular GSH
levels at 6 and 24 h after treatment with diamide plus BCNU were
significantly elevated above the pretreatment value (Fig. 1A
), but these increases in cellular GSH did not alter the
GSH:GSSG ratio (Fig. 1C
).
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Based on these results, subsequent experiments were performed using 30 µM diamide plus 30 µM BCNU treatment to induce mild redox imbalance and to examine its influence on cell proliferation.
Effect of low-dose diamide plus BCNU treatment on cell
proliferation
The effects of 30 µM diamide plus 30 µM BCNU on proliferation
of CaCo-2 cells incubated in serum-containing DMEM are summarized in
Fig. 2
. 3H-Thymidine incorporation in cells treated with
diamide plus BCNU was significantly lower than that in
untreated cells at 6 and 24 h (Fig. 2A
). Notably, total
protein levels (Fig. 2B
) and cell numbers (Fig. 2C
) in untreated cells and cells treated with diamide plus
BCNU were increased at 24 h compared with the values at 6 h.
At 6 h, there was no difference in protein content or cell number
between treated and untreated cells. However at 24 h, these two
parameters were significantly lower in cells treated with the redox
agents. Treatment of cells with 30 µM diamide or 30 µM BCNU
separately had no effect on 3H-thymidine
incorporation (data not shown). Together, the decrease in thymidine
incorporation, protein content, and cell number induced by 30 µM
diamide plus 30 µM BCNU is consistent with a suppression of cell
proliferative activity, which paralleled an early disruption in the
cellular redox status. Diamide plus BCNU did not affect ODC activity, a
common biochemical marker of intestinal proliferation, but overall ODC
activity was significantly lower at 24 h than at 6 h (Fig. 2D
).
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Figure 3
shows the effects of 30 µM diamide plus 30 µM BCNU on proliferation
of CaCo-2 cells incubated in serum-free DMEM. At 6 h,
3H-thymidine incorporation in cells treated with
diamide plus BCNU was significantly lower than that in untreated cells
(Fig. 3A
), which were similar to the observations in cells
cultured in serum-containing media (see Fig. 2A
). However,
ODC activity in cells treated with diamide plus BCNU was suppressed at
6 h compared with untreated cells (Fig. 3B
).
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Modulation of GSH/GSSG status and cell proliferation by NAC
L-NAC is an important precursor of GSH. It is a thiol-containing
compound with reducing activity (29)
. NAC (5 mM) abolished
the decrease in intracellular GSH and the increase in intracellular
GSSG induced by 30 µM diamide plus 30 µM BCNU at 30 min after
treatment (Fig. 4
A, B, respectively). Accordingly, the administration of NAC
resulted in an elevated GSH:GSSG ratio in cells treated with diamide
plus BCNU (Fig. 4C
). In addition, NAC inhibited the
suppression of 3H-thymidine incorporation in
cells treated with diamide plus BCNU at 6 h and reversed the
redox-induced decrease in cell number at 24 h (Fig. 4D
). These results indicate that the disruption of normal
cellular GSH/GSSG status was responsible for the inhibition of
proliferation of cells exposed to diamide plus BCNU.
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Relationship between intracellular GSH-GSSG status and cell
proliferation
BSO is a potent inhibitor of -glutamyl cysteine synthetase, the
rate-limiting step in GSH synthesis (30)
. BSO (5 mM)
markedly decreased intracellular GSH at 30 min, which declined further
at 24 h (Fig. 5
A). Notably, intracellular GSH levels in cells treated with
BSO was significantly lower than the levels achieved after treatment
with 30 µM diamide plus 30 µM BCNU (Fig. 5A
). The
combined treatment with BSO and diamide plus BCNU caused a more severe
decrease in GSH level than BSO treatment alone at 30 min (Fig. 5A
). Figure 5B
shows that BSO also diminished
intracellular GSSG, which is consistent with an overall decrease in
total cellular glutathione levels after BSO treatment of cells.
However, exposure of BSO-treated cells to diamide plus BCNU resulted in
a significant increase in GSSG at 30 min, indicating a potentiation of
oxidative stress under these conditions of severely compromised
cellular GSH status. The degree of GSSG increase after BSO treatment
(without or with diamide plus BCNU) was significantly lower than the
GSSG values in cells without BSO treatment (Fig. 5B
).
Although BSO treatment alone did not change GSH:GSSG ratio at 30 min,
the added stress of diamide plus BCNU resulted in a significant
decrease in the GSH:GSSG ratio to the same extent as that seen with
diamide plus BCNU treatment without BSO pretreatment (Fig. 5C
). However, under these two conditions, the GSH/GSSG redox
status returned to near control values by 24 h.
3H-Thymidine incorporation and changes in cell
number after BSO treatment was unaltered compared with control (Fig. 5D
). However, exposure of BSO-treated cells to diamide plus
BCNU caused a significant decrease in cell number and in
3H-thymidine incorporation compared with control,
treatment with BSO alone, and treatment with diamide plus BCNU (Fig. 5D
). Together, these results indicate that a mere compromise
in the cellular GSH pool per se has minimal influence on cell
proliferative activity. Rather, the data support the notion that an
early and/or abrupt change in the GSH:GSSG ratio shortly (e.g., 30 min)
after oxidative challenge, rather than gradual or later changes (e.g.,
24 h) in redox status, is the important determinant in the
suppression of cell proliferation. Even though the decrease in
intracellular GSH level per se did not inhibit cell proliferation, it
enhanced the susceptibility of cells to redox imbalance-induced cell
arrest.
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Cell viability and cell cycle analysis
Table 1
summarizes the results on cell viability as measured by trypan blue dye
exclusion 6 and 24 h after each treatment. There were no
significant differences among the three treatment conditions,
indicating that the inhibition of cell proliferation was not due to
cell death. Moreover, cell viability as evaluated from the
subG0 fraction by flow cytometry ranged from
93.8% to 98.8% at 6 and 24 h for the three treatment condition
groups (see Fig. 6
), which corroborated the results of cell viability as measured by
trypan blue exclusion. To determine the effect of redox imbalance on
cell cycle changes, we subjected cells to flow cytometric analyses.
Figure 6
illustrates the cell cycle profiles. At 6 h, there were
no significant differences in the ratio of each phase of the cell cycle
(i.e., G0/G1, S, and
G2/M) (Fig. 6)
. On the other hand, treatment with
30 µM diamide plus 30 µM BCNU resulted in a marked increase in the
percentage of cells in the G2/M phase at 24 h. Pretreatment of cells with BSO, followed by diamide plus BCNU,
caused a further increase in the percentage of cells in the
G2/M phase (Fig. 6A
, 6B
).
That the cell cycle did not change at 6 h, even though
3H-thymidine incorporation was already decreased
at this time, suggests that the effect of redox imbalance on cell
proliferation was exerted at the G1-to-S phase
transition. The changes in cell cycle induced by GSH-to-GSSG shifts
were partially reversed by 48 h in cells treated with diamide plus
BCNU alone, but not in cells treated with the combination of BSO,
followed by diamide plus BCNU (Fig. 6A
, 6B
).
Together, it appears that redox imbalance-mediated suppression of cell
proliferation occurred in at least two phases of the cell cycle,
namely, at the G1-to-S phase transition and at
the G2/M phase. Moreover, the results show that a
compromise in intracellular GSH level exaggerated and prolonged the
cell cycle arrest.
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ROS production by diamide, BCNU, and BSO
To verify that the effect of diamide plus BCNU was mediated by
redox shift independent of oxyradical production secondary to decreased
cellular GSH, we quantified oxidant production by measuring the
oxidation of DHR. Oxidant formation was measured as the increase in
fluorescence of rhodamine 123 after DHR oxidation (see Materials and
Methods). Table 2
summarizes the results of intracellular rhodamine 123 accumulation.
There were no significant differences among the three treatment
conditions at 6 and 24 h, indicating that neither treatment of
cells with 30 µM diamide plus 30 µM BCNU nor with 5 mM BSO,
followed by diamide plus BCNU, increased ROS production.
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Effect of EGF on intracellular redox status and cell proliferation
EGF has been shown to exert mitogenic effect on serum-starved
CaCo-2 cells (31)
, but not in serum-replete CaCo-2 cells
(32)
. To test whether redox change is associated with the
mitogenic effect of EGF, CaCo-2 cells were cultured in serum-free DMEM
for 3 days before EGF treatment. Cells were incubated in serum-free
DMEM with 100 ng/ml EGF alone or with EGF and 30 µM diamide plus 30
µM BCNU for 6 h. EGF treatment induced no significant changes in
GSH or GSSG levels and did not change the GSH:GSSG ratio vs. control.
Addition of 30 µM diamide plus 30 µM BCNU to EGF-treated cells
shifted the GSH/GSSG thiol-disulfide balance in favor of GSSG at 30
min, although there was no statistical difference vs. EGF alone
(Fig. 7
A–C). EGF increased 3H-thymidine
incorporation compared with control; this increase was abolished by the
addition of diamide plus BCNU (Fig. 7D
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). At 30 µM, diamide did not change the
intracellular GSH or GSSG level due to the rapid catalytic regeneration
of GSH from GSSG by GSSG reductase. To achieve a more sustained redox
shift, 30 µM BCNU was added to inhibit GSSG reductase and the
conversion of GSSG to GSH (28)
. By the combined treatment
of cells with 30 µM diamide and 30 µM BCNU, we were able to induce
a marked and effective fall in the intracellular GSH:GSSG ratio (Fig. 1)
with no detectable production of ROS (Table 2)
. The results from the
current study have demonstrated a direct relationship between the
induction of mild intracellular redox imbalance and the suppression of
proliferation of the human colonic cancer CaCo-2 cells.
Previous studies have provided a link between intracellular GSH level
and proliferating activity. Actively growing cells were associated with
increases in total cellular GSH, and the decrease in GSH preceded the
slowing of proliferating activity (15
, 18)
. Other studies
showed that maintenance of high GSH level was required for
stimuli-induced cell proliferation (14
, 19)
. However, the
influences of GSSG and/or the ratio of oxidized-to-reduced glutathione
on cell proliferation are poorly understood. In our study, treatment of
cells with 30 µM diamide plus 30 µM BCNU decreased GSH and
increased GSSG, resulting in a decreased GSH:GSSG ratio. Notably, this
redox shift significantly inhibited cell proliferation as measured by
changes in 3H-thymidine incorporation,
protein content, cell number, and cell cycle profiles (Figs. 2
, 3
, 6)
.
The addition of NAC restored the intracellular redox balance and,
correspondingly, cell proliferative activity (Fig. 4)
. Collectively,
these results indicate that intracellular redox status is associated
with cell proliferative activity that is independent of oxyradical
production.
Evidence in the literature shows that a direct effect of depletion of
glutathione level on cell proliferation is unresolved. Several studies
have shown that BSO-induced glutathione depletion was sufficient to
suppress cell proliferation (18
, 33
, 34)
, but others have
reported that the loss of cell glutathione alone had no effect on cell
proliferation (35
36
37)
. Our current study provided the
distinction between the effect of GSH and the effect of GSH:GSSG ratio
on the suppression of cell proliferation. BSO treatment alone markedly
diminished intracellular GSH level but did not change the GSH:GSSG
ratio at 30 min; this had a minimal effect on cell proliferation. On
the other hand, treatment with diamide plus BCNU decreased GSH,
increased GSSG, and lowered GSH:GSSG ratio within 30 min; this
alteration in redox status resulted in inhibition of cell
proliferation. Furthermore, our data show that the magnitude of
suppression of cell proliferation after treatment with diamide plus
BCNU was more severe when cells were first pretreated with BSO,
although the GSH:GSSG ratios did not differ between the two treatment
conditions (Fig. 5)
. These results mean that the initiation of
inhibition of cell proliferative activity was mediated by an early
and/or an abrupt decrease in GSH:GSSG ratio rather than by a depletion
of GSH level. Moreover, whereas GSH depletion per se did not appear to
have a direct suppressive effect on cell proliferation, a compromised
GSH pool exacerbated the suppressive effects of redox imbalance on cell
proliferation. This finding agrees with previous studies by Noelle et
al. (38)
, who reported that diamide-induced inhibition of
thymidine incorporation was more severe in cells with low GSH levels.
White et al. (37)
demonstrated that BSO by itself did not
inhibit cell proliferation in bovine pulmonary artery endothelial
cells, but BSO treatment enhanced the inhibition of cell proliferation
induced by transforming growth factor ß1.
A notable finding in this study is that a compromised intracellular GSH
status renders cells more vulnerable to the suppressive effects of
redox imbalance on cell proliferative activity. Moreover, redox-induced
cell cycle arrest tended to be prolonged in cells with compromised
cellular GSH, as evidenced by the delay in reversal of cell cycle
changes in cells treated with a combination of BSO and diamide plus
BCNU (Fig. 6)
. The reason low GSH exacerbates redox-induced suppression
of proliferation is unclear. One possibility may be that a
substantially decreased cellular GSH pool renders cells vulnerable to
oxidizing events, as they are less able to maintain an intracellular
reducing environment. Another interesting observation is that although
the redox imbalance caused by diamide plus BCNU treatment occurred
within 30 min, redox homeostasis returned to baseline level by 3 h, yet proliferation in these cells was more suppressed than confluent
control cells at 24 h. This suggests that a sustained disruption
of redox balance is neither necessary nor critical to effect
the suppression of cell proliferation. Rather, our kinetic data studies
are consistent with the conclusion that cell arrest was initiated by a
rapid and abrupt fall in the GSH:GSSG ratio within minutes after
oxidative challenge. Once initiated, the recovery of redox balance did
not prevent the progression of cell arrest to its biological end point
at 24 h. Furthermore, even though we found that BSO itself
eventually caused a decrease in GSH:GSSG ratio at 24 h (there was
no change in ratio at 30 min, Fig. 5C
), this has little
effect on cell proliferation. Collectively, the current results suggest
that a transient disruption of redox homeostasis is critical to affect
the proliferative outcome, and the temporal window between the loss of
redox balance and the signal transduction that led to cell cycle arrest
was within the first 30 min of exposure to redox agents. Recent studies
from our laboratory also observed the effect of transient redox
imbalance on apoptosis (6)
. In these latter studies, the
magnitude of redox imbalance was significantly greater than that found
to elicit cell arrest in the current study. These collective findings
demonstrate that the phase transition of cells is differentially
responsive to cellular redox status and that the magnitude of loss of
redox balance may determine cell fate in favor of proliferation,
differentiation, growth arrest, apoptosis, or necrosis.
The influence of redox imbalance in cell cycle progression is unclear.
Recent studies have shown that diethylmaleate, which conjugates with
and depletes GSH, activated the expression of
p21waf1 mRNA and produced
G1 and S phase delays and
G2/M arrest (39)
. It also induces a
rapid dephosphorylation of the retinoblastoma protein, which is
catalyzed by phosphatase independent of p21waf1
accumulation (40)
. In other studies, hydrogen peroxide,
which reacts with GSH and converts GSH to GSSG in a reaction catalyzed
by GSH peroxidase, was found to cause G2 phase
arrest (41)
. In our study, the induction of redox shift by
diamide plus BCNU did not cause a change in cell cycle progression at
6 h, although 3H-thymidine incorporation was
already suppressed at this time. This means that the
G1-to-S phase transition is a likely target site
for redox (GSH/GSSG) control. At 24 h, redox shift clearly induced
cell arrest at G2/M. Together, these results show
that redox imbalance likely exerts its effect at
G1-to-S phase transition and he
G2/M phase of the cell cycle. The mechanism by
which changes in redox status inhibit cell proliferation remains to be
identified. Several transcription factors, such as AP-1, NF
B, and
p53, have been defined whose DNA binding activity and stimulatory
effect on gene expression rely on the redox status of cysteinyl thiol
in their structures (2)
. GSSG may interact in a reversible
manner with protein cysteinyl thiol of many cellular proteins during
oxidative stress (42)
. Indeed, GSSG may act as a protein
phosphatase inhibitor via the formation of protein-disulfide cross-link
in the phosphate transfer domain of the enzyme. Other studies have
shown that GSH controls the onset of tumor cell proliferation by
regulating protein kinase C activity and intracellular pH
(34)
.
In our study, cells treated with diamide plus BCNU exhibited decreased
3H-thymidine incorporation in both
serum-containing medium and serum-free medium. In contrast, ODC
activity was suppressed by diamide plus BCNU treatment only in
serum-free medium. The reason for this interesting observation is
unclear. Typically, ODC is expressed in growing CaCo-2 cells and plays
a major role in cell replication (43)
, and ODC activity
has been shown to be modulated by medium condition in vitro
(43)
or food in vivo (23)
. This difference in
ODC activity in response to redox challenge may be due to stimulatory
effects of trophic factors or different nutrients under serum-replete
but not serum-depleted conditions.
Literature evidence shows that some growth factors affect intracellular
redox status. For example, keratinocyte growth factor was found to
modulate cellular GSH/GSSG redox status in the intestinal mucosa of
food deprived-refed rats (44)
, and platelet-derived growth
factor can increase cellular GSH content (21)
. Smith et
al. reported that growth factors that promote self-renewal, such as
neurotrophin-3 and basic fibroblast growth factor, induce a greater
reduced intracellular redox status. Pharmacological antagonists of
these reducing effects were found to inhibit self-renewal
(8)
. In our current study, EGF stimulated
3H-thymidine incorporation, and this increase was
abolished by induction of redox imbalance with diamide plus BCNU. The
proliferative effect of EGF was not associated with intracellular redox
change, in agreement with previous studies (21)
.
In summary, we have shown that intracellular redox imbalance inhibits cell proliferation independent of ROS production. Depletion of GSH per se has no effect on cell proliferation, but a compromised baseline cellular GSH exacerbated the susceptibility of cells to redox imbalance-induced inhibition of proliferation. Our data support the conclusion that an early and transient fall in GSH:GSSG ratio initiated the suppression of proliferative activity. Notably, cell cycle analyses revealed that the G1-to-S phase transition and the G2/M phase are potential targets for redox regulation. Although EGF stimulated cell proliferation, this effect was unrelated to changes in the intracellular thiol-disulfide status. However, EGF-induced cell proliferation was readily abolished by the induction of cellular redox imbalance.
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ACKNOWLEDGMENTS |
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Received for publication February 22, 2001.
Revision received June 20, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 L. Wang, Y. Tang, D. C. Rubin, and M. S. Levin Chronically administered retinoic acid has trophic effects in the rat small intestine and promotes adaptation in a resection model of short bowel syndrome Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1559 - G1569. [Abstract] [Full Text] [PDF]
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 J. Tian, N. Washizawa, L. H. Gu, M. S. Levin, L. Wang, D. C. Rubin, S. Mwangi, S. Srinivasan, D. P. Jones, and T. R. Ziegler Local Glutathione Redox Status Does Not Regulate Ileal Mucosal Growth after Massive Small Bowel Resection in Rats J. Nutr., February 1, 2007; 137(2): 320 - 325. [Abstract] [Full Text] [PDF]
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 P. Fasanaro, A. Magenta, G. Zaccagnini, L. Cicchillitti, S. Fucile, F. Eusebi, P. Biglioli, M. C. Capogrossi, and F. Martelli Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress FASEB J, June 1, 2006; 20(8): 1242 - 1244. [Abstract] [Full Text] [PDF]
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 K. Jiang, T. Ballinger, D. Li, S. Zhang, and L. Feldman A Role for Mitochondria in the Establishment and Maintenance of the Maize Root Quiescent Center Plant Physiology, March 1, 2006; 140(3): 1118 - 1125. [Abstract] [Full Text] [PDF]
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Y. S. Nkabyo, Y.-M. Go, T. R. Ziegler, and D. P. Jones Extracellular cysteine/cystine redox regulates the p44/p42 MAPK pathway by metalloproteinase-dependent epidermal growth factor receptor signaling Am J Physiol Gastrointest Liver Physiol, July 1, 2005; 289(1): G70 - G78. [Abstract] [Full Text] [PDF]
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A. K. Shoveller, B. Stoll, R. O. Ball, and D. G. Burrin Nutritional and Functional Importance of Intestinal Sulfur Amino Acid Metabolism J. Nutr., July 1, 2005; 135(7): 1609 - 1612. [Abstract] [Full Text] [PDF]
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 X. Chen, G. Moeckel, J. D. Morrow, D. Cosgrove, R. C. Harris, A. B. Fogo, R. Zent, and A. Pozzi Lack of Integrin {alpha}1{beta}1 Leads to Severe Glomerulosclerosis after Glomerular Injury Am. J. Pathol., August 1, 2004; 165(2): 617 - 630. [Abstract] [Full Text] [PDF]
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 C. R. Jonas, L. H. Gu, Y. S. Nkabyo, Y. O. Mannery, N. E. Avissar, H. C. Sax, D. P. Jones, and T. R. Ziegler Glutamine and KGF each regulate extracellular thiol/disulfide redox and enhance proliferation in Caco-2 cells Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2003; 285(6): R1421 - R1429. [Abstract] [Full Text] [PDF]
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 T. Y. Aw Cellular Redox: A Modulator of Intestinal Epithelial Cell Proliferation Physiology, October 1, 2003; 18(5): 201 - 204. [Abstract] [Full Text] [PDF]
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 L. Cicchillitti, P. Fasanaro, P. Biglioli, M. C. Capogrossi, and F. Martelli Oxidative Stress Induces Protein Phosphatase 2A-dependent Dephosphorylation of the Pocket Proteins pRb, p107, and p130 J. Biol. Chem., May 23, 2003; 278(21): 19509 - 19517. [Abstract] [Full Text] [PDF]
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 A. Uc and B. E. Britigan Does Heme Oxygenase-1 Have a Role in Caco-2 Cell Cycle Progression? Experimental Biology and Medicine, May 1, 2003; 228(5): 590 - 595. [Abstract] [Full Text] [PDF]
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Y. S. Nkabyo, T. R. Ziegler, L. H. Gu, W. H. Watson, and D. P. Jones Glutathione and thioredoxin redox during differentiation in human colon epithelial (Caco-2) cells Am J Physiol Gastrointest Liver Physiol, December 1, 2002; 283(6): G1352 - G1359. [Abstract] [Full Text] [PDF]
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E. K. PIAS and T. Y. AW Apoptosis in mitotic competent undifferentiated cells is induced by cellular redox imbalance independent of reactive oxygen species production FASEB J, June 1, 2002; 16(8): 781 - 790. [Abstract] [Full Text] [PDF]
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