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INTRODUCTION |
Oxidative stress has been implicated in various pathophysiological
conditions including neurodegeneration (1-3), which is one of the
characteristics of the human disease ataxia telangiectasia (AT).1 Ions of the toxic
heavy metal cadmium generate reactive oxygen species in cells and
induce oxidative damage in organs such as brain, liver, testis, and
kidney (4-8). Cadmium ions react readily with thiol groups of
proteins, showing a greater affinity for these groups than does
Zn2+, and thereby disrupt the structure of certain cellular
proteins (9). They also induce lipid peroxidation (5-7) and imbalance of cellular sulfhydryl homeostasis (10, 11). Various other physiological effects of Cd2+ have been described. These
include induction of single-strand DNA breakage (12, 13), promotion of
neoplastic transformation both in vitro and in
vivo (14-17), activation and translocation to the nucleus of
protein kinase C (18-20), increase in the intracellular level of
inositol polyphosphates (21) and Ca2+ (22-25), induction
of gene expression of heat shock protein (26, 27), metallothionein (12,
28, 29), heme oxygenase (30, 31), and transcriptional activation of
proto-oncogenes such as c-jun, c-fos, and
c-myc (29, 32-34).
Cells derived from individuals with AT show extreme sensitivity to
ionizing radiation as well as defects in ionizing radiation-induced intracellular signaling (35-37). The gene (ATM) that is
mutated in individuals with AT has been isolated, and the encoded
protein (ATM) is thought to play a role in a variety of cellular
processes including mitogenic signal transduction, cell cycle control,
and repair of DNA damage (38-40). Defects in ionizing
radiation-induced signaling in AT cells include impaired activation of
the transcription factors NF-
B and c-Jun (37, 41-43).
The transcriptional activity of c-Jun is increased by phosphorylation
of Ser63 and Ser73 residues in response to
various stimuli, including ionizing radiation, ultraviolet light,
cytokines, and oxidative stress (44-48). We have previously shown that
the ionizing radiation-induced phosphorylation of c-Jun on these two
residues that is apparent in normal human fibroblasts does not occur in
AT fibroblasts, whereas ultraviolet radiation-induced phosphorylation
of these sites is intact in both types of cells (37). We have proposed
that the c-Jun NH2-terminal kinase (JNK), which is
responsible for the phosphorylation of c-Jun and is present in the
nucleus of both normal and AT fibroblasts, is subjected to negative
regulation that is relieved on exposure of normal cells to ionizing
radiation (37). We now demonstrate a role for ATM in oxidative
stress-induced JNK activation and c-Jun phosphorylation. Our data
indicate that ATM functions as a sensor of ionizing radiation-generated
oxidative stress and as a modulator of intracellular redox regulatory
systems in cells exposed to ionizing radiation or the potent
pro-oxidant CdCl2.
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EXPERIMENTAL PROCEDURES |
Materials--
NAC, BSO, and CdCl2 were obtained
from Sigma. The GST-c-Jun-(1-89) fusion protein was obtained from Cell
Signaling Technology. Antibodies to c-Jun and to HO-1 were from
Transduction Laboratories; those specific for c-Jun phosphorylated on
Ser63 or on Ser73 were from Cell Signaling
Technology; those specific for JNK phosphorylated on Thr183
and Tyr185 were from Promega; and those to ATM were from Calbiochem.
Cell Culture--
Normal human fibroblasts (MRC5CV1) and AT
fibroblasts (AT5BIVA, AT4BIVA, and AT3BIVA) were cultured under 5%
CO2 at 37 °C in Eagle's minimum essential medium
supplemented with 15% (normal cells) or 20% (AT cells) fetal bovine
serum, 2 mM L-glutamine, 1 mM
sodium pyruvate, 0.1 mM nonessential amino acids,
penicillin (100 units/ml), and streptomycin (100 µg/ml).
Hydrocortisone (5 µg/ml) was also included in the culture medium for
AT cells. MRC5CV1/pEAT22 (MRC5CV1 fibroblasts transfected with pEAT22,
which encodes ATM antisense RNA) (49) and AT5BIVA/pMAT1 cells (AT5BIVA
fibroblasts transfected with pMAT1, which contains the full-length ATM
cDNA under the control of the inducible metallothionein II
promoter) (50) were cultured in the presence of hygromycin B (200 µg/ml). All fibroblast cell lines were immortalized with SV40 (51). Before exposure of cells to ionizing radiation or CdCl2,
the culture medium was replaced with serum-free medium for 24 h.
Irradiation was performed at room temperature with a JL Shepherd Mark I
Radiator containing a 137Cs source emitting at a fixed dose
rate of 3.83 Gy/min.
Subcellular Fractionation--
Cells were washed three times
with ice-cold phosphate-buffered saline, harvested by scraping from the
culture dishes into ice-cold equilibration buffer (EB) (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 2 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM EGTA, and protease inhibitors
(52)), and collected by centrifugation at 12,000 rpm (18,000 × g) for 1 min at 4 °C. Cell pellets were resuspended in EB
containing 0.1% Nonidet P-40 and incubated on ice for 15 min. The
cytosolic fraction was then isolated by centrifugation at 14,000 rpm
(25,000 × g) for 1 min, and the resulting pellet was
washed once with EB, resuspended in a nuclear extraction buffer (20 mM HEPES (pH 7.9), 0.42 M NaCl, 1 mM EDTA, 0.1 mM EGTA, 2 mM
dithiothreitol, 20% glycerol, and protease inhibitors), and incubated
on ice for 1 h with occasional vortexing. The extracted nuclear
proteins were isolated by centrifugation at 50,000 × g for 30 min. Protein concentration was determined with the Bradford assay (53), with bovine serum albumin as a standard.
Immunoblot Analysis--
Subcellular fractions were denatured by
boiling for 5 min in SDS sample buffer and subjected to
SDS-polyacrylamide gel electrophoresis. The separated proteins were
transferred to a polyvinylidene difluoride membrane, which was then
incubated for 1 h at room temperature with 5% nonfat dry milk in
phosphate-buffered saline before exposure overnight at 4 °C to
primary antibodies. Immune complexes were detected by enhanced
chemiluminescence (Amersham Phamacia Biotech).
In Vitro Kinase Assay of JNK Activity--
The kinase activity
of JNK was assayed in vitro by incubation of cytosolic or
nuclear proteins for 15 min at 30 °C with 5 µg of
GST-c-Jun-(1-89) in 20 µl of kinase buffer (20 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM MnCl2, 0.1 mM
Na3VO4, 2 mM dithiothreitol, 20 µM ATP). Phosphorylated GST-c-Jun-(1-89) (as well as
phosphorylated endogenous c-Jun) was detected by immunoblot analysis
with antibody specific for c-Jun phosphorylated on
Ser73.
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RESULTS |
Effect of CdCl2 on c-Jun Phosphorylation--
The
intracellular accumulation of Cd2+ generates oxidative
stress (4-8, 10). To investigate whether c-Jun is activated in response to exposure of cells to Cd2+, we first examined
c-Jun phosphorylation on Ser63 and Ser73 in
normal human fibroblasts (MRC5CV1) and AT fibroblasts (AT5BIVA) after
incubation with 10 µM CdCl2 for various
times. Nuclear extracts were prepared and subjected to immunoblot
analysis with antibodies specific for c-Jun phosphorylated on
Ser73 (Fig. 1A). A
biphasic pattern of c-Jun phosphorylation was apparent in both MRC5CV1
and AT5BIVA cells. The first phase of c-Jun phosphorylation was
characterized by a gradual increase that reached a maximum at ~4 h
after exposure to CdCl2 and had decreased to control values by 7 h in both normal and AT cells. Although the extent of the increase in c-Jun phosphorylation at 4 h was similar in both cell types, it was consistently slightly greater in AT5BIVA cells (6-fold) than in MRC5CV1 cells (5-fold). The onset of the second phase of c-Jun
phosphorylation was apparent 12 h after exposure to
CdCl2 and reached a maximum at 16-24 h. In contrast to the
similar pattern of the first phase apparent in these two cell lines,
the pattern of the second phase of c-Jun phosphorylation differed
markedly between the normal and AT cells. Whereas MRC5CV1 cells
exhibited only a 3-fold increase in c-Jun phosphorylation by 24 h,
AT5BIVA cells showed an ~25-fold increase at both 16 and 24 h
after exposure to CdCl2. A similar pattern of c-Jun
phosphorylation on Ser63 was also observed in both MRC5CV1
and AT5BIVA cells in response to CdCl2 (Fig.
1B).

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Fig. 1.
Effects of CdCl2 on c-Jun
phosphorylation in normal human fibroblasts (MRC5CV1) and AT
fibroblasts (AT5BIVA). Cells were incubated with 10 µM CdCl2 for the indicated times, after which
nuclear proteins were isolated and subjected to immunoblot analysis
with antibodies specific for c-Jun phosphorylated either on
Ser73 (A) or Ser63
(B).
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Effects of CdCl2 on JNK Phosphorylation and
Activity--
Activation of JNK is mediated by phosphorylation of the
threonine and tyrosine residues within the TPY motif at positions 183-185 (54-57). Substantial amounts of p46 and p55 isoforms of JNK
are present in both nucleus and cytosol of MRC5CV1 and AT5BIVA cells
(37). Furthermore, the dually phosphorylated form of p46 JNK, but not
p55 isoform, is constitutively present in the nucleus of these cells
(37). Treatment with 10 µM CdCl2 for 16 h had no effect on the abundance of JNK isoforms in either the nucleus or cytosol of these cells (data not shown). However, immunoblot analysis with antibody specific for the active, dually phosphorylated form of JNK revealed that phosphorylation of the p46 isoform present in
the nucleus of AT5BIVA cells was increased within 10 h after exposure to CdCl2 (Fig.
2A). Furthermore,
phosphorylation of the p55 isoform was induced at 16 h after
exposure of these cells to CdCl2 and sustained up to
24 h. These data suggest that both JNK isoforms are activated in
response to CdCl2 in AT5BIVA cells and that the marked,
second phase phosphorylation of c-Jun in AT5BIVA cells (Fig. 1,
A and B) is, at least in part, attributed to the
phosphorylation of p55 isoform of JNK. The phosphorylation of an
unidentified protein that reacted with the antibodies to dually
phosphorylated JNK also appeared to be increased 10 and 16 h after
exposure of AT5BIVA cells to CdCl2 and sustained up to
24 h (Fig. 2A).

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Fig. 2.
Effects of CdCl2 on JNK
phosphorylation and activation in MRC5CV1 and AT5BIVA cells.
A, CdCl2-induced phosphorylation of JNK in
AT5BIVA cells. Cells were incubated with 10 µM
CdCl2 for the indicated times, after which nuclear proteins
were isolated and subjected to immunoblot analysis with antibodies
specific for JNK phosphorylated on Thr183 and
Tyr185. The solid arrows indicate the
phosphorylated p55 and p46 isoforms of JNK; the arrowhead
indicates an unknown immunoreactive protein. B,
CdCl2-induced activation of JNK in the nucleus of MRC5CV1
and AT5BIVA cells. Cells were incubated with 10 µM
CdCl2 for the indicated times, after which nuclear proteins
were isolated and subjected to an in vitro kinase assay with
GST-c-Jun-(1-89) as substrate. The phosphorylated substrate, as well
as phosphorylated endogenous c-Jun, was detected by immunoblot analysis
with antibodies specific for c-Jun phosphorylated on
Ser73.
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We next examined the effect of CdCl2 treatment of MRC5CV1
and AT5BIVA cells for various times to CdCl2 on JNK
activity measured in cytosolic and nuclear fractions by performing
in vitro kinase assay with a glutathione
S-transferase (GST) fusion protein containing residues 1-89
of c-Jun as substrate. Phosphorylated GST-c-Jun-(1-89) was detected by
immunoblot analysis with antibodies specific for c-Jun phosphorylated
on Ser73. Exposure of cells to CdCl2 induced a
biphasic increase in JNK activity in nuclear fraction of MRC5CV1 and
AT5BIVA cells (Fig. 2B). In MRC5CV1 cells, the activity of
JNK in the nucleus, as revealed by the phosphorylation of the
GST-c-Jun-(1-89) fusion protein as well as by that of endogenous
c-Jun, showed two peaks at 4 and 24 h. Cytosolic JNK activity
showed a similar pattern of induction (data not shown). In AT5BIVA
cells, the activity of JNK in the nucleus peaked at 4 and 16-24 h.
Overall, the pattern of JNK activity as revealed by phosphorylation of
GST-c-Jun-(1-89) did not differ substantially between MRC5CV1 and
AT5BIVA cells, suggesting that the greater extent of the second phase
of CdCl2-induced phosphorylation of endogenous c-Jun
apparent in AT5BIVA cells is not attributable to a greater increase in
JNK activity in vitro. These data also implicate that
different isoforms of JNK may participate in different phases for c-Jun
phosphorylation in response to CdCl2. Alternatively,
another mechanism, which involves residues other than amino acids
1-89, is associated with the second phase of c-Jun phosphorylation.
Recently, JNK-independent phosphorylation of c-Jun on serine 63 and 73 residues has been demonstrated by a novel protein complex, which
requires COOH-terminal amino acid residues including DNA binding and
dimerization domains for its activity (58, 59).
Combined Effects of Ionizing Radiation and CdCl2 on
c-Jun Expression and Phosphorylation--
We have previously shown
that ionizing radiation-induced phosphorylation of c-Jun is impaired in
AT fibroblasts (37). We therefore investigated the effect of ionizing
radiation on c-Jun phosphorylation as well as on c-Jun expression in AT
cells that had been exposed to CdCl2, a treatment that
increases c-Jun phosphorylation in these cells. We also examined
further the role of ATM in Cd2+-induced phosphorylation of
c-Jun. For these experiments we used cell lines MRC5CV1, AT5BIVA, and
AT5BIVA/pMAT1, the latter of which corresponds to AT5BIVA cells
transfected with a plasmid (pMAT1) containing the full-length ATM
cDNA under the control of a Cd2+-inducible promoter
(50). Cells were incubated in the absence or presence of 10 µM CdCl2 for 16 h and then exposed to 20 Gy of ionizing radiation. Nuclear proteins were isolated from cells at
various times after irradiation, and c-Jun expression (Fig. 3A) and phosphorylation (Fig.
3B) were examined by immunoblot analysis.

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Fig. 3.
Combined effects of CdCl2 and
ionizing radiation on c-Jun expression and phosphorylation in normal
human fibroblasts and AT cells. The indicated cell types were
incubated in the absence or presence of 10 µM
CdCl2 for 16 h and then subjected to 20 Gy of ionizing
radiation. Cells were harvested at the indicated times thereafter, and
nuclear proteins were isolated and subjected to immunoblot analysis
with antibodies specific for either total c-Jun (both phosphorylated
and unphosphorylated forms) (A) or c-Jun phosphorylated on
Ser73 (B and C).
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Pretreatment of cells with CdCl2 for 16 h markedly
increased the abundance of c-Jun in all three cell lines, and
subsequent exposure to ionizing radiation did not further increase
c-Jun expression (Fig. 3A). Although c-Jun expression was
maximally increased after treatment with CdCl2, such
treatment induced only a small increase in c-Jun phosphorylation in
MRC5CV1 cells (Fig. 3B). Subsequent exposure of these cells
to ionizing radiation further increased c-Jun phosphorylation, although
not to the extent greater than that apparent in MRC5CV1 cells not
pretreated with CdCl2. In AT5BIVA cells, the marked
increase in c-Jun phosphorylation induced by CdCl2
treatment was not further increased by subsequent exposure of the cells
to ionizing radiation (Fig. 3B). Other AT fibroblast lines
(AT4BIVA and AT3BIVA) exhibited a similar pattern of c-Jun
phosphorylation in response to CdCl2 and ionizing radiation (Fig. 3C). In the absence of CdCl2 pretreatment,
ionizing radiation increased both expression (Fig. 3A) and
phosphorylation (Fig. 3B) of c-Jun in MRC5CV1 cells but not
in AT5BIVA cells. In AT5BIVA/pMAT1 cells not treated with
CdCl2, ionizing radiation induced a slight increase in
c-Jun expression (Fig. 3A) but had no effect on c-Jun phosphorylation (Fig. 3B). However, pretreatment of
AT5BIVA/pMAT1 cells with CdCl2, which induces ATM
expression in these cells, resulted in a substantial induction of c-Jun
phosphorylation that was further increased by exposure to ionizing
radiation; subsequent irradiation did not further increase c-Jun
expression in Cd2+-pretreated AT5BIVA/pMAT1 cells. The
increases in c-Jun expression and phosphorylation induced by
CdCl2 pretreatment in AT5BIVA/pMAT1 cells were smaller than
those apparent in AT5BIVA cells.
These data suggest that CdCl2 and ionizing radiation
activate two distinct signaling pathways that converge upstream of
activation of c-Jun and that ATM contributes to the regulation of c-Jun
phosphorylation by both CdCl2 and ionizing radiation. The
combined effects of these two pathways are most apparent in the pattern
of c-Jun phosphorylation observed in AT5BIVA/pMAT1 cells exposed to
both CdCl2 and ionizing radiation.
Role of ATM in c-Jun Phosphorylation Induced by
CdCl2--
To investigate further the role of ATM in
CdCl2-induced c-Jun phosphorylation, MRC5CV1 cells were
transfected with plasmid containing antisense ATM cDNA under the
control of the Rous sarcoma virus promoter (49). Immunoblot analysis
revealed that the amount of ATM in MRC5CV1/pEAT22 cells was reduced to
~40% of that in the parental MRC5CV1 cells (data not shown). The
extent of c-Jun phosphorylation in MRC5CV1/pEAT22 cells at 4 h
after exposure to CdCl2 was similar to that in MRC5CV1 and
AT5BIVA cells (Fig. 4). However, at
16 h after exposure to CdCl2, c-Jun phosphorylation in
MRC5CV1/pEAT22 cells was markedly greater than that apparent in MRC5CV1
cells but less than that observed in AT5BIVA cells.

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Fig. 4.
Role of ATM in CdCl2-induced
phosphorylation of c-Jun. MRC5CV1, AT5BIVA, and MRC5CV1/pEAT22
cells were incubated with 10 µM CdCl2 for the
indicated times, after which nuclear proteins were isolated and
subjected to immunoblot analysis with antibodies specific for c-Jun
phosphorylated on Ser73.
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Role of Oxidative Stress in c-Jun Phosphorylation Induced by
CdCl2 or Ionizing Radiation--
Induction of
c-jun gene expression by Cd2+ is thought to
result from the oxidative stress induced by these metal ions (29). Cadmium ions react readily with thiol groups of proteins and thereby disrupt intracellular sulfhydryl homeostasis (9-11). Ionizing radiation also induces oxidative stress by generating reactive oxygen
species that result from the ionization of intracellular water (30).
Therefore, we examined the role of oxidative stress in c-Jun
phosphorylation induced by ionizing radiation or CdCl2 in
normal and AT cells. Pretreatment of cells with 30 mM
N-acetylcysteine (NAC), an intracellular precursor of
glutathione, markedly reduced the extent of CdCl2-induced
phosphorylation of c-Jun in both MRC5CV1 and AT5BIVA cells (Fig.
5A). Whereas NAC alone had no
effect on c-Jun phosphorylation in MRC5CV1 cells, it induced c-Jun
phosphorylation in AT5BIVA cells, possibly reflecting a difference in
intracellular sulfhydryl homeostasis and basal oxidative stress between
these two cell types (31). Pretreatment of MRC5CV1 cells with NAC also
inhibited the increase in c-Jun phosphorylation induced by ionizing
radiation (Fig. 5B). These data suggest that oxidative stress contributes to the increase in c-Jun phosphorylation in response
to CdCl2 treatment or ionizing radiation exposure.

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Fig. 5.
Effects of NAC and BSO on c-Jun
phosphorylation in response to CdCl2 or ionizing radiation
in normal fibroblasts and AT cells. A, MRC5CV1 and
AT5BIVA cells were incubated first for 1 h in the absence or
presence of 30 mM NAC and then for 4 h with or without
10 µM CdCl2 (in the continuous absence or
presence of NAC). Nuclear proteins were subsequently subjected to
immunoblot analysis with antibodies specific for c-Jun phosphorylated
on Ser73. B, MRC5CV1 cells were incubated for
1 h in the absence or presence of 30 mM NAC and
subsequently exposed to 20 Gy of ionizing radiation. Cells were
harvested at 2 h thereafter, and nuclear proteins were analyzed by
immunoblot analysis as in A. C, MRC5CV1, AT5BIVA, and
AT5BIVA/pMAT1 cells were incubated for 24 h in the absence or
presence of 5 µM BSO and then for 16 h with or
without 10 µM CdCl2 (in the continuous
absence or presence of BSO); cells were exposed to 20 Gy of ionizing
radiation 1 h before the end of the 16-h incubation period.
Nuclear proteins were analyzed for c-Jun phosphorylation as in
A.
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Exposure of cells to L-buthionine sulfoximine (BSO), a
potent inhibitor of
-glutamyl-cysteine synthetase, reduces the
intracellular level of glutathione (29). We therefore examined the
effect of BSO on c-Jun phosphorylation in response to CdCl2
and ionizing radiation in MRC5CV1, AT5BIVA, and AT5BIVA/pMAT1 cells
(Fig. 5C). Incubation with 5 µM BSO alone for
24 h had little or no effect on c-Jun phosphorylation in these
cells. In AT5BIVA cells, however, BSO pretreatment potentiated the
increase in c-Jun phosphorylation induced either by CdCl2
alone or by CdCl2 and ionizing radiation. In contrast,
pretreatment of MRC5CV1 or AT5BIVA/pMAT1 cells with BSO had no effect
on c-Jun phosphorylation induced by CdCl2 either alone or
combined with ionizing radiation. These data are consistent with the
notion that AT cells are more susceptible to disruption of
intracellular redox homeostasis and more sensitive to oxidative stress
than are cells in which ATM function is intact.
Possible Role of ATM as a Sensor of Ionizing Radiation-induced
Oxidative Stress and as a Modulator of Intracellular Redox
Homeostasis--
Heme oxygenase (HO) oxidatively cleaves heme, a
pro-oxidant, into carbon monoxide and biliverdin, which is subsequently
converted to bilirubin, an antioxidant. Three HO isozymes encoded by
three distinct genes have been identified (60, 61). The expression of
HO-1 is induced in a wide variety of mammalian tissues by agents that
elicit cellular injury, and induction of HO-1 has been implicated as a
generalized cellular response to oxidative stress in cultured mammalian
cells (62). We therefore examined the effect of CdCl2 on
HO-1 expression in MRC5CV1, AT5BIVA, and MRC5CV1/pEAT22 cells (Fig.
6). None of these cells expressed HO-1 at
a detectable level under normal physiological conditions. However,
exposure of these cells to CdCl2 resulted in an induction
of HO-1 expression in both nucleus and cytosol (Fig. 6, A
and B). Whereas MRC5CV1 cells showed a low level of HO-1
expression that was first apparent at 7 h after exposure to
CdCl2 and was sustained for up to 24 h (Fig.
6A), AT5BIVA cells exhibited a marked induction of HO-1 that
was first apparent at 4 h and continued to increase for up to
24 h. The extent of HO-1 induction in MRC5CV1/pEAT22 cells, which
express ATM antisense RNA, was intermediate between those apparent in
MRC5CV1 and AT5BIVA cells (Fig. 6B), suggesting an inverse
relation between the level of CdCl2-induced oxidative stress and the abundance of ATM in these cells. These data thus indicate that ATM plays an important role in modulating intracellular oxidative stress generated by the potent pro-oxidant,
CdCl2.

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Fig. 6.
Induction of HO-1 expression by
CdCl2 and ionizing radiation in normal human fibroblasts
and AT cells. A, MRC5CV1 and AT5BIVA cells were
incubated with 10 µM CdCl2 for the indicated
times, after which nuclear and cytosolic proteins were subjected to
immunoblot analysis with antibodies to HO-1. B, MRC5CV1,
AT5BIVA, and MRC5CV1/pEAT22 cells were incubated with 10 µM CdCl2 for the indicated times, after which
the abundance of HO-1 in the nucleus was evaluated as in A. C, MRC5CV1, AT5BIVA, and AT5BIVA/pMAT1 cells were incubated in the
absence or presence of 10 µM CdCl2 for
16 h and exposed (or sham) to 20 Gy of ionizing radiation 1 h
before the end of the incubation. The amount of HO-1 in the nucleus was
then determined by immunoblot analysis.
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Finally, we investigated the role of ATM in modulation of oxidative
stress generated by ionizing radiation in MRC5CV1, AT5BIVA, and
AT5BIVA/pMAT1 cells (Fig. 6C). Ionizing radiation induced a
low level of HO-1 expression in MRC5CV1 cells but had no effect on this
parameter in AT5BIVA or AT5BIVA/pMAT1 cells. Exposure of AT5BIVA/pMAT
cells to 10 µM CdCl2 for 16 h resulted
in an induction of HO-1 expression that was greater than that apparent
in MRC5CV1 cells but smaller than that apparent in AT5BIVA cells.
Furthermore, whereas the induction of HO-1 in AT5BIVA or MRC5CV1 cells
in response to CdCl2 treatment was not further increased by
exposure to ionizing radiation, that apparent in AT5BIVA/pMAT1 cells in
which ATM expression was induced by pretreatment with CdCl2
was potentiated by irradiation. These data suggest that MRC5CV1 cells,
but not AT5BIVA cells, were able to sense and to respond to ionizing
radiation-induced oxidative stress and that expression of recombinant
ATM in AT5BIVA cells restored the cellular response to ionizing
radiation-induced oxidative stress. Taken together, our results suggest
a role for ATM as a sensor of ionizing radiation-induced oxidative
stress and as a modulator of intracellular redox regulatory pathways in
cells exposed to ionizing radiation or CdCl2.
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DISCUSSION |
Intracellular accumulation of Cd2+ results in a
variety of cellular responses including transcriptional activation of
the proto-oncogene c-jun (29, 33) as well as oxidative
damage to cellular macromolecules (4-11). Disruption of intracellular
sulfhydryl homeostasis and reaction with protein thiol groups are
thought to contribute to the Cd2+-induced oxidative stress
(10). Ionizing radiation also damages cellular macromolecules as a
result of the generation of reactive oxygen species (30). We have now
shown that CdCl2 induces a biphasic phosphorylation of
c-Jun on Ser63 and Ser73 residues in both
normal and AT fibroblasts. Furthermore, this Cd2+-induced
c-Jun phosphorylation was mediated, at least in part, through a
signaling pathway activated by oxidative stress. Thus, preincubation of
cells with the antioxidant NAC markedly reduced the extent of
CdCl2-induced phosphorylation of c-Jun. Our data also
revealed that the extent of the second phase c-Jun phosphorylation induced by CdCl2 is much greater in AT cells than in normal
cells, whereas the extent of the first phase is similar in the two
types of cells. Cadmium chloride has previously been shown to induce a
biphasic increase in the abundance of c-jun mRNA in
mouse liver cells (63); whereas the first phase was suggested to result from an early cellular response to Cd2+ itself, the second
phase was attributed to Cd2+-induced cytotoxicity.
Similarly, the exaggerated second phase of c-Jun phosphorylation in AT
cells exposed to CdCl2 may represent a cellular response to
unchecked Cd2+-induced cytotoxicity, presumably
attributable to the excessive generation of oxidative stress.
We have previously shown that the p46 isoform of JNK contributes to the
ionizing radiation-induced phosphorylation of c-Jun in normal human
fibroblasts, and that p55 isoform of JNK, which is constitutively
expressed in both normal and AT human fibroblasts, is not
phosphorylated in response to ionizing radiation (37). We have now
shown that CdCl2 induces a delayed phosphorylation of p55
as well as phosphorylation of p46 in AT cells. Phosphorylation of p55
was not induced by CdCl2 in MRC5CV1 cells (data not shown). However, CdCl2 treatment induced a biphasic increase in JNK
activity in both MRC5CV1 and AT5BIVA cells even though the greater
extent of the second phase of CdCl2-induced phosphorylation
of endogenous c-Jun apparent in AT5BIVA cells is not attributable to a
greater increase in JNK activity in vitro. We interpret
these data to show that different isoforms of JNK, p55 and p46, may
participate in different phases of c-Jun phosphorylation in response to
CdCl2.
Alternatively, the apparent mechanistic dissociation of JNK activity
and c-Jun phosphorylation may be due to another mechanism, which
involves residues other than amino acid 1-89. This observation is
consistent with recent reports demonstrating JNK-independent phosphorylation of c-Jun on serine 63 and 73 residues by a novel protein complex, which requires COOH-terminal amino acid residues including DNA binding and dimerization domains for its activity (58,
59).
Evidence suggests that AT cells exhibit an increased basal state of
oxidative stress. ATM has been suggested to function as a sensor of
reactive oxygen species and oxidative damage to cellular macromolecules
(31, 64, 65). Thus, ATM-deficient mice show evidence of oxidative
damage to proteins and lipids in organs such as the brain and testes,
and they exhibit an increased abundance and activity of HO-1 in various
cell types including Purkinje cells of the cerebellum (65). Induction
of HO-1 expression has been associated with a generalized cellular
response to oxidative stress induced by a variety of stimuli in
mammalian cells (62). We have now shown that CdCl2
generates a greater extent of oxidative stress in AT cells than in
normal cells, and our data suggest that ATM functions as a sensor of
ionizing radiation-induced oxidative stress and as a modulator of
intracellular redox regulatory systems. We examined induction of HO-1
expression as a marker for the generation of and cellular response to
oxidative stress. The extent of HO-1 induction in response to
CdCl2 was markedly greater in AT cells than in normal
cells. The pattern and kinetics of CdCl2-induced expression
of HO-1 in AT cells and in normal cells resembled those of the second
phase of c-Jun phosphorylation in the respective cells, supporting the
notion that the second phase of c-Jun phosphorylation is indeed
mediated by CdCl2-induced oxidative stress. In contrast to
the marked induction of HO-1 by CdCl2 in AT cells, HO-1
expression was not induced by ionizing radiation in these cells.
Irradiation of normal cells induced expression of HO-1 at a very low
level. These observations indicate that AT cells are unable to detect ionizing radiation-induced oxidative stress; however, these cells are
able to detect oxidative stress generated by CdCl2 and
exhibit a hypersensitive response, presumably because of impaired
intracellular antioxidant function.
Expression of full-length wild-type ATM in AT cells corrects various
aspects of the AT phenotype, including extreme radiosensitivity and
impaired activation of JNK in response to ionizing radiation (50).
Conversely, expression of full-length ATM antisense RNA in normal
lymphoblastoid cells was shown to convert the normal phenotype of these
cells to the one resembling that of AT cells (49). Our results with
cells transfected with ATM cDNA in the normal or antisense
orientation further support a role for ATM as a sensor and modulator of
oxidative stress. Thus, AT5BIVA/pMAT1 cells, which express ATM in a
CdCl2-inducible manner, exhibit induction of HO-1
expression in response to ionizing radiation after pretreatment with
Cd2+. Expression of HO-1 was not increased further by
ionizing radiation in CdCl2-pretreated AT5BIVA cells,
indicating that Cd2+-induced expression of ATM was
responsible for the induction of HO-1 in response to ionizing radiation
in AT5BIVA/pMAT1 cells and that ATM functions as a sensor of ionizing
radiation-induced oxidative stress. MRC5CV1/pEAT22 cells, in which the
abundance of ATM is reduced as a result of expression of ATM antisense
RNA, the extent of HO-1 induction in response to CdCl2 was
greater than that apparent in MRC5CV1 cells but smaller than that
observed in AT5BIVA cells. These observations indicate that suppression of ATM function in normal human fibroblasts results in an increased sensitivity to CdCl2-induced oxidative stress. Taken
together, our results indicate ATM as a sensor of ionizing
radiation-induced oxidative stress and as a modulator of intracellular
sulfhydryl homeostasis.
We have shown that ionizing radiation-induced c-Jun phosphorylation in
normal fibroblasts is also mediated, at least in part, by oxidative
stress. Preincubation of cells with NAC markedly reduced the extent of
ionizing radiation-induced c-Jun phosphorylation. AT cells are not able
to sense ionizing radiation-induced oxidative stress and do not exhibit
c-Jun phosphorylation in response to irradiation. Our data indicate
that intact ATM function is required for oxidative stress-mediated
c-Jun phosphorylation in response to ionizing radiation. AT5BIVA/pMAT1
cells in which full-length wild-type ATM is expressed are able to sense
ionizing radiation-generated oxidative stress and exhibit c-Jun
phosphorylation in response to irradiation. Conversely, in
MRC5CV1/pEAT22 cells, which express ATM antisense RNA, the extent of
c-Jun phosphorylation in response to ionizing radiation was reduced
compared with that apparent in MRC5CV1 cells. We also showed that ATM
modulates the oxidative stress and the resulting c-Jun phosphorylation
induced in cells by CdCl2. The excess oxidative stress
generated in AT cells as well as the resulting c-Jun phosphorylation in
response to CdCl2 were markedly reduced in the
ATM-expressing AT cell line AT5BIVA/pMAT1 relative to those apparent in
the parental AT5BIVA cells. Furthermore, the synergistic effect of BSO
on CdCl2-induced c-Jun phosphorylation apparent in AT5BIVA
cells was not observed in AT5BIVA/pMAT1 cells. Conversely,
MRC5CV1/pEAT22 cells showed an increased sensitivity to
CdCl2-induced oxidative stress and an increased extent of
c-Jun phosphorylation in response to CdCl2 compared with
those apparent in the parental MRC5CV1 cells.
ATM has been shown to associate with DNA in normal human cells, and
ionizing radiation-induced double-strand DNA breaks potentiates this
association, suggesting a role for ATM as a sensor of DNA damage (66).
Cadmium ion has been shown to induce single-strand DNA breaks (12, 13).
Therefore, it would be noteworthy to clarify whether a role for ATM in
CdCl2-induced c-Jun phosphorylation was to sense DNA lesion
or to modulate intracellular sulfhydryl homeostasis. Cadmium
ion-induced single-strand DNA breakage has been shown to occur in a
variety of cell systems at relatively high concentrations of
CdCl2 (at 500 µM to 5 mM). A
significant line of evidence suggests that this DNA damage is the
result of generation of reactive oxygen species in cells exposed to
CdCl2 (67-69). The inability of CdCl2 to
induce single-strand DNA breakage under anaerobic conditions, as
measured by alkaline elution technique, and also inhibition of this DNA
damage by antioxidants have been demonstrated (68). However, low
concentrations of CdCl2 (less than 10 µM) can
effectively induce oxidative stress in cultured cells by disrupting
intracellular sulfhydryl homeostasis (10, 11). Recently, inhibition of
proteins involved in the intracellular reduction of protein
glutathionyl-mixed disulfides, such as thiotransferase, GSSG reductase,
thioredoxin, and thioredoxin reductase, has been demonstrated in the
presence of very low concentrations of CdCl2 (70). The
concentration of CdCl2 used in our study was 10 µM. At this concentration, the effect of
CdCl2 on single-strand DNA breakage would be considered as
minimal, if at all (68-70).
Unlike the abnormal c-Jun phosphorylation apparent in AT cells in
response to ionizing radiation or CdCl2, AT cells have been shown to exhibit normal c-Jun phosphorylation in response to UV-C light
(37) and also normal JNK activation in response to anisomycin or UV-C
light (39). There is evidence that ribostressor anisomycin does not
induce oxidative stress in cells (71) and that oxidative damage
generated by UV-C light is shown to be minimal in cells (72).
Furthermore, pretreatment with NAC has been shown to inhibit the
activation of JNK1 by various oxidative stressors but not by UV light
or anisomycin (71). In our study, we have shown that c-Jun
phosphorylation induced by ionizing radiation or CdCl2, which generates oxidative stress in cells, was significantly reduced by
NAC pretreatment. Therefore, our data support that ATM plays a role in
c-Jun phosphorylation in response to ionizing radiation and
CdCl2 through sensing and/or modulating oxidative stress
generated in cells by these stimulants.
In conclusion, our data indicate that ATM is required for the oxidative
stress-mediated signaling that leads to c-Jun phosphorylation in
response to ionizing radiation and that ATM plays an important role in
modulating the oxidative stress and the resulting c-Jun phosphorylation
induced by CdCl2. The biochemical mechanisms by which ATM
senses ionizing radiation-induced oxidative stress and modulates
intracellular sulfhydryl homeostasis remain to be elucidated.