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INTRODUCTION |
The p53 tumor suppressor is a transcription factor that is mutated
in over 50% of human tumors primarily because of missense mutations in
the DNA binding domain (1). In normal cells, p53 is present at very low
levels because of rapid degradation through the
ubiquitin-dependent proteasome pathway. The MDM2
oncoprotein is an important regulator of p53 turnover. Expression of
MDM2 is activated by p53 at the transcription level (2, 3). MDM2 binds
to p53 and promotes its ubiquitination by acting as an ubiquitin E3
ligase (4-6). Gene knockout of MDM2 in mice causes early
embryonic death, which can be rescued by further inactivation of p53
(7, 8). Therefore, MDM2 functions as a major negative feedback regulator to maintain p53 at low levels and protect cells from growth
arrest and apoptosis induced by p53.
About 70-80% of human carcinomas with p53 point mutations in the core
(DNA-binding) domain express mutant p53 protein at high levels (9, 10).
Although a major consequence of p53 mutation is loss of its tumor
suppressor function, maintenance of high level mutant p53 expression
during tumor development suggests that it may also have positive
effects on cell proliferation. Ectopic expression of several p53
mutants (175H, 248W, 273H,
281G) in p53-null cell lines can increase
tumorigenic potential and drug resistance (11-13). Mutant p53 can also
activate the c-myc promoter (281G) and overcome
the mitotic spindle checkpoint in normal human fibroblasts
(175H, 245D, Refs. 14, 15). These observations
suggest that mutant p53 has gain-of-function properties that enhance
cell transformation independent of wild-type p53. Mutational analysis
suggests that amino acids 22 and 23 of p53, which are essential for
transcription activation and MDM2 binding, are also important for the
gain-of-function phenotype of the 281G mutant (16).
MDM2 is overexpressed in certain tumors with wild-type p53 (17),
suggesting that it may contribute to tumor development by inactivation
of p53. However, MDM2 also has p53-independent activities that may play
a role in malignant transformation. Targeted overexpression of MDM2 in
the breast epithelium of p53-null mice can induce abnormal
cell proliferation and aneuploidy (18). Transgenic mice expressing
2-4-fold higher levels of MDM2 develop sarcomas at higher frequencies
in p53-null backgrounds (19). MDM2 can interact with and
inactivate the retinoblastoma tumor suppressor (pRb, Ref. 20).
It can also modulate the activity, stability, and apoptotic function of
E2F1/DP1 transcription factors (21-23). Furthermore, MDM2 can abrogate
the growth arrest function of TGF-
in a p53-independent fashion
(24). Therefore, MDM2 can enhance cell transformation by
p53-dependent and -independent mechanisms.
MDM2 is a transcriptional target of p53 and is often
overexpressed in tumors with wild-type p53 because of enhanced
transcription and translation (2, 25). MDM2 overexpression also occurs frequently through gene amplification (26, 27). In contrast, the status
and function of MDM2 in tumors with mutant p53 is less well
characterized. In cells overexpressing MDM2 because of gene amplification, MDM2 is degraded rapidly with a half-life of about 30 min (28). In this report, we present direct evidence that MDM2 in tumor
cells with high levels of mutant p53 (175H, 241F, 248W, 249S, 266E, 273H,
273C, and 280K) is stabilized by interaction with
p53. Several experimental approaches demonstrate that overexpression of
mutant p53 can prevent MDM2 degradation, resulting in the accumulation of MDM2 in tumor cells to moderate levels. These results suggest a
novel mechanism of MDM2 stabilization and accumulation.
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MATERIALS AND METHODS |
Cell Lines and Recombinant Viruses--
HT1080 (fibrosarcoma, wt
p53), SJSA (osteosarcoma, wt p53, MDM2 amplification), C33A
(HPV-negative cervical carcinoma, p53273R-C) and
DLD-1 (colon carcinoma, p53241S-F) were obtained
from the ATCC. H1299 (lung carcinoma, p53null), Saos2
(osteosarcoma, p53-null), MDA-MB-231 (breast carcinoma, p53280R-K), MDA-MB-435 (breast carcinoma,
p53266G-E, and MDA468 (breast carcinoma,
p53273R-H) were provided by Arnold J. Levine.
Adenoviruses expressing wild-type and p53175H
were provided by Drs. Jiayuh Lin and Bert Vogelstein.
Transfection and Virus Infection--
H1299 and Saos2 cells were
transfected with the pCMV-neo-Bam vector expressing 175H
mutant p53 or human MDM2 cDNA using the calcium phosphate
precipitation method. G418-resistant colonies were selected and pooled
for further analysis. In transient transfections, each 10-cm dish of
H1299 cells was cotransfected with 5 µg of CMV1-MDM2 expression plasmid
and 5 µg of mutant CMV-p53 expression plasmid. Expression of MDM2 and
p53 were analyzed 36 h after transfection. MG132 was added to 40 µM for 5 h where indicated. Recombinant adenovirus
expressing wild-type or mutant p53 were amplified using 293 cells.
H1299 and Saos2 cells were infected with diluted crude viral lysate at
200 plaque-forming units/cell. MDM2 protein levels were determined
24 h after addition of viruses. To determine the half-life of
MDM2, 75 µg/ml of cyclohexamide was added to the cultures, and
samples were collected at different time points for Western blot analysis.
Western Blot Analysis and Immunoprecipitation--
Cells were
lysed in radioimmune precipitation assay buffer (1% sodium
deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM Tris, pH
7.4, 150 mM NaCl, 1 mM phenylmethylsulfonyl
fluoride) and 100-200 µg of protein were fractionated by
SDS-polyacrylamide gel electrophoresis. MDM2 and p53 were detected by
monoclonal antibody 3G9 (29) and DO-1. The filter was developed using
ECL-plus reagent (Amersham Pharmacia Biotech). For
immunoprecipitation-Western blot analysis, cells were lysed in lysis
buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride). Cell lysate (300-600 µg of protein)
was immunoprecipitated using anti-p53 monoclonal antibody Pab421 or
anti-MDM2 monoclonal antibody 2A9 and was washed with lysis buffer.
Immunofluorescence Staining--
Cells cultured on chamber
slides were fixed with acetone/methanol (1:1) for 3 min at room
temperature, blocked with PBS, 10% normal goat serum (NGS) for 20 min,
and incubated with anti-p53 Pab1801 hybridoma supernatant (1:10
dilution) or anti-MDM2 2A9 hybridoma supernatant (1:100 dilution) in
PBS, 10% NGS for 2 h. The slides were washed with PBS, 0.1%
Triton X-100; incubated with fluorescein isothiocyanate-labeled
goat-anti-mouse IgG in PBS, 10% NGS for 1 h; washed with PBS,
0.1% Triton X-100; and mounted.
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RESULTS |
Transient Expression of Mutant p53 Induces MDM2
Stabilization--
Transient cotransfection of wild-type or mutant p53
with MDM2 expression plasmids can result in degradation of p53 (4, 5).
In our assay system, a significant excess of MDM2 plasmid (>5-fold of
p53) was required to achieve p53 degradation (data not shown). After
cotransfection of equal amounts of mutant p53 and MDM2 plasmids into
p53-null H1299 cells, p53 was not degraded. However, the
levels of MDM2 expression in such transfections were significantly
increased (Fig. 1A, compare
lane 3 with lanes 5, 7, 9,
13). Among five p53 hotspot mutants tested, the
175H mutant showed the strongest ability to increase MDM2
expression and the 273H mutant was the weakest. Quantitation
by serial dilution showed that MDM2 expression was induced 8-fold by
the 175H mutant and 3-fold by the 273H mutant
(data not shown). Mutant p53 had no significant effect on the
expression of cotransfected green fluorescent protein GFP (Fig.
1A), suggesting that the increase of MDM2 expression was not
because of enhancement of transfection efficiency.

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Fig. 1.
Stabilization of human MDM2 by mutant p53 in
transient transfections. A, H1299 cells were
transfected with CMV-MDM2, CMV-p53, and CMV-GFP plasmids in a 10:10:1
ratio. Each plate of transfected cells was then split into two plates.
Protein expression was detected by Western blot analysis 36 h
after transfection. MG132 (40 µM) was added to the
duplicate plate 4 h before sample collection. The same filter was
used to detect MDM2, p53, and GFP expression. B, H1299 cells
transfected with MDM2 alone or MDM2 with 175H or wild-type
p53 were treated with 75 µg/ml cyclohexamide (CHX) for the
indicated times. MDM2 levels were detected by Western blot analysis.
C, diagram of p53 and mutants. Location of mutations,
relative to the five conserved regions of p53, and functional domains
are indicated.
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To determine whether the increase of MDM2 level was because of
stabilization, transfected cells were treated with the proteasome inhibitor MG132. The ability of MG132 to induce MDM2 accumulation provides an indication of its degradation rate before treatment. The
results show that MG132 caused less dramatic accumulation of MDM2 that
was coexpressed with mutant p53 (Fig. 1A, compare lane
3 and lanes 4, 5, 6). Therefore, higher MDM2
levels observed in the cotransfection with mutant p53 may be caused by
stabilization of MDM2. Mutant p53 in these experiments were also highly
stable, indicating that they were not degraded by MDM2 at this
expression ratio (Fig. 1A). In MDM2 and wild-type p53
control cotransfection, interpretation of the result was complicated by
the fact that wild-type p53 may repress MDM2 plasmid transcription but
induce endogenous MDM2. However, MG132 treatment showed that both MDM2 and p53 were unstable, suggesting that the stabilizing effect is unique
to mutant p53 (Fig. 1A). MG132 treatment also caused reduction of GFP expression, possibly because of wild-type p53-mediated cell death.
To directly test the stability of MDM2, the
p53175H mutant was transiently cotransfected
with MDM2 into H1299 cells. The transfected cells were treated with the
protein synthesis inhibitor cyclohexamide, and MDM2 levels were
accessed by Western blot analysis. In the absence of mutant p53, MDM2
was degraded to near completion 1.5 h after addition of
cyclohexamide. In contrast, coexpression with the 175H
mutant brought about a stabilization of MDM2 (Fig. 1B). However, coexpression of MDM2 with wild-type p53 did not lead to
stabilization of MDM2 (Fig. 1B). This result indicates that high level expression of mutant p53 can overcome the degradation function of MDM2 and stabilize MDM2.
Stabilization of Endogenous MDM2 by Ectopic Expression of Mutant
p53--
To directly test if stabilization of endogenous MDM2 can be
induced by mutant p53, H1299 cells were stably transfected with CMV
promoter-driven p53175H mutant. Pools of
drug-resistant cells were stained using antibodies against p53 and
MDM2. Nontransfected H1299 cells have very low levels of MDM2
expression (Fig. 2A). However,
stable transfectants expressing p53 mutant (H1299-175H) expressed
increased levels of nuclear MDM2 (Fig. 2A). The increase in
MDM2 level was also confirmed by Western blot analysis (see Fig.
4C). Northern blot analysis was performed to compare the
MDM2 mRNA levels in H1299 and H1299-175H cells. The result shows
that both cell lines expressed similar levels of MDM2 mRNA (Fig.
2B). This suggests that a change in protein stability is
responsible for increased MDM2 level in H1299-175H cells.

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Fig. 2.
Ectopic expression of mutant p53 induces
stabilization of endogenous MDM2. A, H1299 cells stably
transfected with p53175H were stained for p53
and MDM2. B, Northern blot analysis of MDM2 mRNA level.
An identical amount of total mRNA was loaded for each cell line.
C, H1299 cells were infected with adenovirus expressing
lacZ or p53175H mutant for 24 h,
and MDM2 expression levels were determined by Western blot analysis.
D, MDM2 degradation rates in H1299 cells stably transfected
with mutant p53 or infected for 24 h with p53 adenoviruses were
determined after cyclohexamide treatment. Because wild-type p53 virus
induced high level MDM2 expression, protein loading was reduced (10%)
to obtain a similar signal intensity.
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Next, the effect of transiently expressed mutant p53 on endogenous MDM2
level was examined. To achieve uniform expression of mutant p53 in
H1299 cells similar to tumor cells with p53 mutations, the cells were
infected with a recombinant adenovirus expressing p53175H. This approach can achieve expression of
p53 in nearly 100% of the cells, as verified by immunofluorescence
staining (data not shown). The level of MDM2 was significantly
increased 24 h after infection with the 175H virus but
not with a lacZ control virus (Fig. 2C).
To further confirm that the increase in MDM2 level after stable
transfection of 175H or infection with the 175H
virus was due to an increase in stability, the half-life of MDM2 was
determined. H1299 cells were infected with adenoviruses expressing
wild-type p53 or the 175H mutant for 24 h.
MDM2 degradation rate was determined by cyclohexamide treatment. In
uninfected H1299 cells, endogenous MDM2 was rapidly degraded after
cyclohexamide treatment. MDM2 induced by the wild-type p53 virus was
also rapidly degraded with a half-life of ~0.5 h, although its
expression level was much higher because of transcription activation by
wild-type p53 (data not shown). In contrast, MDM2 in cells infected
with the 175H virus or stably transfected with
175H was significantly stabilized, with an estimated
half-life of over 2 h (Fig. 2D). This result indicates
that wild-type p53 induces MDM2 accumulation by stimulating transcription of MDM2, whereas mutant p53 induces MDM2 by
increasing its stability.
Accumulation of Stable Nuclear MDM2 in Tumor Cells with Mutant
p53--
Because many human tumors accumulate high levels of mutant
p53, it is possible that expression of endogenous mutant p53 is sufficient to cause stabilization and accumulation of MDM2.
Immunofluorescence staining of tumor cell lines revealed that in cells
with wild-type p53 (such as HT1080, Fig.
3A), the levels of MDM2
expression were often heterogeneous. A small number of cells in a
population had stronger staining of p53 and MDM2, possibly because of
their spontaneously entering a stressed state. This pattern of MDM2
expression was observed in a panel of over ten cell lines with
wild-type p53 (data not shown). The p53-null H1299 cells
uniformly express very low levels of MDM2, mainly localized in the
nucleoli (Fig. 2A and data not shown). In contrast, C33A
(273C), MDA-MB-231 (280K) and DLD-1
(241F) cells showed uniform and moderate levels of nuclear MDM2 staining (Fig. 3A and data not shown). This uniform
staining pattern was similar to that of mutant p53, which showed high
level expression in nearly 100% of the nuclei (Fig. 3A).
This similarity suggests that elevated nuclear MDM2 staining may be
causally related to the accumulation of mutant p53 in these cells. It
should be noted that although MDM2 staining in p53 mutant cell lines is stronger than in certain p53 wild-type or p53-null cells, it
is significantly lower than the levels in cells with MDM2
gene amplification, such as SJSA (data not shown).

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Fig. 3.
Human tumor cells with mutant p53 have
stabilized MDM2. A, increased nuclear MDM2 level in
cells with mutant p53. MDM2 and p53 were detected by immunofluorescence
staining using monoclonal antibodies 2A9 and Pab1801. B,
MDM2 levels in the cell lines were determined by Western blot analysis
before and after treatment with MG132 for 5 h. An identical amount
of total protein was loaded for each cell line. C, the rates
of MDM2 degradation were determined after treatment with cyclohexamide.
MDM2 Western blot analysis was performed using identical amounts of
total protein.
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Because increased MDM2 expression can also be caused by factors other
than wild-type p53 (such as bFGF and activated ras, Refs.
30, 31), we directly tested whether MDM2 stabilization occurred in the
mutant cell lines. Cells were treated with the proteasome inhibitor
MG132 for 5 h, and the MDM2 levels were detected by Western blot
analysis. MG132 treatment of p53-null H1299 or cells with
wild-type p53 resulted in a significant increase of MDM2 levels (Fig.
3B). This is consistent with rapid degradation of MDM2 in
these cells by proteasomes. In contrast, MDM2 levels in tumor cells
with mutant p53 were only weakly induced after MG132 treatment,
suggesting that MDM2 was not rapidly degraded by proteasomes in these
cells. Densitometry analysis of the films showed that the levels of
MDM2 in p53-mutant cells were at least 4-fold higher than in
the p53-null H1299 cells, which is consistent with the
immunofluorescence staining results. The MDM2 mRNA levels of DLD1
and C33A cells were also similar to that of H1299 (Fig. 2B),
suggesting that mutant p53 induces MDM2 accumulation without stimulating transcription of the MDM2 gene.
To further confirm the stabilization of MDM2 in mutant p53 cell lines,
MDM2 degradation rates were examined after treatment with the protein
synthesis inhibitor cyclohexamide. The half-life of MDM2 in the
p53-mutant DLD-1 cells was significantly longer than that in
SJSA and HT1080 cells, which express wild-type p53 (Fig.
3C). The half-life of MDM2 in SJSA is ~0.5 h, whereas in DLD-1 cells it is over 2 h. Using this assay, we also observed stabilization of MDM2 in C33A, MDA468, MDA231, and T47D cells (data not
shown). Therefore, MDM2 stabilization may be a common event in tumor
cells with mutant p53.
Stabilization of MDM2 by Mutant p53 Requires Complex
Formation--
MDM2 interacts with the N-terminal domain of p53, and
this interaction is not significantly affected by point mutations in the core (DNA binding) domain of p53 (29). To determine whether p53
core domain mutants induce MDM2 stabilization through complex formation, the effect of 175H virus on the stability of an
MDM2 deletion mutant was investigated. In a previous experiment, stable Saos2 cell lines were established that express full-length MDM2 or the
p53 binding-deficient
1-50 mutant (29). These cells were infected
with 175H virus, and the stability of full-length and
1-50 MDM2 mutant were determined. The results show that in contrast
to full-length MDM2, the
1-50 mutant was not stabilized by
175H virus infection (Fig.
4A). In a transient
transfection assay using H1299 cells, 175H also did not
stabilize a
58-89 MDM2 mutant (also deficient for p53 binding, data
not shown). In a reciprocal experiment, MDM2 binding-deficient
p5322Q/23S-281G triple mutant (Fig.
1C) also failed to stabilize MDM2 in transient transfection
of H1299 cells (Ref. 16, Fig. 4B). These results demonstrate
that MDM2 stabilization by mutant p53 requires complex formation.

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Fig. 4.
Mutant p53 stabilization of MDM2 requires
complex formation. A, Saos2 cells stably transfected
with full-length MDM2 or p53 binding-deficient MDM2 1-50 were
infected with 175H adenovirus for 24 h. MDM2
degradation rates were determined after cyclohexamide (CHX)
treatment. B, MDM2 binding-deficient p53 mutant does not
stabilize MDM2. H1299 cells were transiently transfected with MDM2 and
p5322Q/23S-281G triple mutant, MDM2
stability was determined after cyclohexamide treatment. C,
MDM2-mutant p53 complex was detected by immunoprecipitation of 600 µg
of protein using p53 antibody Pab421 followed by anti-MDM2 Western blot
analysis. Total cell lysate (200 µg of protein) was loaded directly
as a control.
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To confirm the presence of the MDM2 mutant p53 complex in cells, lysate
from C33A and stable H1299-175H cells were analyzed by anti-p53 Pab421
immunoprecipitation followed by anti-MDM2 Western blotting. Significant
amounts of MDM2 were coprecipitated with mutant p53 from the cell
lysate (Fig. 4C). MDM2 in the H1299 lysate was not
precipitated by p53 antibody, demonstrating the specificity of the
coprecipitation assay. Therefore, stabilized MDM2 is present in
complexes with mutant p53.
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DISCUSSION |
The results described above demonstrate that expression of p53
core domain mutants in tumor cells causes stabilization of MDM2. We
found that all five p53 hotspot mutants (175H,
248W, 249S, 273H, 281G)
showed the ability to stabilize MDM2 in transfection assays using H1299
lung tumor cells (although with different efficiencies). Furthermore,
solid tumor cell lines carrying 241F, 266E,
273H, 273C, and 280K mutations also
have stabilized MDM2. These mutations are located in the evolutionarily
conserved regions of p53, which are targeted by most of the mutations
found in human tumors (Fig. 1C). Recently, MDM2
stabilization was also observed in a panel of leukemia cell lines with
mutant p53 (32). Therefore, it is possible that this is a rather
general property of tumor cells with p53 core domain mutations. Our
results suggest a mechanism of MDM2 stabilization in such cases. Mutant
p53 is degraded by MDM2 if the latter is overexpressed, such as by
transfection of exogenous MDM2 (4, 5). However, it is apparent that at
the ratio of MDM2 and mutant p53 naturally reached in tumor cell lines, mutant p53 is not efficiently degraded by the endogenous MDM2 but
instead causes stabilization of MDM2.
It should be emphasized that the ability of mutant p53 to cause MDM2
stabilization does not mean that tumors with p53 mutation will
accumulate MDM2 to very high levels. Immunohistochemical staining of
tumor samples has revealed cells that overexpress both MDM2 and mutant
p53, but this is a relatively rare event (27, 33). Our results show
that expression of mutant p53 in H1299 cells can increase MDM2 level by
several-fold. Such a moderate increase probably will not be defined as
overexpression in conventional immunohistochemistry staining of
clinical samples. MDM2 expression levels in tumors will also be limited
by its transcription and translation rates (both are low in the absence
of wild-type p53) and the still unknown threshold of the MDM2/mutant
p53 ratio that will trigger p53 degradation.
RING finger mutations of MDM2 showed that the ability of MDM2 to
promote p53 degradation is tightly linked to its ability to promote
self-ubiquitination and degradation (34). Therefore, MDM2 stabilization
may be caused by loss of its ubiquitin ligase function and may be
related to stabilization of mutant p53 in tumors. Because MDM2
stabilization requires direct binding to mutant p53, it is possible
that mutant p53 inhibits MDM2 ubiquitination after forming a complex.
Mutant p53 may have obtained this capability because of altered
structure or interaction with other cellular factors, such as heat
shock proteins (35). Such a mechanism would suggest that mutant p53 is
stabilized in part because of its ability to inactivate low levels of
MDM2. A model proposed by Midgley and Lane (36) suggests that mutant
p53 is stable because of the loss of MDM2 induction. However, tumor
cell lines express readily detectable amounts of MDM2 bound to mutant
p53, which may be induced by other transcription mechanisms (31). Therefore, reduction in MDM2 transcription after p53 mutation as well
as the ability of mutant p53 to neutralize the function of remaining
MDM2 may both contribute to p53 stabilization.
Mutant p53 has been shown to exhibit gain-of-function properties in the
absence of wild-type p53. Although the precise mechanism of this
phenomenon is still not clear, transcription activation and the
MDM2-binding domain have been implicated by an experiment using the
22/23-281 triple mutant (16). Our results show that the
p5322Q/23S//281G mutant is also deficient in
MDM2 stabilization. Therefore, it is possible that MDM2 stabilization
also contributes to the tumorigenic activity of mutant p53. MDM2
transgenic mouse experiments have shown that a moderate increase
(2-4-fold) of MDM2 expression can significantly increase the incidence
of sarcomas in a p53-null background (19). Therefore,
moderate MDM2 accumulation caused by p53 mutation may have an effect on
tumor proliferation. Further experiments will be needed to test this possibility.