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The Journal of Neuroscience, November 1, 1999, 19(21):9654-9662
Involvement of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
and p53 in Neuronal Apoptosis: Evidence That GAPDH Is Upregulated by
p53
Ren-Wu
Chen1,
Paul A.
Saunders1,
Huafeng
Wei1,
Zhuangwu
Li2,
Prem
Seth2, and
De-Maw
Chuang1
1 Section on Molecular Neurobiology, Biological
Psychiatry Branch, National Institute of Mental Health, National
Institutes of Health, Bethesda, Maryland 20892, and
2 Medical Breast Cancer Section, Medicine Branch, National
Cancer Institute, National Institutes of Health, Bethesda, Maryland
20892
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ABSTRACT |
We recently reported that cytosine arabinoside (AraC)-induced
apoptosis of cerebellar neurons involves the overexpression of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The present study was
undertaken to investigate whether p53 and/or Bax overexpression participates in the AraC-induced apoptosis of cerebellar granule cells
and, if so, the relationship between p53 induction and GAPDH overexpression in these cells.
AraC-induced apoptosis of cerebellar granule cells was preceded by an
increase in levels of p53 mRNA and protein detected between 1 and 8 hr
after treatment. The mRNA level for a p53 target gene, Bax, was also
increased. The increase in GAPDH mRNA lasted longer than that of either
p53 or Bax, and the level of GAPDH protein in the particulate fraction
increased after induction of GAPDH mRNA. The antisense oligonucleotide
to p53 protected granule cells from AraC-induced chromatin
condensation, internucleosomal cleavage, and apoptotic death. The
inhibition of p53 expression by the p53 antisense oligonucleotide not
only blocked the expression of Bax but also partially suppressed the
increased GAPDH mRNA and protein levels. Conversely, the suppression of
GAPDH expression and subsequent attenuation of apoptosis of granule
cells by GAPDH antisense oligonucleotide did not influence the
expression of p53 or Bax. Cerebellar granule cells prepared from p53
knock-out mice were resistant to AraC toxicity, and the p53 gene
knock-out suppressed AraC-upregulated GAPDH expression. Moreover,
infection of PC12 cells with an adenoviral vector containing p53 gene
dramatically increased GAPDH expression and triggered cell apoptosis.
These results suggest that AraC-induced apoptosis of cerebellar granule cells involves the expression of both GAPDH and p53 and that, similar
to Bax, GAPDH is upregulated by p53 after exposure to the apoptotic insult.
Key words:
p53; GAPDH; cerebellar granule cell; PC12; cytosine
arabinoside; apoptosis; adenovirus
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INTRODUCTION |
Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) is an essential glycolytic enzyme that is
expressed in all prokaryotic and eukaryotic organisms. Its role in
cellular metabolism is to convert glyceraldehyde-3-phosphate to
1,3-diphosphoglycerate. Recent studies suggest that GAPDH is a
multifunctional protein endowed with a number of diverse activities
seemingly independent of its role in glycolysis. These activities
include phosphorylating transverse-tubule proteins (Kawamoto et al.,
1986 ), stimulating RNA transcription (Morgenegg et al., 1986),
interacting with microtubules (Huitorel and Pantaloni, 1985),
influencing RNA catalysis by binding to hammerhead ribozyme (Sioud and
Jesperson, 1996), and acting as a diadenosine tetraphosphate
binding protein to influence DNA replication and DNA repair (Baxi and
Vishwanatha, 1995).
Cytosine arabinoside (1- -D-arabinofuranosyl cytosine;
AraC) is usually thought to kill proliferating cells through
incorporation into DNA during replication, followed by chain
termination (Hedley and McCulloch, 1996). However, we and others have
reported that AraC induces apoptosis of postmitotic cerebellar granular
cells cultured from postnatal rats (Dessi et al., 1995; Ishitani and Chuang, 1996; Saunders et al., 1997, 1999). AraC has been shown to
induce apoptosis cerebellar granule cells in vitro in a
concentration-dependent manner only when added within 24 hr after cell
plating (Dessi et al., 1995). Previously, we found that AraC-induced
apoptosis of granule cells can be blocked by inhibitors of
transcription, translation, and deoxyribonuclease (Ishitani and Chuang,
1996). The AraC-induced apoptosis of granule cells is preceded by an increase in the levels of GAPDH mRNA and protein (Ishitani and Chuang,
1996), which is followed by nuclear translocation of the GAPDH protein
(Saunders et al., 1997; Ishitani et al., 1998). Moreover, an antisense
oligonucleotide against GAPDH attenuates GAPDH overexpression and
nuclear translocation and robustly protects granule cells from
apoptosis (Ishitani and Chuang, 1996; Saunders et al., 1997, 1999;
Ishitani et al., 1998), suggesting that GAPDH plays a prominent role in
AraC-induced apoptotic death. It has also been shown that GAPDH nuclear
translocation is involved in apoptosis of non-neuronal and neuronal
cells (Sawa et al., 1997).The role of GAPDH in the pathway and the
events leading to its induction, however, are unknown.
Accumulating evidence supports the view that p53, a nuclear
phosphoprotein, may be necessary for neuronal apoptosis. For example, cerebellar granule cells cultured from p53 null mice are resistant to
treatments with excitotoxins (Xiang et al., 1996) or
DNA-damaging agents, such as irradiation or AraC (Enokido
et al., 1996a,b). Moreover, whole body  -irradiation induces
apoptosis of granule cells in wild-type, but not p53-deficient,
neonatal mice (Wood and Youle, 1995). Other in vivo studies
have shown that knocking out the p53 gene protects hippocampal neurons
from seizure-induced cell death (Morrison et al., 1996), dopamine
neurons from
N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced toxicity (Trimmer et al., 1996) and the brain from ischemia-induced infarction (Crumrine et al., 1994). Conversely,
adenovirus-mediated transfection of the p53 gene in hippocampal or
cortical neurons results in the apoptotic death of these neurons (Xiang
et al., 1996; Jordan et al., 1997). In view of the emerging evidence
suggesting a prominent role for p53 in neuronal apoptosis, this study
examined whether p53 and its responsive gene, Bax, are overexpressed
during AraC-induced apoptosis of granule cells. In addition, we
explored whether GAPDH overexpression is regulated by p53 in granule
cells treated with AraC and in other neurally related cell types.
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MATERIALS AND METHODS |
Primary cultures of cerebellar granule cells and treatment
conditions. Cerebellar granule cells were prepared from 8-d-old Sprague Dawley rat pups or 7-d-old wild-type and p53 knock-out mouse
pups (Donehower et al., 1992) supplied by Taconic Farms (Germantown,
NY), as described previously (Nonaka et al., 1998). Briefly, cerebella
were chopped into 400 µm cubes, and the cells were dissociated by
trypsinization, followed by DNase treatment. The dissociated cells were
resuspended in a basal modified Eagle's medium containing 10% fetal
calf serum, 2 mM glutamate, 50 µg/ml gentamicin, and 25 mM KCl. Granule cells from rat
were seeded at a density of 2.5 × 105 cells/cm2
onto 24-well plates or 100 mm culture dishes and granule cells from
mouse onto 96-well plates or 60 mm dishes precoated with poly-L-lysine. To induce cell apoptosis,
AraC was added to culture medium to a final concentration of 300 µM 12 hr after cell plating. When used,
oligonucleotides were added to the cultures 2 hr before the addition of
AraC. After 24 hr of AraC treatment, viability of granule cells grown
on plates was measured by MTT assays as described previously (Nonaka et
al., 1998). The tetrazolium ring of MTT is reduced by active
dehydrogenase in viable mitochondria, forming a blue-colored
precipitate that was then dissolved in dimethyl sulfoxide and
quantified spectrophotometrically at 540 nm.
Isolation of fragmented DNA. For DNA fragmentation analysis,
cells grown on 100 mm dishes were also harvested after treatment essentially as described by Nonaka et al. (1998). Briefly, cells were
washed with ice-cold PBS and then lysed in a buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 0.5% Triton X-100. Lysates were
centrifuged at 10,000 × g for 10 min at room
temperature. Fragmented DNA in the supernatant was precipitated by the
addition of 400 µl of NaI (7.4 M) and 700 µl
of isopropanol and then isolated by centrifugation at 12,000 × g for 15 min. The DNA pellet was dissolved in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA [Tris-EDTA (TE)]. The DNA
samples were subjected to electrophoresis on 1.2% agarose gel, and the
DNA bands were visualized by ethidium bromide staining.
Northern blotting. Cerebellar granule cells were harvested
after different time lengths of AraC treatment. Total RNA was extracted from cells with guanidine thiocyanate and isolated by cesium chloride gradient centrifugation as described previously (Fukamauchi et al.,
1993), with modifications. After electrophoresis on 1% agarose gel
containing formaldehyde, RNA was transferred to a Duralose membrane
(Stratagene, La Jolla, CA) and hybridized with cDNA probes for Bax,
Bcl-2, p53, and GAPDH, separately. The cDNA probes were labeled with
[ -32P]dCTP by the random priming
method. Hybridizations were performed at 42°C for 16 hr, followed by
two washes at room temperature with 2 × SSC-0.1% SDS and then
two additional washes at 50°C in 0.1 × SSC-0.1% SDS. Northern
blots were quantified by Betascope Model 603 blot analyzer (Betagen,
Waltham, MA).
Western blotting. To measure p53 and Bax protein levels,
cells cultured in 60 or 100 mm dishes at indicated times were detached by scraping and sonicated 30 sec in Laemmli lysis buffer containing 6.25 mM Tris-HCl, pH 6.8, 2 mM EDTA, 15% sucrose, 10% glycerol, 3% SDS,
and 0.7 M -mercaptoethanol. For measurement of
GAPDH immunoreactive protein, the samples were prepared as reported previously (Ishitani and Chuang, 1996), with slight modifications. Briefly, scraped cells were ruptured by sonication in 0.32 M sucrose, and the homogenates were centrifuged
at 2 × 105 g for 30 min.
The particulate fraction (pellet) was resuspended in a small volume of
lysis buffer. Protein concentration was determined by using the BCA
protein assay kit (Pierce, Rockford, IL), and samples containing 10-30
µg of protein were loaded and separated by 12% SDS-PAGE. Proteins
were subsequently transferred to an Immobilon-P membrane (Millipore,
Bedford, MA). After blocking for 1 hr in PBST (1 × PBS and
0.1% Tween 20) containing 5% nonfat dry milk, blots were incubated
for 1 hr at room temperature with antibodies against GAPDH (Advanced
Immunochemical, Long Beach, CA), rat p53 (Pab240), human p53 (DO-1)
(Santa Cruz Biotechnology, Santa Cruz, CA), or Bax (Ab-1; Oncogene
Research, Cambridge, MA) in PBST containing 3% nonfat milk. Blots were
washed four times in PBST during a 40 min period and were then
incubated 1 hr with horseradish peroxidase-conjugated second antibodies
in PBST containing 3% nonfat dry milk. Immunoreactivities of the
protein bands were detected by enhanced chemiluminescent
autoradiography (ECL kit; Amersham Pharmacia Biotech, Arlington
Heights, IL) as instructed by the manufacturer. Western blots were
quantitatively analyzed by capturing images on films using a CCD camera
(Sierra Scientific, Sunnyvale, CA) and the Macintosh NIH Image 1.5 software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD).
Morphological evaluation. Changes in nuclear morphology were
examined by staining cerebellar granule cells with Hoechst 33258 (Nonaka et al., 1998). Cells growing on 35 mm dishes were fixed with
4% formaldehyde. After gentle washing with cold PBS, cells were
stained with Hoechst 33258 (5 µg/ml in PBS) for 5 min at 4°C.
Photomicrographs were taken using an inverted UV illumination microscope.
Oligonucleotide preparations. Antisense and
sense oligonucleotides to GAPDH and p53 were synthesized by Cruachem
(Dulles, VA). The first and last bases of each oligonucleotide were
phosphorathioated. The GAPDH oligonucleotide sequence (GAPDH/AS) was
5'-GACCTTCACCATCTTGTCTA-3', corresponding to a sequence of the rat
GAPDH gene flanking the mRNA translation initiation region (GenBank
GAPDH cDNA clone M17701). The p53 antisense oligonucleotide (p53/AS)
sequence was 5'-GACCTCAGGTGGCTCATACGG-3', corresponding to residues
between 390 and 410 of rat p53 coding sequence (GenBank p53
cDNA clone X13058). The sequences of the GAPDH sense (GAPDH/S) and p53
sense (p53/S) oligonucleotides were the exact inverse of those of their
corresponding antisense oligonucleotides. The stock concentration of
oligonucleotides was 1 mM in TE buffer, pH
7.4.
Construction of adenoviral vectors and transfection into PC12
cells. The construction of adenovirus expressing human wild-type p53 cDNA (AdWTp53), -galactosidase gene (Ad-gal), and adenovirus containing no transgene (AdControl) have been described previously (Katayose et al., 1995). Briefly, the human wild-type p53 gene was
cloned to the site downstream of cytomegalovirus promoter and
upstream of SV40 polyadenylation sequence in the replication-defective recombinant adenovirus. Recombinant adenoviruses were propagated in
human embryonic kidney 293 cells, purified by double cessium chloride
gradient centrifugations, titrated, and stored at  70°C.
PC12 cells were maintained in 100 mm dishes containing RPMI
medium 1640 plus 12% fetal bovine serum and 6% horse serum. Cells were infected with 20 pfu/cell of recombinant adenoviruses at 24 hr
after plating. The infection efficiency of PC12 cells was determined by
counting the -galactosidase-positive cells under a light microscope
at 24 hr after infection with Ad-gal (Li et al., 1998).
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RESULTS |
Levels of GAPDH, p53, and Bax expression are increased during the
AraC-induced apoptosis of cerebellar granule cells
Northern blot hybridization showed that the expression of GAPDH
mRNA in freshly cultured granule cells increased from 1 to 24 hr after
exposure to AraC (Fig. 1). p53 mRNA
levels increased from 1 to 4 hr and then declined, whereas the Bax mRNA
levels were elevated between 2 and 12 hr. Thus, the upregulation of
GAPDH mRNA lasted longer than that of either p53 or Bax. The expression of Bcl-2 mRNA was very low and appeared to be decreased after 4 hr of
AraC treatment.
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Figure 1.
Time course of AraC-induced changes in the mRNA
levels of GAPDH, p53, Bax, and Bcl-2 in cerebellar granule cells. AraC
was added to culture medium to a final concentration of 300 µM 12 hr after plating. At the indicated times after AraC
exposure, cells were harvested for RNA isolation. An aliquot of
 of each sample was used for quantification of total
cellular RNA. The RNA was allowed to migrate ~5 mm from the sample
well into 1% agarose gel, and the total RNA was quantified by image
analysis (Fukamauchi et al., 1993). The same amount (10 µg) of total
RNA from each sample was applied to each well of the agarose gel for
Northern blot analysis. A, Northern blots;
B, quantified results of blots shown in
A.
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Western blots and their quantified results showed that endogenous
cellular p53 protein levels were increased at 1 hr, reached their peak
(265% of the control) at 4 hr, and then decreased to less than the
baseline value by 12 hr (Fig.
2A,B).
Levels of cellular Bax protein were increased at 2 hr and peaked at
~8 hr (155% of the control). Compared with p53 and Bax protein, the
GAPDH protein levels in the particulate fraction of AraC-treated
granule cells increased rather late; the increase was detected at 8 hr,
and reached its maximum (200% of the control) ~24 hr after
exposure.
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Figure 2.
Time course of AraC-induced increases in GAPDH,
p53, and Bax protein levels in cerebellar granule cells. Freshly
cultured cells were treated with AraC as described in Figure 1. At
different time points, cells were collected in lysis buffer and
sonicated for 30 sec. For measurement of GAPDH protein, an aliquot
containing 10 µg of protein from the particulate fraction of the
cells was loaded and separated by SDS-PAGE. For examination of p53 and
Bax protein expression, an aliquot containing 30 µg of total protein
was used for SDS-PAGE. A, Western blots;
B, quantified results of blots shown in
A. Note that protein levels of GAPDH, p53, and Bax were
time dependently changed after AraC treatment.
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p53 and GAPDH antisense oligonucleotides protect cerebellar granule
cells against AraC-induced apoptosis
Cell viability, as measured by MTT assay at 24 hr after treatment
with AraC, was reduced to 40.4 ± 3.2% (n = 4)
(Fig. 3). Photomicrographs showed that
AraC caused condensed and/or fragmented cell nuclei, which are
characteristic of apoptosis (Fig. 4).
Exposure to AraC for 24 hr also caused internucleosomal cleavage of the DNA (Fig. 5). To identify whether the
increase in p53 expression was directly related to the cell death,
granule cells were pretreated with 3 µM
p53/AS before exposure to AraC. The cell viability for the
p53/AS-pretreated group was 72 ± 4.0% of the control
(n = 4), which was significantly greater than that of
AraC alone (p < 0.001) (Fig. 3). Pretreatment
with the corresponding p53/S was ineffective; cell viability was
44.6 ± 3.3% (n = 4; p > 0.05%). p53/AS, but not p53/S, markedly reduced chromatin condensation and nuclear fragmentation (Fig. 4) and essentially blocked
internucleosomal DNA cleavage in AraC-treated cells (Fig. 5). These
results suggest that AraC-induced apoptosis of granule cells is not
only dependent on GAPDH, but on p53 as well. Moreover, similar to the
GAPDH/AS (Ishitani and Chuang, 1996; Saunders et al., 1997), the p53/AS was effective in protecting granule cells from AraC-induced
apoptosis.
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Figure 3.
Antisense oligonucleotide to p53 protects
against AraC-induced neuronal death. Cerebellar granule cells were
pretreated with p53 oligonucleotides (3 µM) for 2 hr
before the addition of AraC (300 µM). After 24 hr
exposure to AraC, neuronal viability was measured using the MTT
colorimetric assay. The results are expressed as percentage of
mean ± SEM of control group from three independent experiments.
CTL, Control. ***p < 0.001 using one-way ANOVA with Fisher's PLSD test.
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Figure 4.
Antisense but not sense oligonucleotide to p53
protects against AraC-induced cell death: morphological studies.
Cerebellar granule cells were treated as described in Figure 3. After
exposure to AraC for 24 hr, cells were fixed with 4% formaldehyde and
then stained with Hoechst dye 33258 (5 µg/ml in PBS) for 5 min at
4°C. Photomicrographs were taken using an inverted UV illumination
microscope.
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Figure 5.
Antisense oligonucleotides to GAPDH and p53 reduce
AraC-induced DNA fragmentation. Cerebellar granule cells were treated
with AraC as described in Figure 3 and pretreated with GAPDH or p53
oligonucleotides (3 µM) for 2 hr. After exposure to AraC
for 24 hr, soluble DNA was extracted from cells and subjected to
electrophoresis as described in Materials and Methods. The DNA size
markers are shown to the left. AS,
Antisense oligonucleotide; S, sense oligonucleotide;
CTL, control.
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Abrogation of p53 expression suppresses GAPDH mRNA and
protein levels
Northern blot results showed that p53/AS, but not p53/S, blocked
AraC-induced upregulation of p53 and Bax mRNA, whereas neither GAPDH/AS
nor GAPDH/S was effective (Figs. 6,
7). AraC-induced GAPDH mRNA upregulation
was blocked by GAPDH/AS and partially suppressed by p53/AS (Fig.
8). p53/AS also concentration dependently suppressed AraC-increased levels of GAPDH protein in the particulate fraction of granule cells (Fig.
9A,B).
At concentrations of 1 and 3 µM, p53/AS
suppressed upregulated GAPDH protein by 37.6 and 74.7%, respectively,
whereas p53/S was ineffective at these two concentrations. At 5 µM, p53/AS completely blocked GAPDH protein upregulation, and p53/S was partially effective. Similar dose-dependent neuroprotective effects of p53/AS and p53/S on AraC-induced apoptosis were observed (data not shown). Therefore, oligonucleotide
concentrations higher than 3 µM were not
recommended. These results suggest that, during AraC-induced apoptosis
of granule cells, GAPDH may function downstream of p53.
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Figure 6.
Antisense oligonucleotide to p53 but not GAPDH
suppresses AraC-induced increases in p53 mRNA. Cerebellar granule cells
were exposed to 300 µM AraC for 12 hr after plating.
Cells were pretreated with GAPDH or p53 oligonucleotides for 2 hr
before the addition of AraC. RNA was extracted from cells after
exposure to AraC for 4 hr. A, Northern blot results of
p53 mRNA levels from a typical experiment. B, Levels of
p53 mRNA expressed as percent of the control quantitated with a
Betascope Model 603 blot analyzer. Each column
represents the mean ± SEM from three independent experiments.
AS, Antisense oligonucleotide; S, sense
oligonucleotide; CTL, control. **p < 0.01; ***p < 0.001 using one-way ANOVA with
Fisher's PLSD test.
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Figure 7.
Antisense oligonucleotide to p53 inhibits
AraC-induced Bax mRNA upregulation. Cerebellar granule cells were
treated with AraC, and RNA was prepared as described in Figure 6.
A, Northern blot results of Bax mRNA levels from a
typical experiment. B, Levels of Bax mRNA expressed as
percent of the control quantitated with a Betascope Model 603 blot
analyzer. Each column represents the mean ± SEM
from three independent experiments. AS, Antisense
oligonucleotide; S, sense oligonucleotide;
CTL, control. **p < 0.01 using
one-way ANOVA with Fisher's PLSD test.
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Figure 8.
GAPDH and p53 antisense oligonucleotides inhibit
AraC-induced GAPDH mRNA increase. Cerebellar granule cells were treated
with AraC, and RNA was prepared as described in Figure 6.
A, Northern blot results of GAPDH mRNA levels from a
typical experiment. B, Levels of GAPDH mRNA expressed as
percent of the control quantified with a Betascope Model 603 blot
analyzer. Each column represents mean ± SEM from
three independent experiments. AS, Antisense
oligonucleotide; S, sense oligonucleotide;
CTL, control. *p < 0.05;
***p < 0.001 using one-way ANOVA with Fisher's
PLSD test.
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Figure 9.
Antisense oligonucleotide to p53 suppresses
AraC-induced GAPDH protein increase in the particulate fraction of
cerebellar granule cells. Cells were treated with AraC as described in
Figure 6. After exposure to AraC for 24 hr, cells were harvested and
the particulate (pellet) fraction was prepared as described in
Materials and Methods. An aliquot of 10 µg of protein from each
particulate fraction was used for Western blot analysis.
A, Western blot results of GAPDH protein levels from a
typical experiment. B, Quantified results of GAPDH
protein levels expressed as percent of mean ± SEM of the
untreated control from three independent experiments.
CTL, Control. *p < 0.05 compared
with the untreated control using one-way ANOVA with Fisher's PLSD
test.
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p53 gene knock-out in mice inhibits AraC-induced apoptosis and
GAPDH expression in cerebellar granule cells
To further explore the role of p53 in mediating GAPDH induction,
we used cerebellar granule cells from p53 knock-out mice. AraC
treatment induced a concentration-dependent decrease in the viability
of granule cells derived from wild type but had little or no effect in
those cells derived from p53/ mice (Fig.
10A). Thus, after
treatment with 1000 µM AraC for 24 hr,
viabilities of granule cells derived from p53+/+ and p53/ mice were
32.1 ± 1.5 and 81.4 ± 5.8%, respectively. These results
confirm that p53 gene knock-out confers resistance to AraC-induced
apoptotic cell death (Enokido et al., 1996b). Importantly, GAPDH
protein levels were markedly elevated by AraC treatment in granule
cells from p53+/+ mice but were only marginally increased in those
cells from p53/ mice (Fig. 10B).
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Figure 10.
Knock-out of p53 gene suppresses AraC-induced
neurotoxicity and GAPDH overexpression in mouse cerebellar granule
cells. Granule cells were prepared from the cerebellum of 7-d-old
wild-type (p53+/+) and p53 knock-out (p53/) mice (Donehower et al.,
1992) according to the procedures described in Materials and Methods.
A, At 12 hr after plating onto 96-well plates, various
concentrations of AraC were added to the cultures, and the cell
viability was determined 24 hr later by MTT assay. Results shown are
mean ± SEM from six independent experiments. B, At
24 hr after plating onto 60 mm dishes, GAPDH protein levels in
wild-type and p53-deficient mouse granule cells were determined by
Western blot analysis. The top represents Western blot
from a typical experiment. The bottom represents
quantified results expressed as mean ± ranges from two
independent experiments. Note that p53 gene knock-out resulted in not
only inhibition of AraC toxicity but also suppression of AraC-induced
GAPDH upregulation. CTL, Control.
***p < 0.001 compared with the corresponding
results in p53-deficient mouse granule cells, using one-way ANOVA with
Fisher's PLSD test.
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p53 gene transfection into PC12 cells upregulates GAPDH protein and
triggers apoptosis
To obtain additional evidence that GAPDH is regulated by p53, we
infected PC12 pheochromocytoma cells with the recombinant adenovirus
containing AdWTp53, Ad-gal, or AdControl. The infection efficiency
determined by counting -galactosidase-positive cells was dependent
on the dose of the recombinant adenovirus and found to be 82.9 ± 4.4% with 20 pfu/cell of Ad-gal (data not shown). The p53 gene
transfection of PC12 cells resulted in a time-dependent increase in the
levels of exogenous p53 protein in the cell homogenate (Fig.
11). Thus, the p53 protein levels were
progressively increased from 12 to 48 hr after AdWTp53 infection. In
contrast, infection with AdControl produced no detectable p53 protein.
Interestingly, the levels of GAPDH protein in the particulate fractions
were also markedly increased at 24 and 48 hr in cells infected with AdWTp53 but not AdControl. The p53 gene transfer into PC12 cells induced a time-dependent loss of cell viability with a detectable decrease at day 1, followed by a progressive decrease to <20% of the
control at days 3 and 5 (Fig.
12A). A dramatic
increase in internucleosomal DNA cleavage was also observed at 2 d
after AdWTp53 infection (Fig. 12B). In contrast,
infection with AdControl induced neither cell death nor detectable DNA
fragmentation throughout the time course studied.
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Figure 11.
p53 gene transfection into PC12 cells induces
GAPDH overexpression. PC12 cells in cultures were incubated at 37°C
for different lengths of time after infection with 20 pfu/cell of
either AdWTp53 or AdControl. Western blotting for p53 was determined at
indicated times as described in Materials and Methods using 30 µg of
cell homogenate proteins. Western blotting for GAPDH was determined
using 7 µg of 2 × 105 g
pellet proteins. Note that infection of cells with AdWTp53, but not
AdControl, induced a dramatic time-dependent increase in p53 and GAPDH
protein. The experiment has been repeated three times with similar
results.
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Figure 12.
p53 gene transfection into PC12 cells induces
delayed DNA fragmentation. Cells were infected with 20 pfu/cell of
AdWTp53 or AdControl as described in the legend to Figure 11. At
indicated times, cells were examined for viability using MTT assay
(A) or for internucleosomal DNA cleavage using
agarose gel electrophoresis (B). Results of MTT
assay are expressed as percentage of mean ± SEM of the respective
zero time control from eight independent determinations. The results of
DNA agarose gel electrophoresis are from a typical experiment of three
such experiments. Note that infection of cells with AdWTp53, but not
AdControl, induced a time-dependent loss of cell viability and an
increase in the level of internucleosomally cleaved DNA in the soluble
DNA preparation. *p < 0.05;
***p < 0.001 using one-way ANOVA with Fisher's
PLSD test.
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DISCUSSION |
The tumor suppressor p53 serves as a critical regulator in the
cell cycle and in the apoptosis of normal cells after exposure to
ionizing radiation or DNA-damaging agents. It has been suggested that
p53 may act as a "guardian of the genome." When DNA damage is mild,
p53 halts cell cycle progression at the G1 phase through the induction
of genes such as p21 and GADD45 (Pellegata et al., 1996) (for review,
see Hughes et al., 1997), allowing DNA repair to occur before advancing
through the cell cycle. However, when DNA damage is extensive and not
repairable, p53 triggers apoptosis (Grasso and Mercer, 1997) (for
review, see Yonish-Rouach, 1996; Hughes et al., 1997). These
biochemical characteristics ensure that p53 plays a key role in
controlling genomic stability (Lowe et al., 1993; Fukasawa et al.,
1997). Elevated expression of p53 may initiate apoptosis by increasing
the expression of the death gene Bax (Miyashita and Reed, 1995),
decreasing the expression of the cytoprotective gene Bcl-2
(Selvakumaran et al., 1994) or inducing the transcription of
redox-related genes that cause oxidative stress (Polyak et al.,
1997).
When DNA is damaged in postmitotic neurons, p53 induction is associated
with mechanisms underlying death rather than recovery (Sakhi et al.,
1994; Wood and Youle, 1995; Enokido et al., 1996a; Jordan et al.,
1997). Transfection studies have shown that overexpression of p53 per
se is sufficient to induce neuronal apoptosis (Xiang et al., 1996;
Jordan et al., 1997). In contrast, p53-deficient neurons are resistant
to apoptosis induced by excitotoxins such as glutamate (Morrison et
al., 1996). The precise mechanism by which p53 modulates neuronal
viability has not yet been determined. However, it has been suggested
that p53 may function by upregulating Bax and/or by downregulating
Bcl-2, whereas generation of the reactive oxygen species and activation
of caspases may be further downstream events of p53-dependent apoptosis
(Johnson et al., 1996; Lotem and Sachs, 1996).
In this study, we extensively characterized AraC-induced apoptosis with
a major emphasis on the role of p53 in the apoptotic process. We found
that levels of p53 mRNA and protein in cerebellar granule cells were
rapidly and markedly increased after treatment with AraC. Pre-exposure
of these cells to p53/AS robustly protected these neurons from
AraC-induced chromatin condensation, nuclear fragmentation, and
internucleosomal DNA cleavage. Concomitant with these effects, the
viability of AraC-treated cells was markedly enhanced. Additionally,
AraC-induced apoptosis was shown to be suppressed in granule cells from
p53 null mice (Enokido et al., 1996b; the present study). These results
suggest that p53 plays an essential role in mediating AraC-induced
apoptotic death of cerebellar granule cells. The overexpression of p53
induced by AraC also suggests that this apoptotic insult involves
extensive DNA damage before p53 induction, as suggested previously
(Tomkins et al., 1994; Enokido et al., 1996a,b). It is interesting to
note that AraC-induced apoptosis of granule cells is preceded by an increase in the levels of both mRNA and protein of p53, suggesting an
involvement of transcriptional regulation of p53.
p53 upregulation was immediately followed by increased expression of
Bax; the latter was blocked by p53/AS but not p53/S, reinforcing the
well established notion that Bax is a p53-responsive target gene and is
involved in p53-mediated neuronal death (Hughes et al., 1997; Xiang et
al., 1998). Importantly, GAPDH mRNA and protein levels were increased
after p53 overexpression, and GAPDH upregulation was effectively
suppressed by p53/AS. Conversely, neither p53 nor Bax overexpression
was affected by GAPDH/AS, which is neuroprotective by blocking GAPDH
upregulation and the subsequent nuclear translocation (Ishitani and
Chuang, 1996; Saunders et al., 1997; Ishitani et al., 1998). Moreover,
our results show that p53 gene knock-out suppressed AraC-induced GAPDH
upregulation in mouse cerebellar granule cells compared with that in
wild-type neurons. These observations suggest that GAPDH overexpression is an event downstream of p53 expression and may function independently of Bax in mediating AraC-induced apoptosis of granule cells. Moreover, our results raise the possibility that GAPDH is a novel target of p53.
In further support of this notion, we found that high-efficiency transfection of p53 gene into PC12 cells by infection with AdWTp53 resulted in dramatic overexpression of not only p53 but also GAPDH protein, and induction of these proteins preceded internucleosomal DNA
cleavage and extensive cell death. In contrast, infection with
AdControl produced neither p53 and GAPDH increase nor apoptosis.
Our results show that levels of Bax mRNA and protein were rapidly and
markedly increased by AraC in cerebellar granule cells. Other studies
have suggested that Bax may produce cell death by induction of the
mitochondrial permeability transition, which can result in disruption
of plasma membrane integrity or release of cytochrome C from
mitochondria (Pastorino et al., 1998; Chen and Chuang, 1999). The
released cytochrome C may bind to Apaf-I, causing caspase cleavage of
substrate proteins and, ultimately, cell death (Liu et al., 1996). The
involvement of Bax in neuronal apoptosis seems to depend on the nature
of stimuli. For example, Bax is required for apoptosis induced by
deprivation of trophic factors and high potassium but is not involved
in NMDA-induced excitotoxicity in some studies (Deckwerth et al., 1996;
Miller et al., 1997).
The precise mechanism underlying the involvement of GAPDH in apoptosis
is unclear. Because overexpressed GAPDH is translocated from the
cytosol to the nucleus in neurons and non-neuronal cells (Saunders et
al., 1997, 1999; Sawa et al., 1997; Ishitani et al., 1998; Shashidharan
et al., 1999), one may surmise that GAPDH-dependent apoptosis is caused
by a perturbation of nuclear functions. In this context, we
found that AraC-induced GAPDH nuclear translocation occurs concurrently
with a nuclear loss of dehydrogenase and uracil glycosylase activities
(Saunders et al., 1999), suggesting that GAPDH undergoes structural and
functional changes after transport to the nucleus. In the cytosol,
GAPDH binds to glutamine repeats in huntingtin, a gene product of
Huntington's chorea, and other polyglutamine-containing proteins
associated with neurodegenerative diseases (e.g., ataxin for
spinocerebellar ataxia) (Burke et al., 1996). Because N-terminal
fragments of huntingtin and ataxin-3 are found in the intranuclear
inclusions of cells in affected brain areas (Davies et al., 1997;
DiFiglia et al., 1997; Paulson et al., 1997), it seems possible that
GAPDH is a "carrier" mediating the transfer of these
disease-related proteins to the nucleus, thus contributing to the
progression of neuronal apoptosis.
Recently, it has been shown that the administration of
R-deprenyl and its derivatives can protect cerebellar
granule cells from AraC-induced apoptosis (Paterson et al., 1998). This
neuroprotective effect could be attributable to the ability of these
drugs to bind overexpressed GAPDH, thereby inhibiting the proapoptotic activity of GAPDH; such an effect was demonstrated in an independent study using neuroblastoma cells (Kragten et al., 1998). Clearly, molecular mechanisms underlying GAPDH-mediated apoptotic death in
neurons and non-neuronal cells require future detailed investigation.
| |
FOOTNOTES |
Received April 2, 1999; revised Aug. 23, 1999; accepted Aug. 23, 1999.
We thank Peter Leeds in our section for his kind assistance
during the course of this study.
Correspondence should be addressed to Dr. De-Maw Chuang, Section on
Molecular Neurobiology, Biological Psychiatry Branch, National
Institute of Mental Health, National Institutes of Health, Building 10, Room 3N212, 10 Center Drive, MSC 1272, Bethesda, MD 20892-1272. E-mail: chuang{at}helix.nih.gov.
| |
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ABSTRACT
INTRODUCTION
