(Stroke. 2001;32:1401.)
© 2001 American Heart Association, Inc.
Original Contributions |
Early Decrease in DNA Repair Proteins, Ku70 and Ku86, and Subsequent DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice
From the Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, Calif.
Correspondence to Pak H. Chan, PhD, Neurosurgical Laboratories, Stanford University, 1201 Welch Rd, MSLS #P304, Stanford, CA 94305-5487. E-mail phchan{at}leland.stanford.edu
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Abstract |
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Background and Purpose—Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway. To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice.
Methods—Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery. Ku protein expression was studied by immunohistochemistry and Western blot analysis. DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL). The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion.
Results—Immunohistochemistry showed constitutive expression of Ku proteins in control brains. The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours. Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P<0.001). DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours. Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining.
Conclusions—These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI.
Key Words: cerebral ischemia, focal • DNA fragmentation • DNA repair • mice
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Introduction |
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Cell death linked to oxidative DNA damage has been implicated in the pathogenesis of ischemia/reperfusion injury. The production of reactive oxygen species (ROS) during reperfusion in the ischemic lesion has been known to cause protein oxidation, lipid peroxidation, and DNA damage.1 Oxidative DNA damage after ischemia/reperfusion was reported to occur in the early stage of reperfusion without cellular injury or DNA fragmentation.2 3 During this process, the DNA repair enzyme prevents ongoing cell death by repairing damaged DNA, but continuous or severe DNA damage beyond the capability of the DNA repair enzyme triggers a cell-death pathway, such as necrosis or apoptosis, which is dependent on the severity of the insult,3 4 suggesting that the DNA repair enzyme may have a role in the deterrence of cell-death pathway induction after ischemia/reperfusion. In recent years, DNA damage and repair have been drawing more attention in the field of ischemic neuronal injury; it has been reported that overwhelming DNA damage against DNA repair could induce apoptosis in several different models.5 6
Ku70 (70-kDa) and Ku86 (80-kDa) proteins are DNA-binding regulatory subunits of the DNA-dependent protein kinase (DNA PK), which is composed of the 470-kDa catalytic subunit and Ku proteins.7 8 Ku70 and Ku86 proteins as regulatory parts of the DNA PK initiate the repair process of DNA double-strand breaks, which produce DNA fragmentation, by activating DNA PK after binding to DNA double-strand breaks.7 In addition to the regulatory function of the Ku proteins in DNA PK, heterodimers of both Ku70 and Ku86 also have independent DNA repair functions, such as single-stranded DNA-dependent ATPase activity and the binding and repair of broken single-stranded DNA, single-stranded nicks, gaps in DNA, and single strand–to–double-strand transitions in DNA.7 8 The Ku-encoding gene is identical to the human x-ray repair cross-complementing gene group 5 (XRCC5), which is the encoding gene of Ku86, and XRCC6, which is the encoding gene of Ku70.9 10 A constitutive expression of Ku mRNA has been found in mice.11 Brief ischemia/reperfusion in rabbit spinal cord induces reversible neurological deficits with increased DNA-binding activity of Ku, which is an indicator of activating DNA PK, whereas severe ischemia/reperfusion causes permanent deficits accompanied by decreased DNA-binding activity of Ku, suggesting that Ku is involved in the initiating step of the cell-death pathway, particularly DNA fragmentation in ischemia/reperfusion injury.12 Recently, we have shown that a decrease in the DNA base excision repair proteins, apurinic/apyrimidinic endonuclease and XRCC1, precedes the occurrence of DNA fragmentation after focal cerebral ischemia (FCI).13 14 Therefore, we assume that Ku may play a role in cell-death mechanisms, especially DNA fragmentation after cerebral ischemia/reperfusion, and that Ku reduction may involve the mechanism of apoptotic cell death. However, this has not been clearly elucidated.
To explore the change in Ku proteins in the cell-death pathway after cerebral ischemia/reperfusion injury, we examined Ku expression by immunohistochemistry and Western blot before and after FCI and evaluated the temporal and spatial relationship between Ku protein and DNA fragmentation.
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Materials and Methods |
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Focal Cerebral Ischemia
All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and were approved by Stanford’s Administrative Panel on Laboratory Animal Care. Male CD-1 mice (35 to 40 g, 3 months old) were subjected to transient FCI by intraluminal middle cerebral artery (MCA) blockade with a nylon suture, as previously described.15 The mice were anesthetized with 2.0% isoflurane in 30% oxygen and 70% nitrous oxide with the use of a face mask. The rectal temperature was controlled at 37±5°C with a homeothermic blanket. Cannulation of the femoral artery allowed monitoring of blood pressure and arterial blood gases, with samples for analysis taken immediately after cannulation, 10 minutes after occlusion, and 10 minutes after reperfusion (Chiron Diagnostics Ltd). After the midline skin incision, the left external carotid artery was exposed, and its branches were electrocoagulated. An 11.0-mm 5-0 surgical monofilament nylon suture, blunted at the end by heating, was introduced into the left internal carotid artery through the external carotid artery stump. After 60 minutes of MCA occlusion, blood flow was restored by the withdrawal of the nylon suture.
DNA Extraction and Gel Electrophoresis
Animals were killed 4 and 24 hours after
ischemia/reperfusion. Ischemic tissue (30 to 50 mg wet
weight) was taken from the third 2-mm section along with homologous
tissue from the contralateral side after the brain was cut coronally.
Samples were incubated overnight in 0.6 mL lysis buffer (0.5% SDS,
10 mmol/L Tris-HCl, and 0.1 mol/L EDTA) with proteinase K (500
µg/mL, Boehringer-Mannheim) at 55°C. The DNA was extracted
with equal volumes of phenol and phenol/chloroform/isoamyl alcohol
(25:24:1) and precipitated overnight in 0.2 mol/L sodium chloride in
100% ethanol at -20°C. The DNA was washed 2 times with 75%
ethanol, air-dried, and resuspended. The DNA concentration was measured
with TO-PRO-1 iodide stain (Molecular Probes) and incubated for 30
minutes at 37°C in 0.1 µg/µL DNase-free RNase
(Boehringer-Mannheim), and 5 µg per lane was loaded onto an
1.8% agarose gel containing ethidium bromide (0.3 g/mL) and
electrophoresed at 100 V for 2 hours. The DNA was visualized and
photographed by using UV transillumination (300-nm
wavelength).
Detection of Oxidative Cellular Injury
After Reperfusion
To confirm the occurrence of oxidative stress, which
produces early DNA injury after ischemia/reperfusion, in situ
detection of oxidized hydroethidine (HEt) was performed 1 hour and 24
hours after reperfusion by modifying a previously described
method.16 Detection of
oxidized HEt is a useful tool to show ROS production because
HEt is oxidized to ethidium mainly by the superoxide anion
radical.17 18 Two
hundred microliters of HEt (stocked concentration, 100 mg/mL in
dimethyl sulfoxide; diluted concentration, 1 mg/mL in PBS with
sonication before use; Molecular Probes) was administrated
intravenously 5 minutes before the induction of
ischemia. The animals were killed 1 hour after reperfusion by
transcardial perfusion with 200 mL of 10 U/mL heparin in 0.9% saline
and 200 mL of 3.7% formaldehyde in 0.1 mol/L PBS. After postfixation
overnight, the brains were sectioned with a vibratome into a
thickness of 50 µm at the level of the mid striatum. The sections
were then counterstained with Hoechst 33258 (Molecular Probes) for 15
minutes. These sections were observed with a microscope under
fluorescent light (HBO, Zeiss). Intensity and expression
patterns of the oxidized HEt and nuclear fragmentation as an indication
of apoptosis were analyzed after taking photographs
with a digital camera (AxioVision, Zeiss) by double exposure to
oxidized HEt and Hoechst 33258.
Western Blot Analysis
Whole-cell protein extraction was performed as
previously described (n=5
each).14 Samples were
obtained from the ischemic core on the ipsilateral side and
from homologous tissue on the contralateral side. Approximately 30 mg
of both the ipsilateral and contralateral striata were cut into pieces
after 4 and 24 hours of reperfusion and put into 10x vol of
Tris-glycine SDS sample buffer (Novex). Samples were then gently
homogenized 20 times in a
polytetrafluoroethylene Dounce
homogenizer (Wheaton). Equal amounts of the samples (10
µL) were loaded per lane. The primary antibodies were either 1:1000
dilution of goat polyclonal antibody against Ku70 and Ku86 (Santa Cruz
Biotechnology) or 1:10 000 dilution of anti–ß-actin monoclonal
antibody (Sigma Chemical Co). For Ku70 and Ku86 detection, Western
blots were performed with horseradish peroxidase–conjugated anti-goat
IgG with a chemiluminescent kit (Amersham International). As the
internal control, Western blot analysis of ß-actin was
performed with horseradish peroxidase–conjugated anti-mouse IgG
reagents (Amersham). After the film was scanned with a GS-700 imaging
densitometer (Bio-Rad), a quantitative analysis was performed
by use of Multi-Analyst software (Bio-Rad).
Immunohistochemistry of Ku Protein
Anesthetized animals were perfused with 200
mL of 10 U/mL heparin in 0.9% saline and 3.7% formaldehyde in 0.1
mol/L PBS (pH 7.4) after 5 minutes and 2, 4, 8, and 24 hours of
reperfusion (n=6 or 7 each), as were normal control animals (n=3). The
brains were removed, postfixed overnight in 3.7% formaldehyde,
sectioned at 50 µm with a vibratome, and processed for
immunohistochemistry. The sections were incubated with blocking
solution as previously
described16 and incubated
with a primary antibody, goat polyclonal antibody for Ku70 and Ku86, at
a dilution of 1:100 (Santa Cruz Biotechnology). The sections were
incubated with biotinylated anti-goat IgG (10 µg/mL, Vector
Laboratories) and incubated with a complex of avidin and biotinylated
horseradish peroxidase (VECTASTAIN Elite ABC Kit, Vector Laboratories).
Ku labeling was visualized by using diaminobenzidine (DAB) and nickel
chloride as previously
described.16 The nuclei were
counterstained with methyl green. As a negative control, sections were
incubated without the primary antibody. We also performed the
preabsorption method by using Ku-blocking peptides (Santa Cruz
Biotechnology) to confirm the specificity of the
antibodies.
Double Labeling With Ku Immunohistochemistry
and TUNEL
To evaluate the relationship between Ku
protein expression and DNA fragmentation, we performed single staining
with terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP nick end-labeling (TUNEL) and double staining with
Ku protein and TUNEL by use of a modified method as previously
described.14 Alternative
sections at the level of the caudate putamen were used for Ku70 and
Ku86 immunohistochemistry. The sections were immunostained
with the Ku70/86 antibody for double staining as described above.
Staining was visualized with the use of DAB (0.25 g/L). For single
TUNEL staining, the above process was omitted. The slides were
incubated for 30 minutes in NeuroPore (Trevigen) for permeation of
tissue and placed in 1x TdT buffer (GIBCO-BRL) for 15 minutes,
followed by reaction with TdT enzyme (GIBCO-BRL) and biotinylated
16-dUTP (Boehringer-Mannheim) at 37°C for 60 minutes. The
slides were then washed twice in 150 mmol/L sodium chloride and
15 mmol/L sodium citrate (pH 7.4) for 15 minutes, followed by a
washing in PBS twice for 15 minutes. Avidin-biotin horseradish
peroxidase solution (Vector) was applied to the sections for 30
minutes. TUNEL labeling was visualized with DAB (0.25 g/L) and nickel
chloride (10 g/L). For single TUNEL staining, the nuclei were
counterstained with methyl green.
Quantification and Statistical
Analysis
The number of Ku70 and Ku86 immunoreactive cells and
methyl green–positive cells was counted in a high-powered field
(x400) and expressed as the percentage of Ku-positive cells, as
previously described.14 The
number of TUNEL-positive cells was counted as previously
described.9 The statistical
comparisons among multiple groups were made by ANOVA, followed by the
Fisher protected least significant difference test, whereas comparisons
between 2 groups were performed by the unpaired
t test (StatView, version 5.
01, SAS Institute). Significance between groups was assigned at
P<0.05.
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Physiological Data and Cerebral Infarction
The preischemic physiological values were as follows: mean arterial blood pressure 71.40±3.03 mm Hg, PaO2 149.25±20 mm Hg, PaCO2 29.05±3.62 mm Hg, and pH 7.34±0.06 (mean±SD). There was no deviation from these values over the period of assessment. An ischemic lesion of the core of the caudate putamen was visible as a pale slightly stained area by cresyl violet staining in the ischemic hemisphere as early as 5 minutes after reperfusion, and it extended to the entire MCA territory at 4 hours by cresyl violet staining (data not shown). The time-dependent increase of infarction in the mouse brains with the use of intraluminal suture blockade is consistent with previous reports that used the same focal stroke model in mice.9
DNA Laddering in Gel Electrophoresis of Genomic
DNA
To confirm nucleosomal DNA fragmentation, which is
characteristic of apoptosis, we analyzed DNA isolated
from both the ischemic hemisphere and the contralateral
hemisphere. DNA laddering was absent in both the control tissue and
ischemic tissue 4 hours after ischemia
(Figure 1
, lanes 2 and 3). A significant amount of DNA
laddering was detected 24 hours after ischemia/reperfusion
(Figure 1, lane 4).
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200 bp 24 hours after ischemia/reperfusion (24H, lane 4) but was not seen 4 hours after reperfusion (4H, lane 3). DNA was labeled with ethidium bromide and electrophoresed on a 1.8% agarose gel. The number of each base pair is shown on the left side. MW indicates molecular weight.
Oxidative Cellular Injury After
Reperfusion
The production of ROS was shown by oxidized HEt
signals as small particles in the cytosol. One hour after reperfusion,
the ischemic lesion showed significantly increased oxidized HEt
signals
(Figure 2A) compared with the nonischemic
hemisphere
(Figure 2B). Under high magnification, cells with intensive
oxidized HEt signals in the cytosol were detected, but cellular injury
was not observed 1 hour after reperfusion
(Figure 2C), and on the contralateral side, cells with
oxidized HEt signals were few, and they were confined to the
perinuclear area
(Figure 2D). Twenty-four hours after reperfusion, cells with
oxidized HEt signals showed nuclear fragmentation, indicative of
apoptosis in the ischemic lesion
(Figure 2E, arrowheads).
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Western Blot Analysis of Ku70 and Ku86
Protein Expression After Reperfusion
Ku70 immunoreactivity was evident as a single band of
molecular mass of 70 kDa in the normal brain and contralateral
nonischemic brain after reperfusion
(Figure 3A, lanes 1 and 3, top). It was decreased 4
hours after reperfusion
(Figure 3A, lane 2, top) and was markedly decreased 24 hours
after reperfusion in the ischemic lesion
(Figure 3A, lane 4, top). Contrarily, a consistent
amount of ß-actin immunoreactivity is seen at the bottom of
Figure 3A, suggesting that the amount of the loaded protein
was consistent. Ku86 expression also showed a decrease at 4
hours and a marked decrease at 24 hours
(Figure 3B). Statistical analysis confirmed the
significant decrease (P<0.001)
of Ku70 and Ku86 after ischemia (optical density of the Ku70
bands was 2.12±0.22, 0.86±0.23, 0.28±0.11 and that of the Ku86 bands
was 2.15±0.28, 0.91±0.31, 0.25±0.16 in the nonischemic
brain, ischemic brain at 4 hours of reperfusion, and
ischemic brain at 24 hours of reperfusion,
respectively).
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Immunohistochemistry and Time Course of Ku
Expression After Reperfusion
Immunostaining without a primary
antibody or after the preabsorption procedure showed no
immunoreactivity
(Figure 4A). Ku70 protein was constitutively expressed
in the entire region of the normal mouse brain. It was mainly expressed
in the nucleus of the cortex
(Figure 4B) and caudate putamen
(Figure 4C). Ku expression in the contralateral hemisphere
after reperfusion was not different from that in the normal brain. Five
minutes after ischemia/reperfusion, reduction of Ku70 was
observed in the lateral caudate putamen
(Figure 4E) but not in the cortex
(Figure 4D). After 4 hours of reperfusion, Ku70 expression
was reduced in the entire MCA territory cortex
(Figure 4F), including the caudate putamen
(Figure 4G). This reduction was sustained up to 24 hours in
the MCA territory cortex
(Figure 4H) as well as in the caudate putamen
(Figure 4I). The changes in Ku86 protein expression before
and after ischemia/reperfusion were similar to those of Ku70.
As shown in
Figure 4J and 4K, the number of Ku70- and Ku86-expressing
cells was significantly reduced 5 minutes after reperfusion in the
caudate putamen (P<0.05) and 2
hours after reperfusion in the MCA territory cortex, including the
caudate putamen (P<0.001).
Approximately 70% to 80% of the cells lost their Ku immunoreactivity
4 hours after reperfusion.
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Relationship Between DNA Fragmentation and
Reduction of Ku Expression
No TUNEL-positive cells were seen in the ipsilateral
caudate putamen 4 hours after reperfusion
(Figure 5A). TUNEL-positive cells were widely
distributed 24 hours after reperfusion, showing apoptotic
characteristics such as condensed or fragmented nuclei with small
particles in the nuclei that resembled apoptotic bodies
(Figure 5B). No TUNEL-positive cells were detected on the
contralateral side
(Figure 5C). Double staining with Ku and TUNEL 24 hours after
reperfusion showed that most TUNEL-positive cells did not show Ku
immunoreactivity
(Figure 5D, arrowheads). In contrast, Ku-immunoreactive cells
were not TUNEL positive
(Figure 5D, stars). At a higher magnification, cells in the
contralateral nonischemic brain showed much stronger nuclear Ku
immunoreactivity
(Figure 5E) compared with the TUNEL-negative cells in the
ischemic lesion
(Figure 5F). TUNEL-positive cells in the ischemic
brain showed condensed and fragmented nuclei without Ku
immunoreactivity
(Figure 5G). Temporal profiles of DNA fragmentation and Ku
expression showed that a reduction in Ku-positive cells preceded an
appearance of TUNEL-positive cells
(Figure 5H).
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The present study shows that early reduction in Ku, a DNA repair enzyme, may underlie, in part, the mechanism of cell demise through the oxidative stress–DNA damage–DNA fragmentation pathway after FCI. This conclusion is based on our findings that a marked DNA fragmentation by genomic DNA analysis (Figure 1
), which was temporally accompanied by obvious ROS
production in apoptotic cells with fragmented nuclei
confirmed by HEt in situ and Hoechst nuclear staining
(Figure 2), was observed in the ischemic lesion 24
hours, but not 4 hours, after reperfusion, whereas Ku expression, which
was detected constitutively in the normal mouse brain, was
significantly reduced in the ischemic lesion as early as 4
hours after reperfusion, and the reduction of Ku remained reduced until
24 hours after reperfusion, determined by Western blot
(Figure 3) and immunohistochemistry
(Figure 4). Furthermore, temporal and anatomic relationships
between the loss of Ku expression and DNA fragmentation were shown by
double staining with Ku and TUNEL
(Figure 5).
This is a first demonstration of the close relationship
between Ku reduction and DNA fragmentation in mice after cerebral
ischemia/reperfusion. Ku is known to play a critical role in
the repair of DNA double-strand breaks by activating DNA PK as a
regulatory component; moreover, Ku itself stabilizes broken DNA ends,
brings them together, and prepares them for ligation and
removal.7 19 On
the basis of these findings, it is conceivable that Ku proteins may
play a role in the mechanism of DNA fragmentation, which is destined to
cause apoptosis, after FCI. However, little is known about the
interaction between Ku and DNA-fragmented cell death after FCI. In the
present study, the expression of Ku in Western blot
analysis was decreased after ischemia/reperfusion,
whereas the expression of ß-actin was without effect
(Figure 3). These data suggest that the reduction of Ku was
not caused by nonselective degradation of proteins after
ischemia. Moreover, double labeling with Ku and TUNEL staining
revealed that all TUNEL-positive cells had completely lost Ku
immunoreactivity. In addition, the quantitative analysis of
TUNEL-positive and Ku-immunopositive cells clarified that the decrease
in Ku-immunopositive cells preceded the increase in TUNEL-positive
cells
(Figure 5). These data suggest that the loss of Ku and the
failure of the DNA repair mechanism might contribute to DNA
fragmentation after FCI in mice. Although there are contradictory
reports indicating that a change in Ku was not
observed20 or that Ku was
significantly decreased in the cells undergoing apoptosis by
potent apoptotic
stimuli,21 a recent study
has shown that Ku-deficient cells in mutant mice are extremely
sensitive to apoptotic stimuli and show more DNA fragmentation
than do cells in wild-type mice because of the deficiency in DNA
double-strand break
repair.22 These data
performed in mutant mice with a Ku deficiency agree with the
present proposal that the loss of Ku may contribute to the
mechanism of DNA-fragmented cell death after FCI.
However, the exact mechanism by which the selective reduction of Ku occurs after FCI is unclear. It has been reported that caspase-3 is responsible for the proteolysis of the 470-kDa catalytic subunit of DNA PK, a catalytic component in the DNA PK complex,23 and that reduction of Ku protein levels and binding activity in the cells undergoing apoptosis were blocked by treatment with a caspase-3 inhibitor.16 These reports suggest that the caspase cascade during apoptosis may involve the selective degradation of Ku. Furthermore, using ischemia/reperfusion in rabbit spinal cord, a recent study demonstrated the reduction of Ku DNA binding activity (which is critical to DNA PK activation) after prolonged ischemia that was accompanied by a reduction in the Ku protein and the proteolysis of poly(ADP-ribose) polymerase, one substrate of caspases.12 This suggests that the reduction of Ku might be involved downstream from the caspase-activating apoptotic pathway after ischemia/reperfusion injury. However, other possible causes of Ku reduction, such as a decrease in Ku protein synthesis, including translational dysfunction or a change in posttranslational regulation after ischemia/reperfusion, remain to be determined.
The present study demonstrated that oxidized HEt
signals, indicative of oxidative stress, were increased in the early
ischemic lesion without morphological cellular changes as early
as 1 hour after reperfusion and that the nuclear fragmented cells with
oxidized HEt signals were detected 24 hours after reperfusion
(Figure 2). This may suggest that early and sustained
oxidative stress is involved in the relatively delayed
apoptotic DNA fragmentation after ischemia/reperfusion.
Furthermore, the most rapid decline in the early reduction of Ku
occurred at 30 minutes to 2 hours in the caudate putamen and at 2 to 4
hours in the MCA territory cortex by the quantitative analysis
of Ku immunohistochemistry
(Figure 4), suggesting that the full-scale loss of Ku may
occur in the ischemic lesion after a short time frame after
reperfusion. It has been reported that oxidative stress and reversible
DNA damage, which is recovered by DNA repair enzymes, occur immediately
after reperfusion in the ischemic lesion and that irreversible
DNA damage follows sustained oxidative DNA injury that exceeds the
controls of the DNA repair
system.2 3 Thus,
the present data suggest that the reduction in Ku and the
subsequent failure of DNA repair may be linked with the mechanism of
irreversible oxidative DNA injury. Although the cause of Ku reduction
is unclear, using mutant mice that overexpress copper/zinc superoxide
dismutase, we have recently shown that the copper/zinc superoxide
dismutase cytosolic antioxidant attenuates the reduction in
apurinic/apyrimidinic endonuclease, another DNA repair protein, and the
subsequent DNA fragmentation after
FCI24 supports this idea.
However, it is still not known whether ROS degrades the Ku protein
directly or secondarily through another oxidative stress–triggered
process, such as caspase activation. Nevertheless, ROS may directly
contribute, in part, to the reduction in the Ku protein after FCI,
inasmuch as ROS could reduce DNA repair
activity25 and directly
damage the DNA repair
enzyme.26 In the case of
severe oxidative DNA damage against DNA repair, the activated
p53 protein has been shown to induce apoptosis directly or
through the activation of other apoptosis-regulating genes,
such as bax.27 28
Moreover, the finding that the reduction in p53 prevented
apoptotic cell death induced by a deficiency in XRCC4, one of
the DNA repair proteins that responds to x-ray–sensitive DNA damage,
in mutant mice29 suggests
that p53 may be involved in the apoptosis cascade triggered by
a defective DNA repair mechanism. As another subcellular mechanism of
apoptosis induced by oxidative DNA damage, it has been proposed
that overactivation of poly(ADP-ribose) polymerase by severely damaged
DNA may trigger apoptosis through excessive energy consumption
during the DNA repair
process.30 31
In conclusion, we have shown that Ku proteins were rapidly decreased as early as 4 hours after ischemia/reperfusion and that DNA-fragmented cell death occurred after the reduction of Ku expression. These results suggest that the loss of Ku proteins may produce incomplete DNA repair and might be responsible for subsequent DNA fragmentation after transient FCI.
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Acknowledgments |
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This work was supported by National Institutes of Health grants NS-14534, NS-25372, NS-36147, and NS-38653. Dr Chan is the recipient of the National Institute of Neurological Disorders and Stroke Javits Neuroscience Investigator Award. The authors thank C. Christensen for editorial assistance, Dr M. Fujimura for advice about surgical techniques, and L. Reola, B. Calagui, and J.O. Kim for technical assistance.
Received June 20, 2000; revision received January 30, 2001; accepted February 26, 2001.
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