From the Department of Biochemistry, University of California, Riverside, California 92521
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ABSTRACT |
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Overexpression of mutant p53 has been reported to
promote tumorigenicity in several cancers. However, despite its
potential importance, the signals regulating mutant p53 protein
expression are not known. Here we show that a form of p53 that is
incapable of binding DNA is overexpressed in the acute promyelocytic
leukemia NB4 cell line. Our results demonstrate that treatment of NB4
cells with bryostatin-1, which induces differentiation in this cell line, leads to hyperphosphorylation of this DNA binding-impaired form
of p53 via mitogen-activated protein kinase. After this
phosphorylation, the p53 protein is degraded by the
ubiquitin/proteasome pathway. Furthermore, we show that inhibition of
p53 hyperphosphorylation blocks p53 protein degradation and cell
differentiation. In addition, inhibition of the ubiquitin/proteasome
pathway also blocks p53 protein degradation and cell differentiation.
These findings suggest a role for mitogen-activated protein kinase in
the degradation of the DNA binding-impaired form of p53 protein and in
the bryostatin-induced differentiation observed in this cell line. The
implications of these results with respect to the functional
significance of p53 phosphorylation and degradation in cell
differentiation are discussed.
Studies of human and mouse p53 have shown that wild-type p53
exerts its antiproliferation function by inducing growth arrest and
apoptosis, whereas mutant p53 loses this function (1, 2). The
biochemical activity of p53 that is required for this relies on its
ability to bind to specific DNA sequences and to function as a
transcription factor (3). The importance of the activation of
transcription by p53 is underscored by the fact that the majority of
p53 mutations found in tumors are located within the domain required
for sequence-specific DNA binding (1, 2). Therefore, it is clear that
this activity is critical to the role of p53 in preventing
proliferation. Although the precise molecular mechanisms by which
mutant p53 loses its antiproliferation functions remain to be
elucidated, three models have been proposed (1). First, mutations in
p53 may result in a loss of tumor suppressor function. Second, mutant
p53 may have a dominant negative effect over wild-type p53 activity.
Finally, mutations in p53 may lead to "a gain of function" such
that it can induce proliferation and promote tumorigenicity of various
cells (4-6).
An important mechanism used to control p53 activity is the regulation
of p53 protein levels. Regulation is primarily achieved via protein
degradation, although p53 levels may also be controlled at the level of
transcription (7) and translation (8). Studies with human papilloma
virus E6 protein and cellular oncogene Mdm2, which interact with and
lead to the degradation of p53, have revealed that p53 is degraded by
the ubiquitin-mediated proteolytic pathway (9-11). Although signals
that target p53 for degradation are not yet fully understood, it is
generally accepted that the phosphorylation status of p53 may be
involved. As an illustration of this, cells treated with the serine
phosphatase inhibitor okadaic acid accumulate high levels of
hyperphosphorylated wild-type p53 (12). Clearly, the regulation of p53
protein level is important to its tumor suppressor function. Therefore,
it follows that the regulation of mutant p53 protein levels may be
important in regulating the oncogenic potential of mutant p53.
In this study we examined the involvement of the p53 pathway in NB4
cell differentiation. The NB4 cell line was originally isolated from a
patient with acute promyelocytic leukemia and is characterized by a
translocation involving chromosomes 15 and 17 (13). It has been used as
a model for studying the mechanisms of cell differentiation, as it can
be terminally differentiated into either mature neutrophilic
granulocytes (14) or monocyte/macrophage-like cells (15) in response to
various treatments. Among the treatments that have been shown to induce
NB4 cells to differentiate into monocytes/macrophage-like cells are
1 To address the question of whether the p53 pathway is involved in
bryostatin-induced differentiation, we asked whether endogenous p53 is
phosphorylated in response to bryostatin treatment. Our results show
that p53 becomes hyperphosphorylated and that the p53 protein is
degraded via the ubiquitin/proteasome pathway after treatment with
bryostatin-1. Furthermore, we demonstrate that inhibition of p53
hyperphosphorylation by a MAPK pathway inhibitor, PD98059, blocks p53
protein degradation and cell differentiation. In addition, inhibition
of p53 protein degradation also blocks cell differentiation. The
correlation between these effects suggests a role for MAPK in p53
degradation and in the bryostatin-induced differentiation in this cell
line. To assess the physiological significance of these
observations, we examined the DNA binding ability of p53 purified from
NB4 cells using anti-p53 antibody, Pab 421. The purified p53
was incapable of binding DNA in electrophoresis mobility shift analysis
(EMSA), indicating that a mutant form of p53 exists in NB4 cells. These
results suggest that following bryostatin treatment of NB4 cells, the
mutant form of p53 is phosphorylated via the MAPK pathway and
subsequently degraded. The implication of these observations with
respect to the functional significance of p53 phosphorylation and
degradation in cell differentiation are discussed.
Cell Culture and Protein Purification--
NB4 cells were
cultured in Dulbecco's modified Eagle's medium/F-12 media
supplemented with 10% fetal bovine serum. The cells were routinely
grown as suspension cultures, and passages 8 to 20 were used for each
assay. Bryostatin-1 (Alexis, CA) was dissolved in ethanol. PD98095
(Calbiochem) and MG132 (Calbiochem) were dissolved in
Me2SO.
p53 was immunopurified from nuclear extracts prepared from NB4 cells
according to the method of Dignam et al. (20). One milliliter of nuclear extract (7 mg of protein/ml) was incubated for
3 h at 4 °C with gentle rotation with 100 µl of packed
protein A-Sepharose beads covalently coupled with anti-p53 antibody Pab 421. Beads were washed twice with 0.5 M KCl D buffer (20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and once with 0.1 M KCl D buffer. p53 was eluted
from the washed beads with 100 µl of 421 epitope oligopeptide
(KKGQSTSRHKK) at 1 mg/ml concentration in 0.1 M KCl D
buffer. Recombinant p53 was prepared from HeLa cells infected with
recombinant vaccinia virus expressing p53 (21) as described above.
Proteins were analyzed by SDS-PAGE followed by Western analysis or
silver-staining to visualize bands.
Detection of p53 Phosphorylation and Protein Level in NB4
Cells--
Two ml of NB4 cells at 106 cell/ml were labeled
with [32P]orthophosphate (ICN) in phosphate-deficient
Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and then
incubated for 1 h followed immediately by bryostatin-1 treatment.
At the end of the treatment, the cells were washed with PBS containing
100 µM vanadate and lysed by adding 0.5 ml of radioimmune
precipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM Na3VO4,
2 mM EGTA, 25 mM NaF, 1 mM
phenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, 1% Nonidet
P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 2 µg/ml pepstatin). The lysate was homogenized by a Dounce homogenizer and
clarified by centrifugation at 14,000 × g for 15 min
at 4 °C. The supernatant were then incubated overnight at 4 °C
with 20 µl of packed protein A-Sepharose beads to which Pab 421, an antibody specific for p53, was covalently linked. The immunoprecipitate was subjected to SDS-PAGE, and the labeled proteins were visualized with a PhosphorImager using Adobe Photoshop software. To determine p53
protein levels, NB4 cells were lysed with radioimmune precipitation buffer as described above, and the protein concentration for each sample was measured. Equivalent amounts of cell lysate were analyzed by
SDS-PAGE followed by Western blot analysis using Pab 421.
Detection of Activated MAPK--
Phosphorylated MAPK was
detected by immunoprecipitation with anti-phosphotyrosine monoclonal
antibody followed by immunoblotting using anti-MAP kinase antibody as
described (18). Briefly, 2 × 106 NB4 cells were lysed
with 0.5 ml of radioimmune precipitation buffer, and protein
concentration was determined with a protein assay kit (Bio-Rad).
Supernatant containing an equivalent amount of protein from each sample
was incubated with 20 µl of packed agarose beads coupled to a
monoclonal anti-phosphotyrosine antibody (PT-66, Sigma) at 4 °C
overnight. The immunoprecipitate was then analyzed by SDS-PAGE followed
by Western analysis with a rabbit anti-p42 MAPK polyclonal antibody
(Pab C14, Santa Cruz). Equal loading of MAP kinase protein was
determined by Western blot analysis using anti-p42 MAPK antibody.
EMSA--
An oligonucleotide probe containing the ribosomal gene
cluster (RGC) p53-binding site was used containing the sequence
5'-AGCTTGCCTCGAGCTTGCCTGGACTTGCCTGGTCGACGC-3'. Binding reactions
contained 60 mM KCl, 12% glycerol, 5 mM
MgCl2, 1 mM EDTA, 10 µg bovine serum albumin,
0.2 µg of poly(d(G·C)), 600 cpm of 32P-labeled probe
and proteins as indicated, and water in a total volume of 12.5 µl.
Reactions were incubated for 30 min at 30 °C and then analyzed on a
3% polyacrylamide gel containing 0.5 × TBE (0.045 mM
Tris borate, 0.045 mM sodium borate, 0.001 mM
EDTA, pH 8.0). The gel was dried, and DNA-protein complexes were
visualized with a PhosphorImager using Adobe Photoshop software.
Northern Blot Analysis--
Total RNA was isolated using TRIzol
reagent (Life Technologies, Inc.) according to the manufacturer's
instructions. Thirty µg of total RNA was subjected to electrophoresis
in a 1.5% agarose gel and transferred to a MAGNA nylon transfer
membrane (Micron Separations Inc.). A 1.3-kilobase pair DNA fragment
corresponding to the full-length human p53 gene was cut from plasmid
pcDNA-p53 (22) and labeled using T7 QuickPrime Kit (Amersham
Pharmacia Biotech) as a probe. Northern analysis was conducted using a
standard procedure (23). The RNA bands were visualized with a
PhosphorImager using Adobe Photoshop software. The membrane was then
stripped and hybridized with a glyceraldehyde 3-phosphate dehydrogenase cDNA probe to normalize for RNA loading.
Studies of Cell Differentiation--
To examine the effect of
bryostatin-1 on cell adherence, 2 × 104 cells were
seeded in a 24-well plate. Cells were pretreated with Me2SO
control or 5 µM PD98059 or 1 µM MG132 for
30 min and then treated with vehicle or 5.6 nM bryostatin-1
for 72 h. At the end of the treatment, cells in suspension
versus those adhered to the culture plate were counted. A
total of 1000 cells were counted for each treatment, and adherence was
expressed as a percentage of adherent cells to the total number of
cells. Phagocytosis was measured by incubation of NB4 cells with latex
beads (3 µm, Sigma) for 5 h after 72 h treatment of cells
with the reagents as described above. Cells were gently washed 4 times
and centrifuged at 125 × g for 5 min to remove the
free beads. A total of 500 cells were counted for each treatment, and
phagocytosis was expressed as the percentage of bead-engulfing cells to
total cells. Both bead-engulfing cells and total cells were counted
under a microscope and photographed.
p53 Hyperphosphorylation and Reduction in NB4 Cell
Differentiation--
To determine whether the p53 pathway is involved
in NB4 cell differentiation, we first investigated whether the
phosphorylation status of p53 was altered in response to bryostatin
treatment. NB4 cells have been characterized with respect to
differentiation induced by bryostatin-1 (Ref. 17; also see Fig. 5 and
Table I). Initial experiments suggested
that treatment with 5.6 and 56 nM bryostatin-1 induced
differentiation. Therefore, NB4 cells were metabolically labeled with
32Pi and immediately treated with 0, 0.56, 5.6, or 56 nM bryostatin-1. From each treatment, lysate
containing equivalent amounts of protein was immunoprecipitated with
anti-p53 antibody. After electrophoresis, the amount of phosphorylation
of p53 was analyzed by autoradiography (Fig.
1A). In the absence of
bryostatin-1, p53 protein showed basal levels of phosphorylation, which
is as expected for a phosphoprotein. The addition of bryostatin-1,
however, resulted in an increase in phosphorylation above this basal
level. The maximum level of phosphorylation was observed after
treatment with 5.6 and 56 nM bryostatin-1 (Fig.
1A). It was also demonstrated that the maximum level of
phosphorylation was obtained 30 min after bryostatin treatment (Fig.
1B). A control Western analysis was performed to ensure the
equivalent amounts of p53 protein present in each sample (data not
shown). Consequently, these results indicate that p53 becomes
hyperphosphorylated after bryostatin treatment in a dose- and
time-dependent manner.
Next we asked whether bryostatin-1 would affect p53 protein levels, as
it has been previously suggested that the phosphorylation status of p53
may be involved in its targeting for degradation. The NB4 cells were
treated with increasing amounts of bryostatin-1 or control vehicle, and
the resulting p53 protein level was analyzed by Western analysis (Fig.
1C). It was anticipated that the hyperphosphorylation of p53
would result in an increase in p53 protein levels as phosphatase inhibitor treatment results in an accumulation of high levels of
wild-type p53 (12). Surprisingly, however, treatment of cells with 5.6 and 56 nM brystatin-1 significantly reduced p53 protein levels. Treatment with 0.056 and 0.56 nM bryostatin-1 had
little effect. Coomassie Blue staining of major representative bands indicated that equivalent amounts of protein were present (Fig. 1C, lower panel). These results suggest that
treatment with bryostatin-1 decreases p53 protein levels in NB4 cells.
Furthermore, the results indicate that the concentrations of
bryostatin-1 sufficient to increase p53 phosphorylation (5.6 and 56 nM) are the same as those required to decrease p53 protein
levels. Similarly, those bryostatin concentrations that had little
effect on p53 phosphorylation were also not effective in reducing the
protein levels. We interpret this correlation to indicate that
hyperphosphorylation of p53 might be involved in the reduction of p53
protein levels. It is particularly interesting to note that treatment
of NB4 cells with bryostatin-1 at concentrations of 5.6 and 56 nM but not at 0.56 nM has been shown to induce
differentiation (Fig. 5 and Table I).2
MAPK Pathway Is Involved in p53 Hyperphosphorylation and Reduction
in p53 Protein Levels--
We have shown that MAPK is activated in NB4
cells after bryostatin treatment,2 which raised the
possibility that the MAPK pathway might be involved in p53
hyperphosphorylation and protein reduction. To address this question, a
specific MAPK pathway inhibitor, PD98059 (24), was employed.
Phosphorylation of MAPK can be blocked in a dose-dependent manner by this inhibitor, but it is maximally effective at a
concentration of 5 µM. Five min after treating NB4 cells
with 5.6 nM bryostatin-1, phosphorylation of MAPK (p44 and
p42) was significantly increased in the absence of inhibitor (Fig.
2A). When 5 µM
PD98059 was included with the bryostatin-1 treatment, no detectable
increase in MAPK phosphorylation was found. This result confirms that
PD98059 can block bryostatin-induced MAPK activation in this cell line.
We next examined whether PD98059 was able to block p53
hyperphosphorylation and protein reduction. Fig. 2B shows
that treatment with 5.6 nM bryostatin-1 increased
phosphorylation of p53 and that concurrent treatment with PD98059
blocked this bryostatin-induced p53 hyperphosphorylation in a
dose-dependent manner. This is consistent with the
hypothesis that MAPK is involved in the bryostatin-induced
phosphorylation of p53. Importantly, treatment with PD98059 also
inhibited bryostatin-induced p53 protein reduction (Fig.
2C). Coomassie Blue staining of major representative bands
indicated that equivalent amounts of protein were present (Fig.
2C, lower panel). Consequently, our data
demonstrate that the MAPK pathway is involved in p53
hyperphosphorylation and in the reduction in p53 protein levels.
Although direct phosphorylation of p53 by the MAPK pathway remains to
be elucidated, it is clear that MAPK is involved. In addition, these
results suggest that p53 hyperphosphorylation might be associated with
a reduction in p53 protein levels.
Reduction in p53 Protein Levels Is Mediated by Ubiquitin/Proteasome
Pathway--
We next asked if the reduction in p53 protein levels was
because of protein degradation. To address this question, we examined the steady state level of p53 mRNA by Northern analysis. NB4 cells were treated with either 5.6 nM brystatin-1 or control
vehicle, and mRNA was extracted at various time points as indicated
(Fig. 3, top). As a control to
ensure equal loading of RNA, a probe specific for glyceraldehyde
3-phosphate dehydrogenase was used (Fig. 3, bottom). Our
results demonstrate that the reduction in p53 protein levels was not
caused by a decrease in p53 mRNA levels. This implied that the
reduction in p53 protein levels might be caused by a decrease in
protein stability.
To test this hypothesis, sodium borohydride (NaBH4), an
inhibitor of ubiquitin COOH-terminal hydrolase, was used to determine whether an inhibition of the ubiquitin/proteasome pathway would block
the bryostatin-induced decrease in p53 protein levels. Ubiquitin COOH-terminal hydrolase is required for the generation of the functional monomeric form of ubiquitin (25) and is suggested to play a
role in bryostatin-induced Reh cell differentiation (26). Our results
show that bryostatin-induced p53 reduction was completely inhibited by
sodium borohydride (Fig. 3B). As proteasomes play a key role
in protein degradation, we then tested whether a proteasome inhibitor,
MG132, could inhibit p53 degradation. Fig. 3B shows that,
like sodium borohydride, MG132 also blocks bryostatin-induced p53
reduction. Taken together, these results suggest that the MAPK-mediated
decrease in p53 protein levels is not caused by decreased gene
transcription but by ubiquitin/proteasome-dependent protein degradation.
p53 from NB4 Cells Is Incapable of Binding DNA--
In an effort
to assess the functional significance of MAPK-mediated p53
phosphorylation and degradation in NB4 cell differentiation, we tested
the ability of p53 purified from NB4 cells to bind DNA using EMSA. p53
was purified from nuclear extracts prepared from NB4 cells using
anti-p53 antibody, Pab 421 (Fig.
4A, lane 2). This
antibody recognizes an epitope in the carboxyl terminus of p53 and is
thought to convert p53 from its latent to its active state and thereby
significantly increase its DNA binding activity (27). Vaccinia virus
expressed epitope-tagged human p53 purified from HeLa cells using the
same antibody was used as a control (vep53, Fig. 4A,
lane 1). When a DNA probe containing the p53 cis element
identified in the ribosomal gene cluster was incubated with vep53, a
shifted band was observed (Fig. 4B, lane 5). This band was supershifted by the addition of anti-p53 antibody N-19 (Santa
Cruz), and the addition of a 100-fold excess of cold ribosomal gene
cluster DNA fragment was sufficient to inhibit its formation, suggesting that this band was p53-specific (data not shown). In comparison to wild-type p53, p53 purified from NB4 cells had a significantly reduced affinity for DNA binding. The inability of this
p53 to bind DNA suggests that either mutations exist within the protein
or that the protein has been inactivated by post-translational modification. Regardless of the nature of the alteration, these results
establish that p53 in NB4 cells is unable to bind DNA. It may be,
therefore, that degradation of this DNA binding-impaired form of p53 is
associated with NB4 cell differentiation.
MAPK-mediated p53 Degradation Is Associated with Bryostatin-induced
NB4 Cell Differentiation--
To demonstrate the physiological
significance of the p53 degradation in NB4 cell differentiation, we
tested whether the proteasome inhibitor, MG132, would block
bryostatin-induced differentiation. Untreated NB4 cells grow as a
suspension and contain nonadherent cells (Fig.
5A). When treated with 5.6 nM bryostatin-1, 35% of the cells become attached to the
surface of the culture plate (Table I), and 21% exhibit a morphology
related to monocyte/macrophages (Fig. 5B), which is the
marker for NB4 cell differentiation. In contrast, concurrent treatment
with MG132 blocked both the bryostatin-induced cell adherence (Table I)
and phagocytotic activities (Fig. 5F), suggesting that
inhibition of the ubiquitin/proteasome pathway blocked NB4 cell
differentiation. Treatment with MG132 alone had little effect on NB4
cell differentiation (Fig. 5E and Table I) or cell viability
(data not shown).
The observation that the MAPK pathway is involved in
phosphorylation/degradation of a mutant form of p53 suggested that the MAPK pathway might be involved in cell differentiation. Therefore, we
tested whether the MAPK pathway inhibitor, PD98059, could inhibit bryostatin-induced NB4 cell differentiation under the same conditions that were sufficient to induce p53 phosphorylation/degradation. Concurrent treatment of NB4 cells with bryostatin-1 and 5 µM PD98059 blocked the bryostatin-induced cell adherence
(Table I) and phagocytotic activities (Fig. 5D), suggesting
that inhibition of MAPK blocked NB4 cell differentiation. Treatment
with 5 µM PD98059 alone had no effect on NB4 cell
differentiation (Fig. 5C and Table I) or cell viability
(data not shown). This observation supports the view that the MAPK
pathway is involved in bryostatin-induced NB4 cell differentiation. The
critical requirement for MAPK in differentiation coupled with the
demonstration that MAPK is involved in phosphorylation/protein degradation of a mutant p53 suggests that degradation of mutant p53 may
possibly play a role in cell differentiation.
A requisite first step toward understanding the molecular
mechanism for cell differentiation and proliferation is to identify the
signal transduction pathways and the cellular targets of those pathways
involved in these processes. In this paper we present biochemical
evidence that indicates that the MAPK pathway, required for
bryostatin-induced cell differentiation, induces p53
hyperphosphorylation, which subsequently reduces p53 protein stability.
This observation is particularly significant because an altered form of
p53, unable to bind DNA in vitro, is overexpressed in NB4
cells. It has been suggested that mutant p53 may gain a function such
that it can promote tumorigenicity in various cells. Consequently,
degradation of mutant p53 protein may result in a reduction of
tumorigenicity. Taken together, our results indicate that an altered
form of p53 is hyperphosphorylated via the MAPK pathway and
subsequently degraded. Although a direct cause and effect relationship
is yet to be established, our findings indicate that a reduction in
mutant p53 levels may contribute to cell differentiation.
Phosphorylation is one potential mechanism by which cells might
regulate p53 protein levels and, hence, its antiproliferation activity.
Experiments in vivo have clearly demonstrated that p53 is a
phosphoprotein on which multiple phosphorylation sites have been
identified (1). In comparison to wild-type p53, literature relating to
the phosphorylation of mutant p53 and the control of its protein level
is limited. In this study we provide in vivo evidence that a
form of p53 which is incapable of binding DNA is hyperphosphorylated
and subsequently degraded via the MAPK pathway, although it is not
clear from these experiments whether MAPK phosphorylates this DNA
binding-impaired form of p53 directly or whether additional kinases are
required. Furthermore, our results suggest that phosphorylation of p53
leads to a reduction in protein stability mediated by the
ubiquitin/proteasome pathway.
The observation of a reduction in protein stability after
phosphorylation is in contrast to previous reports in which
phosphorylation of p53 led to an increase in its stability (12, 28).
However, in agreement with our findings, two proto-oncogene-encoded
transcription factors, c-Jun and BCL-6, have also been shown to be
degraded after phosphorylation by MAPK (29-30). Thus, degradation of
oncogene products, including mutant p53, may represent a general
mechanism by which the MAPK pathway controls cell function. Reduction
of p53 protein levels was observed 12 h after bryostatin
treatment, which is slower than the 2 h reported for BCL and
c-Jun. It is possible that MAPK may mediate a second signal
required for the reduction of p53 protein levels. Nevertheless, our
data show that a strong correlation exists between p53 protein
phosphorylation and stability.
The finding that mutant p53 is phosphorylated via the MAPK pathway and
subsequently degraded has several implications when considering the
model in which mutations in p53 lead to a gain of function that
promotes tumorigenicity. First, overexpression of mutant p53 in pre-B
(31), fibroblast (5), and osteosarcoma cells (32) has been shown to
dramatically enhance the tumorigenicity of these cells. Reduction in
p53 protein levels, therefore, may be of significance in preventing
cell proliferation. Indeed, our data demonstrate a strong correlation
between protein degradation and cell differentiation. Second, the high
frequency of p53 mutations in human cancers warrants a detailed
analysis of the molecular mechanisms of the gain of function of mutant
p53 and the signals that may regulate this function. In this paper we
go some way toward this goal by presenting evidence that the MAPK
pathway is involved in degradation of the DNA binding-impaired form of p53 and by correlating this function with NB4 cell differentiation. Finally, a significant research effort is concerned with finding strategies to inactivate mutant p53. In light of the results presented here, it may also be effective to develop therapies designed to reduce
mutant p53 protein levels.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
,25-dihydroxyvitamin D3 (16) and bryostatin-1 (17).
Although the precise mechanisms involved in this differentiation are
not yet fully understood, it is clear that a mitogen-activated protein
kinase (MAPK)1 pathway is
involved (17, 18). Interestingly, MAPK has been shown to phosphorylate
the amino terminus of p53 in vitro (19). Thus, a possible
mechanism for bryostatin-induced differentiation may involve the
phosphorylation of p53 via the MAPK pathway, resulting in an alteration
of p53 antiproliferation activity.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Effect of PD98059 and MG132 on bryostatin-induced cell differentiation
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Fig. 1.
Treatment with bryostatin-1 induces p53
protein phosphorylation and reduction in NB4 cells. NB4 cells were
labeled with [32P]orthophosphate and were treated with
control or with bryostatin-1 at various concentrations as indicated for
30 min (A) or treated with 5.6 nM bryostatin-1
for various time periods as indicated (B). Cells extracts
were then prepared, and p53 protein was immunoprecipitated using p53
antibody. Protein samples were resolved by electrophoresis on a
SDS-PAGE gel and visualized by autoradiography. C, to study
p53 protein reduction NB4, cells were treated with various
concentrations of bryostatin-1 as indicated for 12 h. Cells
extracts were then prepared, and Western blot analysis was performed
with anti-p53 antibody (top panel). Equal loading of protein
from crude extract was verified by Coomassie Blue staining
(bottom panel).
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Fig. 2.
MAP kinase is involved in p53 phosphorylation
and degradation in NB4 cells. A, after a 30-min
preincubation in the presence or absence of MAPK pathway inhibitor
PD98059, NB4 cells were treated with control or 5.6 nM
bryostatin-1 (Bry) for 5 min. Cells extracts were then
prepared, tyrosine-phosphorylated proteins were immunoprecipitated with
anti-phosphotyrosine antibody, and protein samples were resolved by
electrophoresis on a SDS-PAGE gel. Western blotting was then performed
with anti-MAP kinase antibody (top panel). To determine
total levels of MAPK protein present in the cells, Western blotting
with anti-MAP kinase antibody was performed on cell extracts
(bottom panel). B, NB4 cells were labeled with
[32P]orthophosphate, PD98059 was added at various
concentrations as indicated, either in the presence or absence of 5.6 nM bryostatin-1, and incubation was continued for a further
30 min. Cells extracts were then prepared, and p53 protein was
immunoprecipitated. Protein samples were resolved by electrophoresis on
a SDS-PAGE gel and visualized by autoradiography. C, NB4
cells were preincubated with PD98059 at various concentrations as
indicated, after which control or 5.6 nM bryostatin-1 was
added, and incubation was continued for 12 h. Cells extracts were
then prepared, and Western blotting was performed with anti-p53
antibody. Equal loading of protein from crude extract was verified by
Coomassie Blue staining (bottom panel).
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Fig. 3.
The reduction in p53 protein levels is
because of protein degradation. A, NB4 cells were
treated with control vehicle or with bryostatin-1 at various
concentrations as indicated for 6 or 12 h, after which total RNA
was extracted. Thirty µg of total RNA from each sample were subjected
to Northern blot analysis using a probe specific for p53 mRNA
(upper panel). To ensure equal loading of RNA, the membrane
was stripped and re-incubated with a probe specific for glyceraldehyde
3-phosphate dehydrogenase (GAPDH) mRNA B, to
study the effect of inhibitors of the ubiquitin/proteosome pathway on
bryostatin-1-induced p53 degradation, NB4 cells were preincubated with
sodium borohydride (NaBH4) or MG132 for 30 min, after which
5.6 nM bryostatin-1 was added, and incubation was continued
for 12 h. Cells extracts were then prepared, and Western blotting
was performed with anti-p53 antibody.
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Fig. 4.
p53 purified from NB4 cells fails to bind to
DNA in EMSA. A, vaccinia virus expressed human
p53 (vep53) purified from HeLa cell extracts and endogenous p53
purified from NB4 cell extracts were subjected to electrophoresis on a
SDS-PAGE gel and visualized by silver staining. B, in EMSA,
radiolabeled probe containing the p53 site from the ribosomal gene
cluster was incubated with approximately 50, 75, or 100 ng of p53
purified from NB4 cells (lanes 2-4) or 20 ng of vep53
(lane 5) before electrophoresis on a 3% polyacrylamide gel.
The position of vep53 and p53 expressed from the endogenous genes are
shown. MW, molecular mass.
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Fig. 5.
MAPK-mediated p53 degradation is associated
with cell differentiation. NB4 cells were treated with control
(A), 5.6 nM bryostatin-1 (B), 5 µM PD98059 (C), bryostatin-1 and PD98059
(D), 1 µM MG132 (E), bryostatin-1
and MG132 (F) for 72 h. After each treatment,
phagocytosis and cell adherence, the markers for NB4 cell
differentiation, were measured by incubation of NB4 cells with latex
beads for 5 h, and the cells were photographed with 400×
magnification using a Nikon microscope.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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We thank Dr. Francey Sladek for her valuable comments and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA75180-02 (to X. L.) and DK09012-034 (to A. W. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 909-787-4350;
Fax: 909-787-4434; E-mail: xuan.liu{at}ucr.edu.
The abbreviations used are: MAPK, mitogen-activated protein kinase; EMSA, electrophoresis mobility shift analysis; PAGE, polyacrylamide gel electrophoresis.
2 X-D. Song and A. W. Norman, unpublished data.
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