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The Journal of Neuroscience, January 15, 1999, 19(2):664-673
A Role for MAPK/ERK in Sympathetic Neuron Survival:
Protection against a p53-Dependent, JNK-Independent Induction of
Apoptosis by Cytosine Arabinoside
Christopher N. G.
Anderson and
Aviva
M.
Tolkovsky
Department of Biochemistry, Cambridge University, Cambridge, CB2
1QW, United Kingdom
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ABSTRACT |
The antimitotic nucleoside cytosine arabinoside (araC) causes
apoptosis in postmitotic neurons for which two mechanisms have been
suggested: (1) araC directly inhibits a trophic factor-maintained signaling pathway required for survival, effectively mimicking trophic
factor withdrawal; and (2) araC induces apoptosis by a p53-dependent
mechanism distinct from trophic factor withdrawal. In rat sympathetic
neurons, we found that araC treatment for 12 hr induced ~25%
apoptosis without affecting NGF-maintained signaling; there was neither
reduction in the activity of mitogen actived protein
kinase/extracellular signal-regulated kinase (MAPK/ERK) or
protein kinase B/Akt, a kinase implicated in NGF-mediated survival, nor
was there c-Jun N-terminal kinase (JNK) activation or c-Jun N-terminal
phosphorylation, events implicated in apoptosis induced by NGF
withdrawal. However, araC treatment, but not NGF-withdrawal, elevated
expression of p53 protein before and during apoptosis. Additionally,
araC-induced apoptosis was suppressed in sympathetic neurons from p53
null mice. Although MAPK/ERK activity is not necessary for NGF-induced
survival, it protected against toxicity by araC, because inhibition of
the MAPK pathway by PD98059 resulted in a significant increase in the
rate of apoptosis induced by araC in the presence of NGF. Consistent
with this finding, ciliary neurotrophic factor, which does not cause
sustained activation of MAPK/ERK, did not protect against araC
toxicity. Our data show that, in contrast to NGF deprivation, araC
induces apoptosis via a p53-dependent, JNK-independent mechanism,
against which MAPK/ERK plays a substantial protective role. Thus, NGF
can suppress apoptotic mechanisms in addition to those caused by its
own deprivation.
Key words:
superior cervical ganglion neurons; MAPK/ERK; PKB/Akt; JNK; c-Jun phosphorylation; p53; CNTF; PD98059; signal transduction; DNA damage
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INTRODUCTION |
The nucleoside analog cytosine
arabinoside (araC) is a potent antineoplastic agent for hematopoietic
malignancies (for review, see Grant, 1998 ), but its use is complicated
by toxicity to the nervous system (for review, see Baker et al., 1991).
AraC also induces apoptosis in several types of postmitotic neurons
in vitro (Deckwerth and Johnson, 1993; Tomkins et al., 1994;
Dessi et al., 1995; Sanz-Rodriguez et al., 1997). In mitotic cells,
araC is thought to cause cell death primarily by disrupting DNA
replication (Grant, 1998), but this would not be expected to affect
postmitotic neurons.
Two mechanisms have been proposed for the neurotoxicity of araC. The
first postulates that araC directly inhibits a crucial aspect of growth
factor signaling, effectively mimicking trophic factor withdrawal and
so causing death (Wallace and Johnson, 1989; Martin et al., 1990;
Sanz-Rodriguez et al., 1997). Martin et al. (1990) reported that
although araC was toxic to sympathetic neurons, other arabinose
nucleosides, as well as other inhibitors of DNA synthesis, were
not. They also found that 2'-deoxycytidine, the natural analog of
araC, but not other deoxynucleosides, could inhibit araC toxicity. It
was therefore suggested that araC was interfering with a
2'-deoxycytidine-dependent pathway other than the one involved in DNA
synthesis, which is required for trophic factors to cause neuronal survival.
The second mechanism proposes that araC causes neuronal apoptosis by a
p53-dependent pathway, independent of signaling by a survival stimulus.
This was suggested because cerebellar granule neurons from p53 null
mice were more resistant to araC compared with
p53+/+ or p53+/ neurons,
whereas apoptosis caused by removal of the depolarizing survival
stimulus was independent of the p53 status of the neurons (Enokido et
al., 1996a). This observation fitted with our previous finding that the
rate of apoptosis of sympathetic neurons in the absence of NGF was
accelerated by treatment with araC (Tomkins et al., 1994), suggesting
that araC induces apoptosis by mechanisms other than inhibition of NGF
survival signaling. Additionally, Park et al. (1998) reported that the
caspase inhibitor zVAD-fluoromethylketone could inhibit
apoptosis caused by NGF withdrawal but not that caused by araC,
camptothecin, or UV irradiation (DNA-damaging agents).
In sympathetic neurons, NGF causes the sustained activation of several
signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2) or mitogen activated protein kinases (MAPKs) (Virdee and
Tolkovsky, 1995) and protein kinase B/Akt (Akt), Akt being implicated
in mediating at least part of NGF-induced survival (Crowder and
Freeman, 1998; K. Virdee and A. M. Tolkovsky, unpublished
observations). On the other hand, NGF also suppresses c-Jun N-terminal
kinase (JNK) activity and c-Jun N-terminal phosphorylation, which have
been implicated in the induction of neuronal apoptosis after withdrawal
of survival stimulus (Virdee et al., 1997; Eilers et al., 1998; Watson
et al., 1998).
In this study, we have investigated the effect of araC on
NGF-maintained signaling pathways, in particular those pathways thought
to be involved in the survival and death of sympathetic neurons. We
have also examined the involvement of p53 in apoptosis caused by araC.
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MATERIALS AND METHODS |
Preparation of neurons. Single-cell suspensions of
superior cervical ganglion (SCG) neurons were prepared from <1-d-old
Wistar rat pups as described previously (Buckmaster et al., 1991;
Virdee and Tolkovsky, 1995). Briefly, ganglia were digested in 0.1%
trypsin for ~30 min at 37°C and then triturated through a narrow
bore flame-polished Pasteur pipette. The resulting cell suspension was
then preplated twice on collagen in L15-CO2 medium (Hawrot and Patterson, 1979) containing 5% rat serum in a humidified
atmosphere of 5% CO2-95% air at 37°C. The nonadhering
cells were collected by centrifugation and kept in L15 plating medium
at 4°C until use in experiments.
Culture conditions. Neurons were cultured on
poly-L-lysine-laminin-coated wells in growth medium
(L15-CO2 containing 3% dialyzed rat serum) and additives
as indicated. Rat serum (5 ml) was dialyzed twice against 1 l lots
of 10 mM HEPES, pH 7.4, at 4°C. A 50 mM stock
of 2'-amino-3'-methoxyflavone (PD98059) (Calbiochem-Novabiochem, Nottingham, UK) in DMSO was diluted into culture medium
immediately before use, and its supply was replenished between 6 and 7 hr of treatment (at least 80% of the medium in all conditions was replaced). NGF (2.5S) was prepared from male mouse submaxillary glands (Mobley et al., 1976) and was used at 20-100 ng/ml. Rat ciliary
neurotrophic factor (CNTF) (Peprotech EC, London, UK) was used at 100 ng/ml.
Cell counting and scoring of apoptosis. Neurons were fixed
at the indicated times by the addition of an equal volume of fixing medium (3:1 mix of methanol/acetic acid) to the culture medium. The
fixed cells were usually stored at 4°C before scoring for apoptosis.
Nuclei were visualized using Hoechst 33342 (1.7 µg/ml, final
concentration; Sigma, Poole, UK), and the fixing medium and dye was
replaced by PBS before counting. A minimum of 500 neurons in a
600-µm-wide strip extending through the center of the well were
scored for apoptosis using a Leica (Wetzlar, Germany) DMIL
microscope, with only those neurons which had clearly segmented and
condensed chromatin being counted as apoptotic.
Preparation of cell extracts and immunoblotting. Neurons
were collected at the indicated times, pelleted, and washed with cold
PBS, and then ice-cold lysis buffer was added (20 mM
Tris-acetate, pH 7.5, 0.27 M sucrose, 1 mM
EDTA, 1 mM EGTA, 10 mM
sodium- -glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 0.1% (v/v) -mercaptoethanol, 0.2 mM PMSF, and 1 mM benzamidine). Lysis was
achieved on ice over a period of 20-30 min with occasional vortexing,
after which gel loading buffer was added, and lysates were heated at
100°C for 10 min and then stored at  20°C before processing. In
all experiments, parallel cultures were set up to determine the amount of apoptosis at the time of cell collection.
Cell extracts were resolved on 10% polyacrylamide-SDS gels before
electroblotting onto nitrocellulose membranes (Schleicher & Schuell,
Dassel, Germany). Blots were blocked for 1 hr at room temperature or
overnight at 4°C in Tris-buffered saline-Tween 20 (TBST) (100 mM NaCl, 10 mM Tris, pH 7.5, and 0.1% (v/v)
Tween 20) containing 5% or 2% skimmed milk powder (when using
polyclonal or monoclonal antibodies, respectively). Blots were probed
either overnight at 4°C or room temperature for 1 hr, with the
following antibodies: PhosphoPlus JunII (Ser 63), PhosphoPlus Akt (Ser
473), PhosphoPlus SAPK/JNK (Thr 183/Tyr 185; New England Biolabs,
Beverley, MA), anti-active MAPK (Promega, Madison, WI), anti-p53
(FL-393; Santa Cruz, Santa Cruz, CA); and anti-MAPK (MK12; Transduction Laboratories, Lexington, KY). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA), immunoreactive bands were visualized by chemiluminescence and exposure to Kodak X-OMAT film (Eastman Kodak,
Rochester, NY). In some cases, blots were stripped (in buffer
containing 62.5 mM Tris, 2% SDS, and 0.7% (v/v)
-mercaptoethanol, pH 6.7) for 1 hr at room temperature, followed by
extensive washing in TBST before reblocking and reprobing. Intensity of
bands was analyzed by optical densitometry using the Leica Q500 image
analysis system. To average the data from several experiments, the
intensity of bands for the proteins being investigated was divided by
the intensity of the bands of total ERK1/2. An arbitrary value of 1 was
assigned to the ratio obtained from cultures treated with NGF alone.
For a quantitative analysis of signaling events, a time point of 12 hr
was chosen, which spanned a range of apoptotic values up to ~40%
depending on the conditions of treatment. When apoptosis exceeded 40%
(or the incubation time was much longer than 12 hr) there was often too
much protein loss, which precluded accurate normalization.
p53+/+, p53+/, and p53/ mice.
Founder mice deficient in the p53 gene (p53+/ and
p53/) were a kind gift from Dr. Alan Clarke
(University of Edinburgh, Scotland), and their generation has been
described previously (Clarke et al., 1993). SCG used in experiments
were taken from pups obtained from breeding p53 heterozygote adults.
Litters were taken at 3-4 d postnatally, and the pair of ganglia from
each animal was removed and processed separately. After digestion for 30 min in 0.1% trypsin and trituration with a p200 pipette tip, the
cells were preplated on tissue culture plastic for 4-5 hr in
preplating medium before being plated in growth medium on
poly-L-lysine-laminin-coated wells for experiments. NGF
was used at 100 ng/ml and araC at 100 µM. Cells were
fixed to determine the extent of apoptosis as described above, and a
minimum of 200 cells were counted per well. The genotype of each animal
was determined by PCR only after counting had been completed. The DNA
for PCR was obtained by heating tail tissue at 100°C in 100 mM NaOH for at least 5 min. PCR was initiated using the
primers described by Clarke et al. (1993) (product for wild-type
allele, 642 bp; product for neomycin allele, 510 bp) by hot start
(after 2 min at 94°C), followed by 35 cycles (45 sec at 94°C, 1 min
at 62°C, 2 min 20 sec at 72°C) and then 10 min at 7°C. The
genotype of adult mice was determined by PCR on DNA obtained from hair
roots using the DNAce Clinipure system (Bioline, London, UK).
Statistical analysis. Data for experiments from rat SCG were
analyzed by ANOVA and the Student-Newman-Keuls post
hoc test or by an unpaired two-tailed Student's
t test. Data from experiments on mice SCG were analyzed by
Student's t test for each pair of conditions obtained with
and without araC. The value of n signifies the number of
independent experiments.
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RESULTS |
Matching rates of apoptosis caused by withdrawal of NGF or araC
treatment in the presence of NGF
To address the question of whether araC inhibits NGF signaling,
thereby mimicking NGF withdrawal, we compared changes in signaling processes that occur on NGF deprivation with those induced by araC in
the presence of NGF. To draw direct comparisons between these
death-inducing conditions, a similar rate of apoptosis is required in
both models. We achieved this requirement in newly isolated rat
sympathetic neurons by using 100 µM araC and dialyzed serum (apoptosis at 14 hr: no NGF, 32.4% ± 3.9; NGF and araC, 31.2% ± 7.8; mean ± SD; n = 3) (Fig.
1). Nondialyzed rat serum was found to
reduce cellular uptake of araC and therefore reduce the rate at which
araC kills the neurons (data not shown). It should also be noted from
Figure 1 that apoptosis occurred at an accelerated rate when araC was
added in the absence of NGF compared with the rate with no NGF or araC
added, suggesting that araC can induce apoptosis by mechanisms other
than by inhibiting NGF signaling as suggested previously (Tomkins et
al., 1994).
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Figure 1.
Matching rates of apoptosis caused by NGF
deprivation or araC treatment in the presence of NGF. Newly isolated
neurons were plated into medium without NGF or with NGF (100 ng/ml) or
araC (100 µM) for the indicated times before being fixed,
stained, and counted. The results shown are the mean ± SD of
three independent experiments. See Figure 8 for photomicrographs of
stained nuclei under these conditions.
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AraC does not affect NGF-induced phosphorylation of MAPK/ERK
or Akt
To determine whether araC interferes with NGF-induced
intracellular signaling in the neurons, we first examined whether araC inhibits the activation of three protein kinases whose sustained activity is dependent on NGF, namely the MAPKs p44-ERK1 and p42-ERK2 (ERK1/2) and Akt (Virdee and Tolkovsky, 1995; Virdee and
Tolkovsky, unpublished observations). In sympathetic neurons,
NGF causes the activation of ERK1/2, as long as NGF is present. If the
MAPK pathway is inhibited, however, neuronal survival in the presence of NGF is unaffected (Creedon et al., 1996; Virdee and Tolkovsky, 1996). An NGF-induced pathway that is reported to be involved in
the survival of sympathetic (and other) neurons is the
phosphatidylinositol 3-kinase (PI 3-kinase) pathway with its
downstream target Akt (Dudek et al., 1997; Crowder and Freeman, 1998;
Virdee and Tolkovsky, unpublished observations). The MAPKs ERK1/2 and
Akt need to be phosphorylated on key residues for their activation (Ray
and Sturgill, 1988; Gotoh et al., 1990; Alessi et al., 1996;
Andjelkovic et al., 1996). We therefore investigated whether araC
affected these activation steps using antibodies specific for the
appropriately phosphorylated forms of these enzymes. We have confirmed
that the extent of activation of these kinases measured using
in-gel kinase assays (ERKs) or immunocomplex kinase assays (Akt)
is matched by their phosphorylation, which can be detected by the
anti-phospho-kinase-specific antibodies used in this study (K. Virdee
and A. M. Tolkovsky, unpublished observations). It can be seen in
Figure 2 that during the period of active
cell death, when 24.8 ± 2.5 and 25.3 ± 4.5% apoptosis (at
12 hr: mean ± SEM; n = 6 had occurred in the
presence of araC and NGF or with no additions, respectively, the
phosphorylation of neither Akt nor that of ERK1/2 was affected
(fold-phosphorylation compared with a value of 1 arbitrarily assigned
to the result obtained from cultures treated with NGF alone: Akt,
1.2 ± 0.2; mean ± range; n = 2; ERK1/2,
1.1 ± 0.3; mean ± SD; n = 3). During NGF
deprivation, in contrast, dephosphorylation of all these kinases occurred (Akt, 0.14 ± 0.1; n = 2; ERK1/2,
0.1 ± 0.05; n = 3). The time point of 12 hr was
chosen to enable accurate comparison with experiments shown below in
which >40% of apoptosis was obtained (see Materials and Methods).
However, it should be noted that araC did not affect levels of ERK
phosphorylation after 14 hr, when apoptosis in the presence of NGF and
araC reached ~30% (data not shown).
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Figure 2.
NGF-induced phosphorylation of p44-ERK1, p42-ERK2,
and Akt is not inhibited by araC. SCG neurons were treated for 12 hr
with NGF alone (20 ng/ml) (lane 1), NGF and 100 µM araC (lane 2), and no NGF or araC
(lane 3), and the protein extracts were prepared and
probed as described in Materials and Methods. A,
Top panel shows phospho-Akt probed for phosphorylation
at serine 473 and phospho-ERK1/2 probed with anti-active MAPK antibody;
bottom panel shows total ERKs as an indication of
protein loading. B, Fold-phosphorylation as determined
by optical densitometry (see Materials and Methods). Top
panel, Phospho-Akt; bottom panel, phospho-ERKs;
both normalized to total ERKs. Data for Akt and ERKs are the mean ± range and mean ± SD for two and three independent experiments,
respectively. Numbers below the lanes
give the mean ± SEM of percentage of apoptosis for six
independent experiments.
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AraC does not cause activation of JNK or phosphorylation of c-Jun
at serine 63
To investigate further the effects of araC with respect to
possible inhibition of NGF signaling, we examined whether araC activates similar signaling processes to those which occur after the
withdrawal of NGF and during the subsequent period of apoptosis. The
JNKs are activated within a few hours of withdrawal of NGF and remain
active throughout the period of apoptosis (Virdee et al., 1997; Eilers
et al., 1998). JNK activity has also been implicated in the induction
of neuronal apoptosis (Xia et al., 1995; Eilers et al., 1998; Maroney
et al., 1998). We therefore examined the effect of araC treatment on
the phosphorylation step required for JNK1 activation using a
polyclonal phospho-specific antibody. Figure
3A shows that araC did not
cause any measurable increase in c-Jun N-terminal phosphorylation in
the presence of NGF at the time when apoptosis was occurring (12 hr)
(see above and Fig. 2 for percentages of apoptosis), although, as
expected, JNK was strongly phosphorylated (~10-fold) at its
activation sites because of NGF withdrawal.
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Figure 3.
JNK activation and c-Jun phosphorylation at serine
63 are not required for araC-induced apoptosis. SCG neurons were
treated for 12 hr with NGF alone (lane 1), NGF and 100 µM araC (lane 2), and no NGF or araC
(lane 3), and the protein extracts were prepared and
processed as in Materials and Methods. A, The blot was
probed first for JNK phosphorylated at residues threonine 183 and
tyrosine 185 (top panel) and then for total ERKs
as an indication of protein loading (bottom
panel). For these experiments, NGF was used at 100 ng/ml. The result shown is representative of three independent
experiments. B, In two other independent experiments,
the amount of N-terminal phosphorylation of c-Jun was determined by
probing with an antibody specific for c-Jun phosphorylated at residue
serine 63 (top panel). Immunoblotting for total
ERKs is shown (bottom panel) as an indication of
protein loading. NGF was used at a concentration of 20 ng/ml. See
Figure 2 for percentage of apoptosis for each condition.
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Because the transcription factor c-Jun is reported to be required for
neuronal apoptosis (Estus et al., 1994; Ham et al., 1995) and
particularly its N-terminal phosphorylation at serine 63 (Eilers et
al., 1998; Watson et al., 1998), we investigated whether c-Jun
phosphorylation at serine 63 was elevated in neurons undergoing
apoptosis after araC addition. Figure 3B shows that despite
ongoing apoptosis, phosphorylation of c-Jun at serine 63 was not
stimulated by the addition of araC in the presence of NGF, consistent
with the results showing lack of activation of JNK, whereas intense
phosphorylation of c-Jun at serine 63 (~5- to 10-fold) occurred in
the absence of NGF, consistent with previous reports (Virdee et al.,
1997; Eilers et al., 1998). The lack of significant JNK or c-Jun
phosphorylation observed in some experiments in the presence of NGF
precluded obtaining a reliable quantitative measurement of the change
in activity. However, similar results showing lack of c-Jun
phosphorylation were also obtained after 14 hr of araC treatment (data
not shown). These data indicate that araC does not induce or require
JNK activation and/or c-Jun N-terminal phosphorylation to cause
apoptosis and further suggest that the suppression of JNK activity and
c-Jun phosphorylation by NGF is not affected by araC treatment.
p53 is required in araC-induced apoptosis
It has been reported that cerebellar granule neurons from
p53/ animals were resistant to several
antimitotic agents that also cause apoptosis in neurons, including
araC, in contrast to neurons from p53+/ or
p53+/+ mice (Enokido et al., 1996a,b). We therefore
examined whether apoptosis induced by NGF withdrawal or araC treatment
in sympathetic neurons was dependent on the genotype of p53 by
comparing littermates from p53+/ parents. Figure
4 shows that at 16 hr, no significant
apoptosis occurred because of araC treatment in
p53/ neurons in the presence or absence of NGF
(apoptosis at 16 hr: NGF, 14 ± 6%; NGF with araC, 16 ± 3%; no NGF, 68 ± 10%; no NGF with araC, 63 ± 14%;
mean ± SD; n = 6). When one copy of the p53 gene
was present, however, a significant increase in apoptosis was observed
in response to araC treatment (NGF: no araC, 15 ± 5%; with araC,
29 ± 6%; no NGF: no araC, 68 ± 10%; with araC, 80 ± 7%; mean ± SD; n = 13; p < 0.001 for both comparisons). Similar differences were found in neurons
from p53+/+, mice although the difference in percent
apoptosis between neurons cultured with no NGF, without or with araC,
did not quite achieve significance (0.1 > p > 0.05),
most probably because of the inherent lack of accuracy of scoring
apoptotic neurons when >70% apoptosis has occurred. Figure 4 also
shows that the response of sympathetic neurons to NGF withdrawal was
not dependent on the genotype of p53, consistent with previous reports
(Davies and Rosenthal, 1994; Vogel and Parada, 1998).
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Figure 4.
SCG neurons from p53/ mice
are resistant to apoptosis induced by araC, but not NGF, withdrawal.
SCG neurons were prepared from individual mice from litters born to
p53+/ parents and cultured for 16 hr with or
without NGF (100 ng/ml) and with or without araC (100 µM)
before being fixed, stained, and counted as described in Materials and
Methods. The p53 status of each animal tested was determined after
counting had been completed. The results shown are the mean ± SEM
for 6 (p53+/+), 13 (p53+/), and
6 (p53/) animals, respectively. Student's
t tests gave the significance values as indicated
(***p < 0.001;  0.1 > p > 0.05). Inset, PCR results showing the three
genotypes. Top band (642 bp) represents the presence of
wild-type p53 gene; bottom band (510 bp)
represents the absence of the p53 gene.
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Because it appeared that there was a selective requirement for p53 in
araC-induced apoptosis but not in NGF withdrawal-induced apoptosis, we
determined whether araC caused any increase in the cellular levels of
p53 protein in rat sympathetic neurons before and during induction of
apoptosis, as would be expected if p53 played a causal role in
araC-mediated apoptosis (for review, see Levine, 1997). Figure
5A shows that araC treatment
caused an increase in p53 protein as early as 4 hr after its addition
(approximately threefold), a time point at which there were no
significant differences in the extents of apoptosis between any of the
experimental conditions (Fig. 1). This elevation in the level of p53
protein in araC-treated neurons was maintained for up to 14 hr (when
significant apoptosis was occurring) and occurred irrespective of the
presence of NGF (Fig. 5B). In NGF-maintained cultures, we
observed detectable but low levels of p53 protein, which were not
elevated after NGF withdrawal (Figs. 5,
7B),
in keeping with the results of Sadoul et al. (1996). Thus, there is an
induction of and requirement for p53 in araC-induced apoptosis but not
that caused by NGF-withdrawal.
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Figure 5.
AraC causes an early and sustained elevation of
p53 protein levels. Rat SCG neurons were treated for the indicated
times before being collected, resolved on SDS-PAGE, and electroblotted
as described in Materials and Methods. NGF was used at 100 ng/ml, and
araC was used at 100 µM. A, Lanes
1-4, araC; lanes 1-5, NGF. Top
panel shows the results of probing for p53; bottom
panel shows results for total ERKs as an indication of protein
loading. The results shown are representative of two independent
experiments. B, Cultures were treated for 10 or 14 hr
before collection, processing, and probing as in A.
Lanes 1, 2, 5,
6, NGF; lanes 2, 4,
6, araC. The results shown are representative of three
independent experiments.
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Figure 6.
AraC-induced apoptosis is delayed by NGF but not
by CNTF. Neurons were cultured with 100 ng/ml CNTF, 20 ng/ml NGF, or
with no growth factor additions (none) in the absence
(black bars) or presence (hatched bars)
of 100 µM araC as indicated. The cells were fixed at 14 hr before staining and counting. Results are the mean ± SD of
three independent experiments. A Student-Newman-Keuls multiple
comparisons test gave significant differences (***p < 0.001) for all comparisons, except between NGF versus CNTF
(bars 1, 3), NGF with araC versus no
growth factor additions (bars 2, 5), and
CNTF with araC versus no growth factors with araC (bars
4, 6).
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Figure 7.
Suppression of ERK activity by PD98059 renders
neurons more susceptible to araC: a comparison of signaling pathways.
A, Neurons were cultured in the presence or absence of
NGF (20 ng/ml), araC (100 µM), or PD98059 (50 µM) for 14 hr in the indicated conditions before being
fixed, stained, and counted. All media contained 0.2% DMSO. Results
shown are the mean ± SEM of three to five independent experiments
(**p < 0.002; Student's t test).
See Figure 8 for photomicrographs of apoptotic nuclei.
B, Neurons were treated with 20 ng/ml NGF (lanes
1, 2, 4, 5), 100 µM araC (lanes 2, 5), and
50 µM PD98059 (lanes 4, 5)
for 12 hr and then processed for immunoblotting as in Materials and
Methods. The panels show the results of probing with the
following antibodies (top to bottom):
anti-phospho-Akt (serine 473), anti-phospho-ERK1/2,
anti-phospho-c-Jun (serine 63), anti-p53, and anti-ERK1/2.
Results shown are representative of three independent experiments,
except for anti-phospho-Akt and anti-phospho-c-Jun, which are
representative of two independent experiments. The numbers
below the lanes indicate the percentage of
apoptosis and are the mean ± SD of three independent experiments.
C, Fold-phosphorylation as determined by optical
densitometry (see Materials and Methods): top panel,
phospho-Akt (n = 2); bottom panel,
phospho-ERKs (n = 3); both normalized to total ERK.
Error bars represent the SD and range for ERK and Akt,
respectively.
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NGF protects against araC toxicity via the MAPK/ERK pathway
The survival of newly isolated sympathetic neurons can be
sustained by CNTF for several days when laminin is used as substrate (Nobes and Tolkovsky, 1995; Virdee and Tolkovsky, 1995). Figure 6 shows
that when we used CNTF to support the neurons, the rate of apoptosis
induced by araC was significantly greater than that caused by araC in
the presence of NGF (apoptosis at 14 hr: NGF and araC, 33.1 ± 6.9%; CNTF and araC, 69.6 ± 4.5%; mean ± SD; n = 3; p < 0.001). In addition, the
extent of apoptosis induced by araC in the presence of CNTF at 14 hr
was not significantly different from that obtained when neurons were
treated with araC in the absence of any survival factor (77.3 ± 4.8%; mean ± SD; n = 3) (Fig.
8, corresponding micrographs). These
results suggest that NGF, but not CNTF, stimulates one or more
signaling pathways that protect against araC-induced apoptosis.
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Figure 8.
Nuclear morphologies of SCG neurons under various
conditions as visualized with Hoechst 33342. Neurons were treated with
100 ng/ml NGF (a, d, g),
100 ng/ml CNTF (b, e, h),
or without growth factors (-) (c,
f). AraC was used at 100 µM
(d-f), and PD98059 was used at 50 µM (g, h). Hoechst
33342 was added to the cultures at 14 hr, and photographs were taken
before fixing and counting. Scale bar, 10 µm.
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One difference between NGF and CNTF signaling is that CNTF only
transiently activates the MAPK pathway, with ERK1/2 activity returning
to nonstimulated values by 1 hr (Virdee and Tolkovsky, 1995). The MAPK
pathway, however, is reported not to be a necessary component in
NGF-mediated survival (Creedon et al., 1996; Virdee and Tolkovsky,
1996). To test whether ERK activity may play a role in delaying
araC-induced apoptosis, we used PD98059, an inhibitor of the MAPK
pathway. This compound inhibits activation of the kinase (MEK 1)
immediately upstream of the ERKs, thereby preventing ERK activation
(Dudley et al., 1995). Figure 7 shows the results of scoring for
apoptosis after PD98059 treatment and parallel results obtained from
immunoblots. When PD98059 was added to neurons treated with araC in the
presence of NGF (Fig. 7A), the extent of apoptosis caused by
araC was significantly enhanced (at 14 hr: NGF and araC, 32.7 ± 5; NGF, araC, and PD98059, 63.2 ± 11.2; mean ± SD;
n = 5; p < 0.002), strongly suggesting
a specific role for ERK1/2 activity in protecting against araC-induced
apoptosis. Inhibition of ERK1/2 had no significant effect on either the
ability of NGF to promote neuronal survival or the rate of death in the absence of NGF as shown previously (Creedon et al., 1996; Virdee and
Tolkovsky, 1996). Figure 7B shows that PD98059 inhibited
ERK1/2 phosphorylation in the presence of NGF to a level comparable
with that seen in the absence of NGF (Fig. 2B), but
Akt and c-Jun N-terminal phosphorylation were not significantly
affected, whether or not araC was present. This further supports a role
for ERK1/2 in antagonizing the specific mechanism by which araC causes
apoptosis, because PD98059 had no effects on the other pathways
investigated. In keeping with this observation, we found that PD98059
did not cause apoptosis when the cells were maintained with CNTF, nor
did it further increase apoptosis in the presence of araC (apoptosis at
14 hr: CNTF, 8.9 ± 1.4%; CNTF and PD98059, 8.4 ± 1.3%;
CNTF and araC, 69.6 ± 4.5%; CNTF, araC, and PD98059, 72.1 ± 2.5%; mean ± SD; n = 3). PD98059 also did not
affect p53 levels in the absence of araC and did not impair the
induction of p53 as a result of araC treatment (Fig. 7B),
thus suggesting that ERK1/2 do not act by regulating the araC-induced
elevation of p53 protein levels.
Because toxicity of araC in mitotic cells is dependent on the efficacy
of its uptake and metabolism into triphosphonucleotides (Grant, 1998),
it was possible that ERK activity decreased the uptake and/or
metabolism of araC, so reducing its toxicity. We found, however, that
PD98059 inhibited rather than increased the uptake of
[3H]araC into the neurons by ~50%, without
affecting its relative metabolism into the mono-, di-, and triphosphate
forms (measurements taken after 14 hr of [3H]araC
uptake; data not shown). ERK1/2 may therefore be involved in
controlling the uptake of arabinose nucleosides in some manner, but
this is unlikely to be the cause of the increased toxicity of araC in
the presence of PD98059.
| |
DISCUSSION |
We have compared changes in the levels of activity of signaling
pathways during apoptosis induced by NGF-deprivation or araC treatment
in the presence and absence of NGF to explore the mechanism of araC
toxicity in sympathetic neurons. We found that (1) araC treatment did
not interfere with any of the NGF-maintained signaling pathways
examined, (2) araC treatment did not mimic signaling induced by
NGF-withdrawal, (3) there was a sustained elevation of and requirement
for p53 during apoptosis caused by araC but not NGF withdrawal, and (4)
ERK activity protected against apoptosis caused by araC.
We have already reported some evidence that araC does not interfere
with NGF-mediated signaling in which pretreatment of NGF-deprived neurons with araC for 3 hr did not inhibit the subsequent NGF-mediated induction of c-Fos protein (Tomkins et al., 1994). In this study, we
have investigated the effect of araC on the phosphorylation and hence
activation of the signaling kinases ERK1, ERK2, and Akt at a time when
apoptosis was already occurring. Activation of the ERKs is mediated by
Ras (for review, see Segal and Greenberg, 1996), which is critical for
SCG sympathetic neuron survival (Nobes et al., 1996; Markus et al.,
1997), but ERK activity is not required in NGF-mediated survival
(Creedon et al., 1996; Virdee and Tolkovsky, 1996). Akt is also
activated by NGF, in part via the PI 3-kinase pathway (Park et al.,
1996), and is reported to mediate at least part of the NGF-induced
survival of sympathetic neurons (Crowder and Freeman, 1998; Virdee and
Tolkovsky, unpublished observations; but see Philpott et al., 1997). We
report that araC does not act by inhibiting the pathways leading to
either ERK or Akt activation, because apoptosis occurs without the
reduction of the phosphorylation states of these proteins. Therefore,
neither active Akt nor ERKs are sufficient to maintain survival in the
presence of araC. It is possible that araC directly inhibits the
signaling downstream of Akt or another Ras-dependent survival pathway,
but the acceleration of apoptosis caused by araC in the absence of NGF
suggests another mechanism of action.
After withdrawal of NGF from sympathetic neurons, various events have
been found to take place, such as the induction of c-Jun gene
expression (Estus et al., 1994), a transient increase in reactive
oxygen species (ROS) (Greenlund et al., 1995), and an early and
sustained increase in JNK activity, causing N-terminal phosphorylation
of c-Jun (Virdee et al., 1997; Eilers et al., 1998). It has been
reported that the transcription factor c-Jun is a necessary and
possibly sufficient mediator of apoptosis induced by NGF-withdrawal
(Estus et al., 1994; Ham et al., 1995). It has also been suggested that
JNK activity and particularly phosphorylation of c-Jun at serine 63 (which enhances transcriptional activation by c-Jun) are requirements
for induction of neuronal apoptosis after withdrawal of a survival
stimulus (Eilers et al., 1998; Maroney et al., 1998; Watson et al.,
1998). We have found that araC does not cause the activation of JNK or
the phosphorylation of c-Jun at serine 63 either before or during
apoptosis. Our data therefore suggest that (1) araC does not inhibit
the ability of NGF to suppress JNK activity, and (2) enhancement of the
transcriptional activation of c-Jun is not required for apoptosis
induced by araC.
Although we analyzed the effects of araC on intracellular signaling
when up to 40% of the population had undergone apoptosis, it is likely
that our observations extend to most of the neurons, because (1)
NGF-deprived SCG neurons commit apoptosis stochastically, as predicted
for a homogenous population of cells (Edwards and Tolkovsky, 1994), so
signaling events in early and late dying neurons would be expected to
be similar, and (2) downregulation of ERK and Akt signals (Virdee and
Tolkovsky, 1995; Virdee and Tolkovsky, unpublished observations) and
upregulation in JNK activity-cJun phosphorylation (Virdee et al.,
1997) precedes apoptosis by several hours. If, therefore, araC
caused late death by mimicking NGF-withdrawal, such advance signaling
changes should have already occurred in the yet-to-die population, but
our data indicate that these changes did not occur.
The suggestion that araC causes apoptosis in postmitotic cerebellar
granule neurons in a p53-dependent manner (Enokido et al., 1996a)
prompted us to examine the requirement for p53 in araC-induced
apoptosis in SCG neurons. We found that the response of sympathetic
neurons to NGF withdrawal was independent of their p53 status, as
reported previously (Davies and Rosenthal, 1994; Vogel and Parada,
1998), but that neurons from p53/ mice were
resistant to araC toxicity. One copy of the p53 gene was, however,
sufficient to cause significant susceptibility to the toxin. We
provided further evidence for a requirement for p53 in apoptosis caused
by araC by demonstrating that the level of p53 protein in rat SCG
neurons was elevated in response to araC after 4 hr of treatment and
that p53 protein level remained elevated up to 14 hr (the longest time
point tested), when significant apoptosis had already occurred.
Furthermore, elevation of p53 protein levels by araC was independent of
the presence of NGF, whereas p53 was not elevated by NGF withdrawal as
described previously (Sadoul et al., 1996). Because overexpression of
p53 using an adenovirus vector causes neuronal apoptosis (Slack et al.,
1996; Jordan et al., 1997), we suggest that it is the elevation of
endogenous p53 protein levels by araC that causes apoptosis in SCG neurons.
How araC causes this increase in p53 protein in postmitotic neurons
remains unclear. Levels of the p53 protein may be increased by various
stimuli, such as DNA damage, nucleotide pool imbalances, and hypoxia
(for review, see Levine, 1997), but hypoxia is unlikely under the
culture conditions used. It is also unlikely that araC mediates
nucleotide pool imbalance, because high concentrations of
deoxyribonucleosides, fluorodeoxyuridine, or hydroxyurea (Martin et
al., 1990; Tomkins et al., 1994), which might be expected to affect
nucleotide pool ratios, do not have the same toxic effect. AraC may
therefore cause DNA damage, but the mechanism remains to be identified.
Although NGF and CNTF caused similar levels of survival of sympathetic
neurons, the rate of apoptosis caused by araC in the presence of CNTF
was not significantly different from that caused by araC added without
any growth factors. In comparison, NGF significantly reduced this rate
of apoptosis. This finding implied that one or more components of NGF
signaling that are not shared with CNTF signaling have an inhibitory
effect on the mechanism of araC toxicity. One difference in signaling
pathways induced by these two neurotrophic factors is the duration of
ERK activation; whereas NGF causes sustained activation of ERK
activity, CNTF causes only a transient activation of <1 hr (Virdee and
Tolkovsky, 1995). To test the involvement of the MAPK pathway in
resistance to araC-induced apoptosis, we used the selective MAPK
pathway inhibitor PD98059. We found that the rate of apoptosis caused
by araC in the presence of NGF was substantially increased by PD98059,
thereby suggesting a critical role for the MAPK pathway in NGF-mediated
resistance to araC. This conclusion is reinforced by the lack of
protection against araC toxicity observed when CNTF was the survival factor.
The mechanism of ERK-mediated protection against araC-induced apoptosis
remains unclear. We found that PD98059 did not cause any detectable
change in the level of p53 protein at 12 hr, suggesting that ERK
primarily acts to inhibit araC-induced apoptosis downstream of p53
induction and also therefore downstream of araC metabolism. Because
some of the targets of ERK are transcription factors (Segal and
Greenberg, 1996), it is possible that downregulation of ERK activity
may inhibit transcriptional activation and expression of unstable
proteins involved in survival or protection against araC.
Evidence for an anti-apoptotic role for ERK has been reported in PC12
cells after growth factor withdrawal (Xia et al., 1995) and in
Fas-inducible apoptosis in Jurkat cells (Holmstrom et al., 1998). ERK
has also been reported to function as a suppressor of ROS in which the
addition of PD98059 to SCG neurons caused a detectable but transient
increase in ROS (Dugan et al., 1997). As it has been suggested that
apoptosis caused by p53 is mediated by ROS (Johnson et al., 1996;
Polyak et al., 1997) and araC induces ROS in lymphocytes (Hedley and
McCulloch, 1996), the ability of ERK to suppress ROS would be a
possible mechanism for inhibiting p53-mediated araC-induced apoptosis.
In support, Skaper et al. (1998) showed that the glutamate-induced
ROS-mediated apoptosis of young cerebellar granule cells could be
inhibited by BDNF. This protection was reduced, however, by treatment
with PD98059. The finding that different signaling pathways may be used
to protect against different insults supports results obtained in
fibroblasts (Ullrich et al., 1998) in which NGF suppression of
apoptosis induced by different stimuli was mediated by different
TrkA constructs.
Our results, summarized schematically in Figure
9, provide a clear separation of the
signaling cascades involved in NGF withdrawal-mediated apoptosis and
those involved in araC-mediated apoptosis in sympathetic neurons. The
data presented here also provide evidence for a role for the MAPK
pathway in protection against apoptosis in primary neurons. Thus,
although ERK activity is not required for NGF-mediated survival under
certain conditions in vitro, it may be important for
survival under some physiological or pathological conditions in
vivo. More generally, our findings highlight the importance of
sustaining multiple signaling pathways for conferring protection against a variety of insults.
View larger version (16K):
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Figure 9.
Scheme summarizing proposed interactions between
NGF signaling and apoptotic pathways. NGF induces at least three
signals: the sustained activation of MAPK/ERK and Akt, and suppression
of JNK and c-Jun phosphorylation. In the absence of NGF, JNK is
activated, whereas ERK1/2 and Akt are inactivated. In the presence of
NGF, araC treatment causes elevation of p53 and apoptosis without
affecting NGF signaling. ERK1/2, however, suppresses signaling
downstream of p53, thereby delaying apoptosis. Inhibition of the ERK
pathway with PD98059 primarily eliminates this suppression, suggesting
that the lack of ERK activity caused by NGF withdrawal may therefore
account for the acceleration in the rate of apoptosis induced by
araC.
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FOOTNOTES |
Received Aug. 25, 1998; revised Oct. 28, 1998; accepted Nov. 2, 1998.
This work was supported by a Wellcome Trust toxicology studentship to
C.N.G.A. and a program grant to A.M.T. We thank Andreas Nelsbach (New
England Biolabs, Bedford, MA) for the generous provision of
anti-phospho-kinase antibodies used during the early part of this work,
Alan Clarke (University of Edinburgh, Edinburgh, Scotland) for the kind
gift of p53 null and heterozygote founder mice, Richard Farndale
(University of Cambridge, Cambridge, UK) for help with statistical
analysis, and members of the A.M.T. laboratory for critical discussions.
Correspondence should be addressed to A. M. Tolkovsky, Department
of Biochemistry, Downing Site, Tennis Court Road, Cambridge, CB2 1QW,
United Kingdom.
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REFERENCES |
-
Alessi DR,
Andjelkovic M,
Caudwell B,
Cron P,
Morrice N,
Cohen P,
Hemmings BA
(1996)
Mechanism of activation of protein kinase B by insulin and IGF-1.
EMBO J
15:6541-6551[Web of Science][Medline].
-
Andjelkovic M,
Jakubowicz T,
Cron P,
Ming XF,
Han JW,
Hemmings BA
(1996)
Activation and phosphorylation of a pleckstrin homology domain-containing protein-kinase (RAC-PK/PKB) promoted by serum and protein phospatase inhibitors.
Proc Natl Acad Sci USA
93:5699-5704[Abstract/Free Full Text].
-
Baker WJ,
Royer GLJ,
Weiss RB
(1991)
Cytarabine and neurologic toxicity.
J Clin Oncol
9:679-693[Abstract].
-
Buckmaster A,
Nobes CD,
Edwards SN,
Tolkovsky AM
(1991)
NGF is required for induction of c-fos immunoreactivity by serum, depolarisation, cyclic AMP or trauma in cultured rat sympathetic neurons.
Eur J Neurosci
3:698-707[Web of Science][Medline].
-
Clarke AR,
Purdie CA,
Harrison DJ,
Morris RG,
Bird CC,
Hooper ML,
Wyllie AH
(1993)
Thymocyte apoptosis induced by p53-dependent and independent pathways.
Nature
362:849-852[Medline].
-
Creedon DJ,
Johnson Jr EM,
Lawrence JC
(1996)
Mitogen-activated protein kinase-independent pathways mediate the effects of nerve growth factor and cAMP on neuronal survival.
J Biol Chem
271:20713-20718[Abstract/Free Full Text].
-
Crowder RJ,
Freeman RS
(1998)
Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor dependent sympathetic neurons.
J Neurosci
18:2933-2943[Abstract/Free Full Text].
-
Davies AM,
Rosenthal A
(1994)
Neurons from mouse embryos with a null mutation in the tumour-suppressor gene p53 undergo normal cell death in the absence of neurotrophins.
Neurosci Lett
182:112-114[Web of Science][Medline].
-
Deckwerth TL,
Johnson Jr EM
(1993)
Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor.
J Cell Biol
123:1207-1222[Abstract/Free Full Text].
-
Dessi F,
Pollard H,
Moreau J,
Ben-Ari Y,
Charriaut-Marlangue C
(1995)
Cytosine arabinoside induces apoptosis in cerebellar neurons in culture.
J Neurochem
64:1980-1987[Web of Science][Medline].
-
Dudek H,
Datta SR,
Franke TF,
Birnbaum MJ,
Yao R,
Cooper GM,
Segal RA,
Kaplan DR,
Greenberg ME
(1997)
Regulation of neuronal survival by the serine-threonine protein kinase Akt.
Science
275:661-665[Abstract/Free Full Text].
-
Dudley DT,
Pang L,
Decker SJ,
Bridges AJ,
Saltiel AR
(1995)
A synthetic inhibitor of the mitogen-activated protein kinase cascase.
Proc Natl Acad Sci USA
92:7686-7689[Abstract/Free Full Text].
-
Dugan LL,
Creedon DJ,
Johnson EM,
Holtzman Jr DM
(1997)
Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway.
Proc Natl Acad Sci USA
94:4086-4091[Abstract/Free Full Text].
-
Edwards SN,
Tolkovsky AM
(1994)
Characterisation of apoptosis in cultured rat sympathetic neurons after nerve growth factor (NGF) withdrawal.
J Cell Biol
124:537-546[Abstract/Free Full Text].
-
Eilers A,
Whitfield J,
Babij C,
Rubin LL,
Ham J
(1998)
Role of the Jun kinase pathway in the regulation of c-Jun expression and apoptosis in sympathetic neurons.
J Neurosci
18:1713-1724[Abstract/Free Full Text].
-
Enokido Y,
Araki T,
Shinichi A,
Hatanaka H
(1996a)
p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.
Neurosci Lett
203:1-4[Web of Science][Medline].
-
Enokido Y,
Araki T,
Tanaka K,
Aizawa S,
Hatanaka H
(1996b)
Involvement of p53 in DNA strand break-induced apoptosis in postmitotic CNS neurons.
Eur J Neurosci
8:1812-1821[Web of Science][Medline].
-
Estus S,
Zaks WJ,
Freeman RS,
Gruda M,
Bravo R,
Johnson Jr EM
(1994)
Altered gene-expression in neurons during programmed cell-death: identification of c-jun as necessary for neuronal apoptosis.
J Cell Biol
127:1717-1727[Abstract/Free Full Text].
-
Gotoh Y,
Nishida E,
Yamashita T,
Hoshi M,
Kawakami M,
Sakai H
(1990)
Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen activated MAP kinase of fibroblastic cells.
Eur J Biochem
193:661-669[Web of Science][Medline].
-
Grant S
(1998)
Ara-C: cellular and molecular pharmacology.
Adv Cancer Res
72:197-233[Web of Science][Medline].
-
Greenlund LJS,
Deckwerth TL,
Johnson Jr EM
(1995)
Superoxide-dismutase delays neuronal apoptosis
 a role for reactive oxygen species in programmed neuronal death.
Neuron
14:303-315[Web of Science][Medline]. -
Ham J,
Babij C,
Pfarr CM,
Lallemand D,
Yaniv M,
Rubin LL
(1995)
A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death.
Neuron
14:927-939[Web of Science][Medline].
-
Hawrot E,
Patterson P
(1979)
Long-term culture of dissociated sympathetic neurons.
Methods Enzymol
58:574-584[Medline].
-
Hedley DW,
McCulloch EA
(1996)
Generation of reactive oxygen intermediates after treatment of blasts of acute myeloblastic leukemia with cytosine arabinoside: role of bcl-2.
Leukemia
10:1143-1149[Web of Science][Medline].
-
Holmstrom TH,
Chow SC,
Elo I,
Coffey ET,
Orrenius S,
Sistonen L,
Eriksson JE
(1998)
Suppression of Fas/APO-1-mediated apoptosis by mitogen activated kinase signaling.
J Immunol
160:2626-2636[Abstract/Free Full Text].
-
Johnson TM,
Yu Z-X,
Ferrans VJ,
Lowenstein RA,
Finkel T
(1996)
Reactive oxygen species are downstream mediators of p53-dependent apoptosis.
Proc Natl Acad Sci USA
93:11848-11852[Abstract/Free Full Text].
-
Jordan J,
Galindo MF,
Prehn JHM,
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[Abstract/Free Full Text].
-
Levine AJ
(1997)
p53, the cellular gatekeeper for growth and division.
Cell
88:323-331[Web of Science][Medline].
-
Markus A,
von Holst A,
Rohrer H,
Heumann R
(1997)
NGF-mediated survival depends on p21ras in chick sympathetic neurons from the superior cervical but not from lumbosacral ganglia.
Dev Biol
191:306-310[Web of Science][Medline].
-
Maroney AC,
Glicksman MA,
Basma A,
Walton KM,
Knight EJ,
Murphy CA,
Bartlett BA,
Finn JP,
Angeles T,
Matsuda Y,
Neff NT,
Dionne CA
(1998)
Motoneuron apoptosis is blocked by CEP-1347 (KT 7515), a novel inhibitor of the JNK signaling pathway.
J Neurosci
18:104-111[Abstract/Free Full Text].
-
Martin DP,
Wallace TL,
Johnson Jr EM
(1990)
Cytosine arabinoside kills postmitotic neurons in a fashion resembling trophic factor deprivation: evidence that a deoxycytidine-dependent process may be required for nerve growth factor signal transduction.
J Neurosci
10:184-193[Abstract].
-
Mobley WC,
Schenker A,
Shooter EM
(1976)
Characterization and isolation of proteolytically modified nerve growth factor.
Biochemistry
15:5543-5551[Medline].
-
Nobes CD,
Tolkovsky AM
(1995)
Neutralizing anti-p21(Ras) fabs suppress rat sympathetic neuron survival induced by NGF, LIF, CNTF and cAMP.
Eur J Neurosci
7:344-350[Web of Science][Medline].
-
Nobes CD,
Reppas JB,
Markus A,
Tolkovsky AM
(1996)
Active p21ras is sufficient for rescue of NGF-dependent rat sympathetic neurons.
Neuroscience
70:1067-1079[Web of Science][Medline].
-
Park DS,
Morris EJ,
Stefanis L,
Troy CM,
Shelanski ML,
Geller HM,
Greene LA
(1998)
Multiple pathways of neuronal death induced by DNA-damaging agents, NGF deprivation, and oxidative stress.
J Neurosci
18:830-840[Abstract/Free Full Text].
-
Park EK,
Yang SI,
Kang SS
(1996)
Activation of Akt by nerve growth factor via phosphatidylinositol 3-kinase in PC12 pheochromocytoma cells.
Mol Cells
6:494-498.
-
Philpott KL,
McCarthy MJ,
Klippel A,
Rubin LL
(1997)
Activated phoshpatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons.
J Cell Biol
139:809-815[Abstract/Free Full Text].
-
Polyak K,
Xia Y,
Zweier JL,
Kinzler KW
(1997)
A model for p53-induced apoptosis.
Nature
389:300-305[Medline].
-
Ray LB,
Sturgill TW
(1988)
Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo.
Proc Natl Acad Sci USA
85:3753-3757[Abstract/Free Full Text].
-
Sadoul R,
Quiquerez AL,
Martinou I,
Fernandez PA,
Martinou JC
(1996)
p53 protein in sympathetic neuronscytoplasmic localization and no apparent function in apoptosis.
J Neurosci Res
43:594-601[Web of Science][Medline].
-
Sanz-Rodriguez C,
Boix J,
Comella JX
(1997)
Cytosine arabinoside is neurotoxic to chick embryo spinal motoneurons in culture.
Neurosci Lett
223:141-144[Web of Science][Medline].
-
Segal RA,
Greenberg ME
(1996)
Intracellular signaling pathways activated by neurotrophic factors.
Annu Rev Neurosci
19:463-489[Web of Science][Medline].
-
Skaper SD,
Floreani M,
Negro A,
Facci L,
Giusti P
(1998)
Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway.
J Neurochem
70:1859-1868[Web of Science][Medline].
-
Slack RS,
Belliveau DJ,
Rosenberg M,
Atwal J,
Lochmuller H,
Aloyz R,
Haghighi A,
Lach B,
Seth P,
Cooper E,
Miller FD
(1996)
Adenovirus-mediated gene transfer of the tumor suppressor, p53, induces apoptosis in postmitotic neurons.
J Cell Biol
135:1085-1096[Abstract/Free Full Text].
-
Tomkins CE,
Edwards SN,
Tolkovsky AM
(1994)
Apoptosis is induced In postmitotic rat sympathetic neurons by arabinosides and topoisomerase-II inhibitors in the presence of NGF.
J Cell Sci
107:1499-1507[Abstract].
-
Ullrich E,
Duwel A,
Kauffmann-Zeh A,
Gilbert C,
Lyon D,
Rudkin B,
Evan G,
Martin-Zanca D
(1998)
Specific TrkA survival signals interfere with different apoptotic pathways.
Oncogene
16:825-832[Web of Science][Medline].
-
Virdee K,
Tolkovsky AM
(1995)
Activation of p44 and p42 MAP kinases is not essential for the survival of rat sympathetic neurons.
Eur J Neurosci
7:2159-2169[Web of Science][Medline].
-
Virdee K,
Tolkovsky AM
(1996)
Inhibition of p42 and p44 mitogen activated protein kinase activity by PD98059 does not suppress nerve growth factor-induced survival of sympathetic neurones.
J Neurochem
67:1801-1805[Web of Science][Medline].
-
Virdee K,
Bannister AJ,
Hunt SP,
Tolkovsky AM
(1997)
Comparison between the timing and onset of JNK activation, c-Jun phosphorylation, and onset of death commitment in sympathetic neurones.
J Neurochem
69:550-561[Web of Science][Medline].
-
Vogel KS,
Parada LF
(1998)
Sympathetic neuron survival and proliferation are prolonged by loss of p53 and neurofibromin.
Mol Cell Neurosci
11:19-28[Web of Science][Medline].
-
Wallace TL,
Johnson Jr EM
(1989)
Cytosine arabinoside kills postmitotic neurons: evidence that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis.
J Neurosci
9:115-124[Abstract].
-
Watson A,
Eilers A,
Lallemand D,
Kyriakis J,
Rubin LL,
Ham J
(1998)
Phosphorylation of c-Jun is necessary for apoptosis induced by survival signal withdrawal in cerebellar granule neurons.
J Neurosci
18:751-762[Abstract/Free Full Text].
-
Xia Z,
Dickens M,
Raingeaud J,
Davis RJ,
Greenberg ME
(1995)
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
270:1326-1331[Abstract/Free Full Text].
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M. A. King, C. G. Goemans, F. Hafiz, J. H. M. Prehn, A. Wyttenbach, and A. M. Tolkovsky
Cytoplasmic Inclusions of Htt Exon1 Containing an Expanded Polyglutamine Tract Suppress Execution of Apoptosis in Sympathetic Neurons
J. Neurosci.,
December 31, 2008;
28(53):
14401 - 14415.
[Abstract]
[Full Text]
[PDF]
|
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E. Szatmari, K. B. Kalita, G. Kharebava, and M. Hetman
Role of Kinase Suppressor of Ras-1 in Neuronal Survival Signaling by Extracellular Signal-Regulated Kinase 1/2
J. Neurosci.,
October 17, 2007;
27(42):
11389 - 11400.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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|
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D. P. Sester, K. Brion, A. Trieu, H. S. Goodridge, T. L. Roberts, J. Dunn, D. A. Hume, K. J. Stacey, and M. J. Sweet
CpG DNA Activates Survival in Murine Macrophages through TLR9 and the Phosphatidylinositol 3-Kinase-Akt Pathway
J. Immunol.,
October 1, 2006;
177(7):
4473 - 4480.
[Abstract]
[Full Text]
[PDF]
|
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M. Colon-Cesario, J. Wang, X. Ramos, H. G. Garcia, J. J. Davila, J. Laguna, C. Rosado, and S. Pena de Ortiz
An inhibitor of DNA recombination blocks memory consolidation, but not reconsolidation, in context fear conditioning.
J. Neurosci.,
May 17, 2006;
26(20):
5524 - 5533.
[Abstract]
[Full Text]
[PDF]
|
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|
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A. Wyttenbach and A. M. Tolkovsky
Differential Phosphoprotein Labeling (DIPPL), a Method for Comparing Live Cell Phosphoproteomes Using Simultaneous Analysis of 33P- and 32P-Labeled Proteins
Mol. Cell. Proteomics,
March 1, 2006;
5(3):
553 - 559.
[Abstract]
[Full Text]
[PDF]
|
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K. G. Brywe, A.-L. Leverin, M. Gustavsson, C. Mallard, R. Granata, S. Destefanis, M. Volante, H. Hagberg, E. Ghigo, and J. Isgaard
Growth Hormone-Releasing Peptide Hexarelin Reduces Neonatal Brain Injury and Alters Akt/Glycogen Synthase Kinase-3{beta} Phosphorylation
Endocrinology,
November 1, 2005;
146(11):
4665 - 4672.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
|
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H. K. Wong, M. Fricker, A. Wyttenbach, A. Villunger, E. M. Michalak, A. Strasser, and A. M. Tolkovsky
Mutually Exclusive Subsets of BH3-Only Proteins Are Activated by the p53 and c-Jun N-Terminal Kinase/c-Jun Signaling Pathways during Cortical Neuron Apoptosis Induced by Arsenite
Mol. Cell. Biol.,
October 1, 2005;
25(19):
8732 - 8747.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Landau, K. C. Luk, M.-L. Jones, R. Siegrist-Johnstone, Y. K. Young, E. Kouassi, V. V. Rymar, A. Dagher, A. F. Sadikot, and J. Desbarats
Defective Fas expression exacerbates neurotoxicity in a model of Parkinson's disease
J. Exp. Med.,
September 6, 2005;
202(5):
575 - 581.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Hanamoto, T. Ozaki, K. Furuya, M. Hosoda, S. Hayashi, M. Nakanishi, H. Yamamoto, H. Kikuchi, S. Todo, and A. Nakagawara
Identification of Protein Kinase A Catalytic Subunit {beta} as a Novel Binding Partner of p73 and Regulation of p73 Function
J. Biol. Chem.,
April 29, 2005;
280(17):
16665 - 16675.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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Y. Sun and F. A. Sinicrope
Selective inhibitors of MEK1/ERK44/42 and p38 mitogen-activated protein kinases potentiate apoptosis induction by sulindac sulfide in human colon carcinoma cells
Mol. Cancer Ther.,
January 1, 2005;
4(1):
51 - 59.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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W. B. Jacobs, G. S. Walsh, and F. D. Miller
Neuronal Survival and p73/p63/p53: A Family Affair
Neuroscientist,
October 1, 2004;
10(5):
443 - 455.
[Abstract]
[PDF]
|
|
|
|
|
|
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Y. Murayama, J.-i. Miyagawa, K. Oritani, H. Yoshida, K. Yamamoto, O. Kishida, T. Miyazaki, S. Tsutsui, T. Kiyohara, Y. Miyazaki, et al.
CD9-mediated activation of the p46 Shc isoform leads to apoptosis in cancer cells
J. Cell Sci.,
July 1, 2004;
117(15):
3379 - 3388.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. D. Nair, T. Yuen, C. W. Olanow, and S. C. Sealfon
Early Single Cell Bifurcation of Pro- and Antiapoptotic States during Oxidative Stress
J. Biol. Chem.,
June 25, 2004;
279(26):
27494 - 27501.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I.-J. Kim, K. M. Drahushuk, W.-Y. Kim, E. A. Gonsiorek, P. Lein, D. A. Andres, and D. Higgins
Extracellular Signal-Regulated Kinases Regulate Dendritic Growth in Rat Sympathetic Neurons
J. Neurosci.,
March 31, 2004;
24(13):
3304 - 3312.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Lynch, M. Moore, S. Craig, P. E. Lonergan, D. S. Martin, and M. A. Lynch
Analysis of Interleukin-1{beta}-induced Cell Signaling Activation in Rat Hippocampus following Exposure to Gamma Irradiation: PROTECTIVE EFFECT OF EICOSAPENTAENOIC ACID
J. Biol. Chem.,
December 19, 2003;
278(51):
51075 - 51084.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Gozdz, A. Habas, J. Jaworski, M. Zielinska, J. Albrecht, M. Chlystun, A. Jalili, and M. Hetman
Role of N-Methyl-D-aspartate Receptors in the Neuroprotective Activation of Extracellular Signal-regulated Kinase 1/2 by Cisplatin
J. Biol. Chem.,
October 31, 2003;
278(44):
43663 - 43671.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. G. Besirli and E. M. Johnson Jr.
JNK-independent Activation of c-Jun during Neuronal Apoptosis Induced by Multiple DNA-damaging Agents
J. Biol. Chem.,
June 13, 2003;
278(25):
22357 - 22366.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Wang, H. Wang, L. Xu, D. J. Rozanski, T. Sugawara, P. H. Chan, J. M. Trzaskos, and G. Z. Feuerstein
Significant Neuroprotection against Ischemic Brain Injury by Inhibition of the MEK1 Protein Kinase in Mice: Exploration of Potential Mechanism Associated with Apoptosis
J. Pharmacol. Exp. Ther.,
January 1, 2003;
304(1):
172 - 178.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Fei, E. J. Bernhard, and W. S. El-Deiry
Tissue-specific Induction of p53 Targets in Vivo
Cancer Res.,
December 15, 2002;
62(24):
7316 - 7327.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. P. Roux, G. Dorval, M. Boudreau, A. Angers-Loustau, S. J. Morris, J. Makkerh, and P. A. Barker
K252a and CEP1347 Are Neuroprotective Compounds That Inhibit Mixed-lineage Kinase-3 and Induce Activation of Akt and ERK
J. Biol. Chem.,
December 13, 2002;
277(51):
49473 - 49480.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Bezombes, A. de Thonel, A. Apostolou, T. Louat, J.-P. Jaffrezou, G. Laurent, and A. Quillet-Mary
Overexpression of Protein Kinase Czeta Confers Protection Against Antileukemic Drugs by Inhibiting the Redox-Dependent Sphingomyelinase Activation
Mol. Pharmacol.,
December 1, 2002;
62(6):
1446 - 1455.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Y. Bhatt, T. W. Kelley, V. V. Khramtsov, Y. Wang, G. K. Lam, T. L. Clanton, and C. B. Marsh
Macrophage-Colony-Stimulating Factor-Induced Activation of Extracellular-Regulated Kinase Involves Phosphatidylinositol 3-Kinase and Reactive Oxygen Species in Human Monocytes
J. Immunol.,
December 1, 2002;
169(11):
6427 - 6434.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Tong, G. Singh, and A. J. Rainbow
Sustained Activation of the Extracellular Signal-regulated Kinase Pathway Protects Cells from Photofrin-mediated Photodynamic Therapy
Cancer Res.,
October 1, 2002;
62(19):
5528 - 5535.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhu, G.-Y. Yang, B. Ahlemeyer, L. Pang, X.-M. Che, C. Culmsee, S. Klumpp, and J. Krieglstein
Transforming Growth Factor-beta 1 Increases Bad Phosphorylation and Protects Neurons Against Damage
J. Neurosci.,
May 15, 2002;
22(10):
3898 - 3909.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Cheng, P. Sapieha, P. Kittlerova, W. W. Hauswirth, and A. Di Polo
TrkB Gene Transfer Protects Retinal Ganglion Cells from Axotomy-Induced Death In Vivo
J. Neurosci.,
May 15, 2002;
22(10):
3977 - 3986.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-S. Bae, Y. Kim, J. C. Park, P.-G. Suh, and S. H. Ryu
The synthetic chemoattractant peptide, Trp-Lys-Tyr-Met-Val-D-Met, enhances monocyte survival via PKC-dependent Akt activation
J. Leukoc. Biol.,
February 1, 2002;
71(2):
329 - 338.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. C. P. Somervaille, D. C. Linch, and A. Khwaja
Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax
Blood,
September 1, 2001;
98(5):
1374 - 1381.
[Abstract]
[Full Text]
[PDF]
|
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B. A. Ballif and J. Blenis
Molecular Mechanisms Mediating Mammalian Mitogen-activated Protein Kinase (MAPK) Kinase (MEK)-MAPK Cell Survival Signals
Cell Growth Differ.,
August 1, 2001;
12(8):
397 - 408.
[Full Text]
[PDF]
|
|
|
|
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|
F. C. Barone, E. A. Irving, A. M. Ray, J. C. Lee, S. Kassis, S. Kumar, A. M. Badger, R. F. White, M. J. McVey, J. J. Legos, et al.
SB 239063, a Second-Generation p38 Mitogen-Activated Protein Kinase Inhibitor, Reduces Brain Injury and Neurological Deficits in Cerebral Focal Ischemia
J. Pharmacol. Exp. Ther.,
April 13, 2001;
296(2):
312 - 321.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. R. Hansen, X.-M. Zha, J. Bok, and S. H. Green
Multiple Distinct Signal Pathways, Including an Autocrine Neurotrophic Mechanism, Contribute to the Survival-Promoting Effect of Depolarization on Spiral Ganglion Neurons In Vitro
J. Neurosci.,
April 1, 2001;
21(7):
2256 - 2267.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
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P. Dent and S. Grant
Pharmacologic Interruption of the Mitogen-activated Extracellular-regulated Kinase/Mitogen-activated Protein Kinase Signal Transduction Pathway: Potential Role in PromotingCytotoxic Drug Action
Clin. Cancer Res.,
April 1, 2001;
7(4):
775 - 783.
[Full Text]
|
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|
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|
J. E. Cavanaugh, J. Ham, M. Hetman, S. Poser, C. Yan, and Z. Xia
Differential Regulation of Mitogen-Activated Protein Kinases ERK1/2 and ERK5 by Neurotrophins, Neuronal Activity, and cAMP in Neurons
J. Neurosci.,
January 15, 2001;
21(2):
434 - 443.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Li, X. Wang, M. K. Meintzer, T. Laessig, M. J. Birnbaum, and K. A. Heidenreich
Cyclic AMP Promotes Neuronal Survival by Phosphorylation of Glycogen Synthase Kinase 3beta
Mol. Cell. Biol.,
December 15, 2000;
20(24):
9356 - 9363.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
U. Namgung and Z. Xia
Arsenite-Induced Apoptosis in Cortical Neurons Is Mediated by c-Jun N-Terminal Protein Kinase 3 and p38 Mitogen-Activated Protein Kinase
J. Neurosci.,
September 1, 2000;
20(17):
6442 - 6451.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. C. Fletcher, L. Xue, S. K. Passingham, and A. M. Tolkovsky
Death Commitment Point Is Advanced by Axotomy in Sympathetic Neurons
J. Cell Biol.,
August 21, 2000;
150(4):
741 - 754.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. I. Savitz and J. A. Kessler
Leukemia Inhibitory Factor Requires Concurrent p75LNTR Signaling to Induce Apoptosis of Cultured Sympathetic Neurons
J. Neurosci.,
June 1, 2000;
20(11):
4198 - 4205.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Lawlor, X. Feng, D. R. Everding, K. Sieger, C. E. H. Stewart, and P. Rotwein
Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways
Mol. Cell. Biol.,
May 1, 2000;
20(9):
3256 - 3265.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Sanchez-Prieto, J. M. Rojas, Y. Taya, and J. S. Gutkind
A Role for the p38 Mitogen-activated Protein Kinase Pathway in the Transcriptional Activation of p53 on Genotoxic Stress by Chemotherapeutic Agents
Cancer Res.,
May 1, 2000;
60(9):
2464 - 2472.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
D. S. Park, E. J. Morris, R. Bremner, E. Keramaris, J. Padmanabhan, M. Rosenbaum, M. L. Shelanski, H. M. Geller, and L. A. Greene
Involvement of Retinoblastoma Family Members and E2F/DP Complexes in the Death of Neurons Evoked by DNA Damage
J. Neurosci.,
May 1, 2000;
20(9):
3104 - 3114.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Xue, J. H. Murray, and A. M. Tolkovsky
The Ras/Phosphatidylinositol 3-Kinase and Ras/ERK Pathways Function as Independent Survival Modules Each of Which Inhibits a Distinct Apoptotic Signaling Pathway in Sympathetic Neurons
J. Biol. Chem.,
March 17, 2000;
275(12):
8817 - 8824.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. R. Datta, A. Brunet, and M. E. Greenberg
Cellular survival: a play in three Akts
Genes & Dev.,
November 15, 1999;
13(22):
2905 - 2927.
[Full Text]
|
|
|
|
|
|
|
L. L. Dugan, J. S. Kim, Y. Zhang, R. D. Bart, Y. Sun, D. M. Holtzman, and D. H. Gutmann
Differential Effects of cAMP in Neurons and Astrocytes. ROLE OF B-RAF
J. Biol. Chem.,
September 3, 1999;
274(36):
25842 - 25848.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Hetman, K. Kanning, J. E. Cavanaugh, and Z. Xia
Neuroprotection by Brain-derived Neurotrophic Factor Is Mediated by Extracellular Signal-regulated Kinase and Phosphatidylinositol 3-Kinase
J. Biol. Chem.,
August 6, 1999;
274(32):
22569 - 22580.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Gibson, S. Tu, R. Oyer, S. M. Anderson, and G. L. Johnson
Epidermal Growth Factor Protects Epithelial Cells against Fas-induced Apoptosis. REQUIREMENT FOR Akt ACTIVATION
J. Biol. Chem.,
June 18, 1999;
274(25):
17612 - 17618.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-J. Kim, J.-W. Ju, C.-D. Oh, Y.-M. Yoon, W. K. Song, J.-H. Kim, Y. J. Yoo, O.-S. Bang, S.-S. Kang, and J.-S. Chun
ERK-1/2 and p38 Kinase Oppositely Regulate Nitric Oxide-induced Apoptosis of Chondrocytes in Association with p53, Caspase-3, and Differentiation Status
J. Biol. Chem.,
January 4, 2002;
277(2):
1332 - 1339.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Orike, G. Middleton, E. Borthwick, V. Buchman, T. Cowen, and A. M. Davies
Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons
J. Cell Biol.,
September 3, 2001;
154(5):
995 - 1006.
[Abstract]
[Full Text]
[PDF]
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Top
Abstract
Introduction
Compound via MeSH
