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2003;63:2109-2117.

Cancer Res

Sarita G. Menon, Ehab H. Sarsour, Douglas R. Spitz, et al.

Mouse Embryo Fibroblast Cell Cycle
to S Phase Transition in the
1
Redox Regulation of the G

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[CANCER RESEARCH 63, 21092117, May 1, 2003]
Redox Regulation of the G
1
to S Phase Transition in the Mouse Embryo Fibroblast
Cell Cycle
1
Sarita G. Menon, Ehab H. Sarsour, Douglas R. Spitz, Ryuji Higashikubo, Mary Sturm, Hannah Zhang, and
Prabhat C. Goswami
2
Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, Iowa 52242 [S. G. M., E. H. S., D. R. S., M. S., H. Z., P. C. G.,
and Department of Radiation Oncology, Washington University Medical School, St. Louis, Missouri [R. H.]
ABSTRACT
The hypothesis that intracellular oxidation/reduction (redox) reactions
regulate the G
0
-G
1
to S-phase transition in the mouse embryonic fibro-
blast cell cycle was investigated. Intracellular redox state was modulated
with a thiol-antioxidant, N-acetyl-L-cysteine (NAC), and cell cycle pro-
gression was measured using BrdUrd pulse-chase and flow cytometric
analysis. Treatment with NAC for 12 h resulted in an 6-fold increase in
intracellular low-molecular-weight thiols and a decrease in the MFI of an
oxidation-sensitive probe, dihydrofluorescein diacetate, indicating a shift
in the intracellular redox state toward a more reducing environment.
NAC-induced alterations in redox state caused selective delays in progres-
sion from G
0
-G
1
to S phase in serum-starved cells that were serum
stimulated to reenter the cell cycle as well as to inhibit progression from
G
1
to S phase in asynchronous cultures with no significant alterations in
S phase, and G
2
M transits. NAC treatment also showed a 70% decrease
in cyclin D1 protein levels and a 34-fold increase in p27 protein levels,
which correlated with decreased retinoblastoma protein phosphorylation.
Cells released from the NAC treatment showed a transient increase in
dihydrofluorescein fluorescence and oxidized glutathione content between
0 and 8 h after release, indicating a shift in intracellular redox state to a
more oxidizing environment. These changes in redox state were followed
by an increase in cyclin D1, a decrease in p27, retinoblastoma protein
hyperphosphorylation and subsequent entry into S phase by 812 h after
the removal of NAC. These results support the hypothesis that a redox
cycle within the mammalian cell cycle might provide a mechanistic link
between the metabolic processes early in G
1
and the activation of G
1
-
regulatory proteins in preparation for the entry of cells into S phase.
INTRODUCTION
In recent years, increasing evidence has suggested that intracellular
oxidation-reduction (redox) reactions play a critical role in the regu-
lation of several physiological processes including cell proliferation
(1), senescence (2), differentiation (3), and apoptosis (4). ROS
3
in-
cluding superoxide, hydrogen peroxide, hydroxyl radical, singlet mo-
lecular oxygen, and organic hydroperoxides are believed to be con-
tinuously generated intracellularly as byproducts of O
2
metabolism
and have traditionally been thought of as unwanted and toxic byprod-
ucts of living in an aerobic environment (5, 6). However, results from
several studies suggest that the metabolic production of ROS is tightly
regulated and serves a physiological function during mitogenic stim-
ulation of cultured cells (1, 59). It has been proposed that the
endogenous production of ROS operates as a key signaling process in
the cascade of events leading to cell proliferation after stimulation
with platelet-derived growth factor (7), EGF (8), cytokines and anti-
gen receptors (9), and oncogenic Ras (10). Although these studies
provide evidence for a possible role of redox signaling in the transi-
tion of quiescent cells to proliferation during mitogenic stimulation, it
is currently unknown whether changes in intracellular redox state
influence progression through specific phases of the cell cycle and the
identity of specific cell-cycle-regulatory processes that may be redox-
regulated during the normal cell cycle.
Cellular growth is a tightly regulated sequence of transitions from
G
0
-G
1
to S phase to G
2
to M phases involving the sequential activa-
tion of CDK activities (1114). Progression from G
0
-G
1
to S phase is
largely regulated by the D-type cyclins (in particular cyclin D1) in
association with the CDKs CDK4/6 (14). This involves promoting the
synthesis and stability of the cyclin subunit as well as decreasing the
levels of CDK inhibitors. Cyclin D1/CDK4,6 kinase complex is
activated on removal of inhibitory serine and threonine phosphates
from CDK by Cdc25A phosphatase. Active cyclin D1/CDK4,6 kinase
complex partially phosphorylates the Rb protein, which causes the
release of the E2F family of proteins, initiating the transcription of
E2F-mediated gene expression during the transition from G
1
to S
phase. The inhibition of cyclin D1 expression prevents the transition
of cells from G
1
to S phase, whereas ectopic expression of cyclin D1
accelerates G
1
progression (15, 16). Thus, it is possible that changes
in intracellular redox state could provide a mechanistic link coupling
necessary metabolic processes early in G
1
and the activation of
G
1
-regulatory proteins, leading to the redox-sensitive progression
from G
1
to S phase.
The present study was designed to test the hypothesis that the
intracellular redox state has a specific regulatory role in cell cycle
progression from G
0
-G
1
to S phase and to begin to identify the
specific cell-cycle-regulatory processes amenable to redox regulation.
Results from this study show that progression from (a) G
1
to S phase
in asynchronously growing exponential cultures, and (b) G
0
-G
1
to S
phase in serum-stimulated quiescent cells and cells released from a
NAC-induced G
1
arrest, is associated with a transient increase in
pro-oxidant production that appears to mediate the activation of the
G
1
-regulatory proteins. Furthermore, the inhibition of these signals
using the thiol-reducing agent NAC blocks entry into S phase as well
as blocking the activation of G
1
-regulatory proteins. These results
support the hypothesis that redox-sensitive signaling in early G
1
controls the progression into S phase via the activation of G
1
-regula-
tory proteins.
MATERIALS AND METHODS
Cell Culture and Synchronization. MEF clones [12(1), wild type p53
phenotype] and [10(1), mutant p53 phenotype] were a gracious gift from Dr.
Arnold Levines laboratory Princeton University, Princeton, New Jersey, USA
and were cultured in DMEM supplemented with 10% fetal bovine serum,
nonessential amino acids (Life Technologies, Inc.), and antibiotics (penicillin
and streptomycin). Cells were cultured at 37C in a humidified incubator with
Received 8/27/02; accepted 3/5/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by NIH Grant CA69593 and a departmental support (to P. C. G.), and NIH
Grant HL51469 (to D. R. S.) and CA66081.
2
To whom requests for reprints should be addressed, at Free Radical and Radiation
Biology Program, Department of Radiation Oncology, University of Iowa Medical
School, B180 Medical Laboratories, Iowa City, Iowa 52242. Phone: (319) 384-4666; Fax:
(319) 335-8039; E-mail: goswamip@mail.medicine.uiowa.edu.
3
The abbreviations used are: ROS, reactive oxygen species; BrdUrd, bromodeoxyuri-
dine; NAC, N-acetyl-L-cysteine; NPM, N-(1-pyrenyl) maleimide; MFI, mean fluorescence
intensity; CDK, cyclin-dependent kinase; Rb, retinoblastoma protein; MEF, mouse em-
bryonic fibroblast; BSO, buthionine-(S,R)-sulfoximine; HPLC, high-performance liquid
chromatography; GSH, reduced glutathione; GSSG, oxidized glutathione; GGC, -
glutamyl cysteine; DFH-DA, dihydrofluorescein diacetate; PI, propidium iodide;
FACS, fluorescence-activated cell sorter.
2109
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5% CO
2
. Stock solutions of NAC (Sigma Chemicals) were adjusted to pH 7.0
with sodium bicarbonate, and appropriate aliquots were added to the cell-
culture medium. The toxicity of NAC was determined by single-cell colony-
forming assay, and no toxicity was observed in the concentration ranges (120
mM) used in this study (data not shown). Asynchronously growing cell cultures
were incubated simultaneously with NAC and 1 mM BSO, an inhibitor of
-glutamylcysteine synthase, for the inhibition of glutathione synthesis.
Measurement of Intracellular Glutathione and NAC Levels. Intracellu-
lar GSH and GSSG as well as NAC levels were assayed using previously
published spectrophotometric and HPLC assays (17, 18). Monolayer cells were
scrape-harvested in PBS at 4C and centrifuged, and the cell pellet was stored
frozen at 80C. Cell pellets were homogenized in 50 mM potassium phos-
phate buffer (pH 7.8) containing 1.34 mM diethylenetriaminepenta-acetic acid.
Total glutathione content was determined in sulfosalicylic acid extracts (5%
SSA, w/v) by the method of Anderson (19). GSH and GSSG were distin-
guished by the addition of 2 l of a 1:1 mixture of 2-vinylpyridine and ethanol
per 30 l of sample, followed by incubation at room temperature for 1.5 h
before the addition of the sulfosalicylic acid. Amounts of individual thiols were
normalized per mg protein in whole cell extract.
HPLC (HPLC system; Shimadzu Scientific Instruments, Inc., Columbia,
MD) with fluorescent detection was used to determine intracellular low-
molecular-weight thiol levels after previously published assays (17). Fifty
pmol to 100 fmol of standard thiols (NAC, GSH, GGC, and cysteine) were
derivatized with NPM and resolved using a 15-cm C18 Reliasil column
(Column Engineering, Ontario, CA) to generate standard curves. Retention
times of individual thiols were determined to be 7.7 min (NAC), 20.1 min
(GSH), 24.7 min (GGC), and 30.9 min (cysteine) using this system. Total
cellular protein extracts prepared from control and NAC-treated cells were
treated with NPM and were analyzed by HPLC. All of the biochemical
determinations were normalized to the protein content of whole-cell homoge-
nates by using the method of Lowry et al. (20).
Measurement of Intracellular Prooxidant Production. MEF cells were
trypsinized and pelleted at 200400 g for 5 min and the pellets were
resuspended in 1 ml of PBS supplemented with 5.5 mM glucose at 37C. Cells
were stained with 10 M DFH-DA (Molecular Probes, Eugene, OR) for 15 min
at 37C. DFH-DA stock solutions were prepared in DMSO, and cells that were
stained with 0.2% DMSO alone were included as a negative control for
DFH-DA staining. DFH-DA is a low-molecular-weight nonfluorescent and
nonpolar compound that enters cells freely. On entering the cell, the intracel-
lular esterases cleave the diacetate bond, resulting in a polar and nonfluores-
cent DFH. Intracellular prooxidants oxidize DFH to the fluorescent compound
DF. The intensity of green fluorescence of DF is an indicator of the levels of
prooxidant production. Changes in DFH fluorescence were detected using a
FACS (Becton Dickinson, San Jose, CA) equipped with 15-mW and 488-nm
argon-ion laser. Excitation was performed at 488 nm and emission at 530 nm
and 30-band pass filter. Data from 20,000 cells were collected, and geometric
mean fluorescence was calculated using the Cell Quest software (Becton
Dickinson). MFIs of the unstained cell populations were subtracted from the
MFIs of the stained cell populations, and the variations in geometric MFIs
were calculated relative to control cells at the time of experimental manipu-
lations. The specificity of prooxidant-mediated changes in DFH fluorescence
was determined by staining cells in duplicate dishes with carboxy DCFDA
[C-369, 5-(and-6-)-carboxy-2-7-dichlorofluorescein diacetate (Molecular
Probes, Eugene, OR)]. Carboxy DCFDA is an oxidation-insensitive compound
that is nearly chemically identical to the oxidation-sensitive compound
DFH-DA except that it lacks the oxidation-sensitive site. Thus, it is an
appropriate control for changes in DFH-DA uptake, ester cleavage, or efflux
that might result from various experimental manipulations.
DFH fluorescence was also used to measure prooxidant production during
the transition of cells from the quiescent phase to the proliferation cycle.
Asynchronously growing exponential cultures of MEFs were synchronized by
culturing cells in medium containing 0.1% serum for 24 h and serum stimu-
lated to reenter the cell cycle. Cells were harvested at representative times after
serum stimulation, stained with DFH-DA, and assayed by flow cytometry as
described above. DFH-DA-stained cells were also visualized using a Bio-Rad
MRC-600 laser-scanning confocal microscope (Central Microscopy Research
and Learning Facility at the University of Iowa).
BrdUrd Pulse-Chase Flow Cytometric Assay for Measurements of Cell
Cycle Phase Transits. BrdUrd pulse-chase flow cytometric assay for cell
cycle phase transitions were performed according to previously published
methodology (21, 22). Asynchronously growing cell cultures in 100-mm tissue
culture dishes were pulse-labeled with 10 M BrdUrd for 30 min at 37C in an
incubator. At the end of the pulse labeling, monolayer cultures were washed
with sterile, warm PBS to remove any unincorporated BrdUrd from the
medium. BrdUrd-labeled cells were either collected immediately at the end of
the BrdUrd-labeling or continued in culture in regular growth medium sup-
plemented with 10 M thymidine and cytidine (21, 23). Cells were harvested
at regular intervals by trypsinization, washed once with PBS, and fixed in 70%
ethanol. Fixed cells were stored at 4C before analysis by flow cytometry.
Ethanol-fixed cells were washed with PBS and incubated with pepsin (0.4
mg/ml in 2 N HCl) for 30 min at room temperature. After neutralization with 0.1
M borate buffer, nuclei were isolated by centrifuging the samples at 200400 g
in a Beckman centrifuge set at 4C for 5 min. The pellet was washed once with
PBS and incubated with anti-BrdUrd antibody (1:20; Becton Dickinson Immuno-
cytometry Systems, San Jose, CA) for 1 h at room temperature. At the end of the
incubation, samples were diluted with 1 ml of PBS and centrifuged at 200
400 g. Nuclei were then incubated with FITC-conjugated goat antimouse IgG
for 1 h and were digested with RNase A (0.1 mg/ml) for 30 min at room
temperature. The nuclei were counterstained with 20 g/ml PI for 1 h, and stained
cells were analyzed on a FACS 440 flow cytometer. The FACS 440 flow
cytometer is equipped with a Coherent I90-5 UV laser operating at 300 mW and
at 488 nm excitation wavelengths. Red fluorescence from PI was detected through
a 640-nm long pass filter, and green fluorescence from FITC was detected through
a 525-nm band pass filter. Data from a minimum of 20,000 nuclei were acquired
in list mode and were processed using Cytomation software (Cytomation, Ft.
Collins, CO). The acquired data were displayed as dual-parameter PI versus
log-FITC histograms and three compartments (BrdUrd-positive S-phase cells,
BrdUrd-negative G
1
, and BrdUrd-negative G
2
phases) identified using Cytomation
software. The fraction of cells in G
1
and G
2
were used as a measurement of G
1
and
G
2
transit time. Transit through S phase was measured by determining relative
movement as described earlier by Begg et al. (24). Relative movement was
calculated from the mean DNA content of undivided BrdUrd-positive cell popu-
lation normalized to mean DNA content of G
1
and G
2
population. The fraction of
BrdUrd-positive cells that had divided was also calculated for measurements of the
transit of the cells through S phase, G
2
, and M.
For staining with PI only, cells were harvested by trypsinization and fixed
in 70% ethanol. Approximately 1 10
6
cells were incubated with 100 l of
RNase A for 30 min at room temperature followed by staining with PI (30
g/ml) for 60 min. Samples were analyzed by flow cytometry, and cell cycle
phase distribution was determined using CellQuest software (Becton Dickin-
son Immunocytometry Systems).
Immunoblotting. Total cellular proteins were separated by SDS-PAGE
and transferred to polyvinylidene difluoride membrane (BIORAD Labs.).
Blots were incubated with antibodies to cyclin D1 (G124326; PharMingen),
p27 (sc-1641; Santa Cruz Biotechnology), and Rb (G3245; PharMingen).
Immunoreactive polypeptide was visualized using horseradish peroxidase-
conjugated secondary antibodies and enhanced-chemiluminescence detection
reagents (Amersham Pharmacia Biotech.) following manufacturer-supplied
protocols. Blots were reprobed with antibodies to actin (sc-1615; Santa Cruz
Biotechnology) for comparison of results.
Data Analysis. Experiments were repeated two or three times, and data
were calculated as means, SDs, and SE. Statistical comparisons were carried
out using two-tailed Students t test and ANOVA. Ps were calculated with 95%
confidence intervals.
RESULTS
Redox-sensitive Progression from G
1
to S Phase in Exponen-
tially Growing Asynchronous MEFs. A flow cytometry-based
BrdUrd pulse-chase assay (Fig. 1) was used to monitor the transit of cells
through each phase of the cell cycle. Exponentially growing monolayer
cell cultures were pulse-labeled with BrdUrd, an analogue of thymidine,
for 30 min and were chased in regular growth medium containing
thymidine and cytidine. Thus, cells synthesizing DNA (S-phase cells,
BrdUrd positive; Fig. 1A, box 1) will incorporate BrdUrd during the
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labeling period, whereas both G
1
(Fig. 1A, box 2) and G
2
(Fig. 1A, box
3) phases will show BrdUrd-negative staining. Fig. 1B shows a repre-
sentative histogram 4 h after the BrdUrd-labeling. Two additional groups
of cells were identified: box 4 (Fig. 1B), BrdUrd-positive cells with G
1
DNA content that have completed cell division; and box 5 (Fig. 1B),
BrdUrd-negative G
1
cells entering S phase. Using this assay, we deter-
mined three parameters (23, 24) by measuring the progression of cells
through different phases of the cell cycle (see Fig. 1B):
Entry into BrdUrd-negative S phase

Mean fluorescence of cells in box 5


Mean fluorescence of cells in box 2
(A)
Transit through S phase
4

Mean fluorescence of cells in box 1


mean fluorescence of cells in box 2
Mean fluorescence of cells in box 3
mean fluorescence of cells in box 2
(B)
Transit through S phase, G
2
, and M

1
2 number of cells in box 4
Number of cells in box 1
1
2(number of cells in box 4)
(C)
The three parameters were calculated relative to the 0-h untreated
control.
Fig. 2 represents HPLC measurements of intracellular low-molec-
ular-weight thiol levels in cells cultured in the absence and presence
of NAC. Quantitation of individual thiols (Fig. 2B) was determined by
resolving standard solutions of NAC, GSH, GGC, and cysteine
derivatized with NPM and plotting standard curves. Using this
method, the amounts of intracellular GSH and cysteine in asynchro-
nously growing exponential cultures of MEFs were determined to be
3.3 0.9 (average SD) nmol/mg protein and 0.67 0.16 nmol/mg
protein, respectively (Fig. 2C). Intracellular soluble low-molecular-
weight thiol levels increased 6-fold after a 12-h treatment of expo-
nentially growing MEF cultures with 20 mM NAC (Fig. 2D). NAC-
induced increases in intracellular soluble low-molecular-weight thiol
levels were calculated to be GSH (11.6 2.1 nmol/mg protein),
cysteine (2.6 1.1 nmol/mg protein) and NAC (9.4 0.3 nmol/mg
protein). These results show that treatment of MEFs with NAC shifted
the intracellular redox state toward a more reducing environment.
In addition, intracellular redox state was also determined by flow
cytometric measurements of changes in the fluorescence of a prooxidant-
sensitive chemical DFH-DA. Asynchronously growing cultures of MEFs
were grown in medium containing 20 mM NAC and were harvested at 4-
and 12-h intervals for DFH-DA staining and flow cytometric analysis
(Fig. 3). The relative fold change in MFI, calculated from the geometric
4
Transit through S phase or Relative Movement [Begg et al. (24)].
Fig. 2. Treatment of MEFs with NAC increases intracellular pools of reduced thiols:
HPLC analysis of intracellular thiol levels in exponentially growing cell cultures of MEFs
(C) and 12 h after the addition of 20 mM NAC (D). Total cellular protein extracts were
derivatized with NPM and resolved using a 15-cm C18 Reliasil column. Quantitation of
individual thiols was determined by injecting standards containing known amounts of
NAC, GSH, GGC, and cysteine (Cyst, 50-pmol standards shown in B) and constructing
standard curves of peak area versus concentration of analytes. Peaks attributable to the
derivatizing reagent alone in the absence of the added thiols are shown in A.
Fig. 3. A decrease in prooxidant-sensitive DFH fluorescence after the exposure of
MEFs to NAC. Exponential MEF cultures were treated with or without 20 mM NAC (0,
4, and 12 h) and stained with a prooxidant-sensitive DFH-DA (A) and insensitive C369 (B)
probe. Stained cells were analyzed by flow cytometry, and MFI was calculated using
CellQuest software. Peak 1 in A, autofluorescence in cells without staining, peaks 2 and
3, fluorescence of cells cultured in absence (NAC) and presence ( NAC) of NAC for
12 h. Bottom panels, means and SDs of relative MFI calculated from three separate
experiments. Relative fold-change in MFI in each experiment was calculated relative to
0 h untreated control.
Fig. 1. Flow cytometric BrdUrd-pulse-chase assay for measurements of progression
through the cell cycle: contour plots of DNA content versus log BrdUrd-FITC histograms
of asynchronously growing MEFs and their subdivision into cell cycle compartments. A,
exponentially growing, asynchronous population of MEFs analyzed immediately after a
30-min pulse labeling with BrdUrd. B, BrdUrd pulse-labeled cells were followed in
culture for 4 h in BrdUrd-free regular growth medium before analysis. Five cell cycle
compartments were identified as follows: Box 1, BrdUrd-positive undivided S-phase cells;
Box 2, BrdUrd-negative G
1
cells; Box 3, BrdUrd-negative G
2
cells; Box 4, BrdUrd-
positive G
1
cells that have completed cell division; Box 5, BrdUrd-negative S-phase cells.
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mean fluorescence of cells grown in the presence of NAC, showed an
40% decrease after 12 h of NAC treatment compared with cells grown
in the absence of NAC (Fig. 3A; P 0.05). The decrease in DFH
fluorescence in NAC-treated cells was specific to NAC-induced changes
in intracellular redox state because staining with the oxidation-insensitive
C369 compound did not show any significant difference in MFI between
the cells grown in the absence and the cells grown in the presence of
NAC (Fig. 3B). Therefore, the NAC-induced decreases in the oxidation
of the DFH was representative of a shift in intracellular redox state
toward a more reducing environment and was not caused by changes in
uptake, ester cleavage, or efflux of the compound.
To determine whether changing the intracellular redox state toward a
more reducing environment affected progression through G
1
, S phase,
and G
2
M, a time course study was performed using the BrdUrd
pulse-chase assay. Asynchronously growing exponential cell cultures of
MEFs were pulse-labeled with BrdUrd and were chased in regular
growth medium for 12 h in the absence and presence of NAC. Cell cycle
phase distributions were analyzed using flow cytometric analysis. Results
presented in Fig. 4 show that increasing thiol pools during the first 9 h of
NAC exposure (5, 10, and 20 mM) accelerated entry into S phase by
11.5 h (Fig. 4A; P 0.05). However, progression through S phase,
measured as relative movement (Fig. 4B), was not affected by the
increase in the thiol pools. Similarly, progression through S phase and
G
2
M, measured as a fraction of BrdUrd-positive cells that have divided
(Fig. 4C), also showed no difference.
In contrast, continued treatment with NAC for 12 h resulted in a
significant inhibition of cells entering S phase during both the first and
second generations (Fig. 4, DG). In the untreated control group, S
phase cells at 12 h after BrdUrd labeling in generations 1 and 2 were
20 0.004% and 28 0.015%, respectively. However, the S-phase
fraction in NAC-treated cells decreased 23-fold to 7 0.02% in
generation 1, and 11 0.03% in generation 2 (Fig. 4, F and G). The
inhibition of entry into S phase was also apparent at 24 h of NAC
treatment (data not shown). These results show that increasing the
intracellular reduced-thiol pools with NAC did not affect progression
from late G
1
to S phase to G
2
M in generation 1. However, progres-
sion from early G
1
in generations 1 and 2 seemed to be inhibited.
These results suggest that the NAC-sensitive redox signaling event(s)
initiating progression from G
1
to S phase resides early in G
1
.
To assess whether NAC-induced inhibition in progression from G
1
to S phase is associated with cell death in MEFs, a clonogenic cell
survival assay was performed. Results from the experiment showed
that the concentrations of the NAC used in this study did not affect
cell survival (data not shown), suggesting that the NAC-induced cell
cycle arrest was not a result of cell death.
To determine whether the NAC-induced G
1
arrest could be attrib-
utable to its thiol antioxidant/radical scavenger properties or its ability
to increase cellular glutathione content, glutathione synthesis was
inhibited with 1 mM BSO 1 h before and during the 12-h NAC
treatment. Cell cycle phase distribution was analyzed by PI staining
and flow cytometry. Results from this experiment showed that more
than 75% of the cells reside in G
1
in the NAC-treated cells as well as
cells treated with both BSO and NAC (data not shown). These results
indicate that NAC-induced G
1
arrest is independent of the effect of
NAC on cellular glutathione levels. These results suggest that the
antioxidant properties of NAC rather than its effect on glutathione
levels were the mechanism regulating NAC-induced G
1
arrest. Such
Fig. 4. Treatment with NAC inhibits the progression of cells from G
1
to S phase. Exponentially growing asynchronous cultures of MEFs were pulse-labeled with BrdUrd and chased in
medium containing 0, 5, 10, and 20 mM NAC. Cells were harvested at the indicated times and cell cycle phase transit was analyzed by flow cytometric assay. A, transit through G
1
(Rel. X S

/G
1
)
was measured by monitoring the mean DNA content of cells in the BrdUrd-negative S phase (Box 5 in Fig. 1B) relative to the mean DNA content of cells in the BrdUrd-negative G
1
phase
(Box 2 in Fig. 1B). B, relative movement (RM) of BrdUrd-positive undivided cells was used as a measure of transit through S phase. C, fraction (Fr.G
1

) of BrdUrd-positive G
1
cells (Box
4, Fig. 1B) was calculated to measure transit through S phase and G
2
and M phases. D and E, contour plots of exponentially growing MEFs pulse-labeled with BrdUrd and harvested 12 h after
culturing in absence (D) and presence (E) of 20 mM NAC. F and G, S-phase distributions in generations 1 and 2 after 12 h of NAC exposure compared with control. The difference in G
1
-transit
kinetics in cells cultured in absence and presence of NAC (A) was statistically significant as determined by ANOVA (P 0.05).
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INTRACELLULAR REDOX AND CELL CYCLE
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an antioxidant property of NAC could be directed in the inhibition of
redox reactions occurring on cellular protein thiols.
Redox-sensitive Progression from G
1
to S Phase in Exponen-
tially Growing MEFs Is Independent of Cellular p53 Status. To
determine whether the redox-sensitive progression from G
1
to S phase
depends on p53 status, asynchronously growing exponential cultures
of MEFs [clone 12(1), wild type p53; and clone 10(1) containing
mutant p53] were treated with 120 mM NAC for 12 h and were
pulse-labeled with BrdUrd before harvest. Flow cytometric analysis of
cell cycle phase distribution showed that asynchronous cells in the
absence of NAC had a normal cell cycle distribution with
34 0.014% and 39 0.007% S-phase cells in the wild-type and
mutant p53 cells, respectively (Fig. 5A, upper panels, and B, open bar
graphs). However, both cell lines showed less than 5% S phase cells
after 12 h of 20-mM NAC treatment, which indicated a G
1
delay in
both cell lines (Fig. 5A, lower panels, and B). The fraction of G
1
in
both cell lines increased with 5 mM of NAC and became maximal
(0.70.8%) with 20 mM of NAC (Fig. 5B). The fraction of G
1
in
control 12(1) cells was 0.5 0.014 and increased to 0.7 0.02 and
0.79 0.007 after exposure to 5 and 20 mM NAC, respectively.
Similarly, the fraction of G
1
in control 10(1) cells were 0.45 0.005
and increased to 0.6 0.002 and 0.8 0.009 after incubation with 5
and 20 mM NAC, respectively. These results indicate that the NAC-
sensitive progression from G
1
to S phase in MEF cell cycle is
independent of cellular p53 status.
Prooxidant Production after Release from NAC-induced G
1
Block Is Associated with MEFs Entry into S Phase. To further
investigate the presence of a NAC-sensitive redox event(s) before S
phase entry, exponentially growing asynchronous cultures of MEFs
were incubated with 20 mM NAC for 12 h and were released from the
NAC-induced G
1
block by washing the monolayer with regular cell
culture medium without NAC. Cells were continued in culture in the
absence of NAC and were harvested at regular intervals for analysis
of glutathione and glutathione disulfide, NAC levels, cysteine levels
and DFH fluorescence measurements. Removal of NAC resulted in a
decrease in intracellular glutathione levels from 19.7 4 nmol/mg
protein at the time of release to 16.2 4 nmol/mg protein at 4 h and
8.9 3.1 nmol/mg protein at 8 h after release. The intracellular
cysteine levels decreased from 1.4 0.5 nmol/mg protein at the time
of release to approximately control levels of 0.4 0.2 nmol/mg
protein at 8 h after release from the NAC-induced G
1
block. The
percentage of GSSG was 3.4 0.5% at the time of release from the
NAC treatment, increased to 11.4 0.5% at 4 h (P 0.05) and
decreased to 10.3 0.8% at 8 h (P 0.05) after release from the
NAC-induced G
1
-arrest (Fig. 6B). The transient increase in percent-
age GSSG was also associated with an increase in DFH fluorescence.
The MFI at the end of the NAC treatment decreased 55 0.07%
compared with control (Fig. 6A, 0 h, P 0.05) and subsequently
increased to 78 0.04% of control at 4 h after release from the
NAC-induced G
1
block (Fig. 6A, 4 h). These results indicate a
transient increase in prooxidant production between 2 and 8 h after
cells were released from the NAC-induced G
1
block. After the tran-
sient increase in prooxidant production, 49 0.06% of the cells
entered S phase at 12 h after removal of NAC (Fig. 6, B and C). These
results support the earlier finding of redox-regulated event(s) during
the entry of G
1
cells into S phase (Figs. 4 and 5) and suggest the
presence of a prooxidant-stimulated event during progression from G
1
to S phase in MEF cultures after release from NAC-induced G
1
arrest.
Redox-regulated Event(s) during G
1
Correlates with Changes
in G
1
-Regulatory Protein Levels. To determine whether the redox-
regulated event(s) during G
1
impacts on the abundance of G
1
-regulatory
proteins, asynchronously growing exponential cultures of MEFs were
cultured in medium containing 20 mM NAC and were harvested at
various intervals for immunoblotting. In asynchronously growing control
Fig. 5. Redox-signaling during progression from G
1
to S phase is independent of
cellular p53 status. A, flow cytometric assay of cell cycle phase distribution in asynchro-
nously growing MEFs with wild-type [clone 12(1), wtp53, left panels] and mutant p53
[clone 10(1), mp53, right panels)] phenotypes cultured in absence (top panels) and
presence (bottom panels) of 20 mM NAC. Cells were pulse-labeled with BrdUrd at the end
of a 12-h NAC treatment and were harvested immediately after labeling for flow
cytometric analysis. B, distributions of cell cycle phases in the two cell lines after
treatment with the indicated doses (020 mM) of NAC. The fraction of G
1
in control and
20-mM-NAC-treated cells were as follows: in clone 12(1) wtp53 MEFs, 0.5 0.014
(mean SD) and 0.79 0.007, respectively; in clone 10(1) mp53 MEFs, 0.45 0.005
and 0.8 0.009, respectively.
Fig. 6. Prooxidant production after release from NAC-induced G
1
arrest is associated
with entry of MEFs into S phase. Exponential MEF cultures were treated with 20 mM
NAC for 12 h and washed with regular growth medium without NAC. Cells were
continued in culture in medium without NAC and were harvested at regular intervals for
DFH staining (A), thiol assays (B), and measurements of S-phase distribution (C). A,
relative fold change in MFI of DHF-stained cells at the time of release (0 h) and 4 h after
the removal of NAC was calculated relative to the untreated control. B, percentage GSSG
and fraction of S phase measured in cells at various intervals after the removal of NAC.
The changes in percentage of GSSG was statistically significant as determined by
two-tailed Students t test (P 0.05). C, representative PI versus log BrdUrd-FITC
histograms of cells at the end of the NAC treatment (0 h, left histogram), and 12 h after
removal of NAC (right histogram); arrowhead, position of S-phase cells.
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cells, cyclin D1 protein levels remained high (0 h, Lane 1, Fig. 7A).
However, cells grown in the presence of NAC showed approximately a
70% decrease in cyclin D1 protein levels between 6 and 12 h of NAC
treatment and remained low at 24 h (Lanes 24, Fig. 7A). The decrease
in cyclin D1 protein levels in NAC-treated cells at 12 and 24 h was
accompanied with an increase in the CDK inhibitor p27 protein levels.
Similarly, cyclin D1-dependent Rb phosphorylation, a critical regulatory
step in the release of the E2F transcription factor and initiation of S phase,
was also differentially regulated in NAC-treated cells. In exponentially
growing control cells, hyper- and hypophosphorylated Rb migrated as a
diffuse band (0 h, Lane 1, Fig. 7A). In contrast, a faster migrating
polypeptide band representative of the hypophosphorylated form of the
Rb protein was observed both at 12 and 24 h in cells grown in presence
of NAC (Lanes 3 and 4, Fig. 7A). The alterations in G
1
-regulatory
proteins in cells cultured in the presence of NAC correlated with the
inhibition of the prooxidant event in G
1
as well as the inhibition in
progression from G
1
to S phase (Figs. 3 and 4). These results further
support the idea that early G
1
-specific redox-regulated event(s) that are
inhibited by NAC might stimulate progression from G
1
to S phase in
MEF cell cycle.
In a separate experiment, NAC-treated cells were washed with
regular cell culture medium and continued in culture in the absence of
NAC. Cells were harvested at various intervals after removal of NAC,
and G
1
-regulatory proteins levels were analyzed by immunoblotting.
Results presented in Fig. 7B showed that decreases in the thiol pools
and transient increases in the oxidation of DFH and percentage of
GSSG were followed by an increase in cyclin D1 protein levels
between 0 and 10 h after release from the NAC treatment (Lanes 57,
Fig. 7B). In addition, the protein levels of the CDK inhibitor p27
decreased between 4 and 10 h after release from the NAC-induced G
1
block (Lanes 57). Consistent with these changes, a slower migrating
polypeptide band representing the hyperphosphorylated form of the
Rb protein was observed between 4 and 24 h after release from NAC
treatment (Lanes 78). The reversal of NAC-induced changes in
G
1
-regulatory protein levels accompanies the cells subsequent entry
into S phase (Fig. 6, B and C). These results clearly suggest that the
redox-regulated signaling event(s) during G
1
precedes entry into S
phase and could impact on the regulation of G
1
cell cycle proteins.
The NAC-induced Redox-sensitive Site Responsible for G
1
Ar-
rest Resides Early in G
1
. The NAC-induced redox-sensitive site in
progression from G
1
to S phase was further mapped in synchronized
MEFs by monitoring the G
0
to G
1
to S-phase transition after mito-
genic stimulation of serum-starved cells. Exponentially growing
MEFs were recruited into G
0
by serum starvation for 24 h and then
serum stimulated to reenter the cell cycle. Serum-stimulated cells
were cultured in the presence or absence of 20 mM NAC. Results
presented in Fig. 8 show that 15% of cells in the control group were
synthesizing DNA by 10 h after serum stimulation (Fig. 8A, Control).
In contrast, no cells had entered S phase in the NAC-treated group at
10 h after serum stimulation (Fig. 8B, () panel). In the control group,
the fraction of cells entering S phase increased to 38 0.02% and
84 0.03% at 12 and 14 h, respectively (Fig. 8, A and C). In contrast,
the majority of the cells (90%) in the NAC-treated group remained
in G
1
during the same time interval (Fig. 8, B and C). These results
Fig. 7. Redox-regulated signaling during G
1
impacts on G
1
-regulatory protein levels.
Immunoblot analysis of G
1
-cell-cycle-regulatory protein levels during the NAC treatment
(A) and after release from the NAC-induced G
1
arrest (B). Exponentially growing MEFs
were cultured in the absence (Lane 1) and presence (Lanes 24) of 20 mM NAC, and total
cellular proteins were isolated at the indicated times for immunoblotting. Lanes 58,
immunoblot analysis of G
1
-regulatory protein levels at the time of release from the
NAC-induced G
1
block (Lane 5), and at 4 (Lane 6), 10 (Lane 7), and 24 (Lane 8) h after
the removal of NAC. Rb and pRb, hypo- and hyperphosphorylated forms, respectively, of
the Rb protein. Actin protein levels were used for loading control.
Fig. 8. NAC-induced redox-sensitive site responsible for
G
1
arrest resides early in G
1
. Exponentially growing MEFs
were synchronized by culturing the cells in medium contain-
ing 0.1% serum and were serum-stimulated to reenter the cell
cycle in the absence (A) or presence (B) of NAC. Cells were
pulse-labeled with BrdUrd at regular intervals, and cell cycle
phase distribution was analyzed by flow cytometric assay. A,
representative contour plots of dual parameter PI versus log
BrdUrd-FITC histograms of synchronized cell cultures
grown in absence (Control) and presence (B, NAC) of 20
mM NAC; left panel (A), G
1
, S phase, and G
2
populations are
labeled. C. the fraction of cells in S phase at various hours
during the NAC treatment was calculated from the dual
parameter histograms. Each data point, the mean of three
independent experiments; error bars, SD.
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support the previous studies using asynchronous cell populations (Fig.
4) and map the NAC-sensitive redox-signaling site to early in G
1
phase.
The presence of a redox-signaling event early in G
1
was further
evaluated by DFH staining. Cells were synchronized by serum starvation
and serum stimulated to reenter the cell cycle. Serum-stimulated cells at
various time intervals were stained with DFH-DAfor 15 min and assayed
by flow cytometry to determine whether changes in prooxidant produc-
tion were occurring before entry into S phase. Results presented in Fig. 9,
A and D, show a transient 1.9 0.12 (mean and SD)-fold increase in
DFH fluorescence as measured by MFI of 20,000 cells at 4 h after serum
stimulation (Fig. 9A, peak 3) compared with the MFI of cells at the time
of serum stimulation (Fig. 9A, peak 2). Interestingly, the transient in-
crease in DFH fluorescence decreased to 1.4 0.12-fold at 6 h after
serum stimulation (Fig. 9A, peak 4). DFH fluorescence in serum-stimu-
lated cells was also analyzed by confocal microscopy (Fig. 9C). The
frequency of DFH-stained cells was low in cultures collected after 5 min
of serum stimulation and stained for 15 min (Fig. 9C, left panel). In
contrast, more than 95% of the cells harvested at 4 h after serum
stimulation, and stained for 15 min, showed DFH-positive cells (Fig. 9C,
right panel). Four to six h after this apparent change in DFH oxidation,
2040% cells entered S phase 810 h after serum stimulation (Fig.
9D). In contrast, serum-stimulated cells cultured in the presence of NAC
did not show any significant changes in DFH fluorescence at 46 h after
serumstimulation and demonstrated no significant increase in the fraction
of S-phase cells at 810 h poststimulation (Fig. 9, B and D). These results
show that the transient increase in prooxidant production (as determined
by MFI) after serum stimulation is an early event in G
0
to G
1
transition
that clearly precedes the entry of the cells into S phase. Furthermore,
treatment with a thiol antioxidant, NAC, which suppressed the apparent
increase in prooxidant production, inhibited entry into S phase after
serum stimulation. These results suggest a causal relationship between
the prooxidant production during G
1
and progression of the cells into S
phase.
DISCUSSION
Alterations in intracellular oxidation/reduction (redox) reactions are
believed to play a regulatory role in various cellular and molecular
events including modulation of protein activities, signal transduction,
cell death, and cell proliferation (9, 2533). Depletion of low-molec-
ular-weight thiols such as L-cysteine and glutathione has been shown
to suppress thymidine uptake in natural killer cells (30). Lipid perox-
ide-induced imbalance of GSH/GSSG in human colon cancer CaCo-2
cells is known to arrest cell proliferation in G
0
-G
1
(31). Intracellular
glutathione levels have also been reported to vary during the mouse
fibroblast cell cycle being maximal in mitotic cells (33). Although
these previous studies provide some evidence for a mechanistic link
between intracellular redox reactions and cell proliferation, the direct
role of intracellular redox state during progression through specific
phases of the cell cycle as well as the specific cell-cycle-regulatory
processes that may be redox regulated are not completely understood.
In the present study, a membrane-permeate aminothiol, NAC, was
used to modulate intracellular redox state and to determine whether such
a manipulation affects progression through specific phases of the cell
cycle. NAC is a well-known thiol antioxidant as well as a precursor to
intracellular glutathione synthesis via its ability to augment cysteine
pools. Glutathione is the major low-molecular-weight intracellular thiol
that acts in a redox-buffering capacity and in general is used as an
indicator of changes in intracellular redox state. Treatment of exponential
MEFs with NAC resulted in an increase in intracellular glutathione and
cysteine levels, indicating a shift in intracellular redox state toward a
more reducing environment. NAC-induced changes in intracellular redox
state toward a more reducing environment were also supported by a
decrease in relative MFI of a prooxidant-sensitive compound, DFH-DA.
The shift toward a more reducing environment did not affect the transit of
cells through S phase, G
2
, and M. In fact, cells from mid- to late G
1
appeared to accelerate entry into S phase in the first generation (Fig. 4A).
However, cells after the completion of division were unable to progress
into S phase and showed an early G
1
arrest in the second generation (Fig.
4, DG). These results clearly indicate that an oxidation event early in G
1
may be a critical regulatory step in the progression of cells from G
1
to S
phase.
The mapping of the redox-signaling event(s) to early in G
1
was
further verified in synchronized G
0
cells that were serum stimulated to
reenter the cell cycle. Detection of a transient increase in DFH
fluorescence in serum-stimulated cells at 4 h poststimulation further
Fig. 9. Transient increase in prooxidant production during
G
0
-G
1
precedes entry into S phase. MEFs, synchronized by
serum starvation, were serum stimulated to reenter the cell
cycle and were collected at various intervals for the meas-
urement of prooxidant production by DFH staining and S-
phase entry by flow cytometry. DFH fluorescence at 0 h
(peak 2), 4 h (peak 3), and 6 h (peak 4) after serum stimu-
lation in control (A) and in serum-stimulated cells cultured in
the presence of NAC (B). Peak 1, fluorescence from un-
stained cells. C, confocal microscopy images of DFH fluo-
rescence of serum-stimulated cells at 5 min and at 4 h after
the serum stimulation and at 15 min of DFH staining. D,
relative fold change in MFI of synchronized cells that were
serum stimulated to reenter the cell cycle in the absence ()
and presence (F) of NAC. Relative fold changes in MFI were
calculated relative to control, and statistical significance was
determined using ANOVA (P 0.05). The P for the 4-h time
point was calculated to be less than 0.05. Cells in replicate
dishes were pulse-labeled with BrdUrd, and cell cycle phase
distribution was assayed by flow cytometry. Fraction of S
phase calculated from the dual parameter PI versus log
BrdUrd-FITC histograms: f, cells that were serum stimu-
lated in the presence of NAC; , serum-stimulated control
cells. Error bars (D), mean SD.
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indicated that the redox-sensitive signaling resides early in G
1
(Fig.
9). Inhibiting this redox signaling with NAC inhibited entry into S
phase. The presence of a redox-signaling event before S phase entry
was also observed in cells released from the NAC-induced G
1
arrest.
A transient increase in DFH fluorescence and the percentage of GSSG
was observed between 2 and 8 h after the removal of NAC, and,
subsequently, more than 50% cells entered S phase at 12 h after
release from the NAC-induced G
1
arrest (Fig. 6). These results sup-
port the hypothesis that a transient increase in intracellular prooxidant
levels early in G
1
initiates progression from G
1
to S phase and suggest
that such a redox-signaling event is cyclic in nature in the MEF cell
cycle (Fig. 10). Although a transient increase in prooxidant levels
would initiate progression from G
1
to S phase, antioxidants would
inhibit such a progression. Although the identity of the prooxidant(s)
was not investigated in this study, it is possible that ROS (i.e.,
superoxide and hydrogen peroxide), generated during mitochondrial
oxidative phosphorylation, and ATP production could act as a signal-
ing mechanism early in G
1
in preparation for entry of the cells into S
phase. Under normal conditions of cell growth, cellular antioxidant
defense mechanisms, including enzymatic scavenging systems (super-
oxide dismutases, catalase, glutathione peroxidases, thioredoxin per-
oxidases) and thiol-reducing buffers (small protein- and nonprotein
thiols), would neutralize the transient increase in prooxidant(s) levels
providing a redox balance and continuation of the progression of cells
from G
1
to S phase. In contrast, failure to maintain such a redox
balance would halt progression and, depending on the severity of the
imbalance, could lead to oxidative stress with deleterious effects. In
fact, earlier studies have reported fluctuations in the levels of antiox-
idant enzymes in proliferating cells compared with cells in the qui-
escent phase (32, 33). Increased levels of superoxide dismutase en-
zyme in G
1
and variations in intracellular thiol levels during cell
division (32, 33) support the idea of a regulatory role of an intracel-
lular redox state during progression through the cell cycle.
In many ways such a redox-sensitive regulation in G
1
resembles the
restriction point (34). Whereas growth factors withdrawal after the
restriction point would not affect transit through the remainder of G
1
, S
phase, G
2
, and M phases, withdrawal of growth factors before the
restriction point halts progression from G
1
to S phase. Similarly, manip-
ulating the intracellular redox state toward a more reducing environment
in cells in which the prooxidant event had already occurred would not be
expected to affect cells transit through the remainder of G
1
, S phase, G
2
,
and M phases of the cell cycle (Fig. 10). In contrast, such a manipulation
before the prooxidant event would inhibit progression into S phase.
Interestingly, many of the growth factors (e.g., platelet-derived growth
factor) are known to generate ROS that are believed to be participating in
cell growth (7). Additional mapping experiments are needed to determine
whether the timing of the redox signaling event and the restriction point
overlap. Furthermore, the periodicity of the redox signaling during the
cell cycle (Fig. 10) also supports the idea of a redox cycle within the
mammalian cell cycle that could link the activity of crucial metabolic
processes early in G
1
to the activation of cell-cycle-regulatory proteins
necessary for entry into S phase. Such a redox control in G
1
could
function as an intracellular redox-status-sensitive cell cycle checkpoint
pathway. Perhaps loss of such an intracellular redox-status-sensitive cell
cycle checkpoint would result in uncontrolled cell growth. In fact a recent
study reported a preferential sensitivity of thiol antioxidants toward
transformed and tumor-derived human cells (35). Additional study is
necessary to determine whether this differential response could be caused
by an alteration in the redox-sensitive cell cycle checkpoint pathway.
The mechanismby which NACaffects progression fromG
1
to S phase
is unclear. Because simultaneous treatment of MEFs with BSO and NAC
did not reverse the NAC-induced G
1
arrest both in this study and in
hepatic stellate cells (36), these results suggest that the NAC-mediated
increase in glutathione levels per se does not cause the G
1
arrest. These
results, however, do not rule out the possible role of other intracellular
low-molecular-weight thiols (i.e., cysteine, thioredoxin, glutaredoxin) in
NAC-induced G
1
arrest. Indeed cells released from the NAC-induced G
1
arrest showed a gradual decrease in cysteine levels, which was accom-
panied by a transient increase in percentage of GSSG and MFI before
entry into S phase. Furthermore, because the DFHfluorescence decreased
after NAC treatment (Figs. 3 and 6), and DFH has been shown to be
sensitive to intracellular hydroperoxides, our results indicated that the
NAC-induced cell cycle arrest in MEFs was not caused by auto-oxidation
of NAC (37) to form greater quantities of hydroperoxides. Therefore, it is
possible that the NAC-induced G
1
arrest in MEFs could be attributable to
the antioxidant properties of NAC. Although, such a possibility was ruled
out in hepatic stellate cells (36), it was not clear whether transient
fluctuations in peroxide level after the release from the NAC-induced
G
1
-arrest did occur under those experimental systems. Moreover, the
addition of peroxide to cells that are arrested in G
1
might complicate
interpretation of results because of peroxide-mediated toxicity. Our re-
sults (Figs. 6 and 9) suggest that the redox event early in G
1
is transient
in nature and is required for entry into S phase.
The antioxidant property of NAC could be directly inhibiting redox
reactions on cellular protein thiols present in the G
1
-S phase cell-cycle-
regulatory proteins (Fig. 10). Thus, target proteins modulated by NAC
might contain redox-sensitive cysteine residues that could participate in a
thiol-disulfide intra- or intermolecular reaction. Although critical cysteine
residues in transcription factors (e.g., activator protein and nuclear factor
B) are known to participate in regulating redox-sensitivity of transcrip-
tion factor DNA-binding activity, such a thiol-disulfide reaction is yet to
be clearly identified in G
1
-cell-cycle-regulatory proteins. However, a
recent study has reported a decrease in Cdc25C protein levels after
hydrogen peroxide exposure (38). Cdc25 family of dual specific phos-
phatase regulates activation of CDK activity by removing inhibitory
phosphate groups in CDKs. Whereas Cdc25Cacts on the activation of the
mitotic cyclin/CDK kinase complexes, Cdc25A activates the G
1
-cyclin/
CDK complexes. The authors in this study have shown that double
mutations of cysteines at positions 330 and 377 of Cdc25C phosphatase
resulted in resistance of the redox modulation as well as inhibited its
degradation (38). In the present study, we did not observe any significant
changes in Cdc25A protein levels during the NAC-induced G
1
arrest
Fig. 10. A schematic illustration of a redox cycle within the mammalian cell cycle.
Prooxidants would initiate progression from G
1
to S phase, whereas antioxidants would
inhibit these processes. Once the redox event is initiated, the cells would complete the
present cell cycle and repeat the process in G
1
of the next cell cycle. Target proteins
(cyclin D1, p27, Rb, and so forth), modulated by NAC, may react via the cysteine residues
in these proteins that could participate in a thiol-disulfide intra- or intermolecular reaction.
CKI, CDK inhibitor; E2F, transcription factor; P, phosphate.
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(data not shown). This difference in results could be because, although
hydrogen peroxide exposure caused oxidative stress in the previous
report, subtle changes in intracellular redox state after mitogenic stimu-
lation (our study) may not result in oxidative stress. In fact, the fold
induction in MFI of oxidation-sensitive probe is much lower (24-fold;
Figs. 6 and 9) in nonoxidative stress conditions compared with oxidative
stress conditions (79-fold increase; data not shown). Our results do not
rule out the possibility of intra- and/or intermolecular disulfide reactions
in the regulation of Cdc25Aphosphatase activity during progression from
G
1
to S phase.
NAC-induced inhibition in the redox signaling resulted in de-
creased cyclin D1 protein levels, increased p27 protein levels, and
hypophosphorylation of Rb. In contrast, reestablishment of the redox-
signaling event by release from NAC led to decreases in p27 protein
levels, increases in cyclin D1 protein levels, and an increase in Rb
phosphorylation with subsequent entry into S phase. Whereas addi-
tional studies are needed to understand the precise molecular mech-
anisms associated with redox signaling early in G
1
, it is possible that
one such mechanism could include alterations in the redox status of
redox-sensitive thiol-disulfide reactions at critical cysteine residues in
cyclin D1, p27, and Rb proteins. In fact the B domain of the Rb
protein is a site critical for binding to the LXCXE motif found in the
D-type cyclin (39). The Rb protein has 15 cysteine residues, and three
of these cysteines are located within the B domain (GeneBank acces-
sion no. M33647 J02994). It is not clear whether cysteine in the
LXCXE motif of cyclin D1 forms an intermolecular disulfide linkage
with the cysteines in the Rb B-domain. There are also two cysteine
residues within the cyclin A binding domain of p27 (40). Although
any possible role of these cysteines in redox regulation of G
1
to S
phase is speculative at present, it is reasonable to suggest that thiol-
redox reactions could act as sulfhydryl switches that reversibly
modulate cell-cycle-regulatory protein activity.
In summary, results from this study indicate the presence of a
redox-signaling event early in G
1
, which is required for entry into S
phase. Although shifting of the intracellular redox state toward a more
reducing environment selectively inhibited the entry of early G
1
cells
into S phase, a shift toward an oxidizing environment seemed to
stimulate entry into S phase. The periodicity of such a redox cycle
within the mammalian cell cycle may provide a mechanistic link
between metabolic processes early in G
1
and the activation of G
1
-
regulatory proteins in preparation for the entry of cells into S phase.
ACKNOWLEDGMENTS
We thank Justin Fishbaugh for assisting with the flow cytometric assays.
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