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INTRODUCTION |
It is well established that tumor cells are able to acquire
resistance to chemotherapeutic agents through a variety of mechanisms including enhanced drug metabolism, altered drug accumulation, drug-target amplification, and repair of damaged drug target (1). More
recently, induction of apoptosis has been discovered as a novel
mechanism by which many anti-neoplastic therapies achieve their
therapeutic effects (2-4). Therefore, the inability of a tumor cell to
activate the apoptotic response has been proposed as a pathway of
resistance to antineoplastic therapy (5).
Neuroblastoma (NB)1 is one of
the most common malignancies in childhood. Unlike other tumor types,
nearly all human NBs were found to carry the wild-type p53
gene (6-9). Wild-type p53 protein is expressed in many of the cell
lines derived from NB (10). However, the p53 protein is found to be
restricted primarily to the cytoplasmic compartment of the NB cells
(11). This finding has led to the proposal that the p53-mediated
response in NB cells is impaired because of an unknown cytoplasmic
sequestration mechanism that prevents its translocation into the
nucleus (11, 12). Our laboratory has recently reported a protein kinase
inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H-7) that
possesses novel activities of inducing nuclear accumulation of the p53
protein and triggering p53-dependent apoptosis in several
NB cell lines that carry the wild-type p53 gene (13). H-7,
similar to DNA damaging agents, increased the steady-state level of p53
in NB cells, which at least, in part accounted for the dramatic nuclear accumulation of p53 in these cells (12, 13).
All-trans-retinoic acid (RA) and many of its natural and
synthetic derivatives, collectively known as retinoids, have been investigated extensively as potential therapeutics for human cancer because of their effects in inhibiting cell proliferation, inducing cell differentiation, and promoting apoptosis in many types of cancer,
including NBs (14-17). The activities of retinoids are mediated by a
network of RA receptors (RARs) and retinoid X receptors (RXRs)
(18-21). RA at low concentration activates RARs in vivo (18-21). RA can also be metabolized to several active metabolites in vivo, including 9-cis-RA, the high-affinity
ligand for RXR (22). In addition, RXRs are also known to heterodimerize
with RARs and a subset of other nuclear receptors and co-regulator molecules to participate in a complex array of signaling events (18-21). Besides having a role in differentiation, RA has also been
implicated in mediating apoptosis in NB cells (23). Paradoxically, a
recent study has shown that treatment of NB cells with RA resulted in
the development of resistance to apoptosis induced by chemotherapeutic agents (24).
In this report, we examine the possible contribution of the p53 pathway
in the chemoresistance response rendered by RA treatment in the NB cell
line, SH-SY5Y. RA treatment was found to confer resistance selectively
to p53-dependent apoptotic stimuli in these cells. The
effect was associated with the suppression of H-7-induced nuclear
accumulation rather than the enhancement of protein stability of
wild-type p53 protein in SH-SY5Y cells, suggesting that drug-induced up-regulation and nuclear accumulation of the p53 protein are separable processes.
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EXPERIMENTAL PROCEDURES |
Materials--
Isoquinoline
(1-(5-isoquinolinesulfonyl)-2-methylpiperazine; H-7) and staurosporine
were purchased from RBI.
N-Acetyl-D-erythro-sphingosine was
from Calbiochem. All other chemicals were purchased from Sigma unless
otherwise indicated.
Cells--
The human neuroblastoma cell lines SH-SY5Y, LA-N-5,
and IMR-32 have been described previously (13). RPMI 1640 medium was supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and antibiotics (100 mg/ml streptomycin and 100 IU/ml penicillin, Life Technologies, Inc.). H-7 was dissolved in water;
all the other compounds were dissolved in Me2SO-ethanol (1:1). The cells were grown to subconfluence and then treated with RA
for different durations and with different concentrations of RA. The
medium was replaced every 48 h. When reagents dissolved in
Me2SO-ethanol were used, equal volume of the solvent was
added to the control cells.
Antibodies--
The following antibodies were purchased from
Transduction Laboratories (Lexington, KY): anti-p53 monoclonal antibody
P21020 that recognizes the C terminus of the human p53 protein, anti-RB monoclonal antibody, and anti-ICH-1L monoclonal antibody.
Anti-p53 (DO-1) monoclonal antibody, anti-Bcl-2 (100) monoclonal
antibody, rabbit anti-Bcl-xL/S (S-19), anti-Bax (N-20),
anti-c-Jun/AP-1 (N), and anti-c-Myc (A-14) polyclonal antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p53
(Ab-2) monoclonal antibody was purchased from Oncogene Science
(Uniondale, NY). Horseradish peroxidase-conjugated anti-mouse IgG and
anti-rabbit IgG were from Amersham Pharmacia Biotech (Buckinghamshire, UK).
MTT Assay--
Drug-induced cytotoxicity was assessed by (3-[4,
5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay carried out in 96-well microtiter plates. The cells were treated
with RA for different durations and concentrations, as described in the
figure legends, and then exposed to the apoptotic drugs for additional
24 h. 3 h before the end of drug treatment, MTT was added to
each well to a final concentration of 1 mg/ml and incubated at
37 °C. The reaction was terminated by removing the supernatant and
the dye was dissolved by adding 100 µl Me2SO and 10 µl
of Sorensen glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5), followed by thorough mixing. In a
parallel set of experiments, the cells were exposed to the indicated
amounts of ionizing radiation at a dose rate of 1 Gy/min in a
137Cs
-irradiator and then incubated at 37 °C for
6 h before the MTT assay was performed. The plates were read at
562 nm on a Ceres 900 microtiter plate reader (Bio-tek Instruments
Inc.). The means and standard deviations were derived from six replicates.
DNA Ladder Assay--
Cells were grown on 60-mm tissue culture
dishes (Nunc). After drug treatment, the cells were harvested, washed
once with ice-cold PBS, resuspended in 250 µl of lysis buffer (10 mM Tris-HCl, pH 7.6, 20 mM EDTA, pH 8.0, 0.5%
v/v Triton X-100) in a microcentrifuge tube, and the samples were
incubated at room temperature for 15 min. After centrifugation at
16,000 × g for 5 min, the supernatant was transferred
to another microcentrifuge tube and sequential extractions were carried
out. The samples were extracted once with equal volume of phenol, once
with phenol-chloroform (1:1), and once with chloroform-isoamyl alcohol
(24:1). DNA was precipitated with 0.1 volume of 3 M sodium
acetate, pH 4.8, and 2.5 volumes of ethanol at
20 °C overnight.
The DNA was then pelleted at 16,000 × g for 30 min the
next day. After washing once with 70% ethanol, the samples were
digested with 0.1 µg of RNase A at 37 °C for 30 min before loading
onto 1.5% agarose gels and the DNA was visualized by ethidium bromide staining.
Preparation of Cell Extracts--
Cells were grown in 90-mm
dishes, cultured, and treated as described. To prepare the whole cell
lysates, the medium was removed and cells were washed twice with
ice-cold Tris buffered saline (TBS) (150 mM NaCl, 10 mM Tris, pH 7.6) and lysed with 500 µl of lysis buffer
(10 mM Tris, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 25 mM
-glycerophosphate, 1 mM PMSF, 2 mM benzamidine, 10 mg/ml aprotinin,
10 mg/ml leupeptin, and 1 mM sodium orthovanadate) for 15 min. The lysed cells were transferred into 1.5-ml microcentrifuge tubes, centrifuged at 15,000 × g for 10 min at
4 °C, and the supernatants were collected. To prepare the nuclear
extracts, the cells were washed with cold PBS twice, and detached from
plates by adding 1 ml of detachment buffer (150 mM NaCl, 1 mM EDTA, pH 8.0, 40 mM Tris, pH 7.6) for 5 min
at room temperature. The cells were then transferred to microcentrifuge
tubes and centrifuged at 300 × g for 4 min at 4 °C.
The supernatant was discarded, and the pellet was resuspended in 400 µl of cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 2 mM benzamidine, 10 mg/ml aprotinin,
10 mg/ml leupeptin, and 1 mM sodium orthovanadate) and
incubated on ice for 15 min. 10 µl of 10% Nonidet P-40 was added,
and the mixture was vortexed briefly. Nuclei were pelleted by
centrifuging at 2800 × g for 4 min at 4 °C and then
resuspended in 50 µl of ice-cold buffer B (20 mM Hepes,
pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM
DTT, 1 mM PMSF, 2 mM benzamidine, 10 mg/ml
aprotinin, 10 mg/ml leupeptin, and 1 mM sodium
orthovanadate). The mixture was shaken vigorously for 15 min at
4 °C, centrifuged at 15,000 × g for 5 min, and the
supernatant was collected. The protein concentration was determined by
the Bradford assay (25).
Immunoblotting--
Total cell lysates or nuclear extracts,
volume adjusted according to the protein concentration, were resolved
by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(PAGE), and the proteins were transferred to 0.2-µm pore size
nitrocellulose membrane (Amersham Pharmacia Biotech). Filters were
blocked with TBS containing 3% nonfat dry milk and 0.1% Tween 20 (TBST) for 3 h at room temperature and probed with primary
antibodies diluted according to the supplier's instructions for 1 h in the above buffer. The filters were then washed three times with
TBST containing 0.1% Tween 20, probed with a 1:10,000 dilution of
horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia
Biotech), washed again in TBST, and developed using the enhanced
chemiluminescence (ECL) system (Amersham Pharmacia Biotech). To ensure
identical amounts of protein extracts from different conditions were
loaded, a duplicate set of 10% SDS-PAGE gels was routinely prepared
and the gel was subjected to Coomassie staining to allow a visual inspection of the overall protein staining patterns.
Immunofluorescence Analysis--
Cells were seeded onto glass
coverslips and treated with various reagents after which monolayers
were washed twice with ice-cold PBS, pH 7.4. The immunofluorescence
staining procedure has been described previously (26). Briefly, cells
were fixed for 3 min at 4 °C with absolute methanol (
20 °C).
The washing step with PBS was then repeated once. The cells were
blocked at 37 °C for 30 min with 2% bovine serum albumin, 5% fetal
bovine serum, 5% normal goat serum in PBS. The cells were incubated at
room temperature for 45 min with the monoclonal anti-p53 (Ab-2)
antibody in blocking buffer and washed with PBS. The cells were then
incubated with fluorescein isothiocyanate-conjugated goat anti-mouse
IgG (Molecular Probes Inc., Eugene, OR). After washing, the coverslips
were mounted in 80% glycerol in PBS containing 1 mg/ml
p-phenylenediamine, and examined with a Zeiss Axioplan microscope.
Pulse-Chase Experiments--
Cells were grown in 90-mm plates,
treated with solvent or RA for 4 days and then subjected to 50 µM H-7. The cells were labeled by incubation for 3 h
in methionine-free medium (Life Technologies, Inc.) supplemented with
dialyzed fetal bovine serum and 25 µCi/ml [35S]methionine (NEN Life Science Products). After the
labeling, the cells were washed three times with PBS before refeeding
with fresh medium and then harvested 2, 4, 6, and 8 h later. The
cells were lysed in lysis buffer (10 mM phosphate buffer,
pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate and 0.1% SDS, 1 mM PMSF, and 2 mg/ml
aprotinin) and subjected to centrifugation at 140,000 × g at 4 °C for 20 min. Radioactivities of the supernatants collected were determined by scintillation counting, and protein concentrations were determined. Immunoprecipitation of p53 protein was
performed from samples containing equivalent amount of total cellular
protein. The lysates were incubated with 1 µg of p53 monoclonal
antibody, DO-1, at 4 °C for 1 h. The immune complexes were
bound with protein A (4 °C, 1 h), followed by three washes with
lysis buffer. Finally, samples were denatured by boiling in SDS-loading
buffer (100 mM Tris, pH 6.8, 2% SDS, 0.1% bromphenol blue, 10% glycerol, 25 µM
-mercaptoethanol) and
loaded onto a 10% SDS-polyacrylamide gel. Autoradiography was then
performed on the dried gel. The intensities of the bands were
quantitated with a phosphorimager using ImageQuantTM
(Molecular Dynamics).
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RESULTS |
RA Pretreatment Inhibits H-7- and Etoposide-induced Nuclear
Accumulation of p53 Protein in the Human Neuroblastoma Cell Line,
SH-SY5Y--
To facilitate our investigation into the mechanism
underlying the chemoresistance response in the neuroblastoma cells,
SH-SY5Y, associated with RA treatment (24), we evaluated the effect of RA on cellular levels of a panel of proteins known to have a role in
various apoptosis pathways, with and without H-7 treatment. Many of
these proteins are known activators or suppressors (e.g. Bcl-xL/S, Bcl-2, Bax, c-Myc, RB) of apoptosis, and their
expressions in SH-SY5Y cells have been documented (13, 27, 28). Protein levels were evaluated by Western analyses. With the exception of Bcl-2
and p53, no changes were noted in levels of all the proteins tested in
mock- or RA-treated cells, with or without H7 (Fig. 1A).

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Fig. 1.
RA prevents H-7-induced nuclear accumulation
of p53 in human NB cells, SH-SY5Y. A, immunoblot
analyses of various proteins in apoptosis pathway in SH-SY5Y cells.
Cells were pretreated with solvent (Mock) or 10 µM RA for 5 days before exposure to 100 µM
H-7 for an additional 10 h. Levels of Ich-1L,
Bcl-xL, Bax, and Bcl-2 protein were examined by Western
analysis of total cell lysates. Nuclear extracts were used for
analyzing p53, RB, c-Jun, and c-Myc protein levels. 1,
Ich-1L; 2, Bcl-xL; 3,
Bax; 4, Bcl-2; 5, RB; 6, c-Jun;
7, c-Myc; 8, p53. B, RA treatment
inhibits the effect of H-7 on p53 nuclear accumulation.
Immunofluorescence analysis of p53 in SH-SY5Y cells. The cells were
pretreated for 4 days with solvent or 10 µM RA before
exposure to 50 µM H-7 for an additional 7 h. The
subcellular localization of p53 in the cells with the indicated
treatment was analyzed by immunofluorescence analysis using the
p53-specific monoclonal antibody (Ab-2). C, RA inhibits both
etoposide and H-7-mediated induction of nuclear p53. The cells were
treated for 4 days with solvent or with 10 µM RA before
exposure to 50 µM H-7 or 100 µM etoposide
for an additional 7 h. Nuclear extracts were prepared from the
cells, and the presence of p53 was analyzed by immunoblotting using the
p53-specific mouse monoclonal antibody P21210.
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Similar to the results obtained in the earlier study, the level of
Bcl-2 protein was found to be up-regulated in RA-treated SH-SY5Y cells
(24) (Fig. 1A). However, H-7 treatment did not affect the
Bcl-2 level in either mock- or RA-treated cells (Fig. 1A).
Interestingly, while RA treatment did not seem to affect the level of
nuclear p53 in these cells, it suppressed the H-7-mediated nuclear
accumulation of p53 that was observed in solvent-treated (Mock) SH-SY5Y cells (Fig. 1A).
The effect of RA in preventing nuclear accumulation of p53 mediated by
H-7 in SH-SY5Y cells was further documented by immunofluorescence staining of endogenous p53 protein in these cells subjected to various
treatments. Diffuse staining of p53 in the cytosol of SH-SY5Y cells was
apparent in untreated cells (Fig. 1B). Upon stimulation by
H-7, nuclear p53 signal was dramatically enhanced in the cells treated
with solvent (Mock). However, in RA-treated cells, the
strong nuclear staining was no longer observed upon H-7 treatment (Fig.
1B), supporting the conclusion drawn from Western analysis
that RA treatment blocked H-7-mediated accumulation of nuclear p53 protein.
The ability of RA to block nuclear p53 induction in SH-SY5Y cells was
not restricted to the H-7 paradigm, as essentially identical results
were obtained with etoposide treatment (Fig. 1C), which is
also an established paradigm for the induction of nuclear p53 (29).
RA Blocks p53-dependent Apoptosis in SH-SY5Y
Cells--
The proteins that we have examined in Western analysis
represent only a fraction of all the possible proteins that might have a role in the apoptotic pathway affected by RA. In order to assess whether the up-regulation of Bcl-2 and the suppression of H-7-induced p53 nuclear accumulation by RA had any functional impact on the apoptotic response of SH-SY5Y cells, we compared the apoptotic responses of these cells upon induction by paradigms that are either
p53-dependent or p53-independent. H-7, etoposide, and
-irradiation have been regarded as p53-dependent
apoptotic paradigms in many experimental systems (5, 11-13, 30), while
staurosporine, N-acetyl-D-erythro-sphingosine, and
menadione are believed to act through p53-independent pathways
(31-33). Indeed, treatment of SH-SY5Y cells with staurosporine,
N-acetyl-D-erythro-sphingosine, or
menadione effectively resulted in apoptosis as demonstrated by DNA
ladder assay (Fig. 2A), and
yet none of these paradigms induced nuclear accumulation of p53 under
identical conditions (Fig. 2B), confirming that their
apoptotic effects are likely to involve p53-independent mechanisms. On
the other hand, treatment of SH-SY5Y cells with H-7, etoposide, or
-irradiation induced apoptosis as well as nuclear accumulation of
p53 (Fig. 2, A and B).

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Fig. 2.
Assessment of apoptotic paradigms for their
effects in mediating apoptosis and inducing nuclear accumulation of p53
protein in SH-SY5Y cells. A, DNA fragmentation analysis
of SH-SY5Y cells undergoing apoptosis induced by various treatments.
SH-SY5Y cells were subjected to staurosporine (50 nM),
N-acetyl-D-erythro-sphingosine (20 µM), menadione (2 µM), H-7 (50 µM), or etoposide (50 µM) treatments for
24 h. For -irradiation, SH-SY5Y cells were exposed to 1 Gy of
ionizing radiation. Small molecular weight DNA was extracted from the
SH-SY5Y cells as described under "Experimental Procedures."
B, a subset of apoptotic paradigms induce nuclear
accumulation of p53 protein in SH-SY5Y cells. SH-SY5Y cells were
exposed to various apoptosis inducing treatments as described in
A for 10 h. Nuclear extracts were prepared and
subjected to Western analysis with the p53-specific mouse monoclonal
antibody P21210.
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We next assessed the effect of RA pretreatment on the sensitivity of
SH-SY5Y cells to these apoptotic stimuli. The cells were treated with
either solvent or 10 µM RA for 4 days before exposure to
the different apoptosis-inducing paradigms, and the cell viability was
measured by MTT assay (Fig. 3). RA
treatment did not alter the apoptotic response of the cells to
staurosporine,
N-acetyl-D-erythro-sphingosine, and
menadione (Fig. 3, D-F). Interestingly, RA-treated cells
were considerably more resistant to apoptosis induced by H-7,
etoposide, or
-irradiation (Fig. 3, A-C), as
demonstrated by a shift of the dose-response curves to the right. These
results support the idea that pretreatment of these cells with RA led
to the development of resistance selectively to the
p53-dependent apoptotic stimuli.

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Fig. 3.
RA selectively impairs
p53-dependent apoptosis in SH-SY5Y cells. SH-SY5Y
cells were plated onto 96-well plates and were pretreated for 4 days
with solvent (open squares) or with 10 µM RA (filled squares) before
exposure for an additional 24 h to the apoptosis-inducing
treatments. Viability of the cells was determined by MTT method at the
end of the treatment period, as described under "Experimental
Procedures." The 100% values of viable cells were defined by
measurement obtained from mock- and RA-treated cells in the absence of
H-7 treatment. Values shown are means ± S.D. of six samples. Each
treatment paradigm was repeated at least twice, and similar results
were obtained.
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TPA Pretreatment Up-regulates Bcl-2 Protein Level but Fails to
Confer Resistance to H-7-induced Apoptosis in SH-SY5Y Cells--
In
agreement with a previous study, the protein level of Bcl-2 was found
to be up-regulated by 3-4-fold in SH-SY5Y cells upon RA treatment
(24). Since Bcl-2 has been reported to be an effective suppressor of
apoptosis triggered by a variety of apoptotic stimuli (34-37), it is
therefore possible that the chemoresistance response observed in
SH-SY5Y cells was a consequence of the elevated Bcl-2 level. The
observation that RA-treated cells develops resistance selectively to
only p53-dependent apoptosis argues against the elevated
Bcl-2 levels to have a dominant role in conferring the RA-mediated
resistance response in these cells. To clarify the issue further, we
subjected SH-SY5Y cells to 12-O-tetradecanoyl-13-acetate (TPA) treatment, which is known to up-regulate Bcl-2 in SH-SY5Y cells
(27). Similar to RA, TPA treatment (16 nM × 5 days)
resulted in 3-5-fold induction of the Bcl-2 protein (Fig.
4A). In contrast to RA-treated
cells, TPA-treated cells remained sensitive to H-7 and exhibited
nuclear accumulation of p53 (Fig. 4B). Moreover, TPA was
unable to confer resistance to the p53-dependent apoptotic response mediated by H-7. Despite a rise in Bcl-2 level, SH-SY5Y cells
pre-exposed to TPA remained as sensitive as the solvent-treated cells
(Mock) to H-7-mediated apoptosis, as evidenced by the DNA ladder assay (Fig. 4C).

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Fig. 4.
Effects of TPA on Bcl-2 level and
H-7-mediated apoptosis in SH-SY5Y cells. A and
B, immunoblot analyses of total or nuclear extracts for
monitoring of change in protein level of Bcl-2 or p53, respectively,
upon induction by H-7. The SH-SY5Y cells had been treated with either
solvent (Mock), RA (10 µM), or TPA (16 nM) for 5 days before exposure for an additional 10 h
to 50 µM H-7. C, TPA failed to confer
resistance to H-7-mediated apoptosis. DNA fragmentation analysis of RA-
and TPA-treated SH-SY5Y cells condemned to die by treatment with 50 µM H-7 for 24 h.
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Kinetics of RA-induced Resistance to p53-dependent
Apoptosis--
The effect of RA in conferring resistance to
p53-dependent apoptosis appeared to be time- and
concentration-dependent. The effect was not detectable
within the first 24 h of exposure (Fig. 5A). After 48 h of
exposure to RA, resistance to p53-dependent apoptosis
became apparent and the degree of resistance was enhanced in a
time-dependent manner. The chemoresistance effect of RA was found to be dose-dependent as higher concentrations
produced more pronounced effects (Fig. 5A). RA at
concentration above 10 µM was toxic to the cells and
therefore was not used.

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Fig. 5.
Time and dose dependence of RA-induced
resistance to H-7-mediated apoptosis in SH-SY5Y cells.
A, SH-SY5Y cells were treated for different durations with
solvent (open squares), 100 nM RA
(filled squares), 1 µM RA
(filled circles), or 10 µM RA
(open circles) before exposure to 50 µM H-7 for an additional 24 h, and the cell
viability was assessed by MTT method. The 100% values of viable cells
were defined by measurement obtained from mock- and RA-treated cells in
the absence of H-7 treatment. Values shown are means ± S.D. of
six samples. Each treatment paradigm was repeated at least twice and
gave similar results. B, inhibition of H-7-induced apoptosis
by RA was analyzed by DNA ladder assay. SH-SY5Y cells were pretreated
for different durations with solvent or 10 µM RA and then
exposed to 50 µM H-7 for an additional 24 h. Small
molecular weight DNA was then extracted and separated on 1.5% agarose
gel, as described under "Experimental Procedures."
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In contrast to an earlier study, which showed that SH-SY5Y cells
treated with RA at 10-100 nM for 4 days developed
chemoresistance to adriamycin and cisplatin (24), the resistance effect
studied here appeared to require a relatively higher concentration of RA (1 µM). This raises the possibility that a metabolite
of RA, rather than RA itself, was responsible for this effect.
DNA fragmentation, a hallmark of late stage apoptosis, induced by H-7,
was dramatically reduced only when the SH-SY5Y cells were pretreated
with RA for more than 24 h (Fig. 5B). These data provide evidence to support the hypothesis that the effect of RA on NB
cells was to prevent death resulting from apoptosis. The loss of
sensitivity of SH-SY5Y cells to p53-dependent apoptosis upon RA treatment may represent a gradual metabolic process, which may
involve re-programming of complex signaling processes in the cells.
RA Confers Resistance to H-7-mediated p53-dependent
Apoptosis in Only a Subset of NB Cell Lines--
We further
investigated the effect of RA in preventing the induction of functional
p53 and subsequent apoptosis mediated by H-7 in two additional NB cell
lines. These two cell lines, LA-N-5 and IMR-32, have been previously
demonstrated to be sensitive to the effect of H-7 in mediating nuclear
accumulation of p53 and apoptosis (13). In our experiments, only LA-N-5
cells responded to RA in a similar manner as SH-SY5Y cells. LA-N-5
cells showed a reduction in sensitivity to H-7 in the induction of
nuclear p53 as well as apoptosis upon treatment with 10 µM RA for 4 days (Fig.
6A). In contrast, RA
pretreatment had no effect on H-7-induced nuclear p53 accumulation and
also failed to confer resistance to apoptosis in IMR-32 cells (Fig.
6B).

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Fig. 6.
Differential effects of RA on
H-7-dependent apoptosis in human neuroblastoma cell
lines. Left panels, RA treatment selectively inhibited
the H-7-dependent apoptotic response in only a subset of NB
cells. NB cell lines were treated for 4 days with solvent
(open squares) or with 10 µM RA
(filled squares) prior to exposure to 50 µM H-7 for an additional 24 h, and the cell
viability was assessed by MTT assay as described in Fig. 3. The 100%
values of viable cells were defined by measurement obtained from mock-
and RA-treated cells in the absence of H-7 treatment. Values shown are
means ± S.D. of six samples. Each treatment paradigm was repeated
at least twice and gave similar results. Right panels,
immunoblot analysis of nuclear extracts from NB cell lines, with mock
or RA (10 µM × 4 days) pretreatment, to determine the
level of nuclear p53 protein upon treatment with 50 µM
H-7 for 10 h.
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RA Treatment Does Not Affect H-7-induced Enhancement of the
Steady-state Level of the p53 Protein--
H-7 has been shown to
elevate the steady-state level of p53 in NB cells by increasing the
half-life of the protein (13). The effect may, in part, account for the
nuclear accumulation of p53 in the cells upon exposure to this drug. It
is conceivable that RA treatment in SH-SY5Y cells may affect the
protein stabilizing effect of H-7 on p53 in these cells.
We examined the amount of p53 protein in whole cell lysates and nuclear
extracts of SH-SY5Y cells by immunoblotting before and after exposure
to RA treatment. RA treatment did not significantly affect the low
basal level of p53 detected in the whole cell lysate of SH-SY5Y cells
(Fig. 7, A, lanes 1 and 3, and B). Upon H-7 treatment, however, a
marked increase in p53 level was detected in the whole cell extract of
both mock and RA-treated cells (Fig. 7,A, lanes 2 and 4, and B). Apparently, H-7 was still able to
enhance the steady-state level of p53 in RA-treated cells to a similar
extent as in the control cells (Fig. 7B).

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Fig. 7.
RA does not affect the H-7-mediated increase
in p53 stability in SH-SY5Y cells. A, Western blot
analysis of total and nuclear p53 protein upon H-7 (50 µM × 10 h) treatment in mock- or RA-treated (10 µM × 4 days) SH-SY5Y cells. Extracts from whole cells or nuclei were
prepared and fractionated using SDS-PAGE. Equivalent amounts of lysates
were analyzed by immunoblotting using the p53-specific mouse monoclonal
antibody P21210. B, graphic representation of the data in
A. Data from immunoblotting experiments were quantitated
with a densitometer using ImageTM program, and the -fold
increase in p53 protein was plotted as the ratio of the amount of p53
from H-7 treated cells versus untreated cells. Results from
mock- and RA-treated cells are represented by open and
filled bars, respectively. Values shown are
means ± S.D. of three separate experiments. C,
pulse-chase analysis of p53 protein stability in SH-SY5Y cells. The
cells were pretreated for 4 days with solvent or 10 µM
RA, and the stability of the p53 protein upon H-7 treatment was
determined as follows; cells were pulsed for 3 h in the presence
of [35S]methionine and then chased with non-radioactive
medium for the indicated times. H-7 (50 µM) was added to
the media at the beginning of the pulse period and throughout the chase
period. Whole cell extracts were then subjected to immunoprecipitation
using the anti-p53 antibody DO-1. The immune complexes bound to protein
A were then fractionated on a 10% SDS-polyacrylamide gel followed by
autoradiography.
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The effect of H-7 on nuclear accumulation of p53, however, was
essentially absent in RA-treated SH-SY5Y cells (Fig. 7, A, lane 8, and B). Indeed, pulse-chase experiments
revealed that pre-exposure to 10 µM RA for 4 days had
minimal effect on the H-7-induced stabilization of p53 protein in
SH-SY5Y cells (Fig. 7C), suggesting that the effect of RA on
blocking the H-7-induced nuclear accumulation of p53 is independent
from its effect on the stability of p53 in these cells.
Therefore, RA may modulate the p53-dependent response in
SH-SY5Y cells by inhibiting the nuclear localization process from taking place despite the high level of p53 in the cytoplasm induced by
drug treatments.
 |
DISCUSSION |
In this study, we investigated the role of the p53 pathway in the
development of chemoresistance response to p53-dependent apoptosis in NB cells as a result of exposure to RA. The SH-SY5Y, LA-N-5, and IMR-32 NB cell lines are known to be sensitive to p53-dependent apoptosis pathway regulated by H-7 (13). RA
treatment effectively conferred resistance in SH-SY5Y and LA-N-5 but
not IMR-32 cells to H-7-mediated apoptosis. The differential responses among NB cell lines to RA may be due to a variation of genetic backgrounds in NBs. RA-treated SH-SY5Y and LA-N-5 cells failed to
accumulate nuclear p53 upon H-7 treatment, consistent with the
suggestion that the p53 pathway might be involved in this effect.
Interestingly, RA did not significantly affect the H-7-mediated up-regulation of the cytoplasmic pool of the wild-type p53 protein in
SH-SY5Y cells, raising the possibility that the drug-induced nuclear
accumulation of p53 is a separable process from enhanced protein stability.
It is widely believed that restoring and/or enhancing wild-type p53
functions in tumor cells represent a promising treatment strategy for
human cancers (38). Unlike other tumors, NBs have an extremely low rate
of mutation in their p53 gene (6, 8). Irradiation and DNA damaging
agents are known to induce p53 expression in NB cells harboring the
wild-type gene (12). Recently, we reported that the protein kinase
inhibitor, H-7, has the ability to elevate the p53 level in the nucleus
of NB cells, thus leading to apoptosis (13). H-7 treatment is known to
enhance the level of p53 in SH-SY5Y cells by increasing its stability
(13). As a result, a large increase in p53 protein level in both
cytoplasmic and nuclear compartments was noted upon stimulation (12,
13). However, it is not clear whether the increase in p53 in the
nucleus mediated by drug treatments is an active or passive process and whether it is separable from the effect of the increased stability of
the protein. Indeed, Moll et al. (12) proposed that the
cytoplasmic retention of p53 in NB cells is a saturable process and a
sudden increase in p53 level can saturate the storage capacity of the cytoplasmic pool of p53 leading to nuclear accumulation of the protein.
In this study, RA treatment did not substantially affect the
H-7-induced enhancement of the p53 steady-state level but dramatically
inhibited the nuclear accumulation of p53. This observation leads us to
propose that the nuclear import of p53 is an independent and active
process that can be selectively regulated by RA in a subset of NB cells
that expresses wild-type p53 protein. While the loss of
p53-dependent response has been associated with
chemoresistance in certain tumor cells, the molecular basis of the
resistance has primarily been associated with gene mutation and protein
stability (5, 39). Our current findings represent the first
documentation that the process of nuclear import of wild-type p53
protein can be a target for modulating the chemoresistance response in
some tumor cells.
An earlier study has reported that RA treatment, at 10-100
nM, renders chemoresistance to adriamycin and cisplatin in
SH-SY5Y cells (24). Bcl-2 up-regulation upon RA treatment was proposed as a likely mechanism for the development of the chemoresistance (24).
In agreement with this report, we observed a 3-4-fold up-regulation of
total Bcl-2 protein in SH-SY5Y cells upon RA (10 µM)
treatment (Fig. 4). However, Bcl-2 has been shown to prevent apoptosis
resulting from treatments with various apoptotic stimuli including
sphingosine, staurosporine, and chemotherapeutic agents (34-37). Since
RA treatment did not confer resistance to apoptotic stimuli rendered by
sphingosine or staurosporine in our system, it is therefore
questionable whether the mild up-regulation of Bcl-2 protein could have
a major role in conferring selective resistance to the
p53-dependent apoptosis.
TPA is as effective as RA in up-regulating Bcl-2 in SH-SY5Y cells.
However, only RA is able to confer resistance to
p53-dependent apoptosis in SH-SY5Y cells. These data offer
further evidence arguing against the up-regulation of Bcl-2 alone being
responsible for the RA-mediated chemoresistance response studied here.
However, it remains formally possible that Bcl-2 might have a role in
collaborating with p53 or some other molecule in the apoptotic pathway
to confer the resistance response seen in these cells.
The network of signaling pathways influenced by RA is extremely complex
and diverse (40). Indeed, some synthetic retinoids, which have
selective affinity to a particular RAR or RXR subtype, have been shown
to promote different effects in NB cells in vitro (41, 42).
It is possible that the pleiotropic effect of RA on NB cells is
mediated by a complex interplay of multiple retinoid signaling
pathways. Additional studies involving receptor subtype-specific retinoid ligands are, therefore, necessary to further our understanding of the contribution of individual retinoid signaling pathways to the
processes of differentiation, proliferation, apoptosis, and
chemoresistance in NB cells.