From the Center for Clinical Sciences Research, Department of Radiation Oncology, Stanford University, Stanford, California 94303-5152
Received for publication, December 4, 2002
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ABSTRACT |
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The ATR kinase phosphorylates both
p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of
less than 0.02%). In contrast to ATR, loss of ATM does not affect the
phosphorylation of these or other targets in response to hypoxia.
However, hypoxia within tumors is often transient and is inevitably
followed by reoxygenation. We hypothesized that ATR activity is induced
under hypoxic conditions because of growth arrest and ATM activity
increases in response to the oxidative stress of reoxygenation. Using
the comet assay to detect DNA damage, we find that reoxygenation
induced significant amounts of DNA damage. Two ATR/ATM targets, p53
serine 15 and histone H2AX, were both phosphorylated in response to
hypoxia in an ATR-dependent manner. These phosphorylations
were then maintained in response to reoxygenation-induced DNA damage in
an ATM-dependent manner. The reoxygenation-induced p53
serine 15 phosphorylation was inhibited by the addition of
N-acetyl-L-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen
species. Taken together these data implicate both ATR and ATM as
critical roles in the response of hypoxia and reperfusion in solid tumors.
It is has been hypothesized that tumor hypoxia plays a critical
role in the malignant progression of solid tumors and represents a poor
prognostic indicator for tumor control. The mammalian response to
hypoxia is complex and varies at different oxygen tensions (1). These
include the induction of hypoxia-responsive genes by the transcription
factors early growth response-1 (EGR1), AP-1, and
hypoxia-inducible factor
(HIF)1 (2, 3). HIF is a
heterodimer that consists of Hif1 Histone H2AX has recently been identified as having a
phosphatidylinositol 3-kinase motif (SQ) at serine 139 and is a target for both ATM and ATR (17, 18). Histone H2AX is phosphorylated ( In this report we have further investigated the phosphorylation of p53
serine 15 by ATR in response to hypoxia, and we have shown that like
p53, histone H2AX is also phosphorylated in an ATR-dependent manner in response to hypoxia. Most
importantly, co-localization of p53 serine 15 and Cell Lines and Transfections--
The RKO and H1299 cell lines
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum. GM1526 and GM0536 were maintained in RPMI
supplemented with 15% fetal bovine serum. Both GM1526 (ATM+/+, p53+/+)
and GM536 (ATM Hypoxia Treatment--
The cells were plated in glass dishes and
treatment carried out in a hypoxia chamber (<0.2% O2)
(Sheldon Corp., Cornelius, OR). Unless otherwise noted, the cells were
harvested in the hypoxia chamber using PBS, lysis buffer, and trypsin
where needed that had been equilibrated with the chamber.
N-Acetyl-L-cysteine (NAC) was used at a final
concentration of 30 mM for the duration of the hypoxia treatment.
Immunoblotting--
For immunoblotting the cells were lysed in 9 M urea, 75 mM Tris-HCl, pH 7.5, and 0.15 M Immunofluorescence--
The cells were grown and treated
on 8-well chamber slides. After treatment the cells were fixed in
methanol at Comet Assay--
Comet assays were carried out as
previously described (28, 29). In brief, 1-3 × 104
RKO cells were prepared as a single cell suspension in
magnesium/calcium-free PBS. Three volumes of a 1% low melt agarose,
2% Me2SO solution were added to the cells followed by
mixing. The mixture was placed onto a microscope slide and allowed to
set on a cold surface. When completely set the slide was immersed in
lysis buffer for 1 h (0.03 M NaOH, 1 M
NaCl, 0.1% N-lauroylsarcosine) at room temperature. The
propidium iodide-stained cells (comets) were visualized using a Nikon
Optiphot microscope attached to an Ikegami 4612 CCD camera and
fluorescence image analysis system. Using specially designed software,
the tail moment of each cells was calculated as the product of the
percentage of DNA in the tail multiplied by the length of the comet
tail. 200 comets were scored for each treatment.
The Roles of ATR, ATM, and DNA-PKcs in the Induction of
Histone H2AX has been shown to form foci in response to both damage-
and hydroxyurea-mediated replication arrest (17), although the exact
nature of these foci is as yet unclear. Fig.
2 shows that p53 Ser15 and
Hypoxia is not the only physiological stress present within a tumor.
Tumor cells are also exposed to low pH, increased pressure, lack of
glucose or serum, and osmotic shock (32, 33). It is therefore possible
that these factors have a co-operative role in inducing the
phosphorylation of ATR targets like p53 serine 15 or histone H2AX. To
investigate this possibility, we grew RKO cells in conditions designed
to mimic those present in tumors. The Western blots shown in Fig.
4 demonstrate that neither p53 serine 15 nor H2AX were substantially phosphorylated in response to any of
the stresses tested unless significant apoptosis was also induced. As
previously mentioned, H2AX has been shown to be phosphorylated during
the early phases of apoptosis (21). Interestingly, treatment with
sodium chloride, to mimic osmotic shock, induced one of the highest
levels of apoptosis and yet very little p53 serine 15 or H2AX
phosphorylation.
Reoxygenation Induces DNA Damage and ATM-dependent
Phosphorylation of p53 Serine 15--
We have previously demonstrated
that hypoxia does not induce any detectable DNA damage using the
alkaline comet assay (1). We hypothesized, however, that reoxygenation
would induce significant amounts of damage. This is physiologically
relevant because hypoxia within tumors is often transient, resulting
from transient blockage of poorly developed vasculature or increased
interstitial pressure (34). It is hypothesized that tumor cells are
exposed to continuous cycles of hypoxia and reoxygenation. To
assess the amount of damage associated with reoxygenation, RKO cells
were treated with hypoxia for 16 h and then harvested after
different times after reoxygenation. The relative amounts of DNA damage
were then assessed by comet assay. As a reference point, the cells were
also exposed to 8 Gy of ionizing radiation (Fig.
5). When cells were harvested entirely in
normoxic conditions and hence fully reoxygenated, a significant amount
of DNA damage occurred. Reoxygenation induced DNA damage approximately
equivalent to treating cells with 4-5 Gy of ionizing radiation. We
proposed that this level of damage would subsequently lead to increased
or sustained phosphorylation of proteins that contain ATM recognition
sites. To investigate this, we again made use of the GM1526 (ATM Many previous reports have demonstrated that These data provide the first in vivo evidence for a role of
ATR in tumors. We have demonstrated that ATR does not phosphorylate target molecules like p53 and H2AX until oxygen levels are low enough
to induce a complete stop in DNA synthesis, i.e. replication arrest. Significantly, the finding that both p53 and H2AX are phosphorylated in vivo in the hypoxic regions of tumors
indicates that these extreme levels of hypoxia do indeed occur in
tumors. We have eliminated many other tumor-physiologically relevant
stresses as having a co-operative role in the induction of these
phosphorylation events.
We have previously showed that hypoxia did not induce any DNA
damage detectable by the comet assay. Here, in contrast, we found that
cells taken from severe hypoxia to normoxia had a significant amount of
comet-detectable damage. What was particularly striking about these
findings was the rapid kinetics of DNA damage induction in response to
reoxygenation. We hypothesized that this damage might lead to
subsequent ATM activation and also may have been mediated
by the formation of ROS. We have presented evidence that both of these
hypotheses are indeed valid. In the absence of ATM, the level of p53
phosphorylated at serine 15 slowly decreased, whereas it was maintained
in the presence of ATM for at least 60 min. The addition of the ROS
scavenger NAC inhibited the reoxygenation-induced phosphorylation of
p53 at serine 15 but had no effect on the hypoxia-induced phosphorylation of p53 at serine 15.
We propose that both ATR and ATM have roles to play in tumor
progression but that these roles may be distinct. Fig.
7 shows our proposed model. Initially ATR
responds to replication arrest induced at severe levels of hypoxia
followed by an ATM response to the DNA damage induced when these areas
become reoxygenated. Our data suggest that the activation of one
phosphatidylinositol 3-kinase over another is based on the presence or
absence of DNA damage. We have been unable to detect DNA damage in
cells that have undergone a replication arrest in response to hypoxia
and therefore conclude that damage is not required for the
relocalization of ATR to nuclear foci. We do not exclude the
possibility that damage nondetectable by Comet assay does occur but
would argue that if present it must be at a very low level and
certainly not comparable with the significant damage seen upon
reoxygenation. As previously mentioned, ATM has a much more defined
role in the response to DNA damage, and it is perhaps therefore not
surprising that it has a role to play in the reoxygenation response.
Further insight will come from the identification of ATM- or
ATR-specific targets. Neither Chk 2 or residue 20 of p53 are
phosphorylated in response to extreme
hypoxia,2 but both may be
induced in response to reoxygenation in an ATM-dependent manner. The identification of these damage-specific targets will allow
us to further elucidate the ATM damage response and the ATR replication
response. Hypoxia may well be the ideal if not only model to study this
further because it is unique in the induction of replication arrest
without detectable concomitant damage.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits that bind to the
sequence 5'-RCGTG-3' (4). Under normoxic conditions HIF-1
is rapidly
degraded when it is bound to the von Hippel-Lindau tumor
suppressor protein that targets it for ubiquitination (5, 6). Under
normoxic conditions HIF-1
is hydroxylated at a conserved proline
residue, number 564, by a family of highly conserved 4-prolyl
hydroxylases (7, 8). Under hypoxic conditions the activity of this
oxygen-sensitive hydroxylase is repressed, and HIF-1
is unable to
complex with VHL, which in turn leads to its increase in stability. In
contrast to HIF-1, which is stabilized at 2% oxygen, the protein
product of the p53 tumor suppressor gene also accumulates in hypoxic
cells but requires more stringent hypoxic conditions (1).
Hypoxia-induced p53 activates a cytochrome c-mediated
apoptotic pathway that can act as a selective pressure for the
expansion of tumor cells with either inactive or mutant p53 (9, 10).
The mechanism by which p53 accumulates under hypoxic conditions has
been attributed to both a decrease in mdm2 levels
(11) and increased translation. mdm2 acts a negative
regulator of p53 by targeting p53 for degradation by the
ubiquitin-proteosome pathway. mdm2 is a p53-responsive gene
that acts to keep p53 in check through a feedback loop. However, under
hypoxic conditions, p53 does not seem to transactivate mdm2, and the decrease in mdm2 protein in hypoxic cells is due to
degradation of the protein. It has been reported that p53 protein that
accumulates under hypoxic conditions is transcriptionally impaired and
is unable to induce p21, Bax, or mdm2. This loss of
transactivation potential has been attributed in part to the lack of
association between p53 and the co-activator p300 in hypoxic extracts.
Instead, p53 that accumulates under hypoxic conditions associates with the co-repressor molecule mSin3a, suggesting that can act as a trans-repressor (12). Both p300 and mSin3a have been shown to bind to
the amino terminus of p53 (13). The amino terminus of p53 is also the
site of mdm2 binding and specific stress-induced phosphorylations. We have shown previously that p53 is phosphorylated by ATR in response to hypoxia at residue serine 15 (1). Those studies
also indicated that without this phosphorylation event, p53
accumulation in response to hypoxia was diminished. Several explanations exist for this finding, including the potential masking of
a nuclear export signal by this phosphorylation and the subsequent accumulation of p53 in the nuclear compartment (14). In contrast to
ATR, we found that ATM had no role to play in the phosphorylation of
p53 at serine 15 in response to hypoxia because of a lack of DNA damage
under hypoxic conditions. The link between the ATM kinase and
phosphorylation of p53 in response to DNA damage-inducing stresses is
well established and may well be responsible for suppressing tumor
expansion (15, 16).
H2AX)
in response to genotoxic agents, UV, hydroxyurea-mediated replication
arrest, and at physiological sites of recombination during class
switching (19). During the initiation of DNA fragmentation that occurs
during apoptosis, H2AX is also phosphorylated. This phosphorylation occurs with the appearance of high molecular weight DNA
fragments but before the externalization of phosphatidylserine or the
appearance of internucleosomal DNA fragments (20, 21). Recent studies
have provided some insight into the function of H2AX. Homozygous null
H2AX knockout mice are born with the expected frequency but are
radiation-sensitive, growth-retarded, immune-deficient, and infertile
(22, 23). Elegant foci studies have shown that H2AX null cells had
impaired recruitment of Nbs 1, 53bp1, and Brca1 to the sites of DNA
damage. However, the formation of Rad51 foci in response to DNA damage
was only slightly affected, if at all, in the absence of H2AX (22, 23).
These findings indicate that histone H2AX is needed for genome
stability and efficient DNA repair and in particular the assembly of
specific DNA repair proteins to DNA damage-induced nuclear foci. We
have used
H2AX as a marker of both ATR and ATM activity that can be
readily assayed.
H2AX within
hypoxic regions of tumors indicates that oxygen concentrations within
tumors are low enough to activate ATR. These data indicate that ATR and
ATR-mediated signaling have a physiologically significant role to play
in tumor development. We have also demonstrated that in contrast to
hypoxia, reoxygenation induces a significant amount of DNA damage that can be detected by comet assays. This damage leads to
ATM-dependent phosphorylation of p53 serine 15 and other
ATM targets. Because of the poorly developed vasculature of tumors, the
tumor microenvironment represents a dynamic situation where tumor cells
are exposed to both hypoxia and reoxygenation (24). These studies
suggest that ATR is the principal kinase for the phosphorylation of p53
in response to hypoxia and that ATM is activated by DNA damage during reoxygenation. Thus, ATR and ATM are activated by different stimuli in
the tumor microenvironment.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
isolated from an ataxia-telangiectasia
patient) are Epstein-Barr virus immortalized lymphoblastoid cell lines.
The parental HCT116 cell line and the ATR
/flox derivative
were maintained in McCoy's medium supplemented with 10% fetal calf
serum. Prior to infection with adenovirus-cre, 5 × 105 cells were plated were plated on a 10-cm dish. The
cells were then infected for 48 h with fresh medium, and virus was
added after 24 h. The medium was replaced before hypoxia treatment
(25).
-mercaptoethanol and sonicated briefly. 50 µg
of protein were electrophoresed on 10% polyacrylamide
Tris-Tricine gels. Primary antibodies used were p53 DO-1,
anti-phospho-H2AX (serine 139; Upstate Biotechnology number 07-164),
anti-phospho-p53 (serine 15; Cell Signaling Technology number 9284),
GAPDH (TRK5G4-6C5; Research Diagnostics), Hif1
(H72320;
Transduction Laboratories), and protein G-purified
-ATR (26).
20 °C for 20 min, then rehydrated in PBS, and blocked
in 20% heat-inactivated normal goat serum, 0.1% bovine serum albumin,
0.1% sodium azide in PBS for 30 min. The cells were incubated for
1.5 h at 37 °C in a humidified box with anti-phospho-H2AX
(serine 139) at a final concentration of 1 µg/ml in blocking
solution. The samples were washed with PBS, 0.2% Tween 20 and then incubated with a fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG antibody (Sigma) for 1 h. After washing, the
samples were counter-stained with Hoescht (10 µg/ml) and mounted with
coverslips and an aqueous anti-fade mounting reagent (Vectashield,
Vector Laboratories). Snap frozen tumors were sectioned (14 µm),
fixed, and stained for EF5 as previously described (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
H2AX by
Hypoxia--
Previous work has indicated that H2AX is a
substrate for the phosphatidylinositol 3-kinase family (18). To
investigate the phosphorylation of H2AX by hypoxia, RKO cells were
grown at both 0.02 and 2% oxygen and harvested at different times over
a 24-h period (Fig. 1A). We
examined the changes in HIF-1
and p53 protein levels and p53 serine
15 and
H2AX. HIF-1
accumulated at both 0.02 and 2% oxygen. In
contrast p53 only accumulated at 0.02% oxygen. Histone H2AX was
clearly phosphorylated in response to extreme hypoxia but remained
unaffected at 2% oxygen. These finding suggest that like p53 histone
H2AX might be phosphorylated by a stress-activated phosphatidylinositol
3-kinase. To investigate the kinase responsible for this
phosphorylation, we made use of both ATM and DNA-PKcs matched cell
lines and a conditional ATR knockout cell line (25, 30). Using isogenic
ATM-deficient and reconstituted cell lines (GM1526 and GM0536), we
found that both p53 serine 15 and H2AX are phosphorylated in response
to hypoxia in an ATM-independent manner (Fig. 1B). We also
found this to be true in spontaneously transformed mouse embryonic
fibroblasts from ATM
/
animals. Thus, a deficiency in ATM had little
affect on H2AX phosphorylation. Similarly, cells that lack DNA-PKcs
exhibited similar levels of H2AX phosphorylation as parental wild-type
cells (Fig. 1C). Taken together, these results indicate that
hypoxia does not activate the DNA damage response kinases ATM or
DNA-PKcs. Therefore, we hypothesized that ATR was responsible for
histone H2AX and p53 phosphorylation in response to hypoxia. HCT116
ATR
/flox cells were treated with adenovirus-cre to knock
out the remaining ATR allele from this cell line; the cells were then
exposed to hypoxia and harvested at varying times for protein analysis
(Fig. 1D). The level of ATR in the HCT116
ATR
/flox was significantly reduced when compared with the
parental HCT116 cell line. The level of ATR protein was reduced
further by infection with adenovirus-cre. Treatment with adenovirus-cre
reduced hypoxia-dependent induction of both p53 serine 15 and
H2AX, indicating that the ATR kinase was activated under hypoxic
conditions. Although the levels of ATR in the parental cell line and
the ATR-heterozygous version were significantly different, both p53
serine 15 and H2AX were phosphorylated to the same extent in response
to hypoxia. This implies that the level of ATR in the
heterozygote is sufficient to phosphorylate its targets and that
it is only when ATR levels are reduced below this that differences in
activity can be detected. Interestingly a more significant decrease in
p53 serine 15 was observed when ATR was reduced compared with the
reduction seen in
H2AX levels. Fig. 1D shows a decrease
in p53 serine 15 of 50%, whereas the decrease in
H2AX signal is
reduced by 30%. Similar results were also seen when the ATR dominant
negative was transfected into the RKO cell line and exposed to hypoxia
(data not shown). Using this approach we would not expect to reduce the
phosphorylation of either H2AX or p53 by 100% because there is some
residual ATR protein and hence ATR activity. However, the consistent
discrepancy we see between
H2AX and serine 15 of p53 leads us to
conclude that phosphorylation of these targets by ATR is not
equivalent. A possible explanation for this finding is the different
cellular localizations of p53 and H2AX;
H2AX has been shown to
co-localize with BRCA1, PCNA, and 53BP1 in nuclear foci and therefore
could be phosphorylated first by the remaining ATR in the
adenovirus-cre cells (18). It is also possible that other kinases, for
example ATM, take over the role of ATR in its absence and that these
may act on H2AX preferentially or with faster kinetics.
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Fig. 1.
p53 serine 15 and histone H2AX serine 139 are
phosphorylated in response to oxygen concentrations of 0.02% in an
ATM-independent and ATR-dependent manner.
A, RKO cells were grown at either 0.02% or 2% oxygen and
harvested at the times shown. The total levels of Hif1 , p53, p53
Ser15,
H2AX, and GAPDH are shown. B, GM1526
ATM
/
and GM0536 ATM+/+ were exposed to hypoxia (0.02%
O2) and were harvested at the times indicated. Both p53
serine 15 and histone H2AX serine 139 were phosphorylated in response
to hypoxia in the absence of ATM, indicating that this is an
ATM-independent pathway. C, histone H2AX is also
phosphorylated in spontaneously transformed mouse embryonic
fibroblasts in a DNA-PKcs and ATM-independent manner. D,
HCT116 and HCT116 ATR
/flox were infected with
adenovirus-cre for 48 h and then exposed to hypoxia for 8 h.
The levels of ATR, p53 serine 15,
H2AX, and GAPDH are shown. The
levels for uninfected cells are also shown. Using the cre recombinase,
the level of ATR in the HCT116 ATR
/flox decreased. With
this reduced level of ATR, there were significant decreases in the
hypoxia-dependent induction of p53 serine 15 and
H2AX.
The p53 ser15 was decreased by 50%, and the
H2AX was
decreased by 30%. The relative decreases in signal were determined
using a PhosphorImager. wt, wild type.
H2AX is also present in
foci in hypoxia-treated RKO cells. The lack of detectable DNA damage
associated with hypoxia indicates that these foci are not solely formed
at sites of damage (1). Previous studies have suggested that ATR is
activated by replication arrest under hypoxic conditions. As would
be expected for an ATR-mediated event,
H2AX foci were not detected
in all cells.
H2AX foci were seen in ~28% of cells. In contrast,
under hypoxic conditions when cells were treated with the DNA damaging
agent adriamycin, foci were seen in all cells.
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Fig. 2.
H2AX exists as discreet nuclear
foci in cells treated with hypoxia. RKO cells were grown on glass
microscope slides before being exposed to 0.02% oxygen for 16 h.
The cells were then fixed under hypoxic conditions, and
immunofluorescence for
H2AX was carried out. As a positive control
cells were also treated with a DNA-damaging agent, adriamycin (0.25 µg/ml for 16 h).
H2AX appeared as nuclear foci in ~28% of
hypoxia treated cells. The cells treated with adriamycin had nuclear
H2AX foci in all of the cells.
H2AX Staining Co-localizes with
EF5-positive Regions in Tumors--
To determine whether these
in vitro findings with
H2AX or p53 serine 15 occurred in
hypoxic tumor regions, we grew tumors in mice from the H1299 cell line
expressing tetracycline-inducible p53, which we have previously
described (12). Approximately 107 cells were implanted into
the flanks of nude mice and were allowed to grow until they reached a
diameter of 1 cm. Doxycycline and sucrose were added to the drinking
water of half the mice, whereas the remaining half received sucrose
alone before being sacrificed 24 h later. Prior to sacrifice, the
mice were injected with EF5 to allow the visualization of hypoxic tumor
regions (27, 31). Fig. 3 shows tumor
sections stained for total p53 in mice that had been fed doxycycline
(lower panel) or sucrose alone (upper panel).
There was a clear induction of p53 after the addition of doxycycline.
This was also verified by northern blotting and persisted while
doxycycline was given to the mice (up to 6 days; data not shown).
Generating p53-positive tumors this way results in higher levels of p53
than would normally be seen, which ease detection in vivo.
Fig. 3C shows the staining of serial sections for
H2AX,
p53 Ser15, and EF5. We chose to use serial sections for
these studies because the EF5 stain can bleed through to the
fluorescein isothiocyanate channel. The overlays of both
H2AX and
p53 ser15 with EF5 are shown. Despite the use of serial
sections, there was a clear overlap between staining for EF5 and
H2AX as well as EF5 and p53 serine 15. Perhaps more striking
is the overlap between p53 serine 15 and
H2AX. It should be noted
that not all of the cells within the EF5-positive region stained for
p53 serine 15 or
H2AX, consistent with the S
phase-dependent nature of ATR activation. It was not
possible to visualize individual foci within stained cells. However,
both p53 serine 15 and
H2AX did appear to be nuclear in
localization. These data provide direct evidence that oxygen levels
within tumors can reach levels low enough to induce a replication
arrest and hence ATR, supporting an important role for ATR in tumor
development.
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Fig. 3.
p53 serine 15 and
H2AX co-localize with EF5-positive regions of human
tumors grown in mice. p53 was induced in tumors by giving mice
doxycycline in their drinking water, A shows the level of
p53 in a mouse given plain water, and B shows the induction
of p53 in the tumor of a mouse given doxycycline. p53 was induced
throughout the tumor. C, serial sections from a
p53-expressing tumor were stained for
H2AX, p53 Ser15,
and EF5. There was a clear overlap between the stained regions, shown
in the bottom panels. The overlap is not exact because the
sections are serial. To demonstrate the co-localization of the
H2AX
and p53 serine 15 signals, the p53 Ser15 signal has been
altered to red.
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Fig. 4.
RKO cells treated with stresses associated
with tumor physiology show that hypoxia is the primary effector of both
p53 Ser15 and H2AX. RKO cells
were exposed to the following stresses: low pH, increased pressure,
glucose starvation, serum starvation, osmotic shock, and hypoxia.
Western blots were then carried out and the levels of p53 serine 15,
H2AX, and GAPDH are shown. The levels of apoptosis induced in
response to treatment were determined based on cell morphology, and
where not shown, the level of apoptosis was equal to that seen in
untreated cells.
/
)
and GM0536 (ATM+/+) cell lines. The cells were exposed to hypoxia for
16 h before being harvested after various periods of reoxygenation
(Fig. 6A). The p53 protein was
clearly phosphorylated at serine 15 in response to hypoxia in ATM
wild-type and ATM-deficient cell lines. However, as we predicted, the
levels of phosphorylation were sustained in the ATM+/+ cell line,
whereas they begin to decrease after 10 min of reoxygenation in the ATM
nulls. This suggests that in response to the DNA damage that occurs
upon reoxygenation, ATM becomes activated and is responsible for
maintaining phosphorylation of targets such as p53 serine 15. Reoxygenation leads to the rapid production of ROS (reactive oxygen
species) mostly in the form of superoxide molecules. By pretreating
cells exposed to hypoxia with a chemical scavenger for ROS, we
hypothesized that cells would be protected from the DNA-damaging
effects of these molecules, and consequently reoxygenation-induced
phosphorylation of p53 serine 15 would be inhibited. RKO cells were
exposed to hypoxia in the presence or absence of NAC and then
reoxygenated (Fig. 6B). As was seen in the ATM wild-type
cells (GM0536), the levels of p53 serine 15 in RKOs remained high and
constant during the 35-min period after removal from hypoxia. However
NAC significantly reduced the level of p53 serine 15 during
reoxygenation. In the presence of NAC, the hypoxia-induced p53 serine
15 appears identical to that seen in the absence of NAC, indicating
that the production of ROS during hypoxia treatment either is minimal
or has no role in the phosphorylation of p53 at serine 15.
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Fig. 5.
Reoxygenation after exposure to extreme
hypoxia induces significant levels of DNA damage. RKO cells were
exposed to hypoxia for 16 h and were then harvested for comet
assays. The cells were either harvested under entirely hypoxic
conditions, partially in hypoxia or completely in normoxia. The cells
were also treated with 8 Gy of ionizing radiation
(IR).
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Fig. 6.
p53 is phosphorylated at residue serine 15 in
response to reoxygenation in an ATM-dependent manner.
A, GM0536 (ATM+/+) and GM1526 (ATM /
) cells were treated
with hypoxia for 16 h. The cells were then returned to normoxia
and harvested after the time periods shown. The levels of p53 serine 15 and GAPDH are shown. The cells not reoxygenated are also shown (time
0). B, RKO cells were exposed to hypoxia for 16 h
before reoxygenation in the absence or presence of NAC. Upon
reoxygenation the levels of p53 serine 15 remained high in those cells
not treated with NAC but diminished in the presence of NAC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
H2AX is a rapidly
induced marker of DNA damage; in some cases phosphorylation has been
reported within 10 min of genotoxic insult (35). In contrast,
phosphorylation of histone H2AX occurs with much slower kinetics in
response to hypoxia treatment. We have shown that this phosphorylation
is ATR-dependent. Our data suggest that ATR becomes active,
perhaps mediated by a change in cellular localization, in response to
extreme levels of hypoxia, which induce replication arrest (1). The
induction of this replication arrest is directly proportional to the
amount of oxygen present in the microenvironment. In accordance with
this observation, plating cells in glass dishes, which retain less
oxygen, can increase the kinetics of H2AX and p53 phosphorylation.
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Fig. 7.
Model for the roles of ATR and ATM in the
response to hypoxia and reoxygenation. See text for further
details.
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ACKNOWLEDGEMENTS |
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We are very grateful
to Drs. Baz Smith and Dawn Zinyk for technical assistance. We also
thank Drs. David Cortez and Stephen Elledge for the gift of the HCT116
ATR/flox cell line and the Adeno-cre, and Dr. David Chen
for the mouse embryonic fibroblast DNA-PKcs
/
and mouse
embryonic fibroblast ATM
/
cell lines.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA67166 and CA88480 (to A. J. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
650-723-7366; Fax: 650-723-7382; E-mail: giaccia@stanford.edu.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212360200
2 E. M. Hammond and A. J. Giaccia, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HIF, hypoxia-inducible factor; PBS, phosphate-buffered saline; NAC, N-acetyl-L-cysteine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ROS, reactive oxygen species; DNA-PKcs, DNA-dependent protein kinase catalytic subunit.
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