Article

ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair. EMBO J

Genome Stability Unit, Clare Laboratories, London Research Institute, Cancer Research UK, South Mimms, UK.
The EMBO Journal (Impact Factor: 10.43). 02/2011; 30(3):546-55. DOI: 10.1038/emboj.2010.330
Source: PubMed

ABSTRACT

Ataxia telangiectasia (A-T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A-T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A-T pathogenesis. Here, we show that ATM promotes an anti-oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose-6-phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti-oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM-dependent PPP stimulation increases nucleotide production and that G6PD-deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs.

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ATM activates the pentose phosphate pathway
promoting anti-oxidant defence and DNA repair
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Claudia Cosentino
1
, Domenico Grieco
2
and Vincenzo Costanzo
1,
*
1
Genome Stability Unit, Clare Laboratories, London Research Institute,
Cancer Research UK, South Mimms, UK and
2
CEINGE Biotecnologie
Avanzate and Faculty of Biotechnological Sciences DBPCM, University
of Naples ‘Federico II’, Naples, Italy
Ataxia telangiectasia (A-T) is a human disease caused by
ATM deficiency characterized among other symptoms by
radiosensitivity, cancer, sterility, immunodeficiency and
neurological defects. ATM controls several aspects of cell
cycle and promotes repair of double strand breaks (DSBs).
This probably accounts for most of A-T clinical manifesta-
tions. However, an impaired response to reactive oxygen
species (ROS) might also contribute to A-T pathogenesis.
Here, we show that ATM promotes an anti-oxidant response
by regulating the pentose phosphate pathway (PPP). ATM
activation induces glucose-6-phosphate dehydrogenase
(G6PD) activity, the limiting enzyme of the PPP respon-
sible for the production of NADPH, an essential anti-
oxidant cofactor. ATM promotes Hsp27 phosphorylation
and binding to G6PD, stimulating its activity. We also
show that ATM-dependent PPP stimulation increases nu-
cleotide production and that G6PD-deficient cells are im-
paired for DSB repair. These data suggest that ATM
protects cells from ROS accumulation by stimulating
NADPH production and promoting the synthesis of nucleo-
tides required for the repair of DSBs.
The EMBO Journal advance online publication, 14 December
2010; doi:10.1038/emboj.2010.330
Subject Categories: genome stability & dynamics; cellular
metabolism
Keywords: anti-oxidant; ATM; DNA damage
Introduction
A-T is characterized by increased susceptibility to cancer,
radiosensitivity, sterility, immunodeficiency and neurologi-
cal symptoms (McKinnon, 2004). Most A-T patients die of
recurrent pulmonary infection due to severe immuno-
deficiency or cancer development following chromosome
rearrangements. An early symptom of A-T is ataxia, which
is the lack of movement coordination and inability to
control body posture. Ataxia is caused by neurodegenera-
tion and, in particular, by death of the Purkinje cells (Humar
et al, 2001).
ATM regulates the response to double strand breaks
(DSBs) (Girard et al, 2000; Mirzayans et al, 2006) and the
absence of ATM impairs the repair of DSBs. This defect has
been linked to cancer, sterility, radiosensitivity and neurode-
generation (McKinnon, 2004). It has been suggested that
developmental abnormalities also contribute to the death of
cerebellar neurons. During development ATM is required to
induce apoptosis of cells containing excessive DNA damage
(Herzog et al, 1998). According to this model, ATM-deficient
cells accumulate DNA damage that impairs their function.
This phenomenon might be responsible for the neurodegen-
eration in A-T patients. ATM is also required to trigger DNA
damage response in differentiated neurons promoting cell
cycle re-entry upon genotoxic stimuli, a necessary step to
start the programmed cell death pathway in neurons
(Kruman et al, 2004; Biton et al, 2006). As a consequence
ATM-deficient cells are more resistant to the apoptosis in-
duced by genotoxic stress and accumulate more DNA damage
than normal cells.
However, ATM function is not limited to the response to
DSBs. ATM appears to be involved in multiple pathways
controlling several cellular functions such as the response
to oxidative stress (Shackelford et al, 2001; Ito et al, 2004,
2007), possibly acting as a sensor of reactive oxygen species
(ROS) (Rotman and Shiloh, 1997). Recent studies have docu-
mented the presence of high levels of oxidative damage in
A-T patients (Reichenbach et al, 2002; Russo et al, 2009),
confirming previous observations made in the mouse model
of A-T (Kamsler et al, 2001; Stern et al, 2002). These observa-
tions suggest that an impaired response to ROS in A-T cells
might influence neuronal survival. In particular, it has been
shown that cerebellum cells exhibit low levels of NADPH
(Stern et al, 2002), a major cofactor of anti-oxidant enzymes
such as glutathione reductase and cytochrome p450 reduc-
tase, which, together with superoxide dismutase and cata-
lase, are essential to maintain the cellular redox balance
(Kultz, 2005). Consistent with this, A-T lymphoblasts reduce
glutathione more slowly than normal cells after glutathione
depletion induced by oxidative stress (Meredith and Dodson,
1987), possibly because of the low levels of NADPH.
The main source of NADPH is the PPP, which converts
glucose-6-phosphate to ribose-5-phosphate, the sugar back-
bone of nucleotides. This pathway has an oxidative and non-
oxidative phase (Figure 1A). The oxidative phase converts
glucose-6-phosphate to ribulose-5-phosphate and reduces
NADP þ to NADPH through glucose-6-phosphate dehydro-
genase (G6PD) and 6-phosphogluconate dehydrogenase
(6PGD) (Jain et al, 2003). Similar to A-T cells, G6PD-deficient
Received: 11 August 2010; accepted: 23 November 2010
*Corresponding author. Genome Stability Unit, Clare Laboratories,
London Research Institute, Cancer Research UK, South Mimms EN63LD,
UK. Tel.: þ 44 170 762 5748; Fax: þ 44 170 762 5746;
E-mail: vincenzo.costanzo@cancer.org.uk
The EMBO Journal (2010), 1–10
|
&
2010 European Molecular Biology Organization
|
Some Rights Reserved 0261-4189/10
www.embojournal.org
& 2010 European Molecular Biology Organization The EMBO Journal
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cells are more sensitive to ionizing radiation (IR)-induced
apoptosis (Tuttle et al, 2000), suggesting that G6PD is in-
volved in response to DSBs.
Here, we investigate the link between ATM and the PPP.
We show that ATM stimulates the PPP by inducing G6PD
activity, which in turn promotes NADPH production and
nucleotide synthesis. Induction of G6PD activity requires
Hsp27, which is capable of directly stimulating G6PD.
The activation of G6PD likely promotes a reduced cellular
environment and provides sufficient amounts of nucleotide
precursors required to promote DSB repair.
Results
ATM promotes NADPH production
Considering that ATM null mice show reduced cerebellar
level of NADPH and this condition might contribute to the
impaired oxidative stress response (Stern et al, 2002), we
Glucose-6-phosphate
NADPHNADP+
GS-SG 2G-SH
Glutathione
reductase
Anti-oxidant
activity
Non-oxidative PPP
Ribose
Oxidative PPP
6-phospho-
gluconate
6-phospho-glucono-
δ-lactone
G6PD
Lactonase 6PGD
Ribulose-5-
phosphate
NADPHNADP+
A
GH
Minutes
Minutes
100
50
150
0
0 5 10 15
Prot G+DSBs
ctr
DSBs
Caffeine
ATMi
30
20
10
0
40
0
51015
Prot G+DSBs
2-5-ADP
2-5-ADP+DSBs
Fluorescence intensity
(a.u.)
Fluorescence intensity
(a.u.)
DB
Input
Mock
GDP6
50 kDa G6PD
C
Mock
G6PD
80
60
40
0
20
51015
Minutes
100
–20
Fluorescence intensity
(a.u.)
DSBs
Caffeine +
++
250 kDa
p-Ser1981
+
E
DSBs
ctr
100
50
150
0
0 5 10 15
Minutes
Fluorescence intensity
(a.u.)
F
2
–5
G ADP
G6PD
6PGD
48 kDa
75 kDa
25 kDa
37 kDa
DSBs
+
+
Prot
Figure 1 (A) Schematic representation of the pentose phosphate pathway. (B) Western blot with anti-Xenopus G6PD antibodies on Xenopus
egg extract, which was not depleted (input), mock depleted (mock) or G6PD depleted (DG6PD). (C) Kinetic readings of NADPH fluorescence in
mock (triangles) or G6PD depleted (squares) extracts. Fluorescent intensity is indicated in arbitrary units (a.u.). (D) Xenopus egg extract was
treated with 20 ng/ml of DSBs in the presence or absence of 5 mM caffeine or left untreated. The samples were then loaded on 10% SDS–PAGE
and analysed by western blot with anti-p-Ser1981 ATM. (E) Kinetic readings of NADPH fluorescence in untreated extract (ctr) or DSB-treated
extract (DSBs). (F) Xenopus egg extract was treated with or without 20 ng/ml DSBs for 10 min and then incubated with 2
0
-5
0
-ADP-sepharose
beads or protein G-sepharose as a control. An aliquot of the eluted protein was loaded on a 10% SDS–PAGE and the gel stained with Coumassie
blue. (G) A second aliquot was instead used to measure G6PD activity. (H) Xenopus egg extract was untreated (ctr), treated with 20 ng/ml DSBs
alone (DSBs) or in combination with 10 mM ATMi (ATMi) or 5 mM caffeine (Caffeine). The samples were then subjected to 2
0
-5
0
-ADP-sepharose
pull down. G6PD activity was measured on the proteins eluted from beads after the pull down. Graphs shown in this figure indicate average
values of three or more experiments. Error bars indicate s.d. A full-colour version of this figure is available at The EMBO Journal Online.
ATM activates the pentose phosphate pathway
C Cosentino et al
The EMBO Journal & 2010 European Molecular Biology Organization2
Page 2
investigated the possibility that ATM regulates NADPH levels
acting on the key enzyme of the PPP such as G6PD.
We choose Xenopus egg extract as model system to study
the effects of ATM on the PPP. One of the advantages of
Xenopus egg extract is that ATM activation can be rapidly and
specifically induced by adding fragments of double stranded
DNA. ATM activation can be prevented by incubating the
extract with inhibitors of ATM such as caffeine or the more
specific Ku 55933 (ATMi) (Costanzo et al, 2004).
NADPH can be easily detected in vitro by measuring its
fluorescence emission at 460 nm after supplementation of
egg extract with NADP and glucose-6-phosphate (G6P)
(Nutt et al, 2005). Xenopus egg extract has been shown to
be a good source of G6PD and NADPH (Nutt et al, 2005). To
test the specificity of the G6PD activity assay, we incubated
the extract with control or anti-G6PD antibodies to deplete
the enzyme. The western blot in Figure 1B shows that the
specific (DG6PD), but not the control antibody (mock),
efficiently depleted G6PD from the extract. Both the mock
and the depleted extracts were tested for G6PD activity
in vitro. Figure 1C shows that the level of G6PD activity
was severely reduced in the depleted extract, but not in
the mock depleted extract. These observations indicate that
G6PD activity can be reliably and specifically measured in
Xenopus egg extract.
To study the effects of ATM activation on G6PD activity egg
extract was supplemented with 20 ng/ml of DNA containing
DSBs. ATM activation was monitored by detection of the
phosphorylation status of serine 1981 (Costanzo et al, 2004)
(Figure 1D). ATM activation was prevented by incubating egg
extract with caffeine (Figure 1D) or ATMi. Strikingly, activa-
tion of ATM led to significant increase in G6PD activity as
indicated by the increased production of NADPH over time
(Figure 1E). These data suggest that ATM promotes NAPDH
production through activation of G6PD.
To dissect the mechanism leading to G6PD activation by
ATM and to verify its specificity we performed a pull down
from extracts that were untreated or supplemented with DSBs
using 2
0
-5
0
-ADP-sepharose beads. 2
0
-5
0
-ADP mimics NADP and
specifically interacts with NADP-binding proteins (Bernofsky,
1980; Hunt et al, 1983). 2
0
-5
0
-ADP specifically pulled-down
G6PD and other NADP-binding proteins, such as thioredoxin
and 6PGD as confirmed by mass spectrometry (Figure 1F and
data not shown). We then eluted proteins from the beads
and measured G6PD activity. G6PD activity was significantly
higher in the eluate derived from the extract treated with
DSBs compared to the untreated one (Figure 1G). No relevant
enzymatic activity was detected in the pull down with the
control beads. These experiments indicate that the increase in
G6PD activity induced by DSBs is retained after the pull down
and might be due to a stable modification of G6PD such as a
post-translational modification of the enzyme or the binding
of a factor that increases its activity.
We then verified whether ATM mediates the DSB induced
activation of G6PD. To this end the extract was pre-incubated
for 10 min with ATM inhibitors such as caffeine or ATMi.
G6PD was then isolated from the extract with the 2
0
-5
0
-ADP-
sepharose beads. Both the inhibitors suppressed the DSB-
mediated activation of G6PD (Figure 1H). Taken together
these data show that DSBs induce an increase of G6PD
activity and NADPH production in an ATM-mediated manner.
Taking into account that there is no transcription in Xenopus
egg extract (Garner and Costanzo, 2009) and that the protein
levels in the pull down from the treated and untreated
extracts are equal (Figure 1F), these data show that ATM
directly promotes a stable increase of G6PD activity through
post-translational mechanisms.
ATM promotes activation of G6PD through Hsp27
In order to investigate the mechanisms underlying the in-
crease of G6PD activity upon ATM activation we looked for
the presence of post-translational modifications on G6PD.
However, using mass spectrometry analysis we did not detect
any significant modification that could account for the in-
crease in the enzymatic activity (data not shown). Therefore,
we looked for proteins specifically enriched in the pull down
of G6PD after DSB treatment using mass spectrometry
(Figure 1F, data not shown). This approach led to the
identification of Hsp27 as factor potentially involved in the
increase of G6PD activity. Hsp27 has already been implicated
in the regulation of G6PD in a non-transcriptional manner
(Preville et al, 1999; Yan et al, 2002). In order to verify
whether Hsp27 affects G6PD activity we performed a pull
down with 2
0
-5
0
-ADP-sepharose beads from extract treated
with DSBs in the presence or absence of ATMi. Figure 2A
shows that Hsp27 co-precipitates with G6PD in the presence
of DNA damage. Inhibition of ATM prevented Hsp27 binding
to G6PD. These data were confirmed by the use of phleomy-
cin-treated nuclei, which were used as an alternative source
of DSBs. To confirm this finding in other model systems, we
investigated the association between Hsp27 and G6PD in
irradiated human fibroblast AG02603 by immunoprecipitat-
ing Hsp27 with a specific antibody. The upper panel in
Figure 2B shows that G6PD binds Hsp27 upon IR treatment.
We also performed the reverse experiment, which shows
that Hsp27 co-immunoprecipitates with G6PD upon IR ex-
posure and that the binding is suppressed by ATMi
(Figure 2C).
These results suggest that ATM activation stimulates the
association between G6PD and Hsp27. We then decided to
verify whether this association leads to a direct increase in
G6PD activity. To this end we incubated increasing amounts
(15–50 nM) of recombinant Hsp27 (rHsp27) with recombi-
nant G6PD and we measured G6PD enzymatic rate.
Strikingly, rHsp27 led to a direct increase in G6PD activity
(Figure 2D). It is worth noticing that these concentrations are
below the physiological level of Hsp27, which, in the cell
lines that we analysed is between 2 and 5 mg/mg (Bukach
et al, 2009; Supplementary Figure S2). Taken together these
results suggest that ATM induces a direct stimulation of G6PD
activity by promoting the binding of Hsp27 to G6PD.
The mechanism by which the affinity of Hsp27 for G6PD is
increased by ATM remains to be established. However, this
might involve post-translational modifications of Hsp27.
Hsp27 is phosphorylated following cellular stress by the
p38–MK2 kinase complex on serine 15, 78 and 82
(Kostenko and Moens, 2009). We did, in fact observe that
IR induces Hsp27 phosphorylation on serine 78: 1 h after
exposure to IR, phosphorylation levels of Hsp27 significantly
increased and this could be prevented by ATM inhibition
(Figure 2E and F).
It has been reported that the p38–MK2 pathway, which is
responsible for Hsp27 phosphorylation on serine 78, is
activated by ATM/ATR upon DNA damage (Raman et al,
ATM activates the pentose phosphate pathway
C Cosentino et al
& 2010 European Molecular Biology Organization The EMBO Journal 3
Page 3
2007; Reinhardt et al, 2010). Consistent with this we con-
firmed that Hsp27 phosphorylation upon IR can be inhibited
by p38 inhibitors (data not shown).
ATM-mediated activation of G6PD is conserved
in human cells
It has been previously shown that low doses of IR increase
the activity of anti-oxidant enzymes, G6PD included, con-
tributing to the rapid scavenging of the ROS produced upon
irradiation of human cells (Bravard et al, 1999). It is also well
known that G6PD has a major role in the protection of the cell
from apoptosis during oxidative stress (Tian et al, 1999; Pias
and Aw, 2002; Fico et al, 2004). We therefore decided to verify
whether activation of G6PD by ATM was conserved in human
cells. To this end, we irradiated normal human fibroblasts
and we monitored G6PD activity 10 min after irradiation.
Figure 3A shows that G6PD activity is significantly increased
following irradiation. Pre-treatment of the cells with 10 mM
ATMi prevented G6PD activation. Moreover, we compared
the increase in G6PD activity following IR treatment between
normal cells (GM00024 and GM00558) and cells derived from
A-T patients (GM03395 and GM03382). Both A-T cell lines
showed a weaker G6PD activation compared to the corre-
sponding controls (Figure 3B). To make sure that we were
measuring G6PD activity we verified the specificity of the
assay. In this case we pre-incubated normal cells (GM00558)
for 10 min with 100 mM DHEA, a known G6PD inhibitor (Tian
et al, 1998). Supplementary Figure S1 shows that this treat-
ment prevented G6PD activation, indicating that the assay is
specifically measuring G6PD activity in human cells.
These data indicate that the increase in G6PD activity
induced by ATM is conserved from Xenopus to human
cells. The extremely rapid induction of G6PD in Xenopus
egg extracts and human cells following DNA damage suggests
Hsp27
G6PD
+10 Gy + +
Input Hsp27ctr
IP
G6PD
ctr
phl
phl+ATMi
DSBs
DSBs+ATMi
ctr
phl
phl+ATMi
DSBs
DSBs+ATMi
Input
Hsp27
Pull down
IP
++
––+
++
–– +
10 Gy
ATMi
Hsp27
Input G6PD
+
ctr
Tubulin
50 kDa
15 kDa
γH2AX
25 kDa
p-Hsp27
(ser78)
10 Gy + +
ctr ATMi
25 kDa
50 kDa
50 kDa
25 kDa
25 kDa
A
CD
EF
B
Figure 2 HSP27 binding to G6PD. (A) Xenopus egg extract was left untreated (ctr) or treated for 15 min with 20 ng/ml DSBs (DSBs) in the presence
or absence of 10 mM ATMi (ATMi) or supplemented with 3000 nuclei per microliter treated with 10 mM phleomycin (phl). Hundred microliters
of extract were then diluted in PBS and incubated with 2
0
-5
0
-ADP-sepharose beads. The proteins bound to the beads were eluted in Laemmli
buffer and loaded on a 4–12% SDS–PAGE gel, transferred onto nitrocellulose filter and analysed by western blot with anti-G6PD (upper panel)
and anti-HSP27 (bottom panel) antibodies. (B) AG02603 fibroblast cells were left untreated or irradiated with 10 Gy. After 1 h, cells were
collected and the proteins were extracted. The whole cell lysates were incubated with control (ctr) or anti-HSP27 antibodies. The samples were
then analysed by western blot with anti-G6PD (upper panel) and anti-HSP27 (bottom panel) antibodies. (C) AG02603 cells were treated with
0.1% DMSO or 10 mM ATMi before being irradiated with 10 Gy or left untreated. G6PD was immunoprecipitated with specific anti-G6PD antibodies
and the samples were analysed by western blot with anti-HSP27 antibodies. Non-immune serum was used as control (ctr). (D) In vitro G6PD
activity assay: 300 ng of recombinant G6PD was incubated for 10 min at 301C with the indicated amount of recombinant Hsp27. G6PD activity
was then assessed. The histogram represents average enzymatic activities relative to untreated control (0). Experiment was repeated three times.
Error bars represent s.d. (E) Human fibroblasts were exposed to 10 Gy of IR in the presence or absence of 10 mM ATMi. Total cell lysates were
loaded on SDS–PAGE and then analysed by western blot with anti-phospho-Hsp27 (ser78), anti-tubulin and anti-gH2AX. (F) The histogram
represents the average of three independent experiments in which Hsp27 phosphorylation was determined. Error bars represent s.d.
ATM activates the pentose phosphate pathway
C Cosentino et al
The EMBO Journal & 2010 European Molecular Biology Organization4
Page 4
that the mechanism is post-translational and likely involves
the binding of Hsp27 to G6PD, which is conserved between
human and Xenopus cells (Figure 2A and B).
In order to verify the role of Hsp27 in the activation of
G6PD, we silenced Hsp27 gene (Figure 4A). Figure 4B shows
that in the absence of Hsp27 IR treatment did not induce any
increase in G6PD activity.
This result strongly suggests that Hsp27 is responsible for
the ATM-mediated activation of G6PD.
ATM promotes a global stimulation of the PPP
The PPP is the major source of ribose-5-phosphate, which
constitutes the sugar backbone of nucleotides. We reasoned
that the stimulation of G6PD, besides leading to increased
production of the anti-oxidant cofactor NADPH, could also
promote the synthesis of nucleotides by providing more
nucleotide precursors. This phenomenon could be significant
in DNA repair, where an increased amount of nucleotides
might promote the repair of DNA lesions. To verify this
hypothesis we measured the amount of de novo synthesized
nucleotides incorporated into cellular nucleic acids after IR
treatment by supplementing cells with PPP intermediates
labelled with
14
C.
14
C incorporation in RNA would indicate
conversion of PPP intermediates such as ribose-5-phosphate
into nucleotides and ultimately into RNA.
Cells supplemented with [6-
14
C] glucose-6-phosphate were
left untreated or irradiated with IR. Total RNA was then
isolated and the amount of
14
C incorporated into RNA was
measured. We observed a significant increase in the amount
of
14
C incorporated into RNA derived form normal cells
treated with IR (Figure 5). This increase was absent in A-T
cells. These data suggest that ATM promotes the production
of nucleotide precursors by stimulating the PPP.
G6PD activity is required for DNA repair
Similar to A-T cells, lack of G6PD induces radiosensitivity
(Tuttle et al, 2000; Figure S3), suggesting that G6PD might
also be necessary for DSB repair. We asked whether G6PD
activity was required for DNA integrity and DNA repair.
Figure 3 G6PD activity in human cells. (A) AG02603 cells were treated with or without 10 mM ATMi or 5 mM caffeine before being exposed to
10 Gy of IR. Cells were then lysed and G6PD activity was determined over the time as indicated. (B) Normal fibroblasts (GM0024B), A-T
fibroblasts (GM03395), normal lymphoblasts (GM0558) and A-T lymphoblasts (GM03382) were irradiated with 10 Gy or left untreated. These
cells were then lysed and G6PD activity was determined on whole cell extracts. The histogram represents the average fold increase of G6PD
activity over non-treated corresponding cells. Error bars indicate s.d.
AB
2.0
1.5
1.0
0.5
G6PD activity
(fold increase)
0.0
ctr
ut
10 Gy
siHsp27
siHsp27
50 kDa
10 Gy
Tubulin
Hsp27
25 kDa
+ +
ctr
Figure 4 Silencing of Hsp27 in human fibroblasts. (A) Cell transfected with control or Hsp27 targeting siRNA were lysed. Total protein extract
was loaded on SDS–PAGE and analysed for Hsp27 content (lower panel) and tubulin (upper panel). (B) G6PD activity assay in cells where
Hsp27 was silenced. Graph indicates average values from independent experiments. Error bars indicate s.d.
Figure 5 Incorporation of
14
C into RNA. Human lymphoblasts were
irradiated in the presence of
D-glucose 6-phosphate-UL-
14
C. RNA
was extracted and count per minutes (CPM) obtained for the
irradiated cells were plotted as average fold increase compared
to the correspondent non-irradiated cells. Graph represents
average values derived from independent experiments. Error bars
indicate s.d.
ATM activates the pentose phosphate pathway
C Cosentino et al
& 2010 European Molecular Biology Organization The EMBO Journal 5
Page 5
To this end we monitored the ability of G6PD-deficient
Xenopus egg extract and human cells to repair DSBs.
We found that human cells in which G6PD gene is silenced
have reduced ability to repair DSB induced by IR as shown by
the residual DSBs measured by the neutral comet assay
(Figure 6A and B). A similar defect was observed in
Xenopus egg extract when the nuclei were damaged with
phleomycin (Supplementary Figure S4).
To exclude the induction of apoptosis by these treatments
we monitored the level of caspase-3 activation and found that
caspase-3 was not activated consistent with the absence of
any other sign of apoptosis (Supplementary Figure S5). In
addition, we addressed the repair defect by monitoring the
disappearance of gH2AX foci, a marker of DSBs, upon
exposure to 2 Gy. In normal fibroblasts the number of foci
is efficiently reduced within 3 h and returns to basal levels at
48 h post-IR (Figure 6C and D). However, in G6PD silenced
cells a fraction of gH2AX foci persisted confirming a defect in
DSB repair. A similar defect was observed in cells in which
DNA-PK activity, which is required for optimal DSB repair
was inhibited before irradiation (Supplementary Figure S6;
Zhao et al, 2006).
To verify whether G6PD is also responsible for limiting the
effects of ROS induced after DNA damage we measured ROS
production following IR treatment in normal and G6PD knock-
down cells (Supplementary Figure S7). We found that inacti-
vation of G6PD increases ROS production (Supplementary
Figure S7). Suppression of ROS production by G6PD might
contribute to limit ROS-mediated DNA damage and inactiva-
tion of DNA repair factors such as Ku70/80 (Ayene et al, 2000).
AB
siG6PD
G6PD
Tubulin
48 kDa
48 kDa
ctr
20
15
10
5
0
0
10
30
10 Gy
sictr siG6PD
10 Gy
60 0 10 30 60
Minutes
Mean tail moment
(a.u.)
ctr
siG6PD
0148
H
C
8
6
4
2
0
0123420
H
siG6PD
sictr
30 40 50
D
Foci/cell
Figure 6 DSB repair in human fibroblasts upon G6PD silencing. (A) Cells were transfected with a scrambled siRNA (ctr) and G6PD targeting
siRNA. An aliquot of the cells was collected, lysed and analysed by western blot anti-G6PD (upper panel) and a-tubulin (bottom panel).
(B) Another aliquot of the cells was either left untreated or irradiated with 10 Gy. Residual DSBs remaining after the indicated times were
determined by neutral comet assay. The histogram represents the average comet tail moment. Experiment was repeated three times. Error
bars represent s.d. (C) Cells transfected with control or G6PD-specific siRNA were exposed to 2 Gy of IR. gH2AX foci were visualized by
immunofluorescence. (D) The histogram represents number of foci per cells. The data are the average of three independent experiments.
Error bars indicate s.d.
ATM activates the pentose phosphate pathway
C Cosentino et al
The EMBO Journal & 2010 European Molecular Biology Organization6
Page 6
Discussion
Here, we show that ATM regulates the PPP by inducing G6PD
activity. This pathway is conserved in vertebrate organisms
as shown by experiments performed in Xenopus eggs and
several human cell lines. These observations might help to
understand the molecular basis underlying the pathogenesis
of A-T. In normal cells, ATM might contribute to maintain the
reducing power of the cellular environment by promoting
NADPH production. In the absence of ATM, cells would not
be able to counteract oxidative stress. Consistent with this,
previous studies showed a correlation between redox state,
measured as reduced levels of glutathione and severity of the
A-T phenotype (Buoni et al, 2006; Broccoletti et al, 2008;
Russo et al, 2009). These and our findings are in agreement
with earlier observations reporting defects in A-T cells of re-
synthesis of glutathione, which requires NADPH (Meredith
and Dodson, 1987).
Low levels of NADPH were also found in the cerebellum of
the ATM/ mice. The absence of ATM mediated increase in
G6PD activity could explain the impaired oxidative stress
response in A-T cells (Stern et al, 2002). Considering that the
cerebellum is the area of the brain affected by neurodegen-
eration in A-T patients, it is possible that the absence of ATM
also leads to low levels of NADPH in the human brain.
Neuron metabolism produces high level of ROS, which in
normal conditions might activate ATM directly or indirectly
and in turn promote G6PD activity restoring the redox state of
the cells. In A-T patients this feedback might be compro-
mised, leading to the accumulation oxidative stress.
Consistent with this hypothesis it has recently been shown
that ATM is directly activated by ROS and that a mutation in
ATM, which impairs ATM ability to respond to ROS but not to
DNA damage is responsible for A-T (Guo et al, 2010). On the
other hand deficiency in DSB repair is known to cause defects
similar to A-T. Therefore, it is possible that the A-T phenotype
is the result of a defect in the response to both DNA damage
and ROS.
Interestingly, an X chromosome linked human disease
with partial deficiency in G6PD activity can cause haemolytic
anaemia but not any of the symptoms found in A-T
(Cappellini and Fiorelli, 2008). In this case it is possible
that compensatory mechanisms leading to an increased
production of NADPH are activated in the absence of con-
stitutive levels of G6PD. NADPH can indeed be produced
by additional enzymes not active in erythrocytes, which for
this reason are particularly sensitive to low levels of G6PD
(Cappellini and Fiorelli, 2008). We cannot exclude that these
enzymes are also under the control of ATM. These observa-
tions might explain the absence of A-T symptoms associated
with G6PD deficiency.
We also show that the activation of G6PD is required for
efficient DSB repair. The activation of G6PD correlates with
an increased activity of the PPP and this might be required to
increase the dNTPs pool needed to repair DNA. In yeast, it is
well established that upon DNA damage, cells increase their
dNTPs pool (Lee and Elledge, 2006). In yeast as well as in
mammalian cells, the regulation of the dNTPs pool relies
mostly on the ribonucleotide reductase (RNR), whose expres-
sion is controlled by p53 in mammalian cells (Pontarin et al,
2007) and is dependent on NADPH for its activity (Avval and
Holmgren, 2009). The defect we observe in the repair of DSBs
might be due to an impairment of RNR activity as a conse-
quence of low levels of NADPH and to an imbalance in the
dNTPs pool, whose de novo synthesis depends on the PPP
and RNR.
As far as the activation of G6PD and, in turn, the PPP
is concerned we provide evidence that this is mediated
by Hsp27. A role for the small heat shock protein Hsp27
in oxidative stress response has already been proposed
(Preville et al, 1999). However, the mechanism underlying
Hsp27 action was unclear. In humans, cells treated with
hydrogen peroxide Hsp27 promotes an increase of the
G6PD protein levels (Preville et al, 1999). In mice, instead,
downregulation of Hsp25, the homologue of Hsp27, affects
G6PD activity but not G6PD protein levels (Yan et al, 2002).
In our system G6PD protein levels are unaffected. We instead
observe that ATM activation promotes the interaction be-
tween Hsp27 and G6PD (Figure 2), and that Hsp27 directly
increases G6PD activity in vitro. Importantly, we show that
Hsp27 inactivation abolishes ATM-dependent stimulation of
G6PD activity. It is possible that Hsp27 stabilizes the active
conformation of G6PD.
It is known that human Hsp27 is phosphorylated mainly
on three serines by p38–MK2 complex (Kostenko and Moens,
2009). Hsp27 forms large oligomers. In general, phosphor-
ylation of Hsp27 regulates its oligomerization level and
localization in response to different stimuli (Bruey et al,
2000). A study on human skin fibroblasts showed that
following IR Hsp27 is phosphorylated on serine 78/82 or
ser15, depending on the dose (Yang et al, 2006). In our
system, following exposure of cells to IR, Hsp27 is phos-
phorylated on serine 78, a target of p38–MK2 pathway. p38–
MK2 is responsible for the ‘cytoplasmic’ branch of the ATM-
dependent checkpoint (Reinhardt et al, 2010). Once activated
in the nucleus, p38–MK2 re-localizes to the cytoplasm where
it can phosphorylate cytoplasmic targets such as Hsp27. It is
possible that this phosphorylation increases Hsp27 affinity
for G6PD and in turn increases the activity of the latter. The
phosphomimic mutant of Hsp27 can form large oligomers
and protect the cells from several forms of stress (Rogalla
et al, 1999). ATM might favour the formation of the large
oligomers of Hsp27 by inducing serine 78 phosphorylation
and these large oligomers could mediate G6PD activation
(Figure 7).
ATM-dependent control of cellular metabolism and ROS
production is probably not limited to the events described
here. Previous studies have reported a late activation of the
PPP and a contemporary inhibition of the glycolysis sustained
by p53 through TIGAR and NF-kB (Bensaad et al, 2006;
Kawauchi et al, 2008). ATM-mediated inhibition of glycolysis
might be important to reduce ROS produced by glycolytic
metabolism (Figure 7). Interestingly, cancer cells, in which
ATM is frequently mutated, have a high energetic demand but
nonetheless rely on glycolysis rather than oxidative phos-
phorylation for the ATP supply, even when oxygen is present.
This phenomenon is known as Warburg effect (Hsu and
Sabatini, 2008). It is possible that the molecular mechanisms
underlying this phenomenon include a deficient activation of
the PPP, which normally counteracts glycolysis. In addition to
this, two glycolytic enzymes, glyceraldeyde-3-phosphate de-
hydrogenase and pyruvate kinase M2 were recently found to
be ATM/ATR substrate (Matsuoka et al, 2007; Stokes et al,
2007). These observations, together with our own, indicate
ATM activates the pentose phosphate pathway
C Cosentino et al
& 2010 European Molecular Biology Organization The EMBO Journal 7
Page 7
that metabolic control might be an important event in the
ATM-dependent DNA damage response.
Overall our data indicate that ATM is a major player in the
control of redox metabolism and nucleotide production. The
lack of these ATM-dependent functions might contribute to
the numerous clinical manifestations of A-T disease.
Therapeutic strategies aimed at direct stimulation of anti-
oxidant pathways might be helpful to promote cell survival in
A-T patients bypassing the requirement for ATM protein.
Materials and methods
Antibodies, cDNA clones and chemicals
Xenopus G6PD cDNA was the IMAGE 4965682. The cDNA was
amplified by PCR and subcloned into a pMAL c4x vector (New
England Biolabs). Anti-Xenopus G6PD was produced immunizing
rabbits against the full-length recombinant protein (Harlan). The
antibodies used were anti-human G6PD goat polyclonal (Abcam),
anti-Hsp27 mouse monoclonal (Santa Cruz Biotechnology) for
immunoprecipitation, anti-Hsp27 rabbit polyclonal (R&D) for
western blot and anti-tubulin mouse monoclonal (Sigma Aldrich).
Protein G-sepharose, NAD, glucose-6-phosphate (G6P),
D-glucose-
6-phosphate-UL-
14
C disodium salt, caffeine and DHEA were
purchased from Sigma Aldrich. 2
0
-5
0
-ADP-sepharose was from GE-
Healthcare. 2-Morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (ATMi)
was purchased from Calbiochem. Phleomycin was from Invivogen.
DNA-PK inhibitor Nu7441 was from Tocris Bioscience.
Xenopus egg extract and immunodepletion
Xenopus metaphase II arrested oocytes were collected and
processed as previously described (Errico et al, 2007). For the
immunodepletion 50 ml of control or anti-G6PD serum was
incubated overnight at 41C with 50 ml of slurry protein G-sepharose
in 1 ml of PBS. The day after, 500 ml of extract were incubated with
the antibody/protein G complex at 41C with gently rocking, after
30 min the samples were spun and the supernatant recovered. This
step was repeated three times.
G6PD activity measurement
G6PD activity was measured using a Varian UV–VIS spectrometer
(Varian Inc.). Briefly, 3 ml of Xenopus egg extract or 80 mg of cell
lysate were diluted in 85 ml of 10 mM Tris pH 7.5, supplemented
with 5 mM G6P and 10 mM NADP. The excitation wavelength was
set at 340 nM and the emission wavelength was read at 460 nM.
The samples were placed in quartz submicrocells and read with a
kinetic program for 10 min.
2
0
-5
0
-ADP-sepharose pull down
In total, 500 ml of Xenopus egg extract were incubated with 50 ml
of resin (ADP-sepharose or protein G-sepharose) for 90 min at 41C
with gentle agitation. Where indicated proteins were eluted with
10 mM Tris pH 8.5 containing 10 mM NADP and 2 M NaCl.
Cell culture and RNA interference
Human control fibroblast (AG02603 and GM00024) and A-T
fibroblasts (GM03395 and GM05832) were maintained in EMEM
15% foetal calf serum. Mouse fibroblasts NIH3T3 were grown
in DMEM 10% foetal calf serum. Human control lymphoblasts
GM00558 and A-T lymphoblasts GM03382 were maintained in
RPMI 15% heath inactivated foetal calf serum. All media were
supplemented with penicillin/streptomycin 100 mU/ml and 2 mM
glutamine. AG02603 and GM05823 cells were from Coriell Institute.
To silence G6PD, human fibroblasts were transfected with 50 pmol
of G6PD stealth siRNA or control siRNA (Invitrogen) or 100 mMof
Hsp27 II siRNA (Cell signalling) complexed with lipofectamine
RNAiMax (Invitrogen) according to the manufacturer’s instruction.
The procedure was repeated after 24 h from the first transfection in
order to get the maximum silencing of the gene.
Western blot and immunoprecipitation
Cells were lysed in 10 mM Tris pH 7.5, 10 mM NaCl, 0.2% NP40,
1mM b-glycerophosphate, 1 mM NaF, 2.5 sodium pyrophosphate
and a tablet of protein inhibitors cocktail (Complete, Roche).
Protein concentration was determined with the Bradford assay.
In total, cell lysates containing 500 mg of proteins were incubated
with 5 mg of the indicated antibody overnight at 41C on a rocking
wheel. The day after, the samples were incubated for 45 min with
20 ml of protein A/G plus agarose (Santa Cruz). The beads were then
washed and the protein eluted in Laemmli buffer 2 . Protein
samples were run on 3–12% gradient gels (XT-criterion gel, Bio-
Rad) and transferred onto nitrocellulose. The filter was blocked in
PBS 0.1% Tween, 5% non-fat milk. The membranes were incubated
overnight with the primary antibody (1:1000 in blocking buffer) and
45 min with the secondary antibody (1:10 000 in blocking buffer).
PPP measurement
Cells were irradiated or left untreated in the presence of 3 mCi of
D-glucose-6-phosphate-UL-
14
C disodium salt and then incubated at
371C for 3 h. The total RNA was extracted from cells with guanidine
thiocyanate method and the activity was determined with a
Beckman scintillation counter (Beaconsfield et al, 1965).
Neutral comet assay
Cells were treated as indicated in the text, collected and diluted to
5 10
5
per ml. Ten microliters were taken and diluted in 0.5% low
melting point agarose. The sample was spread on a Trevigen slide
for comet assay. Samples were processed as previously described
Hsp27
ATM
P
G6PD
DNA repairCell survival
GSH GSSG
NADP NADPH
G6P
6-P-glucono-δ-lactone
R5P
GSR
NADP
dNTPs
p53
TIGAR
RNR
P
p38
Glycolysis
ROS
Figure 7 Schematic representation of G6PD regulation and downstream effect. DSBs activate ATM, which in turn promotes the interaction
between Hsp27 and G6PD. This association leads to increased activity of G6PD and stimulation of the PPP. ATM also regulates glycolysis and
PPP at transcriptional level (dashed arrows) through p53-mediated induction of TIGAR. As a consequence of these events, ROS levels are
reduced and the dNTP pool is increased, allowing efficient DNA repair and promoting cell survival.
ATM activates the pentose phosphate pathway
C Cosentino et al
The EMBO Journal & 2010 European Molecular Biology Organization8
Page 8
(Trenz et al, 2008). The slides were analysed with Comet assay IV
software.
cH2AX foci
Cells were plated on BD-Falcon culture slides chamber and treated
as indicated in the text. At the indicated time points, cells were fixed
in 95% EtOH/5% acetic acid and stained with anti-phospho-H2AX
(ser139) clone JBW 301 (Millipore) according to the manufacturer’s
instructions. The nuclei were stained with DAPI at 1 mg/ml
concentration. The image was acquired on a Zeiss Axio Imager
M1 microscope with a 63 oil objective and analysed using the
Volocity 4.3.2 Software (Improvision).
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We thank members of the genome stability lab for their insightful
comments. We thank H Mahbubani and J Kirk for technical
support with Xenopus laevis. We thank M Skehel and his group for
the mass spectrometry analysis. This work was funded by Cancer
Research UK. VC is also supported by the European Research Council
(ERC) start up grant (206281), the Lister Institute of Preventive
Medicine and the EMBO Young Investigator Program (YIP). DG thanks
Associazione Italiana per la Ricerca sul Cancro (AIRC) for support.
Authors contribution: CC performed the experiments, analysed
the data and wrote the paper. DG discussed and analysed the data.
VC planned the experiments, analysed the data and wrote the paper.
Conflict of interest
The authors declare that they have no conflict of interest.
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ATM activates the pentose phosphate pathway
C Cosentino et al
The EMBO Journal & 2010 European Molecular Biology Organization10
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    • "In addition to ATM's well-known functions in cell cycle control and response to DNA damage, ATM has increasingly been found to display a variety of metabolic functions. For example, ATM plays antioxidant roles by stimulating the pentose phosphate pathway [2] and through increasing uptake of dehydroascorbic acid (DHA) [3], the oxidized form of the potent antioxidant ascorbate. Mitochondrial dysregulation has been reported for ATM-null thymocytes, with features including disorganized mitochondrial structure, increased mitochondrial reactive oxygen species (ROS), and decreases in whole-cell ATP despite an increase in mitochondrial volume [4]. "
    [Show abstract] [Hide abstract] ABSTRACT: Aims: There are reports that ataxia telangiectasia mutated (ATM) can activate the AMP-activated protein kinase (AMPK) and also Akt, two kinases that play integral parts in cardioprotection and metabolic function. We hypothesized that chloroquine and resveratrol, both known ATM activators, would also activate AMPK and Akt. Main methods: Phosphorylation of AMPK and Akt was assessed after C2C12 myotubes were exposed to chloroquine or resveratrol. Additional experiments were done in cells expressing shRNA against ATM or in the presence of the ATM inhibitor KU55933. The effects of chloroquine on intracellular calcium were assessed with the fluorescent probe Calcium Green-1 AM. Key findings: 0.5 mM chloroquine increased AMPK phosphorylation by nearly 4-fold (P<0.05), and 0.25 mM chloroquine roughly doubled Akt phosphorylation (P<0.05). Chloroquine also increased autophosphorylation of ATM by ~50% (P<0.05). Resveratrol (0.15 mM) increased AMPK phosphorylation about three-fold (P<0.05) but in contrast to chloroquine sharply decreased Akt phosphorylation. Chloroquine increased AMPK and Akt phosphorylation in myotubes expressing shRNA against ATM that reduced ATM protein levels by about 90%. Likewise, chloroquine-stimulated phosphorylation of AMPK and Akt and resveratrol-stimulated phosphorylation of AMPK were not altered by inhibition of ATM. Chloroquine decreased intracellular calcium by >50% concomitant with a decrease in glucose transport. Significance: These ATM-independent effects of chloroquine on AMPK and Akt and the additional effect to decrease intracellular calcium are likely to partially underlie the positive metabolic effects of chloroquine that have been reported in the literature.
    Full-text · Article · Mar 2016
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    • "In addition to DNA repair pathways, only BPDE activated the pentose phosphate pathway (PPP) and the ATM-dependent DNA damage response. Both pathways were shown to closely cooperate in response to doublestrand breaks (DSB) in DNA: ATM recruits and activates the PPP pathway, which increases the input of nucleotides to be used during the replacement of damaged DNA bases (Cosentino et al. 2011). Thus, our results suggest that in contrast to BaP, where the cell can quickly adapt to increasing levels of genotoxicant, abrupt stimuli induced by BPDE have resulted in extensive DNA damage which triggered enhanced expression of genes necessary for DNA repair in order to protect the cell against future challenges (Lei et al. 2007). "
    [Show abstract] [Hide abstract] ABSTRACT: Benzo(a)pyrene (BaP) is a ubiquitous carcinogen resulting from incomplete combustion of organic compounds and also present at high levels in cigarette smoke. A wide range of biological effects has been attributed to BaP and its genotoxic metabolite BPDE, but the contribution to BaP toxicity of intermediary metabolites generated along the detoxification path remains unknown. Here, we report for the first time how 3-OH-BaP, 9,10-diol and BPDE, three major BaP metabolites, temporally relate to BaP-induced transcriptomic alterations in HepG2 cells. Since BaP is also known to induce AhR activation, we additionally evaluated TCDD to source the expression of non-genotoxic AhR-mediated patterns. 9,10-Diol was shown to activate several transcription factor networks related to BaP metabolism (AhR), oxidative stress (Nrf2) and cell proliferation (HIF-1α, AP-1) in particular at early time points, while BPDE influenced expression of genes involved in cell energetics, DNA repair and apoptotic pathways. Also, in order to grasp the role of BaP and its metabolites in chemical hepatocarcinogenesis, we compared expression patterns from BaP(-metabolites) and TCDD to a signature set of approximately nine thousand gene expressions derived from hepatocellular carcinoma (HCC) patients. While transcriptome modulation by TCDD appeared not significantly related to HCC, BaP and BPDE were shown to deregulate metastatic markers via non-genotoxic and genotoxic mechanisms and activate inflammatory pathways (NF-κβ signaling, cytokine-cytokine receptor interaction). BaP also showed strong repression of genes involved in cholesterol and fatty acid biosynthesis. Altogether, this study provides new insights into BaP-induced toxicity and sheds new light onto its mechanism of action as a hepatocarcinogen.
    Full-text · Article · Aug 2015 · Archives of Toxicology
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    • "ATM is subsequently activated through oxidation at specific cysteine residues [61]. Evidence also shows that ATM can promote an antioxidant response via regulation of the pentose phosphate pathway—one of the primary sources of NADPH [137]. An additional example of a DNA repair pathway protein involved in oxidative stress is human RPA. "
    [Show abstract] [Hide abstract] ABSTRACT: Disruption of redox homeostasis is a crucial factor in the development of drug resistance, which is a major problem facing current cancer treatment. Compared with normal cells, tumor cells generally exhibit higher levels of reactive oxygen species (ROS), which can promote tumor progression and development. Upon drug treatment, some tumor cells can undergo a process of 'Redox Resetting' to acquire a new redox balance with higher levels of ROS accumulation and stronger antioxidant systems. Evidence has accumulated showing that the 'Redox Resetting' enables cancer cells to become resistant to anticancer drugs by multiple mechanisms, including increased rates of drug efflux, altered drug metabolism and drug targets, activated prosurvival pathways and inefficient induction of cell death. In this article, we provide insight into the role of 'Redox Resetting' on the emergence of drug resistance that may contribute to pharmacological modulation of resistance.
    Preview · Article · Jul 2015 · Oncotarget
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