Journal of Neuroscience Research 75:83–95 (2004)

Oxidative Stress Induces p53-Mediated

Apoptosis in Glia: p53 Transcription-
Independent Way to Die
Paolo Bonini,1 Simona Cicconi,4 Alessio Cardinale,2 Cristiana Vitale,3
Anna Lucia Serafino,4 Maria Teresa Ciotti,5 and Lionel N.J-L. Marlier4*
1
Department of Internal Medicine, University of Rome Tor Vergata, Rome, Italy
2
Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy
3
Department of Medical Science, San Raffaele Roma Hospital, Rome, Italy
4
Section of Molecular Medicine, Signal Transduction of Apoptotic Mechanisms Laboratory, National Research
Council (CNR), Institute of Neurobiology and Molecular Medicine (INeMM), Rome, Italy
5
Section of Neuroscience, National Research Council (CNR), Institute of Neurobiology and Molecular
Medicine (INeMM), Rome, Italy

Oxidative stress has been implicated in the pathogenesis
of stroke, traumatic brain injuries, and neurodegenerative
diseases affecting both neuronal and glial cells in the
central nervous system (CNS). The tumor suppressor
protein p53 plays a pivotal function in neuronal apoptosis
triggered by oxidative stress. We investigated the role of
p53 and related molecular mechanisms that support ox-
idative stress-induced apoptosis in glia. For this purpose,
we exposed C6 glioma cells and primary cultures of rat
cortical astrocytes to an H2O2-induced oxidative stress
protocol followed by a recovery period. We evaluated the
effects of pifithrin-␣ (PF-␣), which has been reported to
protect neurons from ischemic insult by specifically in-
hibiting p53 DNA-binding activity. Strikingly, PF-␣ was
unable to prevent oxidative stress-induced astrocyte ap-
optosis. We demonstrate that p53 is able to mediate an
apoptotic response by direct signaling at mitochondria,
despite its transcriptional activity. The z-VAD-fmk-
sensitive apoptotic response requires a caspase-
dependent MDM-2 degradation, leading to p53 mito-
chondrial targeting accompanied by cytochrome c
release and nucleosomal fragmentation.
© 2003 Wiley-Liss, Inc.
Key words: astrocyte; C6 glioma; ischemia; MDM2; cy-
tochrome; mitochondria; caspase
mediator of stress response because it plays an essential role
in the death of many cell types, other than neurons. It is
well known that p53 induces cell death by a multitude of
molecular pathways involving activation of target genes
(i.e., pro-apoptotic Bax) (Miyashita and Reed, 1995; Jor-
dan et al., 1997; Brady et al., 1998; Xiang et al., 1998;
Morrison et al., 2003). Nevertheless, evidence for
transcription-independent pathways of p53-mediated ap-
optosis is accumulating (Gottlieb and Oren, 1998; San-
some et al., 2001), showing that p53 can contribute to
apoptosis by direct signaling at the mitochondria (March-
enko et al., 2000) leading to cytochrome c (cyt-c) release
and caspase activation (Schuler et al., 2000).
A key player in p53 regulation is MDM-2 protein.
MDM-2 can inhibit p53 binding within its transactivation
domain interfering with recruitment of basal transcription
machinery components. Moreover, MDM-2 can lead to
complete elimination of p53 through the ubiquitin-
proteasome pathway (Momand et al., 1992, 2000; Barak et
al., 1993; Wu et al., 1993; Zauberman et al., 1993; Chen et
al., 1996; Haupt et al., 1996). In addition, p53 binds specif-
ically to MDM-2 gene promoter establishing an important
autoregulatory feedback loop (Barak et al., 1993; Wu et al.,
1993; Haupt et al., 1997; Kubbutat et al., 1997).
Recently, a small molecule was isolated for its ability to
reversibly block p53 transcriptional activity. This compound,

Apoptotic cell death plays an important role in brain
development as well as in neuronal injury and disease. It is
now generally accepted that massive neuronal death due to
oxidative stress is a common characteristic of brain
whether in stroke (Culmsee et al., 2001b; Cheng et al.,
2003; Love, 2003) or in neurodegenerative diseases such as
Alzheimer’s disease (de la Monte et al., 1997, 1998),
Parkinson’s disease (Jenner, 2003; Tatton et al., 2003), and
traumatic brain injury (Napieralski et al., 1999). The p53
tumor suppressor protein has been proposed as a key
Contract grant sponsor: San Raffaele Roma Hospital; Contract grant spon-
sor: CNR-INeMM; Contract grant sponsor: the Italian Ministry of Uni-
versity Research, and Technology (MURST).
*Correspondence to: Lionel N.J-L. Marlier, National Research Council,
Institute of Neurobiology and Molecular Medicine (INeMM), Viale Marx
15-43, 00137 Rome, Italy. E-mail: l.marlier@in.rm.cnr.it
Received 10 January 2003; Revised 15 August 2003; Accepted 17 August
2003

© 2003 Wiley-Liss, Inc.

84 Bonini et al.
called pifithrin-␣ (PF-␣), protects neurons from apoptosis
induced by ischemic injury and excitotoxic insults. More-
over, PF-␣ protects mice from the lethal genotoxic stress
associated with anticancer treatment without promoting the
formation of tumors (Komarov et al., 1999). Protection by
PF-␣ was correlated with decreased p53 DNA-binding ac-
tivity, decreased expression of the p53 target gene Bax and
suppression of mitochondrial dysfunction and caspase activa-
tion (Culmsee et al., 2001a,b).
The present study was undertaken to investigate the
molecular mechanisms that support oxidative stress-
induced apoptosis in glial cells. For this purpose, we de-
signed an in vitro model of H2O2-induced oxidative stress
using both rat primary cultures of astrocytes and C6 rat
glioma cell line. We examined the role of p53 on the onset
of apoptosis and investigated whether PF-␣ was able to
prevent glial cell death.
MATERIALS AND METHODS
Ethics
Animals used for astrocyte preparations received humane
care according to the standard criteria regarding animal use in
EEC.
C6 Cell Line
C6 cells were maintained in log-phase growth by regular
passage using Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 2 mM
L-glutamine, 50 IU/ml penicillin, and 50 ␮g/ml streptomycin
(Invitrogen, Milan, Italy). Cells were subcultured twice a week
and maintained at 37°C in an atmosphere of 95% humidified air
and 5% CO2.
Primary Cultures of Rat Astrocytes
Rat primary cultures of astrocytes were prepared from
cortex of embryonic Day 14 (E14) rat embryos as described
previously in detail (Mercanti et al., 1987).
Oxidative Stress/Recovery Protocol
To mimic oxidative stress cells were exposed to 100 ␮M
H2O2/100␮M FeSO4 (Fenton reagent) in Hank’s balanced sa-
line solution (HBSS; Invitrogen) for 1 hr (the stress period)
followed by a recovery period consisting of replacing cells in
normal growth medium (Joseph et al., 1997; Richter-Landsberg
and Vollgraf, 1998; Avshalumov and Rice, 2002). The basal
point refers to the intact cells whereas the T0 time point refers
to the end of the stress period/beginning of the recovery period.
The cells were preincubated 3 hr with zVAD-fmk
(20 ␮M final concentration; Biomol, Plymouth Meeting, PA)
and PF-␣ (100 nM final concentration; Calbiochem, San Diego,
CA) before the stress period and both zVAD-fmk and PF-␣
were then replaced during the recovery period.
PCR Analysis
Total RNA was extracted using the TRIAzol reagent
according to the manufacturer’s recommendation (Invitrogen)
and extracted RNA was used for reverse transcription-
polymerase chain reaction (RT-PCR) analysis. Briefly, 1 ␮g
RNA was reverse transcribed for 1 hr at 42°C using 200 U
Moloney murine leukemia virus (MMLV) reverse transcriptase
(Invitrogen) in the presence of 2.5 ␮M random hexamers and
200 ␮M nucleotides (Amersham Pharmacia Biotech, MI, Italy)
in 20 ␮L final volume. Successively, 2 ␮L of each cDNA were
PCR-amplified using 2.5 U Platinum Taq (Invitrogen) in the
presence of 15 pmol of specific primers as follows:
● Bcl-2 up: 5⬘-TAT AAG CTG TCA CAG AGG G;
down: 5⬘-CTC CCC ACA CAC ATG ACC
● BclxL/S up: 5⬘-TCC CAG AAA GGA TAC AGC;
down: 5⬘-TCA CTT CCG ACT GAA GAG
● Bad up: 5⬘-CCA GAT CCC AGA GTT TGA GC;
down: 5⬘-CTT TCC CCA AAT TTC GAT CC
● Bak up: 5⬘-TCC TGC CTC TCA GGG CAA; down:
5⬘-AGT GGT CAG GGA GGC ACT T
● Bax up: 5⬘-TTC ATC CAG GAT CGA GCA G;
down: 5⬘-TTC TTC CAG ATG GTG AGC G
● Bid up: 5⬘-ACT CTG AGG TCA GCA ATG GC;
down: 5⬘-TAG GGA AGG ATG TCT TCA CCT C
● p53 up: 5⬘-GCC ATC TAC AAG AAG TCA CA;
down: 5⬘-GTC TTC CAG CGT GAT GAT G
● MDM-2 up: 5⬘-CCA ACA TGT CTG TGT CTA
CCG; down: 5⬘-ACA ATG TGC TGC TGC TTC TC
● H4 up: 5⬘-GGT AAA GGG CTT GGG AAA G;
down: 5⬘-TTA ACC ACC GAA GCC GTA G
● GAPDH up: 5⬘-ACC ACA GTC CAT GCC ATC
AC; down: 5⬘-TCC ACC ACC CTG TTG CTG TA
To normalize the cDNA amount to be used during the PCR
amplification, pilot experiments were carried out using histone 4
(H4) specific primers (not shown). For all templates, 30 cycles were
used allowing us to consider the basal level as a real representation
of relative expression levels of genes of interest.
To verify whether after 24 hr reperfusion PF-␣ remained
able to block p53 transcriptional activity, RT-PCR analysis of
mRNA expression levels for H4 (as a housekeeping control
gene) and the glyceraldehyde-3-phosphate dehydrogenase gene
(GAPDH) were carried out and the linear dynamic range ana-
lyzed by densitometry analysis using a computer-based imaging
system (Fluor-S; Biorad, Hercules, CA).
In all experiments, cDNAs were denatured initially for
5 min followed by 30 cycles consisting of: 45 sec denaturation;
30 sec annealing; and 50 sec extension (3 min final extension)
using a Perkin Elmer 9700 thermal cycler (Perkin Elmer, Foster
City, CA).
Cell Survival Analysis
Astrocyte survival was assessed by counting the number of
intact nuclei after lysing cells in detergent-containing solution as
described initially by Soto and Sonnenschein (1985) and mod-
ified successively by Volonte et al. (1994). In all cases, 4 dishes
were scored in 4 independent experiments.
Immunofluorescence Studies
For immunofluorescence studies, both C6 cells and primary
astrocytes were seeded in poly-D-lysine-coated coverslips. Cells
were washed three times in phosphate-buffered saline (PBS) and

fixed in freshly prepared 4% paraformaldehyde-PBS for 10 min at
room temperature. Permeabilization was carried out in PBS/Triton
X-100 (0.2%) for 5 min at room temperature. After three washes in
PBS, cells were exposed to the appropriate dilution of primary
antibodies in incubation buffer (0.2 mg/ml bovine serum albumin
[BSA] in PBS) for 1 hr at room temperature. Cells were labeled for
p53, cyt-c, and MDM-2. After three washes in PBS, cells were
exposed to secondary antibody in the same buffer for 45 min at
room temperature. Preparations were mounted with 70% glycerol
in PBS. Samples were examined with a Leica fluorescence micro-
scope coupled to a CCD camera, equipped with a 100⫻ oil
immersion lens. The dye Hoechst 33258 (Sigma-Aldrich, MI, Italy)
was used at 0.5 ␮g/ml in PBS for 30 sec at room temperature
before the cells were fixed. Primary antibodies used were anti-p53
(1:200) and anti-cyt-c (1:200) from Pharmingen (Becton Dickin-
son, MI, Italy) and anti-MDM-2 (1:200) from Santa Cruz Biotech-
nology (Santa Cruz, CA). Secondary antibody was a Texas Red-
conjugated anti-mouse IgG (1:300; Calbiochem).
Confocal Fluorescent Imaging
For confocal microscopy analyses, primary astrocyte cultures
were incubated for 30 min with MitoTracker Red 580 (200 nM
final concentration; Molecular Probes Europe BV, Poortgebouw,
The Netherlands) at the end of the recovery period then fixed with
freshly prepared 2.5% glutaraldehyde in PBS (Sigma-Aldrich) for
30 min at 4°C. Successively glutaraldehyde-induced autofluores-
cence was quenched by incubating cultures in sodium borohydride
(2 mg/ml for 3 ⫻ 10 min; Sigma-Aldrich). Cells were then
permeabilized and processed for p53 immunofluorescence using a
fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody
(1:500; Sigma-Aldrich) as above. Fluorescently labeled preparations
were observed using a Leica TCS 4D confocal laser scanning
microscope supplemented with an argon/krypton laser and
equipped with 40⫻ 1.00 – 0.5 and 100⫻ 1.3– 0.6 oil immersion
lenses. Confocal sections were taken at intervals of 0.5 ␮m.
Excitation/emission wavelengths employed were 488 nm/510 nm,
for FITC labeling, and 568 nm/590 nm, for MitoTracker dye. The
acquisitions were recorded, employing pseudo-color representa-
tion.
Nucleosomal Formation Assay
Both C6 cells and primary astrocytes were seeded in 24-well
plates. After 48 hr and 8–10 days, respectively, the culture medium
was removed and cells were exposed to the oxidative stress protocol
detailed above. Control cells were incubated with HBSS only
during the stress period. After recovery, cells were lysed and nu-
cleosomal formation was assayed by quantitative in vitro measure-
ment of cytoplasmic histone-associated DNA fragments (Cell
Death Detection ELISAPLUS; Roche, MI, Italy), according to the
manufacturer’s recommendations.
Results are expressed as the enrichment factor obtained
according to the formula:
共关ODexperimental兴 ⫺ 关ODbackground兴兲/共关ODcontrol兴 ⫺ 关ODbackground兴兲
where the experimental point refers to the assay of nucleosomes
present at a given time point during stress or recovery periods,
background consists of OD from ELISA plate dishes where
cellular extracts were omitted, and the control point refers to the
Astrocyte Apoptosis and p53 85
basal apoptosis level, i.e., the assay of nucleosomes present in the
culture immediately before stress (also termed basal).
Data are presented as means ⫾ SEM. Each experiment
was repeated at least 3 times using different batches of cells.
Statistical analysis was carried out by one-way analysis of vari-
ance (ANOVA) followed by Bonferroni’s post-hoc comparison
test. Results were considered statistically significant when P
value was below 0.05.
RESULTS
Analysis of Cell Death, Nuclear Morphology, and
Nucleosomal Formation Assay in Glial Cells After
Reactive Oxygen Species Exposure
Cell survival was analyzed in primary cultures of rat
astrocytes (Fig. 1A). This analysis demonstrated that HBSS
exposure during the stress period did not induce cell death
even after 24 hr recovery. On the contrary, cultures ex-
posed to reactive oxygen species (ROS) exhibited cell
death: after 6 hr in untouched control cultures, there was
53.98 ⫾ 3.98% viable cells; after 12 hr, 45.43 ⫾ 7.41%;
and after 24 hr, 28.91% ⫾ 7.67. This demonstrated that
after 24 hr recovery, more than 70% of the cells were dead.
Similar data were obtained in C6 glioma cells (not shown).
To determine whether cell death was due to apoptosis,
a time course analysis of nuclear morphology using Hoechst
33258 labeling (Fig. 1B) and a nucleosomal formation assay
(Fig. 1C) was carried out. Both C6 glioma cells and primary
astrocytes exposed to HBSS during the stress period did not
enter apoptosis, even after 24 hr recovery, as demonstrated by
the absence of chromatin condensation and the lack of nu-
cleosomal enrichment (Fig. 1B,C). On the contrary, after
12 hr recovery, apoptotic nuclei could be seen clearly in both
cell cultures exposed to ROS, with a marked increase after
24 hr reperfusion (Fig. 1B). The same results were confirmed
by increased nucleosomal formation (Fig. 1C): the enrich-
ment factor increased significantly to 3.20 ⫾ 0.22 in C6 cells
(P ⬍ 0.05 vs. HBSS) and 2.07 ⫾ 0.36 in primary cultures of
astrocytes (P ⬍ 0.05 vs. HBSS) after 12 hr reperfusion. This
increase was much more pronounced after 24 hr of reperfu-
sion, reaching 8.90 ⫾ 0.45 and 6.69 ⫾ 1.30, respectively
(P ⬍ 0.01 vs. HBSS in both cases).
Time Course Semiquantitative RT-PCR Analysis
of Various Bcl-2 Family Members and p53-
Regulated Genes
To test for possible involvement of p53 transcriptional
activity in the onset of oxidative stress-induced apoptosis and,
eventually, a relationship with Bcl-2 family member mRNA
expression levels, we carried out a time course semiquanti-
tative RT-PCR analysis on several potential targets (Fig. 2).
This analysis demonstrated that in C6 glioma cells, Bcl-2,
Bcl-xL, Bax, and Bak mRNA expression remained almost
unchanged throughout the experiment. On the contrary,
Bcl-xS and Bid transcripts were upregulated already during
the stress period and their expression levels remained signif-
icantly higher compared to baseline during the recovery
period. In addition, Bad mRNA tended to increase during
recovery but this increase was more evident for MDM-2 and
86 Bonini et al.
Fig. 1. A: Cell viability assay in primary astrocytes exposed to HBSS or
ROS for 1 hr and followed by a 6-, 12-, or 24-hr recovery period.
After ROS exposure, dead cells represent almost 50% of the population
after 6 hr and reach more than 70% after 24 hr. B: C6 cells and primary
astrocytes exposed to oxidative stress (0.1 mM FeSO4/H2O2 for 1 hr)
undergo apoptosis only during the recovery period. Both cell types
were exposed to ROS (or HBSS alone as control) for 1 hr and then
allowed to recover for 12 and 24 hr. Hoechst staining shows that real
apoptotic signs, such as nuclear shrinking and chromatin condensation,
can be observed only after ROS exposure and 24 hr of recovery. Rare
apoptotic nuclei were also observed in primary astrocytes after 12 hr of
recovery. Arrows point to apoptotic nuclei. C: Nucleosomal formation
assay demonstrating severe DNA fragmentation in both experimental
models, particularly after 24 hr of recovery. *P ⬍ 0.05; **P ⬍ 0.01.
p53 mRNA; the latter was upregulated transiently but sig-
nificantly during recovery.
In primary cultures of astrocytes, similar results oc-
curred for all transcripts studied except Bcl-2, Bcl-xL, and
MDM-2. Indeed, during 24 hr of recovery, Bcl-2 expres-
sion was reduced slightly compared to a progressive in-
crease in Bcl-xL expression. MDM-2 expression levels
remained unchanged.
Genomic Effects of PF-␣ and Role in Apoptotic
Response
We monitored after 24 hr of recovery the genomic
effects of PF-␣ on p53 transcriptional activity. We carried
out a step-cycle RT-PCR analysis of mRNA expression
levels in C6 cells for H4 (as a housekeeping control gene)
and GAPDH was used as a positive control because it is
known to be regulated by p53 (Chen et al., 1999).
This experiment demonstrated that whereas the H4
housekeeping gene expression level remained unaffected
by PF-␣ treatment, the GAPDH expression level was
decreased in a dose-dependent manner (20 –500 nM; Fig.
3A). Similar results were obtained on primary astrocytes
(not shown). We next carried out a nucleosomal forma-
tion assay in parallel on C6 glioma cells and primary
cultures of astrocytes exposed or not exposed to PF-␣
treatment (100 nM) after 24 hr recovery (Fig. 3B). In both
experimental models, 1 hr oxidative stress followed by
24 hr recovery markedly induced nucleosomal formation.
This increase reached 8.56 ⫾ 0.45 in C6 cells and 7.33 ⫾
0.75 in primary cultures of glia.
In the presence of PF-␣, the enrichment factors in
C6 cells and primary cultures of astrocytes were 7.65 ⫾
0.65 and 6.54 ⫾ 0.60, respectively. No significant differ-
ences were noted cells treated with PF-␣ and untreated
cells were compared.
Immunofluorescence Analysis of p53, Cyt-c, and
MDM-2 Intracellular Distribution
Time-course immunofluorescence analysis of p53,
cyt-c, and MDM-2 protein expression combined with
Hoechst labeling of the nuclei showed that p53 was detect-
able almost exclusively in the nucleus until the end of the
stress period in both experimental models (Fig. 4, top). The
expression level of p53 increased progressively over the 24-hr
recovery period, showing mainly a cytoplasmic localization
of the protein in both experimental systems. The increase in
p53 expression level was more pronounced in C6 cells when
compared to that in primary astrocytes.
Cyt-c distribution analysis in both experimental models
(Fig. 4, center) evidenced a typical mitochondrial localization
in intact cells after 1 hr stress; filamentous mitochondria were
discerned clearly. After 3– 6 hr recovery, mitochondrial struc-
ture was altered profoundly, showing the typical apoptotic
organization known as megamitochondria (MG). After 12
and 24 hr of recovery, changes in mitochondrial structure and
apoptotic changes of cells were evidenced clearly and prob-
ably reflected cyt-c release. Hoechst labeling also evidenced
chromatin condensation in both cell systems after 12–24 hr of
recovery.
Time course immunofluorescence analysis of
MDM-2 (Fig. 4, bottom), showed in both models a very
low expression level in control cells and at the end of the
stress period. After 3 hr of recovery, MDM-2 seemed
strongly upregulated, showing a marked nuclear localiza-
tion. In both experimental models, MDM-2 immunore-
activity started to decrease after 6 hr of recovery and after
12 hr was almost undetectable.
 Astrocyte Apoptosis and p53 87

Fig. 2. Semiquantitative PCR analysis of

p53 and Bcl-2 family member mRNA
expression levels. This analysis shows that
concomitantly with p53, several pro-
apoptotic genes such as Bad, Bid and
Bcl-xS are upregulated during oxidative
stress and recovery periods. A concomitant
slight decrease was observed in expression
levels of anti-apoptotic genes such as Bcl-2
and Bcl-xL.

Fig. 3. A: Step-cycle RT-PCR analysis of GAPDH and H4 histone.

PF-␣ treatment leads to a marked shift of the amplification curve,
compared to that of untreated cells, starting at a concentration of
20 nM. On the contrary, the H4 housekeeping gene profile is not
affected by PF-␣ treatment. B: Nucleosomal formation assay. Both cell
types were exposed to 1 hr ROS application followed by 24 hr of
recovery in the presence (ROS ⫹ PF-␣) or absence (ROS) of PF-␣.
Control cells were exposed to HBSS alone. It seems that PF-␣ does not
prevent DNA fragmentation.

Confocal Microscopy Analysis of p53 Intracellular labeling after MitoTracker labeling of mitochondria (Fig.
Distribution 5). For this purpose, primary astrocytes were exposed to
To document further the intracellular distribution of 1 hr oxidative stress followed by 6 or 24 hr of recovery.
p53, we carried out confocal microscopy analysis of p53 Confocal microscopy analysis evidenced a clear mitochon-
88 Bonini et al.
drial localization of p53 after ROS exposure compared to
that in HBSS treated cells.
Effects of zVAD-fmk and PF-␣ on p53 and
MDM-2 Distribution After 6 Hours of Recovery
The p53 regulator MDM-2 contains a caspase-3-like
cleavage site (Chen et al., 1997). We therefore wanted to
monitor whether zVAD-fmk treatment could modulate
MDM-2 expression in our experimental paradigm. For
this purpose, C6 cells and primary astrocytes were exposed
to 1 hr of oxidative stress followed by 6 hr of recovery in
presence or absence of the broad-spectrum caspase inhib-
itors zVAD-fmk or PF-␣. In both experimental models,
Fig. 4. Top: Immunofluorescence anal-
ysis of p53 during oxidative stress-
induced injury. In both C6 and primary
astrocytes, p53 is expressed at very low
levels under basal conditions but in-
creases during stress periods, showing a
nuclear localization. After 24 hr of re-
covery, p53 localization changes mark-
edly, evidencing a translocation to the
cytoplasm. Center: Immunofluores-
cence analysis of cyt-c during oxidative
stress-induced injury. Mitochondrial
structure remodeling and cyt-c release in
primary astrocytes and C6 cells exposed
to oxidative stress-induced injury. Un-
der basal conditions, mitochondria dis-
play a healthy morphology characterized
by filamentous structures. Within 3– 6 hr
of reperfusion, megamitochondria for-
mation is evident. Subsequently (12–24
hr of recovery), mitochondrial disrup-
tion and chromatin condensation can be
observed. Bottom: Immunofluores-
cence analysis of MDM-2 during oxida-
tive stress-induced injury. In both C6
and primary astrocytes, MDM-2 shows a
low expression level at the end of the
stress period and a marked increase is
detectable after 3 hr of recovery, show-
ing a strong nuclear localization. Subse-
quently, MDM-2-like immunoreactiv-
ity decreases (see the 6-hr recovery
point), becoming almost undetectable
after 12 hr of recovery until the end of
the experiment.
zVAD-fmk, but not PF-␣ inhibited MDM-2 degradation
(Fig. 6, bottom). Conversely, a marked reduction of p53
protein level was observed in zVAD-fmk but not in PF-␣
treated cells (Fig. 6, top).
Effects of zVAD-fmk on Nucleosomal Formation
Because caspase inhibition was able to modulate
both p53 and MDM-2 expression levels, we investi-
gated whether this could inhibit apoptosis. We moni-
tored the effects of zVAD-fmk on nucleosomal forma-
tion in both experimental models after 24 hr recovery
(Fig. 7). This assay demonstrated that in both C6 cells
and primary astrocytes, zVAD-fmk protected cells from
 Astrocyte Apoptosis and p53 89

Fig. 5. Confocal microscopy analysis of

p53 redistribution during oxidative
stress-induced injury in primary cultures
of astrocytes after mitochondrial staining
(MitoTracker). HBSS control is pre-
sented on the first column after 24 hr of
recovery. Second and third column
show primary astrocytes exposed to 1 hr
ROS, after 6 and 24 hr recovery, respec-
tively. Immunolabeling of p53 is barely
detectable in HBSS-treated cells com-
pared to that in ROS-exposed astro-
cytes, where p53 can be seen in the
nucleus after 6 hr of recovery. Please
note on overlay lane the almost com-
plete overlay for MitoTracker and p53
labeling in ROS-treated cells, docu-
menting the p53 mitochondrial locali-
zation.

oxidative stress-induced apoptosis: the enrichment fac- DISCUSSION

tor decreased from 7.54 ⫾ 0.70 to 2.93 ⫾ 0.35 (P ⬍
0.01 vs. HBSS) in C6 cells and from 5.26 ⫾ 0.65 to
1.84 ⫾ 0.75 (P ⬍ 0.01 vs. HBSS) in primary astrocytes
after 24 hr of recovery.
Effects of zVAD-fmk and PF-␣ on Cyt-c
Distribution After 24 Hours of Recovery
To document further the effects of caspase blockade
on apoptosis with respect to PF-␣, cyt-c immunolocaliza-
tion was monitored in both C6 cells and primary astro-
cytes exposed to 1 hr of oxidative stress followed by 24 hr
recovery, treated or not treated with zVAD-fmk and
compared to those treated with PF-␣ (Fig. 8). This analysis
evidenced that mitochondrial disruption, cyt-c release,
and chromatin condensation were completely abrogated
by zVAD-fmk treatment, whereas PF-␣ treatment did not
impede these events.
Glial Cells Enter Apoptosis During Recovery After
ROS Exposure
To mimic oxidative stress injury, cultured rat cortical
astrocytes and C6 glioma cells were exposed to ROS in
HBSS for 1 hr followed by a recovery period in which cells
were placed in complete growth medium. Time course
analysis of cell viability, nuclear morphology, (Hoechst 33258
labeling) and a nucleosomal formation assay demonstrated
that both C6 glioma cells and primary astrocytes die when
exposed to ROS but not when exposed to HBSS during the
stress period. Cell death involved up to 70% of the cells
within the 24 hr of recovery and was identified clearly as
apoptotic cell death based on nuclear morphology after
Hoechst staining and enrichment of nucleosome formation.
After 12 hr of recovery, apoptotic nuclei could be seen clearly
in both cell cultures exposed to ROS, with a marked increase
after 24 hr of recovery. This induction of apoptosis due to
90 Bonini et al.
ROS exposure was confirmed by the increase in nucleoso-
mal formation.
Expression Levels of Bcl-2 Family Member
mRNA Are Modulated During Recovery: Possible
Involvement of p53
In recent years, a critical role for mitochondria in
apoptosis induction has been demonstrated (Matsuyama
and Reed, 2000). In particular, these organelles release
proteins into the cytosol such as cyt-c and apoptosis-
inducing factor (AIF), triggering caspase-dependent and
independent apoptotic mechanisms (Green, 1998; Reed et
al., 1998; Susin et al., 1998, 1999). Several pro-apoptotic
proteins of Bcl-2 family, such as Bid, Bak, and Bax play a
pivotal role in this process (Eskes et al., 1998, 2000). In
addition, expression of many members of the Bcl-2 pro-
teins family are regulated by p53 (Kannan et al., 2000).
Fig. 6. Immunofluorescence analysis of
p53 and MDM-2: effects of zVAD-fmk
and PF-␣ treatments. C6 and primary
astrocytes were exposed to ROS fol-
lowed by 6 hr of recovery (second col-
umn) compared to cells exposed to ox-
idative stress but treated with zVAD-fmk
(third column) or PF-␣ (fourth column).
Basal (first column) refers to intact cells.
MDM-2 degradation is inhibited by
zVAD-fmk, leading to concomitant p53
degradation. No p53 immunoreactivity
can be detected in cytosol of cells treated
with zVAD-fmk. PF-␣ did not interfere
with MDM-2 distribution or with p53
mitochondria-like accumulation.
Semiquantitative RT-PCR analysis (Fig. 2) showed
that in both experimental models p53 mRNA was upregu-
lated transiently but significantly during recovery with a
parallel upregulation of pro-apoptotic Bax and Bad genes.
Moreover, two other pro-apoptotic members of the Bcl-2
family, Bcl-xS and Bid, were upregulated already during the
oxidative period. The anti-apoptotic genes Bcl-2 and Bcl-xL
remained almost unchanged in C6 glioma cells whereas in
primary astrocytes, during 24 hr of recovery, Bcl-2 expres-
sion was reduced slightly compared to a progressive increase
in Bcl-xL expression. Finally, MDM-2 mRNA was upregu-
lated in C6 cells, but not in primary astrocytes, during re-
covery. The expression profile of pro-apoptotic genes ob-
served in our system was in agreement with a previous
large-scale expression analysis of nonneuronal models of p53-
induced apoptosis (Polyak et al., 1997).

Interestingly, although most pro-apoptotic genes
were upregulated during oxidative stress simulation (T0
time point, Fig. 2), cells exhibited real apoptotic signs (i.e.,
nuclear condensation) only after several hours of recovery
(Fig. 1A). These data confirm observations published pre-
viously regarding p53 induction in the human glioblas-
toma cell line A172 after exposure to oxidative stress;
however, these authors reported an increase in Bak ex-
pression, which did not occur in our system (Kitamura et
al., 1999).
All these findings suggest a possible role for p53
transcriptional activity on apoptosis onset, leading the bal-
Fig. 7. Nucleosomal formation assay in zVAD-fmk treated cultures.
Both cell types were exposed to 1 hr oxidative stress followed by 24 hr
of recovery in the presence (ROS ⫹ zVAD-fmk) or absence (ROS) of
zVAD-fmk. Control cells were exposed to HBSS alone. Nucleosomal
formation was prevented significantly by zVAD-fmk.
Fig. 8. Immunofluorescence analysis of
cyt-c: effects of zVAD-fmk and PF-␣
treatment. C6 cells and primary astro-
cytes were exposed to ROS followed by
24 hr of recovery (second column) com-
pared to cells exposed to oxidative stress
but treated with zVAD-fmk (third col-
umn) or PF-␣ (fourth column). Basal
(first column) refers to intact cells. In
both primary astrocytes and C6 cells
treated with zVAD-fmk (third column),
mitochondrial structure is preserved,
cyt-c immunoreactivity is not altered,
and chromatin condensation cannot be
observed. In cells treated with PF-␣
(fourth column), no peculiar difference
can be observed when compared to cells
exposed to oxidative stress (second col-
umn).
Astrocyte Apoptosis and p53 91
ance of pro-apoptotic versus anti-apoptotic genes towards
pro-apoptotic genes.
PF-␣ Inhibits p53 Transcriptional Activity but
Does Not Protect Glial Cells Against Oxidative
Stress-Induced Apoptosis
Recent preclinical studies have demonstrated the
efficacy of a p53 inhibitor in models of stroke and neuro-
degenerative disorders, and have suggested that drugs that
inhibit p53 may reduce the extent of brain damage in
related human neurodegenerative conditions (Culmsee et
al., 2001a,b; Lakkaraju et al., 2001; Chung et al., 2002).
Based on these data, we tested the potential protective
properties of PF-␣.
We first carried out a pilot experiment using the
nucleosomal formation assay, to evaluate a potential pro-
apoptotic effect of PF-␣ treatment. We showed that, as
reported previously in neurons (Culmsee et al., 2001b),
PF-␣ was without significant effect on glial cell survival at
concentrations up to 200 nM, although higher concentra-
tions slightly induced apoptosis, as revealed by nucleoso-
mal formation assay (data not shown).
To verify whether after 24 hr of recovery PF-␣
remained able to block p53 transcriptional activity, we
used step-cycle RT-PCR analysis to monitor in C6 cells
the mRNA expression levels for H4, as a housekeeping
control gene, and GAPDH, because its expression is mod-
ulated by p53 as mentioned previously (Chen et al., 1999).
The dose-dependent decrease in GAPDH expression
compared to the lack of H4 housekeeping gene expression
modulation confirmed PF-␣ efficacy at THE genomic
level (Fig. 3A).
We therefore decided to use 100 nM PF-␣ to test its
putative protective effect against oxidative stress-induced
apoptosis, in both rat primary cultures of cortical astrocytes
and C6 glioma cells.
92 Bonini et al.
We analyzed nucleosomal formation in both exper-
imental cell models after 24 hr of recovery in the presence
or absence of PF-␣ (Fig. 3B). Strikingly, we observed that
PF-␣ failed to protect both primary astrocytes and C6 cells
against oxidative stress-induced apoptosis, despite its abil-
ity to inhibit p53 genomic effects. This was an unexpected
result because in neurons, this compound was able to
prevent apoptosis induced by ischemic insult, as reported
previously (Komarov et al., 1999). In addition, the lack of
protection against apoptosis after PF-␣ treatment further
supports the hypothesis that discrepancies in various gene
expression noticed between C6 cells and primary astro-
cytes may not have a primary importance for cell survival.
Intracellular Redistribution of p53 Toward
Mitochondria After Oxidative Stress Injury
Nuclear bypass experiments demonstrated that mi-
tochondrial localization of p53 is sufficient to induce p53-
dependent apoptosis, indicating dual action of this protein
during apoptosis (Ding and Fisher, 1998; Ding et al., 1998;
Marchenko et al., 2000).
Time-course immunofluorescence analysis of p53
protein combined with Hoechst labeling of the nuclei
(Fig. 4), showed that p53 was detectable in both experi-
mental models almost exclusively in the nucleus until the
end of the stress period. The p53 expression level increased
progressively over the 24-hr recovery period, showing an
almost cytoplasmic localization of the protein in both
experimental systems. The p53 expression level seemed
higher in C6 when compared to that in primary astrocytes,
in agreement with RT-PCR analysis.
It has been reported that when present in cytoplasm,
most p53 protein is localized within the mitochondrial
membrane compartment, although a small fraction of the
protein can be found in other organelles. This mitochon-
drial localization of p53 was confirmed by confocal mi-
croscopy analysis in primary astrocytes (Fig. 5).
At the mitochondrial level, p53 exerts its pro-
apoptotic function by favoring cyt-c release and subse-
quent caspase activation (Marchenko et al., 2000; Sansome
et al., 2001; Schuler et al., 2000; Schuler and Green,
2001). To investigate further such a mechanism, we car-
ried out a time course immunofluorescence analysis of
cyt-c distribution. Both C6 cells and primary astrocytes
were exposed to ROS for 1 hr and analyzed within 24 hr
of recovery. As shown in Figure 4 in control cells (basal
point), filamentous mitochondria were discerned clearly
whereas between 3– 6 hr of recovery and concomitantly
with p53 redistribution toward mitochondria (see Fig. 4 at
6 hr recovery), the mitochondrial structure was altered
profoundly, showing a typical apoptotic organization
known as megamitochondria (MG). At this point, cyt-c
release was not yet detectable, leading to a situation that
usually, but not necessarily, precedes apoptosis (Karbowski
et al., 1999; Teranishi et al., 2000). After 24 hr recovery,
changes in mitochondrial structure and apoptotic changes
in cells were marked and showed the swelling of free
radical-induced megamitochondria, which in turn were
responsible for cyt-c release and nuclear shrinking. In
agreement with previous findings, these data suggested
that p53 was able to induce apoptosis through its translo-
cation to mitochondria and the subsequent release of cyt-c
(Schuler et al., 2000).
Mitochondrial Localization of p53 Requires
MDM-2 Degradation via Caspase Activity
MDM-2 protein is a key player in the regulation of
p53. Thus, MDM-2 binds to p53 within its transactivation
domain, interfering with recruitment of basal transcription
machinery components, and within the nucleus MDM-2
can bind p53 targeting newly formed complexes toward
the cytoplasm due to a nuclear export sequence (NES)
present in the MDM-2 protein sequence. This leads to the
degradation of p53 by the ubiquitin-proteasome pathway
(Haupt et al., 1997; Kubbutat et al., 1997).
To investigate the involvement of MDM-2 in the
apoptotic response in our experimental model, we carried
out a time course immunofluorescence analysis. As shown
in Figure 4, MDM-2 was expressed in both C6 and
primary cultures of rat astrocytes at very low levels until
the end of ROS exposure, whereas p53 was upregulated
already. MDM-2 reached its maximum expression level
after 3 hr of recovery with a marked nuclear localization
and seemed barely detectable after 6 hr of recovery. Si-
multaneously, p53 immunoreactivity increased strongly,
showing a marked cytosolic signal. After 12 hr of recovery
MDM-2 immunoreactivity decreased strongly becoming
almost undetectable until the end of the recovery period.
These data suggested a direct relationship between the
increase in cytoplasmic p53 expression, and its mitochon-
drial redistribution, and the corresponding MDM-2 ex-
pression levels.
MDM-2 contains a caspase-3-like cleavage site that
is evolutionarily conserved (Chen et al., 1997), and in
vitro experiments carried out in H1299 cells, an epithelial
cell line expressing a temperature-sensitive human p53,
demonstrated that caspase-3 is able to cleave MDM-2.
The cleaved MDM-2 loses the ability to promote p53
degradation and potentially functions in a dominant-
negative fashion to stabilize p53 (Pochampally et al.,
1999).
To verify a possible relationship between MDM-2
degradation and p53 expression levels, we exposed both
C6 cells and primary astrocytes to oxidative stress injury in
the presence or absence of the broad-spectrum caspase
inhibitor zVAD-fmk. We also analyzed the effect of PF-␣
treatment. We demonstrated that p53 and MDM-2 ex-
pression levels displayed a complementary but opposite
profile after 6 hr of recovery (Fig. 4). As shown in Figure
6, in both C6 cells and primary astrocytes, zVAD-fmk, but
not PF-␣ inhibited MDM-2 degradation, leading to a
marked reduction of p53 protein level and prevention of
its translocation toward mitochondria.
These findings suggest a cause-and-effect relation-
ship between caspase activity, MDM-2 degradation, and
p53 protein stabilization.
 Astrocyte Apoptosis and p53 93

Fig. 9. Proposed mechanism of oxida-

tive stress-induced apoptosis in glia cells.
Dashed arrows represent the physiolog-
ical pathway in which p53 contributes to
MDM-2 transcription. When exported
into the cytoplasm, MDM-2 protein
binds p53, driving its degradation
through ubiquitin-proteasome pathway.
This contributes to maintain low p53
expression level. In the presence of ROS
(large arrows), partial mitochondrial
membrane depolarization leads to cyt-c
release and caspase activation, which in
turn contributes to MDM-2 degrada-
tion. p53 can translocate toward mito-
chondria, leading to a massive cyt-c re-
lease and establishing an apoptotic loop.
Treatment with zVAD-fmk abolishes
onset of the apoptotic loop. PF-␣ is un-
able to protect cells from apoptosis, in-
dicating that p53 transcriptional activity
is not the main pathway involved in our
model of oxidative stress-induced apo-
ptosis.

Pro-apoptotic Mitochondrial Function of p53 Is
Abrogated Completely by zVAD-fmk but not by
PF-␣
We demonstrated that, in our models, p53 redistri-
bution within the cytosol is related to cyt-c release from
mitochondria and cell death execution (Fig. 4). In the
presence of zVAD-fmk, p53 mitochondrial targeting was
abrogated completely by preventing MDM-2 caspase-
dependent degradation. The question then raised was
whether this process was sufficient to prevent apoptotic
cell death. For this purpose, we monitored nucleosomal
formation in both experimental models after to 24 hr of
recovery in the presence or absence of zVAD-fmk (Fig. 7).
This assay demonstrated that in both C6 cells and primary
astrocytes, zVAD-fmk protected cells from ROS
exposure-induced apoptosis; indeed, the enrichment fac-
tor was decreased strongly in both cell cultures after 24 hr
of recovery.
Finally, we investigated whether zVAD-fmk protec-
tion was related to the inhibition of p53-mediated cyt-c
94 Bonini et al.
release. Both C6 cells and primary astrocytes were exposed
to ROS, treated or not with zVAD-fmk and compared to
sisters cultures exposed to ROS not treated with PF-␣.
This analysis evidenced that after 24 hr of recovery, no
cyt-c released could be observed and mitochondria dis-
played a filamentous and healthy morphology in zVAD-
fmk treated cells only.
In conclusion, we have found that in a glial model of
oxidative stress-induced apoptosis, p53 plays a pivotal role
by directly signaling mitochondria despite its transcrip-
tional activity. PF-␣ fails to protect glial cells from ROS
exposure despite its ability to inhibit p53 genomic effects,
contrary to what has been observed in neuronal cells
(Komarov et al., 1999). Our data suggest that in the
apoptotic cascade, three fundamental steps are involved.
The first step, engagement, involves low cyt-c release from
mitochondria within first 3 hr of reperfusion (probably
due to a partial mitochondrial membrane depolarization,
as evidenced by JC-1 mitochondrial potential sensor anal-
ysis; data not shown). This is followed by partial caspase
activation, which in turn acts on early targets such as
MDM-2, leading to a redistribution of p53 toward mito-
chondria. In the second step, commitment, concomitantly
with p53 redistribution into the cytosol, mitochondria
modify their structure by forming megamitochondria
(MG) within 6 hr of recovery. MG formation may be an
adaptive and reversible response to unfavorable environ-
ments at the subcellular level that leads to a decrease in
oxygen consumption and ROS production (Karbowski et
al., 1999). The third and last step, death-warrant, occurs
after 24 hr of reperfusion when a complete disruption of
free radical-induced MG leads to cyt-c release, caspase
activation, and cell death.
It is now accepted generally that massive neuronal
death due to oxidative stress is a regular feature in neuro-
degenerative diseases of the brain. Much less attention,
however, has been given to glial cells death. Several studies
suggest that glial cells in neurodegenerative diseases (i.e.,
Alzheimer’s disease) are affected more than neurons by
apoptotic cell death (Lassmann et al., 1995; Smale et al.,
1995).
In our glial system, we highlight for the first time
that glial cells respond to oxidative stress undergoing ap-
optosis via p53 transcription independent pathways, which
is summarized in Figure 9. Based on these results, drugs
such as PF-␣ that selectively protect neurons from apo-
ptotic stimuli would not be sufficient in the treatment of
neurodegenerative diseases. Therefore, therapeutic ap-
proaches based on caspase inactivation (Cheng et al., 1998;
Robertson et al., 2000, 2001) are likely a more efficient
strategy against neurodegenerative diseases because in cell
death execution, both glial cells and neurons exhibit a final
common pathway in which caspases are the main actors.
ACKNOWLEDGMENTS
We thank Dr. S. Biocca (University of Rome Tor
Vergata) for the gift of C6 cells and for critical discussions.
This work was supported in part by research fellowships
(to P.B., S.C., and A.C. from Ministry of Education,
University and Research, Miur-Italy).
REFERENCES
Avshalumov MV, Rice ME. 2002. NMDA receptor activation mediates
hydrogen peroxide-induced pathophysiology in rat hippocampal slices.
J Neurophysiol 87:2896 –2903.
Barak Y, Juven T, Haffner R, Oren M. 1993. mdm2 expression is induced
by wild type p53 activity. EMBO J 12:461– 468.
Brady HJ, Gil-Gomez G. 1998. Bax. The pro-apoptotic Bcl-2 family
member, Bax. Int J Biochem Cell Biol 30:647– 650.
Chen J, Wu X, Lin J, Levine AJ. 1996. mdm-2 inhibits the G1 arrest and
apoptosis functions of the p53 tumor suppressor protein. Mol Cell Biol
16:2445–2452.
Chen L, Marechal V, Moreau J, Levine AJ, Chen J. 1997. Proteolytic
cleavage of the mdm2 oncoprotein during apoptosis. J Biol Chem 272:
22966 –22973.
Chen RW, Saunders PA, Wei H, Li Z, Seth P, Chuang DM. 1999.
Involvement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
and p53 in neuronal apoptosis: evidence that GAPDH is upregulated by
p53. J Neurosci 19:9654 –9662.
Cheng T, Liu D, Griffin JH, Fernandez JA, Castellino F, Rosen ED,
Fukudome K, Zlokovic BV. 2003. Activated protein C blocks p53-
mediated apoptosis in ischemic human brain endothelium and is neuro-
protective. Nat Med 9:338 –342.
Cheng Y, Deshmukh M, D’Costa A, DeMaro JA, Gidday JM, Shah A, Sun
Y, Jacquin MF, Johnson EM, Holtzman DM. 1998. Caspase inhibitor
affords neuroprotection with delayed administration in a rat model of
neonatal hypoxic-ischemic brain injury. J Clin Invest 101:1992–1999.
Chung YH, Shin CM, Kim MJ, Lee EY, Kim G, Cha CI. 2002. Enhanced
expression of p53 in reactive astrocytes following transient focal ischemia.
Neurol Res 24:324 –328.
Culmsee C, Bondada S, Mattson MP. 2001a. Hippocampal neurons of mice
deficient in DNA-dependent protein kinase exhibit increased vulnerabil-
ity to DNA damage, oxidative stress and excitotoxicity. Brain Res Mol
Brain Res 87:257–262.
Culmsee C, Zhu X, Yu QS, Chan SL, Camandola S, Guo Z, Greig NH,
Mattson MP. 2001b. A synthetic inhibitor of p53 protects neurons against
death induced by ischemic and excitotoxic insults, and amyloid beta-
peptide. J Neurochem 77:220 –228.
de la Monte SM, Sohn YK, Ganju N, Wands JR. 1998. P53- and CD95-
associated apoptosis in neurodegenerative diseases. Lab Invest 78:401–
411.
de la Monte SM, Sohn YK, Wands JR. 1997. Correlates of p53- and Fas
(CD95)-mediated apoptosis in Alzheimer’s disease. J Neurol Sci 152:73–
83.
Ding HF, Fisher DE. 1998. Mechanisms of p53-mediated apoptosis. Crit
Rev Oncog 9:83–98.
Ding HF, McGill G, Rowan S, Schmaltz C, Shimamura A, Fisher DE.
1998. Oncogene-dependent regulation of caspase activation by p53 pro-
tein in a cell-free system. J Biol Chem 273:28378 –28383.
Eskes R, Antonsson B, Osen-Sand A, Montessuit S, Richter C, Sadoul R,
Mazzei G, Nichols A, Martinou JC. 1998. Bax-induced cytochrome C
release from mitochondria is independent of the permeability transition
pore but highly dependent on Mg2⫹ ions. J Cell Biol 143:217–224.
Eskes R, Desagher S, Antonsson B, Martinou JC. 2000. Bid induces the
oligomerization and insertion of Bax into the outer mitochondrial mem-
brane. Mol Cell Biol 20:929 –935.
Gottlieb TM, Oren M. 1998. p53 and apoptosis. Semin Cancer Biol
8:359 –368.
Green DR. 1998. Apoptotic pathways: the roads to ruin. Cell 94:695– 698.
Haupt Y, Barak Y, Oren M. 1996. Cell type-specific inhibition of p53-
mediated apoptosis by mdm2. EMBO J 15:1596 –1606.

Haupt Y, Maya R, Kazaz A, Oren M. 1997. Mdm2 promotes the rapid
degradation of p53. Nature 387:296 –299.
Jenner P. 2003. Oxidative stress in Parkinson’s disease. Ann Neurol
53(Suppl):26 –38.
Jordan J, Galindo MF, Prehn JH, Weichselbaum RR, Beckett M, Ghadge
GD, Roos RP, Leiden JM, Miller RJ. 1997. p53 expression induces
apoptosis in hippocampal pyramidal neuron cultures. J Neurosci 17:1397–
1405.
Joseph JA, Strain JG, Jimenez ND, Fisher D. 1997. Oxidant injury in PC12
cells—a possible model of calcium “dysregulation” in aging: I. Selectivity
of protection against oxidative stress. J Neurochem 69:1252–1258.
Kannan K, Amariglio N, Rechavi G, Givol D. 2000. Profile of gene
expression regulated by induced p53: connection to the TGF-beta family.
FEBS Lett 470:77– 82.
Karbowski M, Kurono C, Wozniak M, Ostrowski M, Teranishi M, Nish-
izawa Y, Usukura J, Soji T, Wakabayashi T. 1999. Free radical-induced
megamitochondria formation and apoptosis. Free Radic Biol Med 26:
396 – 409.
Kitamura Y, Ota T, Matsuoka Y, Tooyama I, Kimura H, Shimohama S,
Nomura Y, Gebicke-Haerter PJ, Taniguchi T. 1999. Hydrogen
peroxide-induced apoptosis mediated by p53 protein in glial cells. Glia
25:154 –164.
Komarov PG, Komarova EA, Kondratov RV, Christov-Tselkov K, Coon
JS, Chernov MV, Gudkov AV. 1999. A chemical inhibitor of p53 that
protects mice from the side effects of cancer therapy. Science 285:1733–
1737.
Kubbutat MH, Jones SN, Vousden KH. 1997. Regulation of p53 stability
by Mdm2. Nature 387:299 –303.
Lakkaraju A, Dubinsky JM, Low WC, Rahman YE. 2001. Neurons are
protected from excitotoxic death by p53 antisense oligonucleotides de-
livered in anionic liposomes. J Biol Chem 276:32000 –32007.
Lassmann H, Bancher C, Breitschopf H, Wegiel J, Bobinski M, Jellinger K,
Wisniewski HM. 1995. Cell death in Alzheimer’s disease evaluated by
DNA fragmentation in situ. Acta Neuropathol (Berl) 89:35– 41.
Love S. 2003. Apoptosis and brain ischaemia. Prog Neuropsychopharmacol
Biol Psychiatry 27:267–282.
Marchenko ND, Zaika A, Moll UM. 2000. Death signal-induced localiza-
tion of p53 protein to mitochondria. A potential role in apoptotic
signaling. J Biol Chem 275:16202–16212.
Matsuyama S, Reed JC. 2000. Mitochondria-dependent apoptosis and
cellular pH regulation. Cell Death Differ 7:1155–1165.
Mercanti D, Luzzatto E, Ciotti MT, Levi G. 1987. Mitogenic effect of a
human placental factor on astrocytes and glial precursors. Exp Cell Res
168:182–190.
Miyashita T, Reed JC. 1995. Tumor suppressor p53 is a direct transcrip-
tional activator of the human bax gene. Cell 80:293–299.
Momand J, Wu HH, Dasgupta G. 2000. MDM2—master regulator of the
p53 tumor suppressor protein. Gene 242:15–29.
Momand J, Zambetti GP, Olson DC, George D, Levine AJ. 1992. The
mdm-2 oncogene product forms a complex with the p53 protein and
inhibits p53-mediated transactivation. Cell 69:1237–1245.
Morrison RS, Kinoshita Y, Johnson MD, Guo W, Garden GA. 2003.
p53-dependent cell death signaling in neurons. Neurochem Res 28:15–
27.
Napieralski JA, Raghupathi R, McIntosh TK. 1999. The tumor-suppressor
Astrocyte Apoptosis and p53 95
gene, p53, is induced in injured brain regions following experimental
traumatic brain injury. Brain Res Mol Brain Res 71:78 – 86.
Pochampally R, Fodera B, Chen L, Lu W, Chen J. 1999. Activation of an
MDM2-specific caspase by p53 in the absence of apoptosis. J Biol Chem
274:15271–15277.
Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. 1997. A model for
p53-induced apoptosis. Nature 389:300 –305.
Reed JC, Jurgensmeier JM, Matsuyama S. 1998. Bcl-2 family proteins and
mitochondria. Biochim Biophys Acta 1366:127–137.
Richter-Landsberg C, Vollgraf U. 1998. Mode of cell injury and death after
hydrogen peroxide exposure in cultured oligodendroglia cells. Exp Cell
Res 244:218 –229.
Robertson J, Beaulieu JM, Doroudchi MM, Durham HD, Julien JP,
Mushynski WE. 2001. Apoptotic death of neurons exhibiting peripherin
aggregates is mediated by the proinflammatory cytokine tumor necrosis
factor-alpha. J Cell Biol 155:217–226.
Robertson GS, Crocker SJ, Nicholson DW, Schulz JB. 2000. Neuropro-
tection by the inhibition of apoptosis. Brain Pathol 10:283–292.
Sansome C, Zaika A, Marchenko ND, Moll UM. 2001. Hypoxia death
stimulus induces translocation of p53 protein to mitochondria. Detection
by immunofluorescence on whole cells. FEBS Lett 488:110 –115.
Schuler M, Bossy-Wetzel E, Goldstein JC, Fitzgerald P, Green DR. 2000.
p53 induces apoptosis by caspase activation through mitochondrial cyto-
chrome c release. J Biol Chem 275:7337–7342.
Schuler M, Green DR. 2001. Mechanisms of p53-dependent apoptosis.
Biochem Soc Trans 29:684 – 688.
Smale G, Nichols NR, Brady DR, Finch CE, Horton WEJ. 1995. Evi-
dence for apoptotic cell death in Alzheimer’s disease. Exp Neurol 133:
225–230.
Soto AM Sonnenschein C. 1985. The role of estrogens on the proliferation
of human breast tumor cells (MCF-7). J Steroid Biochem 23:87–94.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM,
Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett
DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G.
1999. Molecular characterization of mitochondrial apoptosis-inducing
factor. Nature 397:441– 446.
Susin SA, Zamzami N, Kroemer G. 1998. Mitochondria as regulators of
apoptosis: doubt no more. Biochim Biophys Acta 1366:151–165.
Tatton WG, Chalmers-Redman R, Brown D, Tatton N. 2003. Apoptosis
in Parkinson’s disease: signals for neuronal degradation. Ann Neurol
53(Suppl):61–70.
Teranishi M, Spodonik JH, Karbowski M, Kurono C, Soji T, Wakabayashi
T. 2000. Swelling of free-radical-induced megamitochondria causes ap-
optosis. Exp Mol Pathol 68:104 –123.
Volonte C, Ciotti MT, Battistini L. 1994. Development of a method for
measuring cell number: application to CNS primary neuronal cultures.
Cytometry 17:274 –276.
Wu X, Bayle JH, Olson D, Levine AJ. 1993. The p53-mdm-2 autoregu-
latory feedback loop. Genes Dev 7:1126 –1132.
Xiang H, Kinoshita Y, Knudson CM, Korsmeyer SJ, Schwartzkroin PA,
Morrison RS. 1998. Bax involvement in p53-mediated neuronal cell
death. J Neurosci 18:1363–1373.
Zauberman A, Barak Y, Ragimov N, Levy N, Oren M. 1993. Sequence-
specific DNA binding by p53: identification of target sites and lack of
binding to p53–MDM2 complexes. EMBO J 12:2799 –2808.