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INTRODUCTION |
The p53 tumor suppressor protein is normally expressed at low
levels due to rapid degradation by the ubiquitin-proteasome pathway,
but p53 is stabilized in response to various cellular stress stimuli
(1-4), resulting in nuclear translocation and transactivation of
numerous target genes. One of the p53-induced genes is the ubiquitin
ligase HDM2 (5), which acts as a key regulator of p53 activity through
its ability to target the p53 protein for proteasomal degradation (6).
Thus, p53-mediated up-regulation of HDM2 gene expression creates a
negative regulatory loop, whereby HDM2 inactivates p53 and promotes its
destruction, presumably after p53 has exerted its cellular functions.
In addition to its role as a ubiquitin protein ligase for p53, HDM2 has
also been implicated in facilitating p53 nuclear export, an activity
that appears necessary for its ability to degrade the protein (7, 8).
DNA damage-induced nuclear translocation of p53 protects the protein
from the destabilizing cytosolic effects of HDM2. Stabilization of p53
can also occur by inhibition of p53-HDM2 complex formation,
which is proposed to be negatively regulated by p53 phosphorylation at
N-terminal sites (9-11). However, at least two reports demonstrate
normal stabilization of p53 mutated at multiple phosphorylation sites
(12, 13), suggesting that additional mechanisms may also be utilized.
Indeed, HDM2 nucleolar sequestration mediated by p14arf
and stress-induced down-regulation of HDM2 are alternative mechanisms resulting in p53 stabilization (14-17).
There is much interest in studying the pathogenesis of neuroblastoma
(NB),1 since this disease is
among the most common of pediatric malignancies. Unlike the majority of
other tumors, NB is unusual in that the p53 protein is wild type (WT)
in the overwhelming majority of cases (18); however, the protein is
reported to be abnormally stabilized and overexpressed (19). p53 has
also been reported to be primarily cytosolic in NB (20), presumably due
to hyperactive nuclear export (21). Neuroblastoma is additionally
complex, in that tumors are composed of a mixture of neuroblastic
(N-type) and Schwannian (S-type) cell morphologies that differ in
tumorigenicity (22) and in p53 subcellular localization (23). Taken
together, these observations suggest that subversion of p53 function by mechanisms independent of mutation potentially play a role in the
progression of NB.
In this study, we utilized a panel of NB cell lines to examine the
relationship between p53 stability and HDM2 function, and we further
investigated whether the p53 protein present in N- and S-type cells is
equally susceptible to HDM2-mediated degradation. In addition, we
examined the dependence for, and regulation of, HDM2 in the
stabilization of p53 following treatment with the DNA-damaging agent
adriamycin (adr). We conclude that the p53 protein from both NB cell
types is inherently unstable, is sensitive to
HDM2-dependent degradation, and can be further stabilized
by the agents adr, ALLnL, and leptomycin B (LMB). We also provide evidence that N-terminal phosphorylation of p53 and its dissociation from HDM2 may be dispensable for p53 stabilization in NB in response to
DNA damage. Instead, we suggest that direct inactivation of HDM2
ubiquitin ligase function may contribute significantly to the
stabilization of p53.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
NB cell lines were cultured in Dulbecco's
modified Eagle's medium/F-12 (1:1) (Biofluids) supplemented with 10%
fetal bovine serum plus 1× nonessential amino acids and
penicillin/streptomycin and incubated in a humidified chamber at
37 °C with 5% CO2. The cell lines SHEP1, KCN69N, and
KCNBU have been described previously (23) and were kindly provided by
Dr. R. A. Ross and B. A. Spengler (Fordham University, Bronx,
NY). For these experiments, WT p53-containing SHSY5Y was obtained from
ATCC. LAN5 cells were originally obtained from Dr. R. C. Seeger
(Children's Hospital, Los Angeles, CA), and p53 null Saos-2
cells were obtained from the ATCC (Manassas, VA). MDM2-deficient MEF
cells were kindly provided by Dr. Allan Bradley (HHMI, Baylor College,
Houston, TX).
Cell Transfections and Treatments--
Cells were treated with
either 50 µM ALLnL (Sigma), 10 µM
lactacystin (Biomol), 0.2 µg/ml adriamycin (Sigma), or 10 ng/ml LMB
(generously provided by M. Yoshida, University of Tokyo, Japan) for the
indicated times. For GFP transfections, cells were seeded in 75-cm
flasks at 60% confluency 24 h prior to transfection with Fugene
(Roche), according to the manufacturer's specifications, and cells
were lysed as described 24 h following transfection. Where
indicated, 0.5 µg of GFP vector (CLONTECH) was
included to normalize for transfection efficiencies. Expression
constructs expressing either WT p53 (24) or the p5322,23
mutant (25) were subcloned into the GFP expression vector to create
GFPp53 fusion proteins with the GFP tag at the N terminus of p53.
p53 Half-life Determination--
To measure p53 stability, cells
were plated into six-well plates and treated with 40 µg/ml
cycloheximide for the indicated times. For half-life determination
following adriamycin treatment, cells were treated for 6 h with
0.2 µg/ml adriamycin, at which time cycloheximide was added for the
indicated times. Densitometric analysis of the p53 signal was
determined, and protein half-life was calculated using NIH Image
software. The quantitated values were graphed on a semilog scale to
derive the half-life for p53.
Metabolic Labeling--
Cells were washed with methionine-free
medium containing dialyzed fetal bovine serum and then incubated in
this serum for 1 h, whereupon 150 µCi/ml
[35S]methionine/cysteine (Tran[35S]label;
ICN) was added for 2 h. After the labeling period, CHX (40 µg/ml) was added, as indicated, and the cells were lysed as described
after the designated times.
Immunoprecipitation and Immunoblotting--
Cells were
solubilized in 1× lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). Lysates were sonicated, clarified by
centrifugation, and quantitated for protein, and 30-40 µg of protein
was subjected to immunoblot analysis. Blots were incubated with the
appropriate primary antibody and developed with a 1:3000 dilution of
horseradish peroxidase-conjugated secondary antibody (Amersham
Pharmacia Biotech) followed by ECL detection (Pierce). For endogenous
p53 detection, a 1:1000 dilution of anti-p53 monoclonal antibody DO-1
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used. For p53
immunoprecipitations, lysates were incubated overnight with a 1:1
mixture of agarose-conjugated 1801 and DO-1 antibodies (Santa Cruz
Biotechnology). For HDM2 immunoprecipitation and detection, 1 mg of
lysate was incubated with agarose-conjugated SMP14 (Santa Cruz
Biotechnology) overnight. The resultant blots were developed with
either a 1:200 dilution of 4B11 hybridoma supernatant, kindly provided
by Arnold Levine (The Rockefeller University, NY) or a 1:500 dilution
of Ab-1 (Oncogene Science). For p53 detection following p53 or HDM2
immunoprecipitation, reducing agent was omitted to prevent interference
of the IgG antibody heavy chain. GFP antibody
(CLONTECH) was utilized at a 1:1000 dilution.
Phosphorylation-specific p53 Antibodies and Phospho-p53
Detection--
Rabbit polyclonal antibodies specific for p53
phosphorylated at Ser6, Ser9,
Ser15, Thr18, Ser20,
Ser33, Ser37, Ser46,
Ser315, and Ser392 have been described
previously (26-28) Cells were either untreated (control) or
continuously treated for 8 h with 0.2 µg/ml adriamycin and lysed
in ice-cold buffer, and 1.2 mg lysate was immunoprecipitated with p53
antibodies as described (26). The resulting samples were split into
four aliquots, each subject to separate immunoblot analysis.
Cell Fractionation--
Cells were treated as described, washed
with cold PBS, overlaid with low salt lysis buffer (20 mM
HEPES, 5 mM NaCl, 5 mM MgCl2, 0.1%
Nonidet P-40, 1 mM dithiothreitol, protease inhibitors), and incubated on ice for 5 min. The cells were scraped into
microcentrifuge tubes and spun at 3000 rpm 4 min. The cytoplasmic
supernatant was removed, and the nuclear pellet was washed with low
salt lysis buffer. The nuclei were lysed in hypertonic lysis buffer
(1× lysis buffer containing 400 mM NaCl) and sonicated,
and both fractions were clarified by centrifugation. To verify the
integrity of the fractionation scheme, the blots were probed with a
1:500 dilution of topoisomerase II antibody (Oncogene) and a 1:2000
dilution of tubulin antibody (Sigma), representative of nuclear and
cytosolic proteins, respectively.
Immunofluorescence--
Cells were grown on chamber slides and
fixed with cold acetone as described (23). For p53 immunofluorescence,
antibodies were used at a concentration of 1 µg/ml and incubated with
Cy3-conjugated secondary antibody (Sigma) diluted 1:750. For
visualization of nuclear fluorescence, slides were treated for 30 min
with 100 µg/ml RNase, incubated for 2 min with 20 µM
TOPRO-3 (Molecular Probes, Inc., Eugene, OR), washed with
phosphate-buffered saline, and mounted. Confocal fluorescent images
were collected with a Bio-Rad MRC 1024 confocal scan head mounted on a
Nikon Optiphot microscope with a × 60 lens. Images were processed
using LaserSharp software (Bio-Rad). The total magnification is × 60 (objective) times 53 times the zoom factor (1 or 1.5, as
specified), and scale bars are shown.
In Vitro Ubiquitin Ligase Assay--
Cells were treated for
6 h with either ALLnL or adr, and 2.5 mg of protein was
immunoprecipitated with SMP14, as described. As a negative control,
MDM2 immunoprecipitations were performed from MDM2 null MEF cells. To
examine intrinsic HDM2 activity, reactions were carried out in the
presence of E1 enzyme (Calbiochem), bacterially expressed wheat
UbcH5b (E2), ubiquitination buffer, and either unlabeled or
32P-labeled ubiquitin, as described (29). The samples were
subjected to SDS-PAGE and visualized by either autoradiography or
immunoblot analysis. For detection of high molecular weight
ubiquitinated HDM2 species, Ab-1 (Calbiochem) was used at a
concentration of 1 µg/ml.
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RESULTS |
Proteasome Inhibitors Stabilize WT p53 in NB--
In NB, the p53
protein is reported to be stabilized (19) and overexpressed (20) in the
absence of mutation, suggesting a dysregulation in its degradation
pathway. Furthermore, our prior work suggested that p53 in neuroblastic
N-type cells might be more stable and highly expressed than p53 from
Schwannian S-type cells (23). We have since sequenced genomic p53 from
the stabilized N-type lines SHSY5Y and BE1N, and we unexpectedly
discovered point mutations. The details of this study will be presented
elsewhere. For the current report, we employed only WT p53-containing
cell lines, and we further confirmed that the p53 in LAN5 is WT by sequencing exons 2-11. To determine whether the p53 protein in N- and
S-type lines is equally capable of being efficiently degraded by the
proteasome, we treated two matched S/N pairs with the general proteasome inhibitor ALLnL. As shown in Fig.
1A, the p53 protein from both
cell types was maximally induced by a 4-h exposure to ALLnL. To confirm
that this stabilization was mediated by the proteasome, we treated the
same cell lines plus the neuroblastic LAN5 line with the highly
specific proteasome inhibitor lactacystin (30). As shown in Fig.
1B, the p53 protein from all of the cell lines was readily
stabilized by this treatment, thereby demonstrating that the p53
protein in NB is indeed a proteasomal substrate, in accordance with a
recent report (31).

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Fig. 1.
p53 turnover is regulated by the proteasome
in NB. Cells were treated with proteasome inhibitors for the
indicated times and subjected to Western analysis as described.
A, immunoblot analysis of p53 from the matched pairs (S-type
(S) and N-type (N)), SHEP1 (S), SHSY5Y
(N); KCNBU (S), and KCN69N (N),
following treatment with 50 µM ALLnL. Immunodetection of
p53 was performed with DO-1. B, immunoblot of p53 from the
above lines, in addition to LAN5 (N), following treatment
with 10 µM lactacystin.
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Multiple p53-stabilizing Agents Promote Nuclear Accumulation of
p53--
The p53 protein in NB has been reported to be primarily
cytosolic (32), presumably due to hyperactive nuclear export (21). The
prevailing notion is that nuclear translocation of p53 following cellular stress allows the protein to escape from MDM2-mediated degradation, which requires the cytosolic localization of both proteins
(7, 8). Therefore, it was of interest to determine whether p53
stabilization in NB occurs concomitantly with nuclear translocation.
Cells were treated with ALLnL or LMB, an antibiotic that inhibits CRM1
receptor-mediated nuclear export of a large number of proteins,
including p53 (33, 34). By examining p53 levels in purified nuclear and
cytoplasmic subcellular fractions, we observed that both ALLnL and LMB
significantly increased the nuclear accumulation of p53 in all cell
lines relative to untreated controls (Fig.
2A), in accordance with
previous reports (21, 35). We did not anticipate the nuclear
translocation of p53 following ALLnL, since this agent is not
thought to mediate a DNA damage response, although similar data have
very recently been reported in NB (36) and other cell lines (37). We
also examined the effect of the DNA-damaging agent adr on
nuclear p53 accumulation and obtained results similar to that with LMB
(Fig. 2B and data not shown). These results demonstrate that
the p53 stabilizing agents ALLnL, adr, and LMB all promote the nuclear translocation and accumulation of p53 in both cell types.

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Fig. 2.
p53 stabilizing agents induce the nuclear
accumulation of p53 in NB lines. A, cells were treated
with the indicated agents for 6 h (lane 1,
control; lane 2, 50 µM ALLnL;
lane 3, 10 nM LMB) and separated as
described into nuclear (N) and cytosolic (C)
fractions, and p53 immunodetection was performed as in Fig.
1A. To normalize for p53 levels, the blots were reprobed for
topoisomerase II (nuclear fraction) or tubulin (cytosolic fraction).
B, the p53 monoclonal antibody DO-1 detects nuclear
p53 in NB. Cells were treated with 0.2 µg/ml adr or 10 ng/ml LMB for
8 h, fixed, incubated with the appropriate primary antibody, and
processed as in Fig. 2B. Nuclei were visualized with TOPRO-3
as described. The smaller N-type cells are enlarged × 1.5 relative to the S-type cells. Specificity of the staining is
demonstrated in a (Saos-2 p53-null cells) and b
(SKBR3 breast carcinoma cells overexpressing mutant p53).
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To confirm these biochemical observations, the subcellular localization
of p53 was examined by fluorescence microscopy following treatment with
either adr or LMB (Fig. 2B). We and others have previously
demonstrated the apparently cytosolic localization of p53 in
neuroblastic lines when utilizing the monoclonal antibody PAb1801 (21,
23, 32). Although this antibody was able to detect p53 nuclear
translocation in S-type cells, it did not efficiently detect nuclear
p53 in N-type cells (data not shown), in contrast to our immunoblot
data (Fig. 2A). This result suggests that certain p53
epitopes may be inaccessible or masked in certain cell lines, as has
been reported for p53 in NB (38). In support of this notion, at least
two reports demonstrate nuclear p53 in NB when an antibody other than
PAb1801 is used (39, 40). Therefore, we repeated this experiment using
the monoclonal antibody DO-1, which recognizes an alternative
N-terminal epitope of p53. As shown in Fig. 2B, DO-1 clearly
detects the up-regulation and nuclear accumulation of WT p53 in both N
and S cell types. Specificity is demonstrated by the lack of staining
in the p53 null line Saos-2 (Fig. 2B, a) and the
strong signal detected in mutant p53-containing SKBR3 cells (Fig.
2B, b). These data demonstrate that various p53-stabilizing agents facilitate the nuclear accumulation of WT p53,
which can be readily detected with the DO-1 antibody. In addition, we
demonstrate that antibody choice can dramatically influence the
apparent subcellular localization of the protein in NB.
The Rapid Degradation of p53 in NB Is Inhibited by
Adriamycin--
At least one previous report suggests that the p53
protein in NB is inherently stabilized, with a half-life on the order
of several hours (19). However, our results in Fig. 1B
suggest that a 2-h treatment with lactacystin is sufficient to
significantly elevate p53 levels. In general, the ability of several
agents to stabilize p53 suggests that the protein is normally labile. To further examine this issue, cells were treated with the protein synthesis inhibitor CHX to determine the half-life of p53. This methodology was chosen over that of radioisotopic labeling because the
latter method has been reported to elicit a DNA damage response (41,
42), resulting in a stabilized p53 protein. As shown (Fig.
3A), the p53 protein in all NB
lines decreased rapidly after even a short incubation (10 min) with
CHX. The half-life of p53 in these lines fell within the range of
10-25 min, and by 1 h, the majority of the p53 protein was
degraded.

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Fig. 3.
Endogenous p53 is subject to rapid
degradation in NB. A, cells were treated with CHX for
the indicated times, and lysates were subjected to Western analysis as
described for Fig. 1A. Levels of p53 were quantitated and
plotted. B, the same cells in A were pretreated
6 h with adr, treated with CHX for the indicated times, and
subjected to Western analysis as in A. Film exposure times
in A were ~10 times longer than in B. C, LAN5 cells were pretreated 2 h with 150 µCi/ml
[35S]methionine, at which time CHX was added as in
B, and the lysates were subjected to Western analysis.
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We next sought to verify that the data obtained utilizing CHX treatment
was an accurate representation of the half-life of p53 in NB. To test
this, we treated cells with adr, which elicits a DNA damage response
(43) and elevates p53 levels in NB (Fig. 2B). We presumed
that the high expression of p53 following adr resulted from an increase
in half-life, which should be detected by CHX treatment. As shown in
Fig. 3B, while the p53 in untreated cells was rapidly
degraded, a 6-h pretreatment with adr extended the half-life of the
protein to over 3 h.
We have previously reported the half-life of p53 in SHEP1 to be almost
2 h (23), which is in marked contrast to the value of 19 min as
determined with CHX treatment. However, the former calculation was
determined following prolonged exposure to radioisotope. We therefore
considered whether the addition of radioisotopic label to cells was
sufficient to induce a DNA damage response, resulting in the
stabilization of p53. We chose to analyze LAN5 cells, since the p53
from this cell type was the most unstable of all of the examined NB
lines (Fig. 3A), yet its half-life as determined by
metabolic labeling has been reported to be well over 6 h (19).
Therefore, LAN5 cells were treated for 2 h with 150 µCi/ml
[35S]methionine, which corresponds to the incubation
period utilized for the half-life determination both for our prior work
(23) and for the previous report (19). Following this incubation period, cells were treated with CHX as in Fig. 3B.
Immunoblot analysis of p53 reveals that the presence of radioisotope
significantly prolonged the half-life of LAN5 to over 4 h. From
these data, we conclude that the p53 protein in NB is subject to rapid
turnover and that DNA-damaging agents or metabolic labeling stabilize
the protein by extending its half-life severalfold.
Adriamycin Inhibits the Ubiquitination of p53 in NB--
The
stabilization of p53 in response to adr indicates that mechanisms exist
to confer resistance to HDM2-mediated degradation in NB. To investigate
this issue, we examined whether the p53 in NB was capable of being
ubiquitinated and targeted for degradation by HDM2. Cells were treated
with the proteasome inhibitor ALLnL, and p53 immunoprecipitates were
immunoblotted with p53 (Fig.
4A). We show that while
ubiquitinated p53 ladders are barely detectable from p53
immunoprecipitates in control cells, high molecular weight p53 ladders
become prominent following ALLnL treatment. The ability of p53 in NB to
become ubiquitinated in the presence of ALLnL is in agreement with a
previous report (31) and further supports the notion that HDM2 is
responsible for p53 degradation. We confirmed that these bands
represented ubiquitinated species by probing the immunoprecipitated p53
with an anti-ubiquitin antibody (data not shown).

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Fig. 4.
Adriamycin treatment prevents p53
ubiquitination. A, cells were treated with ALLnL for
6 h, and 1 mg of lysate was immunoprecipitated with an equal
mixture of anti-p53 PAb1801 and DO-1. Blots were probed with DO-1 and
overexposed to visualize the high molecular weight p53-ubiquitin
conjugates. B, cells were treated for 6 h with the
following p53-stabilizing agents: control (lane
1), ALLnL (lane 2); adr
(lane 3), and LMB (lane 4).
Hdm2 was immunoprecipitated with SMP14, and p53 was immunodetected with
DO-1. C, cells were treated for 6 h with ALLnL
(lane 1), adr (lane 2), or
ALLnL + adr (lane 3), and p53 was immunodetected
as in A.
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The stabilization of p53 following genotoxic stress often correlates
with decreased ubiquitination of the protein, as has been observed
following UV radiation (44). Therefore, we first sought to investigate
whether the adr-mediated stabilization of p53 correlated with its
decreased ubiquitination. Cells were treated with ALLnL, adr, or LMB,
and the levels of ubiquitinated p53 protein associated with HDM2 were
analyzed. Since this approach detects ubiquitinated species associated
with HDM2, it serves to more directly assess the ability of HDM2 to
ubiquitinate p53 in response to various treatments. As depicted in Fig.
4B, the p53 protein co-precipitated with HDM2 in SHEP1 and
SHSY5Y is visibly ubiquitinated following treatment with ALLnL
(lane 2) relative to untreated controls
(lane 1), and this is comparable to the
levels of ubiquitinated p53 from p53 immunoprecipitates (Fig.
4A). The amount of ubiquitinated WT p53 observed following
LMB treatment is also increased relative to untreated cells (Fig.
4B, lane 4), although the levels are less than that observed with ALLnL. This suggests either that some of
the residual cytosolic p53 is susceptible to proteasome-mediated degradation in the presence of LMB, a contention supported by the
inability of LMB to completely inhibit p53 degradation in the presence
of HPV E6 (7), or that a portion of the p53 is ubiquitinated in the
nucleus, as several reports have suggested (45, 46). However, in marked
contrast to ALLnL and LMB, treatment of cells with adr almost
completely inhibited p53 ubiquitination (Fig. 4B,
lane 3). To verify this result and to ensure that
the adr-mediated decrease in ubiquitinated p53 was not due to p53-HDM2 complex dissociation, this experiment was repeated with p53
immunoprecipitates. As shown in Fig. 4C, while treatment
with ALLnL resulted in ubiquitinated p53 species (lane
1), treatment with either adr alone (lane
2) or adr in combination with ALLnL (lane
3) significantly diminished the levels of ubiquitinated
protein. These data therefore support the hypothesis that adr
stabilizes p53 by preventing its ubiquitination.
Adriamycin-mediated p53 Stabilization Is Not Due to HDM2
Down-regulation and Is Not Dependent upon Dissociation of p53-HDM2
Complexes--
We next explored the mechanism for the adr-mediated
stabilization of p53 in NB and investigated the potential role of HDM2 in this pathway. Inhibition of p53 ubiquitination following adr could
be explained by a down-regulation of HDM2 protein levels, which has
been demonstrated following UV treatment (16, 47), hypoxia (48), or
exposure to topoisomerase inhibitors (15). We therefore examined HDM2
expression levels in N- and S-type NB cells in response to the
stabilizing agents used in Fig. 4B. As shown in Fig.
5A (top
panel), levels of HDM2 are very low in untreated NB cells
but generally increase in response to ALLnL (lane
2), adr (lane 3), and LMB
(lane 4). The one exception occurs in KCNBU
following adr, which was unexpected, since we previously demonstrated
up-regulation p53 activity following IR in these cells (23). However,
in four of five cell lines tested, the stabilization of p53 paralleled
an increase in HDM2 levels.

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Fig. 5.
Adriamycin-mediated p53 stabilization does
not depend upon p53-HDM2 dissociation. Cells were treated as in
Fig. 4B, and 1 mg of lysate was immunoprecipitated with
SMP14. A, the blots were divided and probed with either
anti-HDM2 antibody 4B11 (upper panel) or anti-p53
antibody DO-1 (middle panel). Equal amounts of
these lysates were subjected to Western analysis (lower
panel) to quantitate total levels of p53 in response to
treatments (control (lane 1), ALLnL
(lane 2), adr (lane 3), or
LMB (lane 4)). B, SHSY5Y cells were
pretreated for 6 h with either ALLnL or adr and then incubated
with CHX for the indicated times. Hdm2 was immunoprecipitated from
equal amounts of lysate (500 µg), and HDM2 was immunodetected with
4B11. C, Hdm2 is required for p53 degradation in NB. SHSY5Y
cells were transfected with GFPp53 or GFPp5322,23 and
cotransfected with a GFP control vector to normalize for transfection
efficiency. Cells were either left untreated (lane
1) or treated with ALLnL (lane 2) or
adr (lane 3) for 6 h. Equal amounts of
protein were subjected to Western blot analysis with GFP antibody. The
lower panel shows expression of GFP.
D, cells were either left untreated or treated for 6 h
with ALLnL. Immunoprecipitations were performed from equal amounts of
lysate (250 µg) with either p53 (upper panel)
or HDM2 (lower panel), and p53 from resultant
blots was detected with DO-1 using identical exposure times.
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Dissociation of p53-HDM2 complexes following DNA damage has also been
recognized as a mechanism resulting in p53 stabilization (9-11, 49).
However, the data in Fig. 4B suggest that the adr-mediated p53 stabilization observed in SHEP1 and SHSY5Y occurs without significant p53-HDM2 complex dissociation. To further explore this
relationship, we determined the relative amount of p53 associated with
HDM2 for each of these treatments. As shown in Fig. 5A
(middle panel), essentially no p53 is detected
from HDM2 precipitates in untreated cells (lane
1), due to rapid HDM2-mediated p53 degradation. However,
there is a marked increase in the p53 associated with HDM2 following
ALLnL treatment (lane 2). In fact, ALLnL
treatment produced the maximum amount of p53 complexed with HDM2 in all lines tested. LMB treatment also increased the amount of p53 bound to
HDM2 (lane 4) relative to control samples. The
situation is more complex for the adr-treated samples (lane
3). While significant levels of p53 from SHEP1, SHSY5Y, and
KCNBU remained associated with HDM2, there was significant dissociation
of p53 from HDM2 in both KCN69N and LAN5 following adr. For comparison,
the amount of p53 immunoprecipitated from whole lysates is shown in the
lower panel. These results suggest that
adr-induced p53 stabilization may be accompanied by, but does not
require, HDM2 dissociation.
The amount of p53 co-precipitated with HDM2 following treatment with
adr (Fig. 5A, middle panel,
lane 3) was less than the amount co-precipitated
following treatment with ALLnL (lane 2); however,
these treatments stabilized and elevated p53 levels to approximately
the same extent (Fig. 5A, lower panel,
lanes 2 and 3). Another possibility to
explain this difference, independent of p53-HDM2 dissociation, would be
differential stabilization of HDM2. To explore this possibility, SHSY5Y
cells were treated with either ALLnL or adr for 6 h, followed by
treatment with CHX (Fig. 5B). As shown, while adr treatment
(lower panel) elevated HDM2 to levels comparable
to those following treatment with ALLnL (upper
panel), the stability of HDM2 in the adr-treated cells was
considerably shorter than from the ALLnL-treated cells. These results
suggest that less p53 is associated with HDM2 from adr-treated cells
relative to ALLnL-treated cells due to the increased lability of HDM2
in the former.
The data thus far support the notion that p53 turnover in NB is quite
rapid and that this lability depends upon HDM2. However, a recent
report suggests that the p53 in NB is resistant to HDM2-mediated degradation (31), despite the formation of p53-ubiquitination conjugates following proteasome inhibition. This interpretation was
based on data derived from cotransfection of WT p53 and either mutant
or WT HDM2. We therefore sought to establish that normal HDM2 function
is a requirement for p53 degradation in NB. To circumvent potential
difficulties arising from suboptimal ratios of DNA introduced during
cotransfection, SHSHSY cells were transfected with either GFPwtp53 or
GFPmtp5322,23 expression constructs, the latter of which is
unable to associate with HDM2, resulting in a stabilized protein (49).
As shown in Fig. 5C, the transfected WT protein is barely
detectable in control cells (upper panel,
lane 1), while the mutant protein is expressed at
much higher levels. In addition, while the transfected WT protein is
stabilized following adr or ALLnL treatment, the mutant protein is not
further stabilized by these treatments, resulting in high constitutive
expression. Taken together, these data demonstrate the functionality of
endogenous HDM2 in these cells, that HDM2 activity is required for the
rapid turnover of p53 in nonstressed NB cells, and that inhibition of this activity leads to protein stabilization.
Our results demonstrating HDM2-dependent activity in the
degradation of p53 are in disagreement with results demonstrating that
introduction of excess HDM2 does not reduce the levels of cotransfected
p53 (31). While this discrepancy may appear difficult to reconcile, one
possibility involves the relative amount of p53 associating with HDM2
at any given time. For instance, if only a small percentage of p53 is
complexed with endogenous HDM2 at steady state, then introduction of
excess exogenous HDM2 should have no further effect upon p53 levels,
since HDM2 levels would not be the rate-limiting step for p53
degradation. Indeed, when we compared the percentage of p53 associated
with HDM2 following ALLnL treatment relative to the total amount of p53
in the cell, we found that ALLnL significantly increased the overall
levels of total immunoprecipitated p53 from cells (Fig. 5D).
However, HDM2 immunoprecipitation of equivalent amounts of protein
revealed that only a small percentage (less than 25%) of total
cellular p53 is actually associated with HDM2 following this treatment.
N-terminal p53 Phosphorylation following Adriamycin Treatment Does
Not Correlate with Dissociation from HDM2--
The results thus far
suggest that HDM2 is functional and responsible for the degradation of
endogenous p53 in NB. However, arguments have been made suggesting that
the p53 protein in NB is subject to posttranslational modification,
resulting in a more acidic protein (31). Such modification of p53 has
been suggested to be responsible for p53's inability to be further
degraded by exogenous HDM2. Since the phosphorylation of p53 can both
increase protein acidity and modulate protein stability, we chose to
further examine this parameter in NB.
Several reports document a correlation between DNA damage-mediated p53
stabilization and N-terminal p53 phosphorylation. This notion has
gained acceptance, because it has been shown that HDM2 binds to the N
terminus of p53 (50) and phosphorylation of the Ser20
residue of p53 has been demonstrated to interfere with HDM2 association (10, 11). Other reports also implicate Ser15 (9) and
Thr18 (51, 52) in this process, further illustrating the
complexity involved. In light of our results demonstrating variable
p53-HDM2 dissociation following adr (Fig. 5A), we suspected
that some of this variability might be due to specific p53
phosphorylation events. To test this hypothesis, we performed a
comprehensive analysis of site-specific phosphorylation in untreated
and adr-treated cells (Fig.
6A). p53 from the three cell
lines demonstrating significant p53-HDM2 complex formation following
adr (SHEP1, SHSY5Y, and KCNBU; see Fig. 5A) exhibited
different phosphorylation in each cell line. While the p53 from SHEP1
and KCNBU was extremely responsive to adr and showed a large degree of
phosphorylation at N-terminal sites Ser15,
Ser18, and Ser20, p53 from the N-type SHSY5Y
was weakly phosphorylated at Ser15 and Ser20,
and not phosphorylated at Thr18. An analysis of the
phosphorylation pattern in the two cell lines exhibiting dissociation
of p53-HDM2 complexes (KCN69N and LAN5) revealed that p53 from both
lines exhibited some degree of phosphorylation at Ser15,
while no phosphorylation of Ser18 was evident, and
phosphorylation of Ser20 occurred only in KCN69N. These
results suggest that dissociation of p53 from HDM2 in response to adr
does not correlate with or depend upon phosphorylation of p53 at
specific N-terminal sites.

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Fig. 6.
Wild type p53 in NB is multiply
phosphorylated in response to adriamycin. A, cells were
treated for 8 h with 0.2 µg/ml adr, p53 was immunoprecipitated
with a mixture of PAb1801 and DO-1, and immunoblot analysis was
performed with phosphospecific p53 antibodies as described.
B, cells were either left untreated (lane
1) or treated with ALLnL for 8 h (lane
2) or with adr for 8 h (lane 3),
and p53 was immunoprecipitated as in A.
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One of the more prominent differences in the phosphorylation pattern in
N- and S-type cells is the tendency for p53 from the S-type cells to be
highly phosphorylated at the newly identified residue Ser46
(27). Phosphorylation at this site is proposed to increase p53
half-life and play a role in UV-mediated apoptosis. Taken together, our
data demonstrate that p53 from the two S-type cell lines (SHEP1 and
KCNBU) exhibits more extensive N-terminal phosphorylation relative to
the protein in the N-type lines. While the significance of these
phosphorylation differences is unclear, these modifications do not
appear to impart enhanced stability to p53 in NB.
Because the samples in Fig. 6A were not normalized for equal
p53 loading, a second analysis was performed to ensure that the lack of
phosphorylation observed in control cells was not due to low levels of
p53. For the experiment in Fig. 6B, SHEP1 and SHSY5Y were
either left untreated or treated with ALLnL or adr, and p53 was
detected as in Fig. 6A. It is clear from the signal obtained
with DO-1 that the levels of cellular p53 induced by either ALLnL or
adr are roughly comparable. However, despite the elevation of p53
levels produced by ALLnL treatment, essentially no phosphorylation of
p53 at any sites is observed. This confirms the notion that the absence
of signal at multiple sites in control cells reflects the
hypophosphorylated status of the endogenous protein and further
demonstrates that a DNA damage signal is necessary for the induction of
p53 phosphorylation in NB.
p53 Stabilization following Adriamycin Correlates with HDM2
Inactivation--
The data thus far suggest that there are no unusual
phosphorylation events that might render the p53 protein inherently
resistant to HDM2-mediated degradation in NB. We therefore turned our
attention to the activity of HDM2 in these cells. While our data
strongly suggest that HDM2 is responsible for the degradation of p53 in NB, it remains unclear how the p53 protein escapes HDM2-mediated ubiquitination following adr. We speculated that adr itself may inhibit
the function of HDM2, resulting in decreased ubiquitinated p53 species
(Fig. 4, B and C). There is precedent for this,
given a recent report demonstrating decreased HDM2 ligase activity
correlating with increased p53 stability following UV treatment (16).
In addition, another recent report has documented the rapid
phosphorylation of MDM2 following IR (53), which may modulate its
function prior to the onset of p53 stabilization.
Therefore, we directly examined HDM2 ubiquitin ligase activity in
ALLnL- or adr-treated cells. First, endogenous HDM2 was immunopurified
from control or treated SHSY5Y cells. To ensure that equal amounts of
HDM2 were immunoprecipitated, the levels of HDM2 were quantitated by
immunoblot analysis. As shown in Fig. 7A (bottom
panel), comparable amounts of HDM2 were immunoprecipitated from treated cells. A longer exposure of this same blot
(upper panel) reveals high molecular weight HDM2
species in ALLnL-treated cells that are virtually absent in the
adr-treated sample. These high molecular weight ladders are suggestive
of HDM2 ubiquitin conjugates and are consistent with HDM2's ability to
ubiquitinate itself (29). To further demonstrate that these bands
represent autoubiquitinated HDM2, identical aliquots of immunopurified
HDM2 from SHSY5Y served as the E3 enzyme in an in vitro
ubiquitination assay to which purified E1, E2 (UbcH5b), and unlabeled
ubiquitin protein were added. As shown in Fig. 7B, the
addition of ubiquitin significantly increased the high molecular weight
HDM2 species, indicative of autoubiquitination, when HDM2 was
immunopurified from ALLnL-treated SHSY5Y cells, while there was no
signal from MDM2-deficient MEF cells. Strikingly, the addition of
ubiquitin to HDM2 immunopurified from adr-treated cells was not able to promote the appearance of high molecular weight HDM2 ladders, providing
compelling evidence that adr treatment inhibits the ubiquitin ligase
activity of HDM2. This experiment was repeated in LAN5 cells, since,
unlike in SHSY5Y, adr facilitated p53-HDM2 complex dissociation in this
line. Similar to SHSY5Y, the presence of adr markedly inhibited HDM2
autoubiquitination in LAN5.

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Fig. 7.
Adriamycin impairs the endogenous HDM2
ubiquitin ligase activity in NB. A, to examine the
in vivo autoubiquitination of HDM2, cells were treated with
either ALLnL or adr for 6 h, endogenous HDM2 was
immunoprecipitated from 2.5 mg of cellular lysate, and the blot was
probed with HDM2 Ab-1. The lower panel represents
a shorter exposure of the same blot, demonstrating equivalent HDM2
immunoprecipitation. B, SHSY5Y or LAN5 cells were treated
with either ALLnL or adr, and HDM2 was immunodetected as in
A. These immunoprecipitates were utilized in an in
vitro ubiquitin ligase assay, as described, in the presence or
absence of unlabeled ubiquitin and purified E1 and E2 proteins. Hdm2
MEFs were utilized as a negative control. C, the experiment
in B was repeated using [32P]ubiquitin, and
ubiquitinated species were visualized by autoradiography.
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To confirm that these high molecular weight HDM2-immunoreactive bands
were reflective of intrinsic HDM2 ubiquitin ligase activity, we
repeated the in vitro ubiquitination assay using
32P-labeled ubiquitin in place of unlabeled ubiquitin, and
the incorporation of radioactive ubiquitin into immunopurified HDM2 was
visualized by autoradiography. As demonstrated in Fig. 7C,
while no intrinsic ligase activity was observed in control MDM2-null
MEF cells, a high level of activity was observed in ALLnL-treated
SHSY5Y cells. This experiment was repeated with HDM2 from LAN5, with
similar results. Importantly, adr treatment inhibited incorporation of radioactive ubiquitin into immunopurified HDM2 by more than 70% in
each instance, further supporting the hypothesis that adr directly inhibits the ubiquitin ligase activity of HDM2. The correlation between
inhibition of HDM2 activity and elevated p53 levels support the notion
that adr stabilizes the p53 protein in NB by inhibiting HDM2 ubiquitin
ligase activity.
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DISCUSSION |
The main purposes of this study were to elucidate the involvement
of HDM2 in p53 turnover in NB and to characterize the mechanism(s) promoting p53 stabilization following DNA damage. While exploring these
fundamental questions, we were also interested in determining whether
these stabilization mechanisms were similar in both N- and S-type cell
lines. Our data demonstrate that the p53 protein from both N- and
S-type NB cells is inherently unstable due to efficient
proteasome-mediated degradation that is dependent upon normal HDM2
function. First, we detect essentially no WT p53 complexed with HDM2 in
untreated cells, supporting our contention that p53 is rapidly degraded
by HDM2 in these cells. Second, we find maximal levels of ubiquitinated
cellular p53 as well as ubiquitinated p53 complexed with HDM2 in the
presence of ALLnL, thereby demonstrating that these unstable
ubiquitinated species are normally targeted for rapid degradation and
can be detected only upon inhibition of the proteasome. Third,
transfected mutant p53 unable to bind HDM2 is intrinsically stabilized,
while transfected WT protein is further stabilized by either ALLnL or
adr, thereby demonstrating a requirement for HDM2 function in p53
turnover in NB. Finally, treatment of cells with adr concomitantly
inhibits both p53 ubiquitination and HDM2 ligase activity, thereby
further demonstrating a requirement for HDM2 activity in the turnover
of p53 in NB.
We recognize that our present results are in contrast to some of our
prior work (23) as well as to two other reports (19, 31), and we offer
several explanations to account for these differences. One resolved
issue concerns the p53 genetic status of the SHSY5Y cells used in our
prior work (23). As previously mentioned, the p53 gene from cells used
in those experiments contained a previously unrecognized point mutation
that rendered the protein stable. Another issue involves the discrepant
values for p53 half-life in NB. In the work reported herein, the
half-life of p53 in all cell lines examined was less than 25 min;
however, we previously reported the half-life of SHEP1 to be on the
order of 120 min (23). In the present study, p53 half-life was
determined using CHX to inhibit protein synthesis, whereas in the
former work (19, 23), p53 half-life was determined by radioisotopic
labeling. We demonstrate here that the use of radioisotope in NB leads
to a DNA damage response, as has been well documented for other cell types (41, 42). Therefore, previous studies of p53 half-life in NB have
been influenced by the effects of radioisotope. However, our results do
not preclude the possibility that the endogenous levels of p53 are
higher compared with other cell lines due to stabilized and/or elevated
levels of mRNA, as suggested by the fact that NB cells contain
abundant p53 mRNA (19), or to better translation efficiencies.
Our results also support the notion that the rapid degradation of p53
in NB is absolutely dependent upon normal HDM2 function. These results
differ from those of another report (31). Zaika et al. (31)
propose that p53 in NB is resistant to HDM2-mediated degradation based
on the result of the failure of exogenously added MDM2 to further
degrade p53. However, in that work, only the steady state levels of p53
were examined, whereas calculation of half-life would provide a more
quantitative measure of the effect of excess HDM2 upon p53 stability.
In this work, we determined that the p53 in NB is not phosphorylated at
any of the known sites, thereby eliminating this as a possible
mechanism for any potential resistance of p53 to HDM2. Second, in
agreement with Zaika et al. (31), we detect efficient
HDM2-mediated ubiquitination of p53, suggesting that HDM2 can target
p53 for degradation. While it is possible that a defect in the
degradation of p53 could exist in the step following HDM2-mediated
ubiquitination, prior to recognition by the proteasome, this
possibility seems unlikely based on the rapid degradation of p53 in the
NB lines and on the rapid response of p53 to proteasome inhibition. We
instead propose that HDM2 is continuously degrading the p53 protein in
NB; however, at any given time, only a small percentage of the total
cellular pool of p53 protein is associated with HDM2. Therefore, if the
concentration of endogenous HDM2 is not rate-limiting, introduction of
excess exogenous HDM2 would not be expected to further diminish p53 levels.
In addition to examining the stability of p53 in nonstressed NB cells,
we explored the mechanisms involved in the DNA damage-mediated stabilization of p53 in response to adr. Our previous data (23) agree
with results from several groups (32, 40, 54) in demonstrating elevated
p53 levels following IR, suggesting that the p53 in NB is competent to
respond to DNA damage and that the protein can be further stabilized.
We now demonstrate that adr treatment effectively elevates p53 levels
in all NB cell lines tested and that this correlates with a severalfold
increase in p53 half-life. Interestingly, adr-mediated stabilization of
p53 is associated with inhibition of p53 ubiquitination in all lines.
Two separate mechanisms that may account for this result are 1)
inhibition of HDM2 association via p53 phosphorylation, or 2) direct
disruption of HDM2 function by posttranslational modification or
interaction with cellular proteins. Our data do not support the first
possibility, since the WT p53 protein from five NB lines is uniformly
stabilized following adr treatment, although significant dissociation
from HDM2 is only observed in two of these five lines. These data
strongly suggest that dissociation of HDM2-p53 complexes is not the
only mechanism that mediates p53 stabilization following DNA damage. Even more surprising was the observation that in the NB lines undergoing adr-mediated p53-HDM2 complex dissociation, p53 is not
hyperphosphorylated at any N-terminal sites proposed to play a role in
this process, further limiting the importance of N-terminal p53
phosphorylation as a regulator of HDM2 association. At
least two other reports have demonstrated p53 stabilization in response to DNA damage without a necessity for phosphorylation at multiple N-terminal sites (13, 15), further emphasizing that these sites are not
necessary for regulation of p53-HDM2 interactions in all cell lines.
Our data demonstrating decreased p53 ubiquitination following adr
support the hypothesis that direct inhibition of HDM2 activity serves
as an additional mechanism to mediate p53 stabilization and further
emphasize the necessity for HDM2 activity in the turnover of p53
in NB. Hdm2 function can be inhibited by interaction with a growing
list of cellular proteins such as p14arf (55-57), c-Abl (58),
Rb (59), MdmX (60), and the estrogen receptor (61). Alternatively, HDM2
activity can be inhibited by its nucleolar localization (14, 17) or by
down-regulation of HDM2 protein levels in response to cellular stimuli
such as UV or IR (16, 47), hypoxia (48), and topoisomerase inhibitors (15). Our data utilizing endogenous HDM2 in an in vitro
ubiquitin ligase assay clearly show that HDM2 isolated from
ALLnL-treated NB cells is enzymatically active, while HDM2 isolated
from adr-treated cells exhibits significantly less activity.
Interestingly, adr inhibited HDM2 ligase function in LAN5 cells, in
which dissociation of p53-HDM2 complexes was evident, as well as in
SHSY5Y cells, which retain p53-HDM2 complexes. This suggests that
inhibition of HDM2 may be as important or more important a determinant
of p53 stabilization than its dissociation from p53. This finding thus
uncovers a previously unrecognized mechanism by which DNA damage can
regulate p53 stabilization.
To our knowledge, only one previous report has implicated direct
effects of DNA damage upon HDM2 ubiquitin ligase activity (16). This
study demonstrates a correlation between UV irradiation and decreased
SUMO-1 conjugation to HDM2, resulting in a shift from p90 to p75 HDM2
species (16). Decreased sumoylation of HDM2 is also presumed to enhance
the degradation of HDM2 by rendering the protein more susceptible to
ubiquitination. However, our results suggest that a mechanism other
than sumoylation is involved in regulating the adr-mediated inhibition
of HDM2. We show that p53 stabilization following adr is accompanied by
neither p90 HDM2 down-regulation nor increased HDM2 self-ubiquitination
and degradation. In fact, our results demonstrate that elevated levels
of p90 (presumably sumoylated) HDM2 may coexist with stabilized p53,
implicating a direct inhibition of HDM2 activity by an alternative
pathway. It is not yet clear whether the adr-induced inhibition of HDM2 ubiquitin ligase activity is due to a postranslational modification of
HDM2 or to stimulation of its interaction with another cellular protein. Regardless of the precise mechanism, inhibition of HDM2 ligase
activity following adr offers a unifying explanation for our
observations that p53 can be stabilized without a strict dependence upon its phosphorylation state or its association with HDM2.