From the
Ottawa Health Research Institute,
Neuroscience Group, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada, the
Department of Experimental Medicine, University
of Genoa, Viale Benedetto XV, Genoa, Italy, and the Departments of
¶Biochemistry and
||Pathology, Queen's University, Kingston, Ontario
K7L 3N6, Canada
Received for publication, March 19, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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Recent work has shown that the topoisomerase 1 inhibitor camptothecin causes apoptotic death of cultured cortical neurons (610). The tumor suppressor p53 is an important component of the numerous death pathways activated by DNA damage. We (11, 12) and others (13, 14) have shown that p53 is up-regulated prior to death commitment and is required for activation of the conserved death program consisting of Bax translocation (12), cytochrome c release (10, 15), and caspase activation (10, 15). However, the mechanism by which p53 is activated by DNA damage in neurons is not fully understood.
Calpains are calcium-dependent neutral proteases that have been implicated in a variety of physiologic and pathological conditions, including regulation of cell cycle progression, neuronal plasticity, and initiation of neuronal cell death (16). Calpains µ and m are the two most ubiquitously expressed forms of calpains, and they require interaction with a small regulatory calpain subunit encoded by the gene capn4 to function properly. The importance of calpains is underscored by the observation that mice deficient in the calpain small regulatory subunit die embryonically, perhaps due to cardiovascular defects (17, 18). With respect to calpain involvement in neuronal death, pharmacological calpain inhibitors have been shown to protect neurons from a variety of death stimuli, including ischemic/excitotoxic insults, both in vitro (1921) and in vivo (2224). However, the inhibitors utilized are known to inhibit targets other than calpains, and, therefore, the role of calpain in neuronal death has been unclear and controversial.
The regulation of calpain is complex and includes a requirement for calcium
(16), the endogenous cellular
calpain inhibitor calpastatin
(25), and translocation to
membranous compartments (26,
27). Calpains are reported to
modulate a wide variety of intracellular signaling pathways by targeted
cleavage of substrate proteins, including the Cdk5 activator p35
(28,
29), the NFB inhibitor
I
B (30), immediate
early genes such as c-Fos and c-Jun
(31), and the structural
proteins fodrin (32) and
spectrin (33). Calpains are
also thought to cleave and inactivate caspases, the executers of the death
signal in apoptosis (34). The
diversity of the substrates cleaved by calpain highlights the varied nature of
calpain-mediated signals and demonstrates its ability to regulate various cell
functions, possibly by modulating different proteins under different
contexts.
We currently examined the requirement for calpain activation in the death of cultured cortical neurons evoked by DNA damage. Multiple lines of evidence based upon studies of neuronally differentiated calpain-deficient stem cells and calpastatin overexpression, as well as multiple pharmacological calpain inhibitors, strongly suggest the involvement of calpains in neuronal death induced by DNA damage. Moreover, we find that calpain inhibition reduces p53 activation and consequent mitochondrial death effector signals. This evidence implicates p53 as a critical step by which calpains transduce the death signal.
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EXPERIMENTAL PROCEDURES |
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Generation of Recombinant AdenovirusThe recombinant adenoviral vectors were constructed using the AdEasy system as described previously (35). Calpastatin (RNCAST104), subcloned previously into a pAdLox (36), was excised using BamHI and EcoRI. The sequence encoding an enhanced green fluorescent protein (EGFP)1 was excised from pEGFP-C (Clontech) using AfeI and SalI. Both fragments were ligated into pShuttle-CMV to produce pShuttle-CMV-EGFP-Calpastatin. Calpastatin and the control EGFP adenovirus were amplified in 293 cells and purified over CsCl gradients. For primary cell infections, virus (multiplicity of infection 10) was added to neural cultures at the time of plating.
Culture and Survival of Cortical NeuronsMouse cortical
neurons were cultured from embryonic day 15 mice as described previously
(11). Neurons were plated into
24-well dishes (200,000 cells/well) or 6-well dishes (24 million
cells/well) coated with poly-D-lysine (100 µg/ml) in serum-free
medium (N2/Dulbecco's modified Eagle's medium (1:1) supplemented with 6 mg/ml
D-glucose, 100 µg/ml transferrin, 25 µg/ml insulin, 20
nM progesterone, 60 µM putrescine, and 30
nM selenium. One to two days after initial plating, the medium was
supplemented with camptothecin (10 µM) alone or with calpain
inhibitors as indicated in the text and figures. At appropriate times of
culture, cells were lysed, and the numbers of viable cells were evaluated.
Briefly, cells were lysed in 200 µl of cell lysis buffer (0.1 x PBS,
pH 7.4, containing 0.5% Triton X-100, 2 mM MgCl2, and
cetyldimethylethylammonium bromide (0.5 g/100 ml), which disrupts cells but
leaves the nuclei intact. Ten microliters of sample from each culture were
loaded onto a hemacytometer, and the number of healthy intact nuclei was
evaluated by phase microscopy. Nuclei that displayed characteristics of
blebbing, disruption of nuclear membrane, phase-bright apoptotic bodies, and
chromatin margination were excluded. All experimental points are expressed as
a percentage of cells plated on day 0. Alternatively, cells were collected and
analyzed for biochemical analyses as described below.
Culture and Survival of Stem Cell-derived NeuronsMice
heterozygous deficient for the small subunit of calpain (Capn4+/-)
were bred, and the embryos were isolated at embryonic day 10.5. Mouse
forebrain stem cells were cultured from embryonic telencephalons as described
previously (Ref. 37;
modified). Embryos were genotyped as described previously
(17). Stem cells were plated
at a density of 5 x 104 cells/ml and allowed to form
neurospheres in Dulbecco's modified Eagle's medium/F-12 medium supplemented
with 20 µg/ml basic fibroblast growth factor as described previously
(37). To passage, cells were
triturated to obtain a single cell suspension using a flame-polished glass
pipette. To differentiate the neural stem cells, they were plated into 24-well
dishes (50,000 cells/well) or 6-well dishes (one million cells/well)
coated with poly-L-ornithine (Sigma, P4957) in Neurobasal medium
(Invitrogen) supplemented with 0.5 mM glutamine, 50 units/ml
penicillin-streptomycin, l% N2 and 2% B27 supplements (Invitrogen), and 1%
nondialyzed fetal bovine serum. Seven days after initial plating, the medium
was supplemented with camptothecin (10 µM). At appropriate times
of culture, cells were fixed in methanol for 15 min and assessed for neuronal
survival as described below. Alternatively, cells were collected and analyzed
by Western blot analyses as described below.
Western Blot AnalysesCortical neurons or differentiated
stem cell cultures were dissociated and cultured as described above. Cortical
neurons were washed twice in PBS and harvested in SDS-loading buffer
(12). Differentiated stem
cells were washed twice in PBS, harvested in IP buffer (50 mM
HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1
mM dithiothreitol, 0.1% Tween 20, 10% glycerol, 0.1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 20 units/ml
aprotinin, 10 mM -glycerophosphate, 1 mM NaF, and
0.1 mM sodium orthovanadate), and sonicated at 4 °C for 10 s.
Samples containing 10 µg of protein were loaded onto SDS-polyacrylamide
gels and transferred onto nitrocellulose membrane as described previously
(11). Blots were probed with
anti-p53 1C12
[PDB]
(Cell Signaling Technology; 1:2000), calpain-cleaved spectrin
derived from a calpain-cleaved spectrin epitope as reported previously
(33), MDM2 (Santa Cruz
Biotechnology; 1:1000), or anti-
-actin (Sigma; 1:3000) primary
antibodies, followed by horseradish peroxidase-conjugated secondary antibody
(Bio-Rad; 1:3000) as indicated.
ImmunohistochemistryNeurons cultured as described above in
6-well dishes were untreated or treated with camptothecin with and without
calpain inhibitor cotreatment. Neuronal cultures were fixed in 4%
paraformaldehyde in phosphate buffer solution for 30 min at 4 °Cas
described previously (10,
15). The cells were incubated
with anti-cytochrome c (BD Biosciences; 1:400) or anti-p53 1C12
[PDB]
(Cell
Signaling Technology; 1:2000) primary and CY-3 (Jackson ImmunoResearch
Laboratories) secondary antibodies. To distinguish neuronal phenotypes from
glia in differentiated stem cell cultures, we used anti--III tubulin
primary (Berkeley Antibody; 1:500) and streptavidin Alexa Fluor (Molecular
Probes 1:200) secondary antibodies. Finally, cultures were incubated with
Hoechst 33258 (0.25 µg/ml) for 10 min. Cells were visualized under
fluorescent microscopy. The percentage of neuronal survival in stem cell
cultures was obtained by whole well counting of shrunken and condensed nuclei
of
-III tubulin-positive cells. Similarly, in cortical cultures infected
with adenovirus expressing calpastatin and/or GFP, the percentage of shrunken
nuclei and the expression of p53 and cytochrome c of infected cells
were assessed by randomly counting a total of 150 cells/well. Each data point
is the mean ± S.E. from three cultures.
Caspase ActivityCortical neurons were harvested at the indicated times for caspase activity. Briefly, cells were washed twice in PBS and collected in caspase lysis buffer as described previously (15). The cells were incubated on ice for 20 min and briefly sonicated for 3 s. The extracts were then centrifuged for 15 min at 12,000 rpm in an Eppendorf tabletop centrifuge. The supernatants were collected and assayed for protein concentration by Bradford reagent (Bio-Rad). Equal amounts of protein (5 µg) were incubated with the caspase-3 substrate DEVD-AFC as described previously, and fluorescence was measured using a fluorometer (400 nm excitation, 505 nm emission), as described previously (38).
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RESULTS |
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Calpain Inhibition and DNA Damage-induced DeathWe next
determined the functional consequences of calpain activation following DNA
damage-induced neuronal death. To examine this, we first utilized the
pharmacological calpain inhibitors MDL 28170
(39) and PD 150606
(40). As shown in
Fig. 2, A and
B, both inhibitors blocked neuronal death with in
vivo IC50 values of 50 and 25 µM,
respectively, at 14 h following camptothecin exposure. However, assessment of
protection at later points could not be performed due to toxicity of the
agents (data not shown). Neurons protected with the calpain inhibitors have
well defined, healthy somas, whereas those treated with camptothecin alone
show phase bright apoptotic bodies (Fig.
2C). Although neuritic processes were present in both PD
150606- and MDL 28170-treated cultures, they were generally fewer in number
relative to untreated cells. These observations are consistent with reports
linking calpains to process outgrowth
(41). Although the protection
offered by these inhibitors is concordant with the involvement of calpains in
neuronal death, there are possible alternative explanations for these effects.
Importantly, these inhibitors are known to block other targets, which include
cathepsins (40) and
calcineurins (43). These
potentially confounding effects have made interpretation of pharmacological
inhibitor experiments difficult. To overcome this, we explored two additional
and more targeted ways of calpain inhibition.
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In the first strategy, we infected cortical cells with an adenovirus
expressing calpastatin, the endogenous calpain inhibitor. Cortical neuronal
cultures were infected at the time of plating with recombinant adenovirus
expressing either GFP and calpastatin or GFP alone. After 24 h, the cultures
were exposed to camptothecin for 14 h. As shown in
Fig. 3, neurons overexpressing
calpastatin had healthy somas, intact nuclei, and reduced death when compared
with GFP-expressing controls similarly treated with camptothecin (90%
survival with calpastatin versus 50% with GFP).
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To provide further evidence for the involvement of calpain in cell death,
we explored whether calpain deficient neurons were also resistant to DNA
damage-induced death. Previous reports have indicated that cells deficient in
the small calpain subunit (Capn4) lack both calpain µ and m activity
(17). Because,
Capn4-/- mice do not survive beyond embryonic days 1012
(17), it is not possible to
obtain fully differentiated cortical neurons. To circumvent this problem, we
isolated neuronal stem cells from the forebrain region of Capn4+/+,
Capn4+/-, and Capn4-/- embryonic day 10.5 embryos.
Neuronal stem cells were then exposed to differentiation medium as described
previously (37), treated with
camptothecin, and fixed. Neuronally differentiated cells were identified by
staining with the neuronal marker -III tubulin, and death was assessed
by nuclear Hoechst staining. As shown in
Fig. 4, there was significantly
more survival in calpain-deficient cells when compared with litter mate
controls. Taken together, these data show that calpain inhibition by multiple
means inhibits neuronal loss and provide strong evidence for the importance of
calpains in neuronal death evoked by DNA damage.
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Calpains Mediate p53 InductionWe next examined the pathways
by which calpains may mediate DNA damage mediated neuronal death. We
(11) and others
(14) have reported previously
that p53 deficiency blocks death following camptothecin treatment. As shown in
Fig. 5, cellular levels of p53
protein were elevated by 2 h and increased up to 8 h after camptothecin
treatment. Little or no p53 was detectable in control cultures. We next asked
whether calpains might modulate p53 induction. Interestingly, there was a
reduction and a significant delay in p53 induction in cultures cotreated with
the MDL 28170 inhibitor (Fig. 5, A
and C). No p53 was detected at 4 h, and only 50% of
p53 levels was present at 8 h in comparison with neurons treated with
camptothecin alone. Similar results were obtained with the PD 150606 inhibitor
(Fig. 5B).
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To confirm these results and rule out the possibility that inhibition of p53 was due to nonspecific effects of the pharmacological calpain inhibitors, we also examined the effects of calpastatin expression on p53 induction evoked by camptothecin. Neuronal cultures were infected with GFP and calpastatin or GFP-only adenovirus. Following camptothecin exposure, neurons were fixed, and GFP-positive neurons assessed for p53 induction by immunofluorescence. As shown in Fig. 6, 85% of GFP-expressing neurons were p53 positive. In contrast, only 6% of neurons expressing calpastatin were positive for p53. Consistent with these results, differentiated stem cell cultures from Capn4-/- embryos also showed less p53 induction when compared with litter mate controls in response to camptothecin exposure (data not shown). Taken together, the pharmacological and molecular evidence indicates that p53 induction is mediated through calpain activation.
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MDM2 has been implicated in the stability of p53 (see Ref. 44 for review). Because we determined that calpains regulate p53 induction, we next examined whether MDM2 levels were modulated during camptothecin-induced death. However, as shown in Fig. 7, MDM2 levels did not change appreciably following camptothecin treatment, and calpain inhibitor cotreatment did not affect expression of MDM2. These results indicate that calpains do not regulate p53 by modulating MDM2 protein levels.
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Calpain Inhibition Blocks Activation of the Mitochondrial Death
SignalWe have shown previously that p53 is required for activation
of the mitochondrial pathway of death, which includes release of cytochrome
c from mitochondria and caspase activation
(10,
12,
15). Also in this paradigm,
inhibition of caspases, either by general caspase inhibitors or caspase 3
deficiency, transiently protects neurons from camptothecin-induced apoptosis
(10,
15). If calpains indeed act
upstream of p53, one would anticipate that the mitochondrial pathway of death
would also be inhibited. To test this expectation, we determined the effects
of calpain inhibition on cytochrome c release and caspase 3-like
activation. As we have shown previously in cortical neurons, cytochrome
c is localized to punctate mitochondrial compartments when visualized
by immunofluorescence (10,
15) (see also
Fig. 8A). However when
cortical cultures are treated with camptothecin for 12 h, cytochrome
c staining is lost, and nuclei become condensed and fragmented.
Cotreatment with the MDL28170 calpain inhibitor prevented both the loss of
cytochrome c staining and the nuclear fragmentation associated with
apoptosis (Fig. 8). Similar
results are obtained with the PD150606 inhibitor. However, in this case, the
nuclear morphology was not completely normal and displayed a slightly more
rounded appearance. This may reflect the change in general cellular morphology
and neuritic retraction, which is more evident with PD 150606. Nevertheless,
cytochrome c staining was still present in these neurons. Cytochrome
c-positive neurons are quantified in
Fig. 8B. As indicated,
85% of neurons in control cultures were positive for cytochrome c
versus
35% in camptothecin-treated cultures. Importantly,
cotreatment with the MDL or PD compounds resulted in an increased number of
cytochrome c positive neurons (
6575%).
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In accordance with these results, we also determined that calpastatin expression inhibited cytochrome c release in response to camptothecin. Cultures were infected with recombinant adenovirus expressing either GFP or GFP-calpastatin. As shown in Fig. 8C, only 19% of neurons expressing GFP control and treated with camptothecin were positive for cytochrome c. In contrast 73% of neurons expressing calpastatin showed intact cytochrome c labeling.
Cytochrome c release is required for activation of the apoptosome
complex and subsequent downstream effector caspases such as caspase 3
(45). Accordingly, we would
predict that caspase 3-like activity should also be inhibited with calpain
inhibition. Consistent with this notion, cotreatment of cultures with either
calpain inhibitor significantly inhibited the activation of caspase 3-like
activity by 70% as measured by DEVDAFC cleavage activity
(Fig. 9). Taken together, these
findings indicate that calpain proteolysis mediates the up-regulation of p53
and consequent events, including the release of cytochrome c and the
activation of caspases, that result from DNA damage.
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DISCUSSION |
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The role of calpains in neuronal death has been suggested but remains controversial. In addition, the manner by which calpains mediate neuronal death evoked by DNA damage is also unclear. In this paper, we provide multiple lines of molecular and pharmacological evidence that calpains do participate in neuronal death induced by the DNA-damaging agent camptothecin and, furthermore, that they act to regulate the p53 signaling axis.
Calpain Requirement following DNA DamageAs evidenced by the
accumulation of calpain-cleaved spectrin, induction of calpain activity is an
early event that precedes the commitment point of death, which we have
previously established to be 6 h following camptothecin exposure
(12). This observation
suggests that calpain activation is an upstream mediator of death in this
model. Supporting this hypothesis, we show that two distinct pharmacological
inhibitors of calpains block the death of neurons exposed to camptothecin.
Although these results support the proposed role of calpain-mediated death
signals in this death model, we could not exclude the possibility that these
calpain inhibitors may be affecting other signals. In this regard, MDL-28170
and PD inhibitors have been reported to inhibit cathepsins
(43) and calcineurins
(40) respectively, although at
higher concentrations. This is important, because other properties associated
with pharmacological calpain inhibition, such as regulation of cell division,
have not been observed in calpain-deficient cells, suggesting that calpain
inhibitors may act through alternative mechanisms
(17). Accordingly, we examined
two additional molecular means of calpain inhibition. In this regard, our
observation that calpastatin expression as well as calpain deficiency
significantly inhibit death indicates that calpains do participate in the DNA
damage-induced death signal caused by camptothecin. Interestingly, neuronally
differentiated calpain-deficient cells displayed less resistance than that
observed in cortical neurons expressing calpastatin or treated with
pharmacological inhibitors. This may be due to differences in the calpain
involvement between neuronally differentiated stem cells and cortical neurons
obtained from embryonic day 15 mice. Importantly, it is clear from all the
calpain functional data presented that this protease participates in, but does
not solely regulate, DNA damageinduced neuronal death (see below for further
discussion). Finally, it must be noted that the present study examines DNA
damage evoked only by camptothecin. Participation of calpains in other forms
of DNA damage will have to be determined empirically.
Calpains and Regulation of p53How do calpains regulate DNA damage-induced neuronal death? Our results indicate that this occurs through the p53 signaling axis. p53 levels are elevated early and prior to death commitment, and p53 deficiency prevents neuronal death evoked by DNA damage (11, 54). p53 is also an absolute requirement for activation of the distal death effector pathway (Bax translocation, cytochrome c release, and caspase activation) (10, 15, 54). We show that calpain inhibition by pharmacological agents, knockouts, or calpastatin overexpression significantly blocked the activation of p53 following camptothecin treatment. If calpains act upstream of p53, one prediction would be that calpain inhibition would also block the events downstream of p53 activation. Consistent with this idea, the inhibition of calpain activity by calpastatin expression as well as pharmacological inhibition prevented the release of cytochrome c and the activation of caspases.
Although it is unclear how calpain activation modulates p53 levels, it is
unlikely that it has a direct effect on p53 stability. First, MDM2, a
regulator of p53 levels, does not change appreciably during camptothecin
treatment. This observation rules out the possibility that calpain cleavage of
MDM2 accounts for the elevation in p53. Secondly, and in contrast with our
observations, p53 has been proposed to be a substrate of calpains under
certain conditions (58,
59). Calpain inhibitors have
been shown to up-regulate p53 levels and lead to cell cycle arrest and
apoptosis in proliferating cell systems
(60,
61). These effects are
opposite to what is reported here. These differences may be due to the
cellular context of proliferating cells versus neuronal systems.
Interestingly, calpains are known to cleave IB, a negative regulator of
the NF
B pathway (62,
63). NF
B, in turn, can
activate p53 through direct transcriptional means
(64). We have shown that
NF
B is activated by DNA damage and also regulates p53
activation.2
Therefore, an attractive hypothesis is that calpain-mediated activation of the
NF
B pathway may, in turn, regulate p53. However, validation of this
model will require further study.
The transient nature of p53 suppression by calpain inhibition also suggests
that there may be alternative/multiple mechanisms by which p53 stability is
regulated in neurons. Although p53 regulation is complex, three pathways
relevant in this death paradigm will be considered. First, cyclin-dependent
kinases (CDKs) can directly phosphorylate p53, resulting in its enhanced
stability (65). This report is
interesting, because we have previously implicated CDKs as activated and
required for death
(79).
Secondly, p19 ARF has been implicated in mediating oncogenic up-regulation of
p53. In this case, deregulated E2F1 has been shown to up-regulate p19ARF,
which then inhibits the ability of MDM2 to degrade p53
(66). Interestingly, E2F
family members have also been implicated in camptothecin-induced death. For
example, expression of dominant negative DP1, an obligate binding partner to
E2F members, inhibits death in this paradigm
(9). These observations raise
the possibility that CDK activation regulates p53 either directly or through
the Rb/E2F1/ARF19 pathway. However, our observation that inhibition of CDKs
has no effect on p53 levels makes these two possibilities unlikely
(12). The third pathway of p53
stability involves the phosphatidylinositol 3-kinase-like ATM/ATR family of
kinases. Previous reports indicated that these kinases phosphorylate p53
directly on Ser-15 or indirectly on Ser-20 through activation of Chk2
(42,
6771).
In this regard, our results indicate that ATM but not Chk2 also modulates the
stability of p53 and consequent death.2 Therefore, it is likely
that multiple signals relating to calpains, NFB, and ATM regulate p53
activation, and it will be important to explore how these signals ultimately
coordinate the p53 pathway. Taken together, our observations suggest that
calpain-mediated events regulate p53 activity and provide one explanation of
how calpain inhibition is an effective neuro-protectant in p53-mediated death
paradigms.
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FOOTNOTES |
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** To whom correspondence should be addressed. Tel.: 613-562-5800 (ext. 8816); Fax: 613-562-5403; E-mail: dpark{at}uottawa.ca.
1 The abbreviations used are: EGFP, enhanced green fluorescent protein; PBS,
phosphate-buffered saline; GFP, green fluorescent protein; CDK,
cyclin-dependent kinase; ATM, ataxia telangiectasia-mutated protein kinase;
ATR, ATM and Rad-3-related protein kinase.
2 D. P. Park, unpublished data.
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REFERENCES |
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