(Received for publication, June 6, 1995; and in revised form, August 17, 1995)
From the
Incubating human cells in diethylmaleate (DEM) depletes the
intracellular pool of reduced glutathione (GSH) and increases the
concentration of oxidative free radicals. We found that DEM-induced
oxidative stress reduced the ability of p53 to bind its consensus
recognition sequence and to activate transcription of a p53-specific
reporter gene. Nevertheless, DEM treatment induced expression of
WAF1/CIP1 but not GADD45 mRNA. The fact that N-acetylcysteine,
a precursor of GSH that blocks oxidative stress, prevented WAF1/CIP1
induction by DEM suggests that WAF1/CIP1 induction probably was a
consequence of the ability of DEM to reduce intracellular GSH levels.
DEM induced WAF1/CIP1 expression in Saos-2 and T98G cells, both of
which lack functional p53 protein. DEM treatment did not produce an
increase in membrane-associated protein kinase C, but ERK2, a
mitogen-activated protein kinase, was phosphorylated in a manner
consistent with ERK2 activation. DEM treatment also produced a
dose-dependent delay in cell cycle progression, which at low
concentrations (0.25 mM) consisted of a G/M arrest
and at higher concentrations (1 mM) also involved G
and S phase delays. Our results indicate that oxidative stress
induces WAF1/CIP1 expression and arrests cell cycle progression through
a mechanism that is independent of p53. This mechanism may provide for
cell cycle checkpoint control under conditions that inactivate p53.
The accumulation of oxidative damage is believed to be an
important cause of aging and may contribute to the increased incidence
of cancer in older individuals(1) . Reactive oxygen radicals
are capable of damaging many cellular components including
DNA(2, 3) . A wide variety of DNA damages result in
the activation of mechanisms that arrest cell cycle progression at
specific checkpoints, presumably to allow time for the damage to be
repaired. Activation of the G checkpoint mechanism by DNA
damage requires the function of the p53 tumor suppressor protein, which
transiently accumulates in cells after exposure to several agents that
damage DNA (4, 5) . p53 is a transcription factor that
binds to specific DNA sequence elements and activates transcription;
among the genes whose transcription is activated by p53 are WAF1/CIP1
and GADD45(6, 7, 8) . p53 also suppresses
transcription from other genes that do not have p53-specific binding
elements(9, 10) . WAF1/CIP1 encodes a potent 21-kDa
(p21) inhibitor of cyclin-dependent kinase activities that are required
for progression from the G
into the S phase of the cell
cycle(11, 12) . In vitro experiments have
shown that p53 is sensitive to oxidation and that the oxidized form of
p53 is unable to bind its cognate DNA
cis-element(13, 14) .
If the p53 protein is
similarly sensitive to oxidation in vivo, then inactivation
would render it incapable of protecting a cell from the DNA damages
provoked by oxidizing radicals. To address this possibility, we asked
whether p53 DNA binding and transactivation are affected by treatment
with diethylmaleate (DEM), ()an agent that increases the
intracellular concentration of free radicals by depleting the cellular
store of reduced glutathione (GSH).
Figure 1:
Effects of DEM on
the DNA binding efficiency of p53. COS-2 and Hep3B cells transfected to
induce the expression of wild-type p53 protein were incubated without
or with 1 mM DEM for 3 h before harvesting. Nuclear extracts
were examined by electrophoretic mobility shift assay for the ability
to bind a double-stranded, P-labeled oligonucleotide
containing the RGC sequence. Lane a,
P-labeled
RGC-containing oligonucleotide alone; lane b, RGC-containing
oligonucleotide plus nuclear extract from mock transfected COS-2 cells; lane c, nuclear extract from COS-2 cells transfected with
pCMV-p53 human p53 cDNA; lane d, as in lane c but
preincubated with a 20-fold molar excess of cold RGC oligonucleotide; lane e, as in lane c but preincubated with a 20-fold
molar excess of an unrelated oligonucleotide (Sp1); lane f,
nuclear extract from COS-2 cells transfected with pCMV-p53 and exposed
to 1 mM DEM; lane g, nuclear extract from Hep3B cells
transfected with pCMV-p53; lane h, nuclear extract from Hep3B
cells transfected with pCMV-p53 and exposed to 1 mM DEM. The arrow designates the shifted complex that is
p53-specific.
To determine if treatment of cells with DEM also impaired the ability of wild-type p53 to activate transcription in vivo, we examined the effect of DEM treatment on the transcription of a reporter gene whose transcription was dependent on wild-type p53 function (Fig. 2). Saos-2 cells, a human cell line that does not express an endogenous p53, and COS-2 cells were co-transfected with two plasmids: pCMV-p53 to provide wild-type p53 and PG13-luciferase, a reporter plasmid in which luciferase expression is driven by 13 direct repeats of the p53-binding sequence 5`-CCTGCCTGGACTTGCCTGG-3` placed upstream of a basal SV40 early promoter (see ``Materials and Methods''). Transfection with both plasmids resulted in high levels of luciferase expression, whereas after transfection with the PG13-luciferase reporter alone, very little luciferase was produced in Saos-2 cells, and expression in COS-2 cells also was modest. In cells co-transfected with both plasmids, luciferase activity was significantly decreased by exposing cells for 6 h beginning 24 h after transfection to either 0.5 mM DEM or 1 mM DEM. Expression from two p53-independent promoters, pSV2-CAT and RSV-luciferase, was not affected by DEM treatment (Fig. 2); thus, DEM does not act as a general inhibitor of transcription, nor is it an inhibitor of luciferase activity. We conclude that oxidative stress impairs the ability of p53 to function as a transcriptional activator in vivo in a dose-dependent fashion. Together with studies showing that exposing p53 to oxidizing conditions in vitro impairs its sequence-specific DNA binding ability(13, 14) , this experiment and the gel shift experiments described above suggest oxidative stress may block the ability of p53 to activate transcription in vivo by inactivating its ability to bind specific DNA sequences.
Figure 2: Effects of DEM on p53-mediated transactivation. COS-2 and Saos-2 cells were co-transfected with the PG13-luciferase or pRSV-luciferase reporter plasmids with pCMV-p53 and 24 h later were treated with 0, 0.5, or 1 mM DEM for 6 h. Luciferase activity is reported as the percentage of activity in mock treated COS-2 or Saos-2 cells co-transfected with both plasmids (see ``Materials and Methods''). The results represent the mean of three independent experiments; the error bars show one standard deviation from the mean. Black bars, COS-2 cells; hatched bars, Saos-2 cells.
Figure 3:
Effects of DEM on WAF1/CIP1 and GADD45
mRNA expression. Total RNA from HeLa cells left untreated (N)
or treated (DEM) with 1 mM DEM for 3 h was analyzed
by the Northern blot method. The hybridization probes were P-labeled human WAF1/CIP1 and GADD45 cDNAs. A,
the right panel shows a photograph of an ethidium-stained gel
to confirm equivalent RNA loading. B, effect of N-acetylcysteine on WAF1/C1P1
expression.
Figure 4:
Effects of DEM on WAF1/CIP1 and GADD45
mRNA expression in cells lacking functional p53 activity. Total RNA
from Saos-2 (lanes a-d) and T98G (lanes
a`-d`) cells exposed to different concentrations of DEM for
3 h was analyzed by the Northern blot method. The hybridization probes
were P-labeled WAF1/CIP1 and GADD45 cDNAs. a and a`, untreated cells; b and b`, 0.25 mM DEM; c and c`, 0.5 mM DEM; d and d`, 1 mM DEM. The lower panel shows
a photograph of the ethidium-stained gel to confirm equivalent RNA
loading.
Figure 5: Induction of WAF1/CIP1 protein expression by DEM. T98G cells were exposed to increasing concentrations of DEM for 6 h, and extracts were analyzed by the Western blot method for WAF1/CIP1 expression. a, 0 mM; b, 0.25 mM DEM; c, 0.5 mM DEM; d, 1 mM DEM.
Figure 6: Effect of DEM Treatment on protein kinase C activity. Saos-2 (A) and HeLa (B) cells were exposed to 20 ng/ml TPA or 1 mM DEM for 2 h, then harvested, and fractionated as described under ``Materials and Methods'' to provide a membrane preparation (white bars) and a cytosolic fraction (hatched bars). Protein kinase C activity was assayed with the Life Technologies, Inc. assay kit (61) .
Growth factors and DNA damage-inducing agents activate the MAP kinase cascade(32, 33, 34) , which, in turn, activates the transcription of specific genes through phosphorylation of immediate early transcription factors including c-Jun. Activation of the MAP kinase ERK2 is accomplished by its phosphorylation by a MAP kinase kinase, and this phosphorylation decreases the mobility of the ERK2 polypeptide during SDS-polyacrylamide gel electrophoresis(32) . Thus, ERK2 activation can be accessed by monitoring ERK2 mobility by Western blot analysis. Fig. 7shows that exposing HeLa cells to 1 mM DEM produced a significant increase in the phosphorylated (active) form of ERK2. The increase in mobility was similar to that induced by TPA; furthermore, the change was prevented by the pretreatment of cells with N-acetylcysteine. We conclude that the DEM-induced reduction in GSH concentration activates the MAP kinase cascade in a manner similar to the way it is activated after exposing cells to DNA damaging agents such as UV light.
Figure 7: Effect of DEM Treatment on ERK2 activity. HeLa cells were exposed to TPA, DEM, NAC, or NAC and DEM, and the cell extracts were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting for phosphorylated (upper band) and unphosphorylated ERK2 as described under ``Materials and Methods.'' N, untreated cells; TPA, cells treated with 20 ng TPA for 30 min; DEM, cells treated with 1 mM DEM for 30 min; NAC, cells treated with 30 mM NAC for 2 h; NAC + DEM, cells treated with 30 mM NAC for 2 h and then with 1 mM DEM for 30 min.
Figure 8:
Effects
of DEM on cell cycle progression of T98G cells. T98G cells were exposed
to DEM (0.25-1 mM) for 12 h in the presence (A)
or the absence (B) of nocodazole (0.4 µg/ml). Cell cycle
progression was assessed by flow cytometry as described under
``Materials and Methods.'' Values shown are the percentage of
cells in the G, S, or G
/M phases from a
representative experiment in which at least 15,000 cells were analyzed
for each point.
The p21 protein product of WAF1/CIP1 is a potent inhibitor of
cyclin-dependent kinase activities that are required for progression
from the G phase of the cell cycle into S
phase(11, 12) . Transcription of the p21 gene is
induced following DNA damage as a consequence of the accumulation of
wild-type p53, and the elevated concentration of p21 protein mediates,
at least in part, p53-induced growth arrest in response to DNA
damage(7, 12) . These recent findings have evoked
considerable interest because the p53 gene is mutated in many human
tumors, and alternate methods of inducing p21 expression could provide
approaches for controlling tumor growth and influencing the survival of
tumor cells that have lost functional p53.
In this paper we show that p21 is rapidly induced in response to DEM, an agent that increases intracellular oxidative damage by decreasing the intracellular pool of glutathione. p21 induction by DEM was prevented by pretreatment with N-acetylcysteine, a precursor of reduced glutathione, indicating that the DEM-mediated induction of p21 is a consequence of oxidative damage. Induction of p21 by DEM was independent of p53 because it occurs in cell lines that do not express p53 protein (e.g. Saos-2) and that express mutant p53 proteins (e.g. T98G) that cannot activate transcription in response to DNA damage or overexpression.
The induction of p21 following DEM exposure was
associated with arrest of T98G cells in the G and S phases
of the cell cycle (Fig. 8). Arrest at these points in the cell
cycle corresponds well with the proposed actions of p21 on components
of the cell cycle machinery. Arrest in G
phase is probably
related to inhibition of cyclin/Cdk kinase activity, which is required
for progression of cells through the G
restriction
point(35) . Arrest in the S phase following DEM treatment
probably reflects the ability of p21 to inhibit DNA synthesis by
binding to and blocking PCNA function(37) . Indeed,
overexpression of the PCNA binding domain of p21 is sufficient to
inhibit DNA synthesis when transfected into mammalian
cells(38) .
Recently, p21 expression was shown to be induced
in several cell lines by agents that cause terminal differentiation
through a p53-independent mechanism. p21 mRNA expression was elevated
after exposure to TPA, butyrate, trans-retinoic acid, and
MeSO in HL-60, K562, and U937 cells, human hematopoietic or
hepatoma-derived lines that express no or mutant
p53(29, 30, 31) . p21 expression also is
induced by serum, PDGF, FGF, EGF, TPA, and okadaic acid in quiescent
fibroblastic cell lines derived from transgenic mice lacking a
functional p53 gene(26, 29) . The mechanism by which
these agents induce p21 expression is unknown, but induction is
insensitive to cyclohexamide, suggesting that it is a consequence of
the activation of pre-existing transcription factors. The ability of
mitogenic growth factors and TPA to induce p21 expression suggests the
involvement of protein kinase C in modulating its expression.
Consistent with this suggestion, TPA-resistant variants of HL-60 cells
exhibit altered responses in immediate early gene expression including
c-fos and c-jun (39) , and these cells display an altered
protein kinase C isozyme profile, are incapable of translocating
protein kinase C from the cytosol to the membrane fraction, and exhibit
altered protein phosphorylation after TPA
treatment(39, 40) . Further support for the
involvement of protein kinase C comes from the observation that
adriamycin, which activates protein kinase C(41) , is a potent
inducer of p21 in cells containing wild-type p53(35) , and, at
higher concentrations, induces WAF1/CIP1 transcription in p53 null
cells(26) . In contrast to these results, exposing HeLa and
Saos-2 cells to DEM had no effect on the amount or the distribution of
protein kinase C activity (Fig. 6), indicating that the major
forms of protein kinase C are not activated by DEM. Instead, DEM
treatment produced an increase in the phosphorylated form of ERK2 (Fig. 7), a member of the mitogen-activated kinase family;
furthermore, ERK2 phosphorylation, which is required for its
activation, was prevented by pretreating cells with NAC.
Recently,
it has become clear that DNA damage-inducing agents, including
oxidizing agents, may activate immediate early gene expression through
pathways that do not directly involve DNA damage (32, 33, 42, 43) . Treatment of
cells with UV light, ionizing radiation, and HO
all cause a rapid increase in membrane-bound tyrosine kinase
activity, protein kinase C activity, and MAP kinase
activity(32, 44, 45) . These kinases activate
several transcription factors including c-jun, EGR1, and
NF-[kappa]B, and transcription factor and MAP kinase
activation were prevented by pretreating cells with
NAC(46, 47, 48, 49) , suggesting
that in each case activation is mediated through oxidative damage.
Although other mechanisms could account for the accumulation of
WAF1/CIP1 mRNA in DEM treated cells, our finding that DEM treatment
activates ERK2 and that activation is prevented by pretreatment with
NAC is consistent with the hypothesis that oxidative damage induces
transcription of the WAF1/CIP1 gene through the activation of a
transcription factor(s) other than p53. A serum response element that
overlaps the proximal p53 recognition element in the murine WAF1/CIP1
promoter recently was identified(50) . However, serum-mediated
activation of MAP kinase is not prevented by NAC(49) ; thus,
the DEM-induced MAP kinase activation seems to take place downstream of
the membrane receptor growth signal initiating machinery. A possibility
to be explored is the DEM-induced inhibition of the MAP kinase
phosphatase, which is inhibited by oxidants as are other tyrosine
phosphatases(51) . This possibility is consistent with the
observed induction of MAP kinase (52) and of WAF1 (26) as a consequence of okadaic acid treatment.
Other important findings of this study are that the DEM treatment was incapable of inducing transcription of the endogenous GADD45 gene and that the transcription of an exogenous p53-regulated reporter gene was impaired in cells treated with DEM. Furthermore, the p53 from DEM-treated cells exhibited a decreased ability to bind a consensus recognition sequence. Previous studies had shown that the specific DNA binding ability of p53 is sensitive to oxidation in vitro(13, 14) ; this effect can be traced to the oxidation of cysteines that are critical for DNA binding(55) . A similar sensitivity to oxidation is exhibited by transcription factor Sp1 (56) and by the glucocorticoid receptor(20) . Our study extends these observations and suggests that because of its sensitivity to oxidation, p53 may be incapable of activating a response to oxidative stress in vivo. Oxidants generated through normal aerobic metabolism and by inflammatory reactions appear to be a major cause of damage to DNA and other cellular components. Exposure to oxidative stress can cause neoplastic transformation as well as a loss of proliferative capacity that resembles cellular senescence, and it was suggested that the accumulation of oxidative damage with age might lead to senescence through a p53-mediated activation of p21(57) . Recent studies indicate, however, that p53 levels are elevated only modestly in senescent cells (58) . Our results with those of others suggest that p21 induction in response to oxidative stress is mediated through another mechanism, possibly involving activation of the MAP kinase cascade.
Interestingly, low
concentrations of DEM also induced a G arrest in T98G
cells. Arrest at this junction in the cell cycle does not appear to
require p21 (36, 59) and consistent with these earlier
studies we found no appreciable induction of p21 with low doses of DEM (Fig. 5). G
arrest following DNA damage has been
linked to a failure to remove inhibitory phosphorylations from the
ATP-binding domain of the Cdc2 kinase(59, 60) ,
suggesting that DEM could block the G
/M transition by a
mechanism independent of p21 induction. Elucidation of the G
arrest mechanism evoked by DEM could provide a useful insight
into additional mechanisms governing cell cycle progression during
periods of oxidative stress.