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INTRODUCTION |
The p53 tumor suppressor can be activated by DNA damage (1),
deregulated oncogenes (2), and hypoxia (3). Activation of p53 results
in growth arrest or apoptosis by induction of downstream target genes
(4, 5) and possibly transcription-independent mechanisms (6, 7).
The activity of p53 is regulated by the MDM2 oncoprotein, which binds
to p53 and inhibits the transcription activation function (8). MDM2
binding also promotes p53 degradation through the
ubiquitin-dependent proteasome pathway (9, 10). MDM2
expression is induced by p53 (11, 12), thus forming a negative feedback
loop that maintains p53 at low levels in the absence of stress. Loss of
MDM2 results in embryonic lethality due to activated p53 (13, 14), and
overexpression of MDM2 in tumors results in suppression of p53
transcription function and apoptosis (15). Increasing evidence suggests
that regulation of the interaction between p53 and MDM2 by
phosphorylation (16-18), and inhibition of MDM2 by the tumor
suppressor p19ARF are important mechanisms of p53 activation (19,
20).
Activation of p53 can lead to growth arrest or apoptosis, depending on
cell types (21-23). The cell cycle-arrest activity of p53 is largely
mediated by induction of the p21WAF1 gene (24). Despite the
identification of several p53-inducible genes that can regulate
apoptosis, such as Bax (25) and KILLER/DR5 (26),
the apoptotic mechanism of p53 is not completely understood. Furthermore, it is unclear why different tumor cells or different cell
lines of the same tumor type often exhibit either a growth arrest or
apoptosis response to p53, which may be clinically important. Recent
studies using cell fusion revealed that the apoptotic response phenotype is dominant over growth arrest (23), suggesting that expression of a specific factor may confer an apoptotic response to p53.
Apoptosis typically involves the activation of proteolytic cascades of
caspases and cleavage of vital cellular proteins (27). Fas/CD95- and
tumor necrosis factor-
-induced apoptosis involve activation of
caspase 8 through receptor-mediated oligomerization (28). Caspase 8 can
directly activate effector caspase 3 or induce mitochondria cytochrome
c release and activation of the Apaf1-caspase 9 complex
(29), which then activates effector caspases. Because p53-induced
apoptosis also involves activation of effector caspases (23, 30), it is
conceivable that certain upstream signaling events may be activated by
p53. The ability of p53 to induce KILLER/DR5 expression and to promote
Fas/CD95 export to the cell surface (31) provides a possible apoptotic pathway. Whether this is a general mechanism of p53-mediated apoptosis is still not clear.
During p53-induced apoptosis, the MDM2 oncoprotein is also cleaved by a
caspase after the aspartic acid residue 361 (30, 32). Proteolytic
cleavage of MDM2 has been proposed to play a role in eliminating its
p53-inhibitory effect and possibly other cell survival functions of
MDM2. However, due to the rapid and non-synchronous nature of
p53-induced apoptosis, whether MDM2 cleavage is an early event that can
contribute to p53 regulation has not been determined. Furthermore, the
caspase cleavage fragment of MDM2 can still bind to p53 and inhibit its
transcription function when overexpressed (30). Thus the functional
significance of the cleavage is still unclear.
We recently observed that certain tumor cell lines express high levels
of a 60-kDa isoform of MDM2 protein (p60) similar to the caspase
cleavage fragment of MDM2 (33). The expression of p60 can be inhibited
by culturing cells in caspase inhibitor
Z1-VAD-FMK, can be produced
from a transfected full-length MDM2 cDNA, and is blocked by point
mutation of residue 361 (33). Cells can produce high levels of p60 in
normal culture conditions, with no detectable apoptosis and cleavage of
another apoptosis substrate PARP. These results suggest that
caspase-mediated processing of MDM2 can occur in the absence of
apoptosis; MDM2 is unique compared with other known apoptotic
substrates in being targeted by a caspase in non-apoptotic cells. The
variable levels of p60 in different cell lines suggest that this
caspase may be regulated by unknown factors and may play a role in the
regulation of MDM2 and p53.
In this report, we describe the characterization of MDM2 cleavage using
a cell line that undergoes p53-mediated growth arrest followed by
delayed, partial apoptosis. This system enabled us to examine
p53-induced events before the onset of apoptosis. We found that
p53-induced growth arrest results in the activation of a unique caspase
that is specific to MDM2, resulting in the production of p60 after p53
activation. Activation of the MDM2-specific caspase occurs before the
activation of apoptotic caspases. p60 is deficient in promoting p53
degradation and can stabilize p53 by a dominant-negative mechanism.
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MATERIALS AND METHODS |
Cell Lines and Plasmids--
JAR, SJSA, and HT1080 cells were
obtained from the American Type Cell Collection and maintained in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Both
cell lines contain MDM2 gene amplifications (34, 35). Human
p53-null cell line H1299 was provided by Arnold J. Levine. A Bluescript
vector containing human p53 cDNA with an 138 alanine-to-valine
mutation was kindly provided by Ute Moll of the State University of New
York at Stony Brook. The p53 valine 138 cDNA was cloned into the
pCMV-neo-Bam expression vector driven by the CMV promoter (36). CMV
promoter-driven Flag-MDM2 cDNA was kindly provided by
Douglas Tkachuk of the Seattle Veterans Medical Center.
Escherichia coli expression vectors of caspase 6 and 8 were
kindly provided by Dr. Emad Alnemri of Thomas Jefferson University.
Transfection--
In a stable transfection, 2 × 106 cells were seeded into 10-cm dishes for 24 h and
transfected with 15 µg of plasmid using the standard calcium
phosphate precipitate protocol. Two days after transfection, cells were
cultured in medium containing 750 µg/ml G418 for 2 weeks, and
G418-resistant colonies were cloned or pooled. For secondary stable
transfections, H1299-Val-138 cells were transfected with a mixture of
10 µg of pBP100-luciferase or pSG-HDM1B plasmid (encoding Flag-MDM2)
with 5 µg of pGK-hygro plasmid. Cells were selected with a medium
containing 750 µg/ml G418 and 200 units/ml hygromycin. Double
drug-resistant colonies were pooled for analysis.
Western Blot--
Cells were lysed in RIPA buffer (1% sodium
deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM Tris, pH
7.4, 150 mM NaCl, 1 mM phenylmethylsulfonyl
fluoride), centrifuged for 5 min at 10,000 × g, and
the insoluble debris discarded. Cell lysate (100-200 µg of protein)
was fractionated by SDS-polyacrylamide gel electrophoresis and
transferred to Immobilon P filters (Millipore) using a semi-dry electro-blotting apparatus. The filter was blocked for 5 min with phosphate-buffered saline (PBS) containing 5% non-fat dry milk, 0.1%
Tween 20, and then incubated for 1 h with 1/10 dilution of anti-MDM2 monoclonal antibody 3G9 or 4B11 (37) in PBS containing 5%
non-fat dry milk. For detection of p21WAF1, the
filter was incubated with 1:500 dilution of anti-p21 antibody (Oncogene
Research Product) as recommended by the supplier. p53 was detected by
incubating with monoclonal antibody DO-1. The filter was then washed
four times (5 min each) with PBS containing 0.1% Tween 20. Bound
primary antibody was detected by incubating for 1 h with protein
A-peroxidase or horseradish peroxidase goat anti-mouse IgG (for p21)
diluted in PBS containing 5% non-fat dry milk. The filter was washed
with PBS containing 0.1% Tween 20 and developed using the ECL-plus
reagent (Amersham Pharmacia Biotech). For detection of PARP,
105 cells were lysed in sample buffer (50 mM
Tris-Cl, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.003%
bromphenol blue, 5% freshly added 2-mercaptoethanol), sonicated,
boiled, and loaded onto 10% SDS-polyacrylamide gel electrophoresis.
Fractionated proteins were transferred onto Immobilon P filters. The
filter was incubated with an anti-PARP monoclonal antibody
(PharMingen), washed, incubated with horseradish peroxidase goat
anti-mouse IgG, and developed using the ECL-plus system.
Protease Cleavage Assay--
Peptide substrates Ac-DEVD-AMC,
Ac-WEHD-AMC, and Z-VAD-AMC were purchased from Bachem. The
MDM2-specific substrate Ac-DVPD-AMC was synthesized by Dr. Chi Yang
(Synpep Corp., Dublin, CA) by solution phase chemistry. To measure
caspase activities, cytosol was prepared using a procedure described by
Nicholson et al. (38). The cell pellet (~107
cells) was suspended in 0.15 ml of cytosol buffer (10 mM
Hepes/KOH, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µM pepstatin A, 50 µM leupeptin, and 10 µg/ml aprotinin). The mixture was homogenized by 30 strokes in a
Dounce homogenizer with a type B pestle and centrifuged at 15,000 × g for 5 min at 5 °C, and the pellet was discarded.
Fluorogenic assays for caspase activities contain 100 µl of cytosol
buffer, 20 µg of cytosolic protein, 20 µM
tetrapeptide-AMC substrate. After incubating for 2 h at 37 °C,
free AMC concentration was measured using a fluorometer with a 380 nm
excitation filter and 460 nm emission filter.
Purification of recombinant polyhistidine-tagged caspase 3, 6, and 8, and cleavage of in vitro translated MDM2 were performed as
described previously (30). Caspase inhibitors Ac-DEVD-CHO and
Ac-YVAD-CHO (Bachem) were used at 1-2 µM. Z-VAD-FMK
(Bachem) was added to cell culture medium at 30 µM for
6-8 h before the cells were harvested.
Viability Assay--
H1299-Val-138 cells were cultured to 60%
confluency at 39 °C and shifted to 32 °C for various time points.
Cells were trypsinized and counted. Two hundred cells were plated in
each 10-cm dish and incubated for 10 days at 39 °C. The plates were
stained, and visible colonies were counted. The experiment was
performed three times, each with duplicate plates for each time point.
Cytochrome c Release Assay--
Cytochrome c release
from mitochondria was determined as described by Yang et al.
(39). Cells were washed once with ice-cold phosphate-buffered saline
and resuspended in 5 volumes of buffer A (20 mM Hepes-KOH,
pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 250 mM sucrose). Cells were disrupted by 10 strokes in a Dounce homogenizer using a type B pestle and centrifuged twice at 1,000 × g for 6 min each at 4 °C, and the supernatant was
centrifuged at 10,000 × g for 15 min at 4 °C. The
pellet was saved as the mitochondrial fraction. The supernatant was
centrifuged at 100,000 × g for 1 h, and the
supernatant was saved as cytoplasmic soluble fraction. Cytochrome
c was detected by Western blot using an antibody from PharMingen.
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RESULTS |
The Human p53 Val-138 Mutant Is Temperature-sensitive--
A CMV
promoter-driven human p53 cDNA construct containing an amino acid
138 alanine-to-valine point mutation (Val-138) was stably transfected
into the p53-null human lung carcinoma cell line H1299 (40). The
Val-138 mutation is equivalent to the murine p53 codon 135 alanine-to-valine mutation (Val-135) that renders the protein
temperature-sensitive (41). H1299 cells were transfected at 39 °C,
and a stable cell line (H1299-Val-138) expressing the p53 protein was
selected and maintained at 39 °C. Incubation of H1299-Val-138 cells
at the permissive temperature of 32 °C resulted in growth arrest at
the first 48 h, very few cells displayed apoptotic morphology
(Fig. 1). About 84 h after
temperature shift, a small subset (<10%) of cells displayed membrane
blebbing and detached from the plate. The appearance of cells with
apoptotic morphologies was associated with a strong increase in a
caspase activity specific to the DEVD sequence (see below). The
remaining cell population was stable for up to 10 days, when revertant
colonies of proliferating cells began to appear. Therefore, the
phenotype of H1299-Val-138 cells at 32 °C is growth arrest, followed
by delayed and incomplete apoptosis.

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Fig. 1.
Morphology of H1299-Val-138 cells at
32 °C. The human p53 138 alanine-to-valine mutant (Val-138) was
stably transfected into H1299 cells at 39 °C. Cells were grown to
~50% confluency at 39 °C and shifted to 32 °C. The same plate
of cells was photographed at the indicated time points. After 120 h, cell morphologies remain similar for up to 240 h.
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Western blot analysis of two p53-inducible products,
p21WAF1 and MDM2, showed that both were strongly
induced after incubation at 32 °C (Fig.
2A), without significant
change in the level of p53. This is consistent with the Val-138 mutant
protein being activated at 32 °C by a conformational change. When
the H1299-Val-138 cells were further stably transfected with the
p53-responsive reporter BP100-luciferase, up to 270-fold activation of
luciferase expression was detected after 24 h at 32 °C and
persisted through the 96-h observation period (Fig. 2B).
These results confirm that the human p53 Val-138 mutant is
temperature-sensitive in its transcription activation function, similar
to its murine p53 counterpart.

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Fig. 2.
The human p53 Val-138 mutant is
temperature-sensitive. A, Western blot of p53, MDM2,
and p21WAF1 expression in the H1299-Val-138 cell
line. Cells maintained at 39 °C were shifted to 32 °C for
indicated times. p53 and p21 levels were determined by direct Western
blot. MDM2 levels were determined by immunoprecipitation with 2A9
followed by Western blot with 3G9. B, activation of a stably
transfected p53-inducible reporter BP100-luciferase in H1299-Val-138
cells. Cells were shifted to 32 °C for different time points, and
luciferase activity was determined and normalized to protein
concentration. The basal luciferase activity in 39 °C culture was
set as 1-fold. Each point is the average of two experiments.
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MDM2 Is Cleaved into a p60-kDa Product after p53
Activation--
Previous studies showed that MDM2 can be cleaved into
a 60-kDa fragment (p60) by caspase in cells undergoing apoptosis
(30, 32). We have also found that MDM2 is cleaved in certain tumor cell
lines (e.g. JAR) in the absence of apoptosis (33),
suggesting that significant cleavage of MDM2 can also be carried out by
a caspase that is active in non-apoptotic cells. This appears to be a
property unique to MDM2. To determine whether cleavage of MDM2 can be
regulated by p53 before the onset of apoptosis, H1299-Val-138 cells
were incubated at 32 °C for up to 72 h, and MDM2 protein was
detected by Western blot using monoclonal antibody 3G9 (detects both
full-length MDM2 and the N-terminal cleavage product p60) or 4B11
(detects the C-terminal fragment) (37). At early time points up to
24 h at 32 °C, only high levels of full-length MDM2 were
detected. Both 60- and 30-kDa cleavage fragments were detectable 30 h after p53 activation (Fig.
3A). The production of p60
fragment was partially inhibited by treatment with Z-VAD-FMK, a
cell-permeable broad spectrum caspase inhibitor. The timing of p60
production correlates with the timing of p53 transcription activity
reaching maximum (Fig. 2B).

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Fig. 3.
Production of a 60-kDa MDM2 fragment after
p53 activation. A, H1299-Val-138 cells were grown to
80% confluency at 39 °C and shifted to 32 °C in the absence or
presence of 30 µM Z-VAD-FMK. MDM2 was detected by direct
Western blot using the 3G9 monoclonal antibody that detects both
full-length MDM2 and the N-terminal fragment p60. B,
detection of the MDM2 C-terminal cleavage fragment. Western blot was
probed with the anti-MDM2 monoclonal antibody 4B11, which recognizes
full-length MDM2 and the C-terminal fragment, but not p60. The 46-kDa
band is a cross-reacting background. C, p60 is not produced
before p53 activation. H1299-Val-138 cells were stably transfected with
a CMV-driven Flag-tagged MDM2 cDNA. Flag-MDM2 was detected by
anti-Flag immunoprecipitation and 3G9 anti-MDM2 Western blot. JAR cells
stably transfected with Flag-MDM2 was used as positive control for
Flag-p60 production.
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The delayed production of p60 could be caused by the slow accumulation
of p60 generated by a protease that is constitutively active or due to
the activation of a caspase by p53. To distinguish between these
possibilities, the cleavage status of basal MDM2 was examined. H1299
cells express very low levels of MDM2 at 39 °C, when p53 is not
active. To facilitate the detection of MDM2 cleaving activity,
H1299-Val-138 cells were stably transfected with a Flag epitope-tagged
human MDM2 driven by a CMV promoter. The constitutively active CMV
promoter directed expression of high levels of epitope-tagged MDM2 at
both 39 and 32 °C (Fig. 3C). When Flag-MDM2 was examined
by anti-Flag immunoprecipitation and anti-MDM2 Western blot, only
full-length MDM2 was detected at 39 °C. Significant production of
Flag-p60 occurred after 30 h incubation at 32 °C (Fig.
3C). As a control, JAR cells (p53 wild type) stably
transfected with Flag-MDM2 produced high levels of Flag-p60, consistent
with our previous observation that JAR cells constitutively produce p60
from the endogenous MDM2 gene (33). These results suggest
that high level p53 activity in H1299 cells leads to the activation of
a caspase and cleavage of MDM2. Additional experiments using H1299
cells at 32 °C also excluded the possibility of a simple temperature
effect (see below).
Detection of Caspase Activity after p53 Activation--
To test
directly the activation of caspase by p53, cytosol was prepared from
H1299-Val-138 cells cultured at 32 °C at different time points and
incubated with commercially available caspase fluorogenic substrates
Z-VAD-AMC, Ac-DEVD-AMC (detects caspase 3 and 8), and Ac-WEHD-AMC
(caspase 1). Use of these substrates failed to detect a significant
increase in cleavage activity in growth-arrested H1299-Val-138 cells
(incubated at 32 °C for up to 48 h) (Fig.
4A). An activity that rises
strongly at 84 h was detected using Ac-DEVD-AMC (representing the
apoptotic substrate PARP), correlating with the appearance of
apoptosis in a subset of cells.

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Fig. 4.
Detection of caspase activity in
H1299-Val-138 cells. A, activation of a DEVD-specific
activity at late time points after p53 activation. Cytosol prepared
from H1299-Val-138 cells were incubated with Ac-DEVD-AMC, Z-VAD-AMC,
and Ac-WEHD-AMC. The release of free AMC was determined by fluorometry.
B, induction of a DVPD-specific activity after p53
activation. H1299-Val-138 cytosol was incubated with the MDM2 cleavage
site substrate Ac-DVPD-AMC. Parental H1299 cells incubated at 32 °C
were used as a control. C, comparison of net increase of
DVPD and DEVD-specific activity after p53 activation. Results in
A and B were shown after basal activities in
39 °C cytosol were subtracted. D, relative levels of
DVPD-specific activity in cell lines. Identical amounts of cytosol from
JAR (high level p60) and SJSA (low level p60) were incubated with
peptide fluorogenic substrates. The results in A,
B, and D are averages of two experiments. The
same H1299-Val-138 cytosol samples were used to obtain the results
in A and B.
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Since the cleavage of Flag-MDM2 after temperature shift gave a strong
indication that an MDM2-cleaving enzyme was activated before 30 h,
a MDM2-specific substrate Ac-DVPD-AMC was synthesized based on the
caspase cleavage site of MDM2 codon 361. Incubation of Ac-DVPD-AMC with
H1299-Val-138 cytosol revealed an activity that is present at a very
low basal level in 39 °C cells but is induced over 3-fold after
28 h at 32 °C and increased further at 84 h (Fig.
4B). As a control for temperature effect, H1299 cells
incubated at 32 °C did not show an increase in Ac-DVPD-AMC activity
up to 48 h. The increase at later time points is associated with
overcrowding of the cultures since H1299 cells continue to proliferate
at 32 °C. These results confirm that p53 activation leads to the
induction of a caspase activity at early time points that
preferentially reacts with the MDM2 cleavage site.
H1299-Val-138 samples collected at early 32 °C time points also
showed a weak increase in DEVD-specific activity (Fig. 4A). Therefore, the net increases in DEVD- and DVPD-specific activities were
also compared after subtracting the background activity of 39 °C
samples (using data from Fig. 4, A and B). This
conversion showed that the net increase of DEVD-specific activity is
only 30-50% that of DVPD-specific activity at early time points (Fig. 4C) but is 300% that of DVPD at the 84-h time point
(compare Fig. 4, A and B). This is consistent
with the interpretation that the caspase-cleaving DVPD at early time
points is different from the DEVD-cleaving protease.
The p60 MDM2 fragment was found to be produced at high levels in JAR
cells but not in SJSA cells, both overexpressing full-length MDM2
(33-35). When directly compared for their caspase activities, both
cell lines showed similar activities with three commercial substrates,
whereas JAR cytosol cleaved Ac-DVPD-AMC with 10-fold higher efficiency
than SJSA (Fig. 4D). The level of DVPD-specific caspase
activity in JAR cells is comparable with H1299-Val-138 cells incubated
for 28 h at 32 °C (Fig. 4C), consistent with the relative ratio of full-length MDM2 and p60 in these cells. These results confirm the presence of a MDM2-specific caspase activity at
various levels in different cell lines, correlating with the levels of p60.
The p53-inducible Caspase Activity in H1299 Is Distinct from
Caspase 8--
Since activation of p53 may activate the KILLER/DR5 or
Fas signaling pathways (31, 42), both capable of activating caspase 8 (FLICE), we tested whether caspase 8 may be activated in H1299-Val-138 cells. Recombinant caspases 3 and 8, tagged by polyhistidine, were
expressed in E. coli and purified using
Ni2+-chelating chromatography. When incubated with in
vitro translated MDM2, caspase 8 was able to cleave wild type MDM2
but not the 361 aspartic acid to alanine point mutant (Fig.
5A), suggesting that MDM2 can
be a substrate of caspase 8, and the cleavage is dependent on the DVPD
sequence at residue 361. Caspase 6 only has weak activity against
in vitro translated MDM2 (data not shown), whereas caspase 3 was highly effective in cleaving MDM2 (Fig. 5A), as reported
previously (30).

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Fig. 5.
The p60-producing protease is distinct from
caspase 8. A, recombinant caspase 8 is capable of
cleaving MDM2 after DVPD361. In vitro
translated, 35S-labeled MDM2 and the non-cleavable 361 aspartic acid to alanine point mutant were incubated with purified
caspases produced in E. coli. The reaction mixtures were
fractionated by SDS-polyacrylamide gel electrophoresis, and MDM2
fragments were detected by autoradiography. B, relative
efficiencies of recombinant caspases in the cleavage of peptide-AMC
substrates. Identical amounts of each purified recombinant enzyme were
incubated with substrate, and the release of free AMC was determined by
fluorometry. The amounts of each enzyme were adjusted such that
Ac-DEVD-AMC was cleaved at similar rates.
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When assayed using the peptide fluorogenic substrates, recombinant
caspases 3, 6, and 8 cleaved Ac-DEVD-AMC more efficiently than
Ac-DVPD-AMC (Fig. 5B). Caspase 6 was nearly inactive against Ac-DVPD-AMC. These substrate specificities are opposite that of the
H1299-Val-138 32 °C cytosol, which showed a preference for Ac-DVPD-AMC. Therefore, the protease that is initially activated by p53
appears to be distinct from caspases 3, 6, and 8. Western blot of
H1299-Val-138 cells at 32 °C did not reveal a change in the level of
caspase 8 proenzyme (data not shown), consistent with a lack of caspase
8 activation in this system. A previous study also ruled out caspase 1, which cannot cleave MDM2 (32).
p53 Induces Caspase Activation before Commitment to
Apoptosis--
H1299-Val-138 cells cultured for 30-48 h at
32 °C did not show obvious signs of apoptosis, despite significant
cleavage of MDM2. To determine whether the cells were indeed
non-apoptotic, the cleavage status of an apoptotic caspase substrate
PARP (43) was examined. Western blot analysis of PARP using a
monoclonal antibody capable of detecting full-length 117-kDa and the
large 87-kDa fragment of PARP showed that in adherent H1299-Val-138 cells incubated at 32 °C for up to 72 h, PARP was not cleaved (Fig. 6A). As a positive
control, H1299-Val-138 cells were treated with DNA-damaging agents
camptothecin or adriamycin at 32 °C for 24 h. This resulted in
apoptosis accompanied by the cleavage of PARP. Therefore, the
H1299-Val-138 cells with significant cleavage of MDM2 are not at the
end stage of apoptosis characterized by PARP cleavage. This result also
excluded the possibility that the p60 we detected at early 32 °C
time points was contributed by a small number of apoptotic cells.

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Fig. 6.
Absence of early apoptosis in H1299-Val-138
cells after p53 activation. A, cleavage status of PARP
in H1299-Val-138 cells incubated at 32 °C was determined by Western
blot. H1299-Val-138 cells treated with 10 µM camptothecin
or adriamycin for 24 h at 32 °C were used as positive controls.
B, viability of H1299-Val-138 cells cultured at 32 °C.
Cells incubated for indicated time points at 32 °C were trypsinized
and seeded into duplicate plates at low density. Colony formation
efficiencies were determined after incubation at 39 °C for 10 days.
Data are averages of three experiments. C, absence of
cytochrome c release after p53 activation. Soluble
cytochrome c in H1299-Val-138 cells were determined after
incubation at 32 °C for 30 h by Western blot. Apoptotic JAR
cells induced by staurosporine were used as a positive control.
P, pelleted mitochondrial fraction; S, soluble
cytoplasmic fraction.
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Next, the viability of the growth-arrested cells after returning to
39 °C was determined. After incubation at 32 °C, adherent H1299-Val-138 cells were trypsinized; 200 cells were plated in each
10-cm dish and incubated for 10 days at 39 °C before visible colonies were counted. The results showed that after 30 h at
32 °C, there was no decrease in cell viability compared with cells maintained at 39 °C (Fig. 6B). Trypan blue exclusion
tests also did not reveal the appearance of dead cells from the
adherent population (not shown). Therefore, the majority of
H1299-Val-138 cells that have undergone p53 activation, caspase
activation, and MDM2 cleavage have not permanently lost viability.
Cytochrome c release from mitochondria plays an important
role in the amplification of apoptosis signals by activation of the
Apaf1-caspase 9 complex and is regulated by the apoptosis suppressor
Bcl-2 (29, 39, 44, 45). Cytochrome c release was determined
in H1299-Val-138 cells at 32 °C. As a positive control, apoptosis
was induced in JAR cells by treating with the broad spectrum protein
kinase inhibitor staurosporine for 5 h (33). This resulted in a
significant increase of soluble cytochrome c in the cell
extract devoid of mitochondria (Fig. 6C). In contrast, H1299-Val-138 cells incubated at 32 °C for 30 h did not show an increase in soluble cytochrome c. Therefore, activation of
the MDM2-cleaving protease occurs without the release of cytochrome c from mitochondria.
Caspase Cleavage of MDM2 Inactivates Its p53 Degradation
Function--
Previous experiments showed that the p60 fragment of
MDM2 was still capable of binding to p53 (30), consistent with its intact p53 binding domain. A recent report suggested that the C
terminus of MDM2 may be important for its ability to direct ubiquitination of p53 (46). Therefore, we tested the ability of p60 to
promote p53 degradation. A plasmid expressing p60 was cotransfected
with a plasmid encoding the inactive p53 mutant 175R-H, which
eliminated the interference from an apoptotic response. Transient
cotransfection of p53 with full-length MDM2 resulted in a significant
reduction of p53 level; however, this effect was not apparent with the
p60 MDM2 fragment (Fig. 7A),
suggesting that p60 is not capable of promoting p53 degradation.

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Fig. 7.
A, the MDM2 p60 fragment is deficient
for p53 degradation. H1299 cells were cotransfected with a p53-His-175
mutant plasmid (2 µg) and an MDM2 expression plasmid (10 µg).
pCMV, vector; Wt, wild type MDM2;
361D-A, a non-cleavable MDM2 point mutant.
1-361, a cDNA encoding p60. The p53 protein level was
determined by Western blot 36 h after transfection. B,
p60 overexpression stabilizes p53. HT1080 cells were stably transfected
with MDM2 expression plasmids. Pooled colonies were analyzed for MDM2,
p53, and p21WAF1 levels by Western blot. The
levels of p53 bound to MDM2 were determined by anti-MDM2
immunoprecipitation with 2A9 antibody and anti-p53 Western blot. The
control is untransfected HT1080.
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Since p60 is still capable of binding to p53 with similar efficiency as
full-length MDM2, it is possible that it can function in a
dominant-negative fashion to prevent p53 degradation by full-length MDM2. To test this possibility, a cDNA expression plasmid encoding p60 was stably transfected into a human fibrosarcoma cell line HT1080,
which contains wild type p53. p53 protein levels in a pool of stable
transfectants was analyzed by Western blot. Overexpression of p60
induced strong accumulation of p53 (Fig. 7B). A similar result was also observed when p60 is transiently expressed in HT1080
cells (not shown). Immunofluorescence staining of p60-overexpressing cells revealed an increase in nuclear p53 (not shown). This result suggests that p60 binding to p53 can protect p53 from degradation by
full-length MDM2. Therefore, the p60 fragment has lost the ability to
target p53 for degradation and may be able to regulate p53 turnover by
a dominant-negative mechanism.
Because p60 was overproduced in transfection experiments, the
stabilized p53 was not transcriptionally active. Thus, there was no
obvious alteration in cell proliferation and the level of
p21WAF1 expression (Fig. 7B). High
levels of p53 were coprecipitated with p60 (Fig. 7B),
indicating that most p53 molecules were sequestered in a complex with
p60. Further experiments are needed to determine whether producing
physiological levels of p60 can result in increase of p53 level and activity.
 |
DISCUSSION |
The data presented here suggest that p53 can regulate a
proteolytic activity specific for the caspase cleavage site of MDM2, before the onset of apoptosis and activation of other apoptotic caspases. The p53-induced protease activity is less efficient against
peptide substrates for caspases 1, 3, and 8, suggesting the presence of
a distinct MDM2-specific protease. The ability of this protease to
cleave after the aspartic acid residue in the caspase cleavage site of
MDM2 (DVPD361), the partial sensitivity to caspase
inhibitor Z-VAD-FMK, and resistance to conventional serine and cysteine
protease inhibitors suggest that it is a caspase.
By using the MDM2 cleavage site substrate Ac-DVPD-AMC, we also detected
an activity that is present in JAR cells, which constitutively produce
cleaved MDM2 (p60) (33). Furthermore, this activity is nearly absent
from SJSA cells, which lack significant p60 (33). In contrast, use of
other fluorogenic peptide substrates revealed similar levels of
cleavage activities in both cell lines. This further supports the
presence of a protease that preferentially targets MDM2. The
variability of the MDM2-specific protease activity suggests that it may
be a regulatory rather than an effector protease in apoptosis. Thus,
JAR cells with a significant level of MDM2 cleavage proliferate
normally without committing to apoptosis (33).
Cleavage after DVPD361 results in the separation of the
highly conserved MDM2 C-terminal Ring finger domain from the p53
binding domain. It has been shown that cysteine 464 is important for
the ubiquitin ligase activity of MDM2 in vitro (46). p60
does not promote p53 degradation; furthermore, it can function in a
dominant-negative fashion to protect p53 from degradation by
full-length MDM2. These results suggest that the 362-491 region of
MDM2 is an important functional domain for promoting p53 degradation.
The C-terminal cleavage fragment also contains the RNA binding activity
of MDM2 (30, 47), which may also be associated with the ability of MDM2
to promote p53 degradation.
The ability of p53 to activate an MDM2-specific caspase suggests a
signaling pathway for p53-mediated apoptosis. A threshold level of p53
may induce cleavage of MDM2 before commitment to cell death, resulting
in a positive feedback effect on p53 level and further activation of
the protease. In addition to cleavage of MDM2, the protease may be
responsible for subsequently activating the DEVD-reactive caspase,
forming signal transduction and amplification cascades that lead to
commitment to apoptosis. This model is consistent with the
dosage-dependent apoptotic effect of p53 (48). Certain tumor cell lines undergo growth arrest after p53 activation; it will be
interesting to determine whether these cells fail to activate the
MDM2-cleaving caspase.
During apoptosis induced by survival factor withdrawal in the mouse
myeloid cell line M1, p53 activity is required for caspase cleavage of
pRb (49). The pRb-cleaving caspase can also be activated during the
preparation of cytosol (49). However, pRb is not cleaved in
H1299-Val-138 cells producing p60, or by cytosol prepared from 32 °C
H1299-Val-138 cells does not cleave in vitro translated pRb,2 suggesting important
differences between the caspases involved. The activation of MDM2 and
pRb-cleaving caspases may be related events in a p53-regulated pathway;
the pRb-cleaving caspase may be activated in a step more proximal to
the commitment to apoptosis.
In addition to caspase 3, MDM2 is also efficiently cleaved by
recombinant caspase 8, suggesting that signaling pathways involving caspase 8 may be able to regulate p53 stability by modification of
MDM2. Two cell death-promoting factors that act by activation of
caspase 8, i.e. tumor necrosis factor-
and FasL, are
known to induce p53 accumulation (50, 51). It is possible that caspase 8 cleavage of MDM2 contributes to the stabilization of p53 by these
factors. Proteolytic modification of MDM2 may provide a mechanism for
other signaling pathways to interact with the p53 pathway.
Activation of DEVD-specific caspases in the absence of apoptosis
have been observed in mitogen-activated lymphocytes undergoing active
proliferation, before the appearance of apoptosis (52). Our
results provide further evidence for the existence of a novel caspase
active in proliferating or growth-arrested cells, prior to commitment
to cell death.
In summary, this study revealed the presence of a unique p53-regulated
caspase that mediates the pre-apoptotic cleavage and inactivation of
MDM2. This caspase activity may function in a positive feedback
mechanism of p53 and may also have a role in the apoptotic signaling by
p53. The protease is constitutively active in certain tumor cell lines,
suggesting that it may also have a function in non-apoptotic
conditions. Identification of the MDM2-specific caspase may shed light
on the regulation of MDM2 and the apoptotic mechanism of p53.