Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and...

Nitric oxide-induced cell death of cerebrocortical murine astrocytes

is mediated through p53- and Bax-dependent pathways

Hong-Wa Yung,1 Anna K. Bal-Price,1 Guy C. Brown and Aviva M. Tolkovsky

Department of Biochemistry, University of Cambridge, Cambridge, UK

Abstract

We have investigated the mechanism by which nitric oxide

(NO) induces the death of mouse astrocytes. We show that

NO (from donor diethylenetriamine-NO adduct) induces

death with several features of apoptosis, including chroma-

tin condensation, phosphatidylserine exposure on the outer

leaflet of the plasma membrane, Bax translocation to the

mitochondria and cytochrome c release, but no caspase

activation or nuclear fragmentation is observed. Nitric oxide

also elevates p53 expression, causing a concomitant

increase in p53 serine 18 phosphorylation and p53 trans-

location from the cytoplasm to the nucleus. Activation of

Bax and p53 is important for NO-induced apoptosis-like cell

death because Bax- or p53-deficient astrocytes are much

more resistant than wild-type cells to the same NO treat-

ment. We further demonstrate that LY294002-sensitive

kinases are responsible for controlling serine 18 phos-

phorylation of p53, thereby regulating the pro-apoptotic

activity of p53 in astrocytes. While apoptosis is suppressed

in the presence of LY294002, however, death by necrosis is

increased, suggesting that LY294002-sensitive kinases

additionally suppress a latent necrotic response to NO. We

conclude that NO-induced death in astrocytes is mediated

by p53- and Bax-dependent mechanisms, although full

manifestation of apoptosis is aborted by concomitant inhi-

bition of caspase activation. More generally, our data sug-

gest that apoptotic mediators should be evaluated as the

cause of cell death even in cases where a full apoptotic

phenotype is lacking.

Keywords: apoptosis, DETA/NO, LY294002, mitochondria,

necrosis, phospho-p53.

J. Neurochem. (2004) 10.1111/j.1471-4159.2004.02395.x

In the brain, nitric oxide (NO) at physiological levels derived

from neuronal or endothelial NO synthase acts as an intercel-

lular messenger between neurones and between neurones and

other brain cells (Garthwaite and Boulton 1995; Prast and

Philippu 2001). However, under pathological conditions the

expression of inducible NO synthase (cNOS) may be induced

in glia (particularly astrocytes and microglia) by inflammatory

mediators (Murphy 2000). The high levels of NO production

by glia may result in neuronal dysfunction during inflamma-

tory, infectious, ischaemic and neurodegenerative diseases

(Heales et al. 1999; Murphy 2000; Brown and Bal-Price

2003). In culture, neurones are very sensitive to NO-induced

cell death by mechanisms that may include activation of

poly(ADP-ribose) polymerase followed by NAD+ and ATP

depletion, induction of glutamate release from neurones and

astrocytes resulting in excitotoxic damage and inhibition of

mitochondrial respiration (Heales et al. 1999; Chen et al.

2001; Brown and Bal-Price 2003). However, little is known

about the role of NO in astrocyte cell death.

Astrocytes express inducible NO synthase in response to

hypoxia, prions, b-amyloid, bacterial or viral components,

proinflammatory cytokines and a wide range of pathologies

(Murphy 2000). Astrocytes activated by inflammatory agents

that induce expression of inducible NO synthase produce

high levels of NO (about 1 lM in vitro) for a sustained period(Brown et al. 1995; Murphy 2000; Bal-Price and Brown

2001). Nitric oxide inhibits mitochondrial respiration in

astrocytes as it does in neurones (Brown et al. 1995;

Almeida et al. 2001; Bal-Price and Brown 2001) and this

inhibition may be one means by which NO induces cell death

Received September 4, 2003; revised manuscript received October 21,

2003; accepted November 9, 2003.

Address correspondence and reprint requests to Guy C. Brown/Aviva

M. Tolkovsky, Department of Biochemistry, University of Cambridge,

Tennis Court Road, Cambridge CB2 1QW, UK.

E-mail: [email protected]/[email protected] authors contributed equally to this article.

Abbreviations used: ATM, ataxia telangiectasia mutated; ATR, ATM

and Rad3-related; DETA/NO, diethylenetriamine-nitric oxide adduct;

Hoechst, 33342, 2¢-(ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5¢bi-1H-benzimidazole; NO, nitric oxide; PBS, phosphate-buffered saline; PI,

propidium iodide.

Journal of Neurochemistry, 2004 doi:10.1111/j.1471-4159.2004.02395.x

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 10.1111/j.1471-4159.2004.02395.x 1

(Heales et al. 1999; Almeida et al. 2001; Bal-Price and

Brown 2001; Brown and Borutaite 2002; Brown and Bal-

Price 2003). However, NO-induced mitochondrial inhibition

in astrocytes would not cause immediate ATP depletion, as

astrocytes are able to maintain ATP levels by glycolysis, in

contrast to neurones where NO-induced ATP depletion is

very rapid (Almeida et al. 2001; Bal-Price and Brown 2001).

As the level of ATP is critical for determining whether cells

die by apoptosis or necrosis (Eguchi et al. 1997; Leist et al.

1997), the ability of astrocytes to maintain ATP upon

inhibition of mitochondrial respiration could enable an

apoptotic mode of death to be induced in them by NO.

Few studies have been conducted on the non-necrotic

mechanisms involved in NO-induced cell death of astrocytes

(Mitrovic et al. 1994; Robb et al. 1999; Suk et al. 2001,

2002; Takuma et al. 2002).

A number of studies in other cell types suggest that NO-

induced apoptosis occurs through a p53-dependent pathway

(Messmer et al. 1995; Brune and Schneiderhan 2003; Wang

et al. 2003). p53 is one of the crucial proteins involved in the

regulation of apoptosis and is pivotal in controlling whether

an injured cell should undergo apoptosis or cell cycle arrest

in order to allow DNA repair. There are multiple mechanisms

implicated in the regulation of p53 activity, one of them

being phosphorylation (reviewed by Meek 1999; Appella

and Anderson 2001). Phosphorylation of p53 at serine 15 (in

human p53, equivalent to serine 18 in mouse p53), located in

the transactivation domain, has been shown to be crucial for

p53 activation and degradation (Shieh et al. 1997; Lambert

et al. 1998; Dumaz and Meek 1999) as well as its pro-

apoptotic function (Unger et al. 1999). Whether p53 activity

is regulated by NO in astrocytes is unknown, nor is it

established whether NO-induced death requires the medi-

ation of Bax, a key pro-apoptotic Bcl-2 family member

(Scorrano and Korsmeyer 2003).

In the present study, we have investigated the mechanisms

of NO-induced death of astrocytes, focusing in particular on

the roles of Bax and p53 in this process.

Materials and methods

Primary culture of mouse cerebrocortical astrocytes

Cortical astrocytes were prepared from newborn 129/Ola strain mice

or, in the case of bax null mice, from a C57B/CD1 mixed

background, as described previously (Yung and Tolkovsky 2003).

Briefly, meninges-freed cortices were cut into small cubes

(< 1 mm3) and digested with 0.1% trypsin in versene [phosphate-

buffered saline (PBS)/EDTA 0.53 mM] for 30 min at 37�C with

occasional mixing. Trypsin was inhibited by addition of Dulbecco’s

modified Eagle’s medium containing 10% foetal bovine serum

(HyClone, Logan, UT, USA) followed by mechanical trituration

with a Pasteur glass pipette. Cell suspensions were sieved through a

40-lm cell strainer (Falcon, BD Biosciences, Oxford, UK) and the

filtrate was seeded at a density of approximately one cerebrum per

three 75-cm2 tissue culture flasks (Falcon). Cultures were incubated

in 5% CO2/95% air and 100% humidity at 37�C. After culturesreached confluence (12–14 days), they were shaken in an orbital

shaker at 180 r.p.m. for approximately 16 h to remove contamin-

ation from other cell types. The purity of these astrocyte cultures

was assessed by staining for the astrocyte marker glial fibrillary

acidic protein and was approximately 98% (data not shown).

Cultures were then washed twice with Dulbecco’s modified Eagle’s

medium, trypsinized with 0.25% trypsin-EDTA for 15 min, disso-

ciated into a single cell suspension in Dulbecco’s modified Eagle’s

medium containing 10% foetal bovine serum and plated on poly-

L-lysine-coated slides (four chamber; Labtek, Nunc. Naperville, IL,

USA) at a density of 15 000 cells/cm2. After 48 or 72 h of replating,

cells were incubated with 0.5–1 mM of the NO donor diethylene-

triamine-NO adduct (DETA/NO) (Sigma, Poole, Dorset, UK or

Alexis, Lausen, Switzerland) for up to 72 h. LY294002 was from

Biomol (Plymouth Meeting, PA, USA) and U0126 and SB203580

were from Calbiochem (Merck Biosciences, Nottingham, UK).

Genotyping of genetically modified p53 and bax mice

Wild-type and p53- or bax-deficient astrocytes were derived from

littermates from matings of heterozygous parents and prepared by

the same method. Founder 129/Ola strain p53-deficient mice were a

kind gift from Alan Clarke (University of Cardiff, Wales) (Clarke

et al. 1993). The genotype of newborn pups was determined by

PCR as described by Clarke et al. (1993) (product for wild-type

allele, 642 bp; product for neomycin allele, 510 bp) using DNA

extracted from tail tissue using the DNAce Clinipure kit

(Bioline, London, UK). Bax-deficient mice were a kind gift of

Dr S. Korsmeyer and were obtained after extensive crossing into the

CD1 strain from Dr Alun Davies [Royal (Dick) School of Veterinary

Sciences, Edinburgh, Scotland]. The PCR was performed using the

Taq DNA polymerase (recombinant) kit from Invitrogen (10342;

Renfrew, Scotland) and 1/10–1/20 of the extracted DNA. A hot start

(2 min at 94�C) was followed by 35 cycles (45 s at 94�C, 1 min at62�C and 1 min at 72�C) and then 10 min at 72�C. The PCRreaction mixture contained 1· PCR buffer, 1.5 mM MgCl2, 500 nMdNTPs, 0.05% W-1 and 1.25 U DNA Taq polymerase. For Bax

genotyping, the primers used were IN5R, 5¢TTGACCAGAGTGGCGTAG3¢; EX5F, 5¢GCTGATCAGAACCATCATG3¢ and NeoR,5¢GCTTCCATTGCTCAGCG3¢ (product for wild-type allele,

307 bp; product for neomycin allele, 507 bp). A hot start (after

2 min at 94�C) was followed by 40 cycles (45 s at 94�C, 45 s at52�C and 1 min at 72�C) and then 10 min at 72�C using the samereaction components except that MgCl2 was used at 1.25 mM. The

genotype of adult mice was also determined by PCR on DNA

obtained from hair roots using the same DNAce Clinipure system.

Assessment of cell viability

After DETA/NO treatment, astrocytes were incubated with 5 lg/mLeach of 2¢-(ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5¢bi-1H-benzimidazole (Hoechst 33342) and propidium iodide (PI) (both

from Sigma) for 5 min at 37�C and the number of Hoechst 33342-positive cells with normal or abnormal nuclei (stained blue) or

Hoechst 33342- and PI-positive cells with abnormal nuclei (stained

red) were counted separately under UV fluorescence using a Leitz

DM1L microscope, Leica, Milton Keynes, UK. Blue-stained cells

showing chromatin condensation and/or an abnormal shape or

2 H.-W. Yung et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 10.1111/j.1471-4159.2004.02395.x

fragmentation were scored as being apoptotic-like while cells with

homogeneously stained (light blue) nuclei were scored as being

viable. All cells with red nuclei were scored as being necrotic

irrespective of the shape of the nucleus.

Annexin V staining

Translocation of phosphatidylserine from the inner to the outer

leaflet of the plasma membrane was assessed with an annexin V

labelling kit (Roche Diagnostics, Lewes, UK) according to the

manufacturer’s instructions. After exposure to DETA/NO, astro-

cytes were double stained with annexin V and PI and then

examined under a fluorescence microscope. Cells positive for both

annexin V and PI were scored as being necrotic, while cells that

were annexin V positive but PI negative were scored as being

apoptotic-like.

Immunocytochemistry

The subcellular location of p53 and cytochrome c was assessed by

immunocytochemical staining with anti-p53 and anti-cytochrome c

antibodies. Cells were fixed with 3% paraformaldehyde/PBS and

permeabilized with 1% saponin (Sigma) in PBS containing 1%

bovine serum albumin (Sigma) followed by incubation with anti-

cytochrome c (Pharmingen, BD Biosciences, Oxford, UK) or anti-

p53 (FL-393) antibody (Santa Cruz Biotechnology, Santa Cruz,

CA, USA) for 1 h at room temperature. After extensive washing

with saponin buffer, cells were incubated with secondary

antibodies conjugated with Cy-3 (Jackson Immuno-Research,

West Grove, PA, USA) or Alexa Fluor 488 (Molecular Probes,

San Diego, CA, USA) for 1 h at room temperature. Cells were

extensively washed in saponin buffer followed by a 10-min

staining with 5 lg/mL Hoechst 33342. After a brief wash in

water, cells were mounted onto glass slides in an antifade solution

(Vector Laboratories, Burlingame, CA, USA) and examined using

an UltraView LCI confocal imaging system (Perkin Elmer Life

Sciences, Boston, MA, USA). The cultures used for cytochrome c

immunocytochemistry were pre-incubated with 500 nM Mito-

Tracker Orange� (Molecular Probes) before DETA/NO treatment

so that the imprint of mitochondria could be recorded even after

cytochrome c release.

Subcellular fractionation

The method of isolating mitochondria was described by Desagher

et al. (1999). Cells were rinsed twice with cold PBS and scraped

into cold MSHE buffer containing 210 mM mannitol, 70 mM

sucrose, 10 mM Hepes-KOH (pH 7.4), 1 mM EGTA, 1 mM EDTA,

150 lM spermine and 750 lM spermidine, with 5 mM dithiothreitol,2 lg/mL leupeptin, 2 lM benzamidine-HCl and 1 lg/mL pepstatinadded just before the experiment. Cells were then homogenized in a

homogenizer Dounce with 25 strokes on ice. Nuclei and unlysed

cells were separated by centrifugation at 500 g for 12 min at 4�C.The supernatant fluid was then centrifuged at 9500 g for 9 min at

4�C to pellet the mitochondria. After collection of the supernatantfluid (considered as the cytosolic fraction), the mitochondrial pellet

was washed once in MSHE buffer and intact mitochondria were

pelleted by centrifugation at 8500 g for 9 min at 4�C. Themitochondrial pellet was resuspended in MSHE buffer. Both

cytosolic and mitochondrial fractions were frozen at ) 80�C until

analysis.

Western blotting

Cells were washed with ice-cold PBS, scraped off in lysis buffer

(250 mM sucrose, 50 mM Tris, pH 7.6, 1% Triton X-100, 1 mM

EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 0.1 mM phenyl-

methylsulphonyl fluoride, 1 lg/mL leupeptin and 2 lg/mL benz-amidine) and transferred to an Eppendorf tube. Cells were incubated

on ice for 20 min with occasional vortexing and centrifuged at

10 000 g for 5 min. The supernatant fluid was kept at )80�C untilanalysis. A bicinchoninic acid kit (Sigma) was used to determine

protein concentration. Proteins were resolved by sodium dodecyl

sulphate–polyacrylamide gel electrophoresis, blotted onto nitrocel-

lulose (0.2 lm; Schleicher and Schuell, Dassel, Germany) and

analysed by enhanced chemiluminescence (reagents from Amer-

sham Biosciences, Amersham, UK or prepared in the laboratory)

using X-OMAT AR film (Kodak, Rochester, NY, USA). The

following antibodies were used: anti-phospho Akt (serine 473) and

anti-phospho p53 (serine 15) polyclonal antibodies (New England

BioLabs, Beverly, MA, USA); anti-Bax monoclonal antibody 5B7

(Neomarkers, Freemont, CA, USA); anti-ERK (MK12) monoclonal

antibody (Transduction Laboratories, Lexington, KY, USA) and

anti-p53 polyclonal antibody CM5 (a gift from Dr David Lane,

University of Dundee, Scotland). Secondary antibodies were from

Jackson Immuno-Research.

Statistical analysis

Pairwise comparisons were performed using unpaired two-tailed

Student’s t-test.

Results

Nitric oxide induces apoptotic-like cell death of

astrocytes without caspase activation

To determine whether NO is able to induce cell death in

cultured astrocytes, cells were exposed to the NO donor

DETA/NO (0.5 or 1 mM) for 24, 48 or 72 h. Nuclear

morphology was assessed at each time point by Hoechst

33342 staining and plasma membrane integrity by PI

staining. Figure 1(a) shows that incubation with DETA/NO

caused a dose- and time-dependent increase in the percentage

of cell death that was largely apoptotic-like. After exposure

to 1 mM DETA/NO for 72 h, 58.5 ± 12.0% (mean ± SD,

n ¼ 3) of the cells showed apoptotic-like cell death (com-

plete chromatin condensation) while only 11.7 ± 5.0% (mean

± SD, n ¼ 3) of the cells were necrotic (PI positive). The

observed cell death was caused by NO released from DETA/

NO because death was almost completely blocked by

100 lM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-

oxide (a NO scavenger) and the residual product (DETA/

NO kept at room temperature or exposed to light for a few

days) did not cause any significant cell death in any of the

experiments.

Most of the condensed nuclei observed after exposure to the

NO donor displayed an unusual morphology, many being

crescent-shaped with condensed chromatin deposited all

along the nuclear membrane but without showing any

NO-induced cell death of cultured astrocytes 3


fragmentation, which was observed very rarely (Fig. 1b). To

further characterize to what extent these cells were undergoing

a form of apoptosis, translocation of phosphatidylserine from

the inner to outer leaflet of the plasma membrane (an early

feature of apoptosis) was studied using fluorescein-annexin V

staining, while cytochrome c release from mitochondria was

investigated by immunocytochemistry. After 72 h of treat-

ment with DETA/NO, many astrocytes were stained with

fluorescein-annexin V but not with PI, indicating a transloca-

tion of phosphatidylserine without necrosis (Fig. 1c). No

annexin V positive staining was observed in the control

cultures. To study cytochrome c release from mitochondria,

MitoTracker Orange�was pre-loaded into the cells for 30 min

to fluorescently label the mitochondria and track them even

after loss of cytochrome c. In control cultures, there was clear

colocalization of cytochrome c and Mitotracker Orange�

(Fig. 1d, upper panel). However, 48 h after incubation with

1 mM DETA/NO, cytochrome c was already clearly released

from the mitochondria in about 40% of the cells (Fig. 1d,

lower panel). Some cells in which cytochrome c was released

still maintained a normal nuclear morphology, consistent with

current evidence that cytochrome c release precedes the onset

of nuclear condensation and fragmentation. However,

although mitochondrial cytochrome c clearly decreased, there

was no apparent increase in cytosolic cytochrome c at the time

point analysed. This finding may indicate rapid degradation,

oxidation or extrusion of cytosolic cytochrome c as described

previously in neurones (Xue et al. 2001) and other cells

(Luetjens et al. 2001; Renz et al. 2001).

Cytochrome c is crucial for formation of the apoptosome

(consisting of apoptotic protease-activating factor-1 and

caspase 9) which initiates activation of caspase cascades.

To examine whether DETA/NO treatment also activated

caspases, caspase activity was assessed by immunocyto-

chemical staining and immunoblotting using an anti-active

caspase 3 antibody. Surprisingly, both these methods gave a

negative result, suggesting that there was no caspase 3

activation, although active caspase 3 was detected using both

methods after 4 h of oxygen and glucose deprivation

followed by addition of glucose for 24 h (Yung, Ph.D.

Thesis, University of Cambridge, UK). Moreover, apoptosis-

like death was not prevented by the cell-permeable caspase

inhibitors benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluor-

omethyl ketone (100 lM) or 100 lM Boc-Asp(O-methyl)-

fluoromethyl ketone, further suggesting that NO-induced

apoptosis-like cell death was not mediated by caspase

activation. Lack of caspase activation may explain

why DETA/NO-treated astrocytes died without nuclear

(a)

(b)

(d)

(c)

Fig. 1 Nitric oxide (NO) induces apoptotic-like cell death in astrocytes.

(a) Cultured astrocytes were exposed to either 0.5 or 1 mM diethy-

lenetriamine-NO adduct (DETA/NO) for 24, 48 or 72 h. Cell death was

scored after costaining with 5 lg/mL Hoechst 33342 and propidium

iodide (PI) under a fluorescent microscope. PI-positive cells were

scored as being necrotic and PI-negative cells with nuclear abnor-

malities were scored as apoptotic-like. Results show mean ± SD from

three independent experiments. (b) Micrographs showing the mor-

phology of nuclear abnormalities typical of apoptotic-like cell death.

Insert (bottom right) indicates typical apoptotic nucleus found in the

culture. (c) DETA/NO induces translocation of phosphatiylserine from

the inner to the outer leaflet of the plasma membrane prior to mem-

brane rupture. Astrocytes treated with DETA/NO for 72 h were

costained with fluorescent annexin V and PI. Over 90% of the annexin

V-positive cells were PI-negative. (d) DETA/NO induces cytochrome c

release from mitochondria. Astrocytes were pre-loaded with 500 nM

MitoTracker Orange� (MitoTracker, red) for 30 min followed by a 48 h

incubation with 1 mM DETA/NO. Cells were then fixed in 3% para-

formaldehyde followed by immuocytochemical staining with anti-

cytochrome c primary antibody and Alexa Fluor 488 (cyto c, green)-

conjugated secondary antibody. Hoechst 33342 staining was used to

indicate the nuclei (DNA, blue). Images were visualized using a con-

focal microscope, captured with a CCD camera and merged using

Ultraview software.

4 H.-W. Yung et al.


fragmentation rather than by the classical morphology of

apoptosis. To conclude, the NO donor induced apoptosis-like

cell death in primary astrocytes with phosphatidylserine

translocation, cytochrome c release and nuclear condensation

but no caspase activation. However, the finding that

cytochrome c is released from mitochondria suggests the

possibility that Bax, whose translocation to mitochondria

promotes cytochrome c release from the mitochondria

(Desagher et al. 1999), is involved in this death.

Nitric oxide induces Bax translocation and Bax is

required for cell death

To determine whether cytochrome c release was mediated by

Bax-dependent mechanisms, Bax protein expression and

translocation of Bax to the mitochondria were assessed by

subcellular fractionation and immunoblotting after 48 h of

treatment with 1 mM DETA/NO. Figure 2(a) shows a mild

increase of about 1.7-fold in total Bax protein expression in

the DETA/NO-treated sample compared with control (similar

results were obtained in two independent experiments). For

analysis of Bax translocation to the mitochondria, mitoch-

ondrial and cytosolic fractions were prepared from untreated

and DETA/NO-treated cells by cell fractionation. The entire

mitochondrial fraction from each extract was loaded onto the

gel but only one-third of the cytosolic fraction from each

sample was analysed. Figure 2(b) shows that the relatively

small amount of Bax found in the mitochondrial fraction of

the control sample increased about 10-fold (10.3 ± 2.5, mean

± range, n ¼ 2) in mitochondria from the DETA/NO-treated

cells, indicating a translocation of Bax from the cytosol.

There was no apparent reduction of Bax protein in the

cytosolic fraction, which may be due to the relatively high

abundance of Bax in the cytosol as well as to the small

increase in Bax protein expression generally, masking the

small reduction that occurs during the translocation process.

To establish whether Bax translocation is essential for NO-

induced cell death in astrocytes, primary Bax-deficient

astrocytes (from Bax–/– mice) were treated with 1 mM

DETA/NO for 72 h and cell viability was assessed by

double staining with Hoechst 33342 and PI. In wild-type

astrocytes, 1 mM DETA/NO induced a fourfold increase

from about 8% to 33.7 ± 6.3% (mean ± SD, n ¼ 3) in the

percentage of apoptotic-like cells compared with an increase

of about twofold, to 15.7 ± 2.9% (mean ± SD, n ¼ 3), of

apoptotic-like profiles in the bax-deficient astrocytes

(Fig. 2c), a highly significant reduction (p < 0.001). The

lower percentages of cell death observed in wild-type

astrocytes after 72 h of DETA/NO treatment in these

experiments compared with the previous experiments is

probably because the astrocytes from the CD1 strain of mice

are generally more resistant to insults compared with

astrocytes from the p129/Ola strain of mice (Yung,

unpublished observations). From the above results, it appears

that Bax is an important mediator of NO-induced cell death.

As Bax is one of several known target genes of p53, and

given the increase in Bax protein expression induced by

DETA/NO, it was of interest to examine whether p53 was

involved in NO-induced cell death of astrocytes.

Nitric oxide-induced cell death is mediated by p53 which

accumulates, is phosphorylated at serine 18 and is

translocated to nucleus

To test whether p53 is involved in NO-induced death of

astrocytes, we studied the expression of total p53 and its

(c)

(b)

(a)

Fig. 2 Bax is required for diethylenetriamine-nitric oxide adduct

(DETA/NO)-induced cell death in astrocytes. (a) DETA/NO increases

Bax protein expression. Astrocyes were treated with 1 mM DETA/NO

for 48 h, total cellular protein was then extracted and equal amounts of

protein were analysed by western blotting with an anti-Bax antibody.

(b) Bax translocates to the mitochondria after exposure to DETA/NO.

After treatment with 1 mM DETA/NO for 48 h, astrocytes were fract-

ionated into mitochondrial and cytosolic fractions. Equal amounts of

cytosolic protein or mitochondrial protein (comprising the entire mit-

ochondrial fraction but only one-third of each cytosolic fraction) were

analysed by western blotting with an anti-Bax antibody. Ponceau

staining indicates equal protein loading between control (con) and

treated samples in each of the two fractions. The graph shows a

quantitative analysis of mitochondrial Bax from two independent

experiments. (c) Bax-deficient astrocytes are more resistant to DETA/

NO-induced cell death. Both wild-type (wt) and bax-deficient (bax–/–)

astrocytes were prepared from littermates of heterozygous parents.

Astrocytes were treated with 1 mM DETA/NO for 72 h and cell death

was scored after costaining with 5 lg/mL Hoechst 33342 and PI under

a fluorescent microscope. Data show mean ± SD from four inde-

pendent experiments. p < 0.001, unpaired Student’s t-test.



phosphorylation at serine 18 by western blotting. As

translocation of p53 from the cytosol to the nucleus is

required for regulation of its target genes, its subcellular

location in response to DETA/NO was also investigated by

immunostaining. In the presence of DETA/NO, there was a

clear increase in the expression of p53 protein and in that of

phospho-p53(ser18) (Fig. 3a) beginning at 12 h after onset

of treatment and continuing to increase up to 72 h. Moreover,

after 48 h of exposure to 1 mM DETA/NO, intense nuclear

immunoreactivity of p53 was observed with concomitant loss

of the diffuse staining observed in the cytoplasm of non-

stimulated cells, indicating nuclear accumulation of most of

the p53. In contrast, there was little nuclear staining of p53 in

control cells (Fig. 3b). To further examine the roles of p53 in

NO-induced cell death, primary astrocytes were prepared

from p53-deficient mice and these were exposed to the NO

donor. As p53 may also mediate necrotic cell death (Schafer

et al. 2003), both necrotic and apoptotic-like cell death were

investigated. Figure 3(c) shows the results obtained after

72 h. Compared with wild-type astrocytes, in which 70.2%

cell death (11.7% necrosis and 58.5% apoptosis-like) was

observed, there was only 35.6% overall cell death (24.7%

necrosis and 10.9% apoptosis-like) in p53-deficient astro-

cytes, confirming the crucial role of p53 in the mediating

NO-induced cell death. Interestingly, there was slightly more

necrotic death in p53-deficient astrocytes (24.7 ± 5.9%,

mean ± SD, n ¼ 3) than in wild-type astrocytes (17 ± 5%,

mean ± SD, n ¼ 6), which may indicate that p53-deficient

astrocytes are more rather than less susceptible to

NO-induced necrosis, perhaps because p53 induces

antioxidant enzymes (Tan et al. 1999).

LY294002-sensitive kinases phosphorylate p53

at serine 18

An increase in phospho-p53(ser18) indicates that activation of

upstream kinases is likely to have been induced. A number of

studies have demonstrated that multisite phosphorylation and

acetylation of p53 are key events in the regulation of p53

activity in response to genotoxic agents or hypoxia. To

determine which protein kinase was responsible for the

phosphorylation of p53 at serine 18, astrocytes were exposed

for 48 h to DETA/NO (1 mM) in the presence or absence of

the MEK inhibitor U0126 (50 lM), which inhibits ERK1/2and ERK5 activities, the p-38 kinase inhibitor SB203580

(10 lM) or LY294002 (75 lM), an inhibitor of PI-3 kinasesand PI-3 kinase-like [ataxia telangiectasia mutated (ATM),

ATM and Rad3-related (ATR) and mTOR] protein families.

The phosphorylation of p53 at serine 18 was not mediated by

ERK1/2 or p38 kinase because the inhibitors of these kinases

did not block the phosphorylation of p53 induced by the NO

donor (although they inhibited the respective kinase activities;

Yung, data not shown). In contrast, Fig. 4(a) shows that

LY294002 substantially reduced NO-induced phospho-

p53(ser18) suggesting an involvement of LY294002-sensitive

kinases in serine 18 phosphorylation. Total p53 expression

was also reduced, perhaps because the protein was destabil-

ized due to the inhibition of its phosphorylation. Phosphory-

lation of Akt at serine 473 was almost eliminated in the

presence of LY294002, showing the drug’s efficacy in

inhibiting PI-3-kinases upstream of this signalling pathway.

0 12 24 48 72Time (h)

DETA/NO

Phospho-p53 (ser18)

p53

ERK1/2

Control DETA/NO

p53

0%

20%

40%

60%

80%wt

p53-/-

Necrotic Apoptotic-like

p<0.05

p<0.001

Cel

ldea

th

(a)

(b)

(c)

Fig. 3 A p53-dependent pathway mediates apoptotic-like cell death

induced by diethylenetriamine-nitric oxide adduct (DETA/NO) in ast-

rocytes. (a) DETA/NO promotes p53 phosphorylation at serine 18 and

total p53 protein accumulation. Astrocyes were treated with 1 mM

DETA/NO for the indicated time intervals. Proteins were then extrac-

ted and analysed by western blotting with antibodies against phospho-

p53(ser18) and total p53 protein. The anti-ERK1/2 antibody is used to

indicate equal protein loading. (b) Nuclear translocation of p53 after

DETA/NO treatment. After treatment with 1 mM DETA/NO for 48 h,

astrocytes were fixed and subjected to immunocytochemical analysis

with an antibody against p53 and a Cy-3-conjugated secondary anti-

body. Images were visualized using a confocal microscope and cap-

tured with a CCD camera. (c) p53-deficient astrocytes are more

tolerant to DETA/NO-induced cell death. Wild-type (wt) and p53-defi-

cient (p53–/–) astrocytes prepared from littermates of heterozygous

parents were treated with 1 mM DETA/NO for 72 h and cell death was

scored after double staining with 5 lg/mL Hoechst 33342 and PI under

a fluorescent microscope. Data show the mean ± SD from three

independent experiments. p < 0.05, unpaired Student’s t-test.

6 H.-W. Yung et al.


To determine whether reduction of total p53 and serine 18

phosphorylation by LY294002 also reduced NO-induced cell

death, staining with Hoechst 33342 and PI was used again to

assess the percentage of cell death after 24, 48 and 74 h of

cotreatment with LY294002 and DETA/NO. Figure 4(b)

shows that the apoptotic-like component of cell death was

significantly reduced at 48 h (p < 0.01) and 72 h (p < 0.02)

by about 60%when the astrocytes were exposed to LY294002

and the NO donor. However, there was a significant increase

in necrotic cell death in cells cotreated with DETA/NO and

LY294002 (56.8 ± 7.5%, mean ± SD, n ¼ 3) compared with

control cells (15.5 ± 2.5%, mean ± SD, n ¼ 3, p < 0.001).

This effect was not due to a generalized toxicity of LY294002

as its addition alone did not cause any toxicity up to 72 h and

there was also no increase in necrosis 24 h after treatment

with the NO donor together with LY294002 (Fig. 4b). To

examine whether the necrotic death was p53-dependent, the

same experiment was conducted in p53-null astrocytes. In the

absence of the NO donor, necrosis was 8.4% in astrocytes

from wild-type mice and 11% in p53 null astrocytes while, in

the presence of the NO donor, LY294002 increased necrosis

to 31% in wild-type astrocytes and 30% in the p53-deficient

cells (averages of duplicate plates). Hence, it appears that the

apoptotic component of cell death may be dependent on

stabilization of p53 through LY294002-dependent phos-

phorylation but LY294002-induced necrosis in the presence

of DETA/NO is probably due to other LY294002-mediated

mechanisms.

Discussion

Nitric oxide can induce cell death by a variety of mechanisms

that differ in different conditions and cell types (Bosca and

Hortelano 1999; Murphy 1999; Bal-Price and Brown 2000;

Borutaite et al. 2000; Ignarro 2000; Brown and Borutaite

2001, 2002; Chung et al. 2001). Reasons for this variety and

complexity of mechanisms may include: (i) conversion of

NO into various derivatives such as peroxynitrite, NO2,

N2O3 and S-nitrosothiols differentially; (ii) NO and its

derivatives are differentially reactive and react directly with a

wide range of cell components and (iii) NO and its

derivatives can be cytotoxic, cytoprotective or both, depend-

ing on conditions (Ignarro 2000; Chung et al. 2001). These

factors cause the analysis of NO-induced cell death to be

complex.

Our own results with astrocytes indicate that, under the

conditions used, NO induces a form of cell death that cannot

be straightforwardly classified as apoptosis although the

death is clearly not necrotic. Features consistent with

apoptosis were the presence of chromatin condensation,

annexin V staining without necrosis, p53 phosphorylation

and accumulation, p53-dependent cell death, Bax transloca-

tion to the mitochondria, cytochrome c release from mito-

chondria and Bax-dependent cell death. However, certain

classical features of apoptosis were missing, such as nuclear

fragmentation, caspase 3 activation and there was no

protection against cell death by caspase inhibitors. Our

finding that cytochrome c released from the mitochondria

fails to be detected in the cytoplasm (Fig. 1d) may suggest

that NO is modifying the cytochrome c (by S-nitrosylation or

tyrosine nitration) and/or speeding its degradation and/or

extrusion (Luetjens et al. 2001; Renz et al. 2001; Xue et al.

2001). Atypical apoptosis and lack of caspase activation

might be due to the known ability of NO to block apoptosis

at various points, in particular by causing S-nitrosylation and/

or oxidation of the active site cysteine residue of caspases

(a)

(b)

Fig. 4 LY294002-sensitive kinase(s) regulate p53 serine 18 phos-

phorylation and total p53 protein accumulation. (a) Astrocytes were

treated with 1 mM diethylenetriamine-nitric oxide adduct (DETA/NO)

for 48 h in the presence or absence of 75 lM LY294002. Proteins were

extracted and analysed by western blotting with antibodies against

phospho-p53 (ser18), p53 protein or phospho-Akt (serine 473). Anti-

ERK1/2 antibody was used to indicate equal protein loading. (b)

LY294002 treatment in the presence of DETA/NO inhibits apoptotic-

like death but facilitates necrotic cell death in astrocytes. Cells were

treated with 1 mM DETA/NO for 72 h in the presence or absence of

75 lM LY294002. The amount of cell death was scored after double

staining with 5 lg/mL Hoechst 33342 and PI under a fluorescent

microscope. Data show the mean ± SD from three independent

experiments. Apoptotic-like death at: 24 h, no difference; 48 h,

p < 0.01; 72 h, p < 0.02. Necrotic death at: 24 h, no difference; 48 and

72 h p < 0.001.



(Chung et al. 2001). Another possible explanation is that

NO-induced energy depletion (due to respiratory and/or

glycolytic inhibition) prevents caspase activation (Bal-Price

and Brown 2000). Whatever the explanation for the lack of

caspase activation, NO appears to cause a form of cell death

in astrocytes that has many more features of apoptosis than

necrosis.

Bax has previously been implicated in mediating

NO-induced apoptosis. For example, Gordon et al. (2001)

found that NO increased expression of both Bax and p53 in

thymocytes and Kim et al. (2002) found that NO-induced

accumulation of p53 caused expression of Bax and caspase-

mediated apoptosis in primary chondrocytes. Ghatan et al.

(2000) reported that NO induced Bax translocation to

mitochondria in cortical neurones and a neuroblastoma cell

line and that Bax-deficient cortical neurones were resistant to

NO-induced cell death. Our own results indicate that, in

astrocytes, NO causes Bax to translocate to mitochondria and

NO-induced cell death is largely, although not completely,

Bax dependent as cell death is reduced by 65% in the Bax–/–

cells (Fig. 2). As Bax expression was also mildly increased

by NO in the astrocytes, and p53 is known to increase Bax

expression in other cells, we investigated whether p53 might

also be involved in NO-induced cell death of astrocytes.

p53 has been implicated in mediating NO-induced apop-

tosis in some cell types and conditions but not others. For

example, Messmer and Brune (1995) found that inducible

cNOS induction or the NO donor S-nitrosoglutathione

caused a marked accumulation of p53 in the RAW 264.7

macrophage cell line that preceded DNA fragmentation.

Transfection of these cells with plasmids encoding p53

antisense RNA decreased (but did not eliminate) S-nitro-

soglutathione-induced DNA fragmentation but S-nitroso-

glutathione caused a similar level of DNA fragmentation in

U937 cells which lack p53 (Messmer and Brune 1996).

Nitric oxide-induced apoptosis was reported to be reduced

and/or delayed in thymocytes from p53 null mice (Gordon

et al. 2001), human lymphoblastoid cells with mutant p53

(Li et al. 2002) and in primary chondrocytes expressing a

dominant negative p53 (Kim et al. 2002). However, Kibbe

et al. (2002) reported that aortic vascular smooth muscle

cells from p53–/– mice were more sensitive to NO-induced

apoptosis than wild-type vascular smooth muscle cells,

suggesting that p53 can be protective. In these cells,

NO-induced apoptosis was blocked by inhibitors of p38

MAPK or MEK1/2. Thus, NO can induce apoptosis by

p53-dependent or p53-independent mechanisms depending

on cell type and conditions.

In human lymphoblastoid cells that, like astrocytes, are

protected from NO-induced death in the absence of p53, Li

et al. (2002) found that mitochondrial membrane depolar-

ization and cytochrome c release were induced in both p53-

expressing and p53-deficient cells. However, elevation of

apoptotic protease-activating factor-1 (Apaf-1) protein and

reduction of X-chromosome-linked inhibitor of apoptosis

protein (XIAP) were observed only in the p53 wild-type

cells. This finding suggests that p53 may sensitize cells to

NO-induced apoptosis by up-regulating apoptotic protease-

activating factor-1 and/or decreasing X-chromosome-linked

inhibitor of apoptosis. Our own results are consistent with

p53 activation sensitizing cells to apoptosis induced by NO

via p53-dependent and -independent mechanisms as apopto-

sis was reduced by 83% in the p53–/– cells after 3 days of

treatment but was not entirely eliminated.

A number of kinases can phosphorylate serine 15 of

human p53 (equivalent to serine 18 in mouse), including

ATM, ATR, p38 and DNA-PK. Wang et al. (2003) reported

that the serine 15 phosphorylation and accumulation of p53

in response to NO still occurred in ATM–/– cells but was

abolished when the ATR kinase was inactivated by mutation

or caffeine. However, Hofseth et al. (2003) reported that

NO-induced phosphorylation at serine 15 was reduced in

ATM–/– fibroblasts and was further reduced by caffeine,

implicating both ATM and ATR, but there was no effect of

p38 or DNA-PK inhibitors. In primary chondrocytes,

NO-induced p53 activation was apparently mediated by

p38 MAPK, both directly via p38 phosphorylation of serine

15 and indirectly via activation of NFjB causing p53

expression (Kim et al. 2002). Our own results indicate that,

in astrocytes, NO-induced p53 phosphorylation at serine 18

is independent of p38 MAPK but dependent on kinases

inhibitable by LY294002, which may include ATM and ATR.

Although LY294002 prevented NO-induced p53 phos-

phorylation and decreased apoptosis-like death, it did not

prevent NO-induced cell death as it increased NO-induced

necrosis, although it was not toxic by itself (Fig. 4b). This

activity of LY294002 was not due to ATP depletion (Eguchi

et al. 1997; Leist et al. 1997) as necrotic death due to ATP

depletion occurs within about 6 h (Yung, data not shown). It

may be that, in addition to inhibiting PI-3 kinase family

kinases implicated in p53 phosphorylation, LY294002

inhibits other PI-3 kinases that are protecting the astrocytes

against NO (for example, the class I PI-3 kinase implicated in

Akt phosphorylation). In keeping with this idea, Matsuzaki

et al. (1999) found that LY294002 and wortmannin

enhanced NO-induced death of hippocampal neurones

apparently by acting as PI-3 kinase inhibitors, thereby

preventing growth factor-induced protection of the cells.

Yamaguchi et al. (2001) further reported that over-expressed

Akt inhibits the transcriptional activity of p53 during

NO-induced neuronal apoptosis without causing any reduc-

tion in p53 protein accumulation, its overall phosphorylation

or its nuclear localization. It was suggested that the increased

survival mediated by Akt antagonized p53-dependent Bax

expression, although the effect of this reduction on Bax

function was not investigated. As phospho-Akt levels

remained high during the NO-induced rise in p53 and

concomitant cell death, yet lack of p53 protected the

8 H.-W. Yung et al.


astrocytes from NO-induced death, it is unclear whether Akt

plays any role in protecting astrocytes from NO-induced

death. Our own results are consistent with LY294002 having

a dual action, preventing NO-induced phosphorylation

of p53 but also blocking pathways protecting against

NO-induced necrosis perhaps mediated by Akt. However,

this interpretation remains to be tested.

We conclude that NO causes p53 activation in astrocytes

via LY294002-sensitive kinases and induces an apoptosis-

like form of cell death that is p53 and Bax dependent but

caspase independent. LY294002-sensitive kinases also sup-

press NO-induced necrosis. A possible scheme of how these

events are related is depicted in Fig. 5. Our data show that a

lack of caspase activity does not necessarily indicate that

apoptosis has not been triggered and that treatment against

early facets of apoptosis, most particularly elimination of

Bax translocation, may still provide protection against pro-

oxidant insults.

Acknowledgements

We are grateful to Dr D. Lane for providing the anti-p53 CM5

antibody, Dr A. Clarke for providing founder p53 null mice and Drs

S. Korsmeyer and A. Davies for providing founder Bax null mice.

We thank Helen Bye for help with mouse breeding and mainten-

ance. Work from the Tolkovsky laboratory was supported by a

Wellcome Prize Studentship and Training Fellowship (HWY) and a

Wellcome Trust Programme Grant (AMT). Work from the Brown

laboratory was supported by a Medical Research Council cooper-

ative component grant.

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10 H.-W. Yung et al.


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