From the Department of Genetics, Yale University School of
Medicine, New Haven, Connecticut 06520 and the
Medical Breast Cancer Section, Medicine Branch, National
Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892
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INTRODUCTION |
DNA damage agents are mutagens that induce genetic lesions and
cause cancer. Mammalian cells respond to DNA damage signals by
activating cell cycle checkpoints which arrest the cell cycle in both
G1 and G2 phases or by inducing programmed cell
death (apoptosis) (1, 2). It is well established that the p53 tumor
suppressor is responsible for both DNA damage-induced G1 cell cycle arrest and apoptosis (3). The DNA damage-induced G1 cell cycle arrest is in part caused by the
p53-dependent transcriptional activation of
p21Cip1/Waf1 (4), a potent inhibitor of the cell cycle
kinases (cyclin/CDKs) that binds to various cell cycle kinases (5, 6).
In vivo, a major fraction of p21 also associates with the
proliferation cell nuclear antigen
(PCNA),1 a replication and
repair factor (7, 8). The binding of p21 has been shown to inhibit the
replication, but not repair, activity of PCNA in vitro (9,
10). Although it remains unclear the in vivo significance of
the association between p21 and PCNA, the co-localization of p21 with
PCNA at the DNA repair foci, and the fact that p21 can associate with
active cell cycle kinases suggest that p21 may play a role during the
DNA damage response (8, 10, 11).
Activation of p53 also induces apoptosis and dysfunction of p53 leads
to cellular resistance to genotoxic stress such as DNA damage (3).
However, it is still unclear how p53 induces apoptosis in certain
cells but causes cell cycle arrest in other cells. Several observations
suggest that failure to express sufficient levels of p21 converts the
normal cell cycle arrest into apoptotic cell death (12, 13). In human
cancer cells that normally respond to DNA damage by cell cycle arrest,
deletion of p21 genes by homologous recombination leads to the loss of
cell cycle checkpoint control and cells die through apoptosis (12). The
expression of p21 is also controlled by mitogenic growth factors (13).
Growth factor withdrawal reduces p21 expression. DNA-damaged cells
cultured under growth factor starvation conditions die through
apoptotic death instead of normal cell cycle arrest (15). Conversely, it has been reported that ectopical expression of p21 prevents the
p53-mediated apoptosis in human melanoma cells (16).
Genetic and biochemical studies indicate that programmed cell death or
apoptosis is triggered by activation of the members of the
CED-3/caspase protease family (17, 18). These proteases preferentially
cleave protein substrates at certain aspartic acid residues. Activation
of caspases during apoptosis converts the inactive, pro-enzyme form of
capases into the active, processed form which in turn cleaves
downstream substrates, leading to the appearance of apoptotic
morphologies such as condensation of nucleoplasm, blebbing of nuclear
and cytoplasmic membranes, and fragmentation of cells to form apoptotic
bodies, which are accompanied with biochemical events such as DNA
fragmentation (2). In mammals, a number of caspases have been
identified. Among them, caspase-3 (formerly CPP32, Yama, or apopain)
has been implicated in playing a critical role during apoptosis (19,
20). So far, a number of caspase substrates have been identified. These
include poly(ADP-ribose)polymerase, nuclear lamin,
DNA-dependent protein kinase (DNA-PK), and the U1
RNA-associated 70-kDa protein (21-24). However, it remains unclear whether these proteins are the critical substrates that cause the
manifestation of apoptotic morphology and the eventual apoptotic death
of the cells.
To investigate how cells control the conversion between cell cycle
arrest and apoptosis during DNA damage response, we analyzed the
molecular events of cell cycle regulation during
-irradiation. We
report here that in cells that are prone to apoptosis, p21 is rapidly
induced by DNA damage but is selectively cleaved by a caspase-like
activity. p21 thus appears to be a critical checkpoint target for both
cell cycle arrest and apoptosis.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Metabolic Labeling--
The ML-1 (human
myeloblastic leukemia) cells were a gift from Dr. David Beach (Cold
Spring Harbor Laboratory) and were cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum. WI38 (human lung
fibroblasts), HCT116 (human colon carcinoma), and DLD-1 (human colon
adenocarcinoma) cells were purchased from ATCC. They were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum at 37 °C. For metabolic labeling of cells with
[35S]methionine, ML-1 cells were irradiated with a
137Cs irradiator at various doses and then cultured in the
methionine-deficient medium supplemented with 10% dialyzed fetal
bovine serum (Life Technologies, Inc.) in the presence of 250 µCi/ml
[35S]methionine (25). For the pulse and chase
experiments, the cells were labeled for 2 h in medium containing
[35S]methionine. The labeling medium was then removed and
the cells were washed twice with warm RPMI 1640 medium. The chase was
performed in the regular RPMI 1640 medium in the presence of 1 mM methionine.
Antibodies and Immunological Procedures--
The rabbit
polyclonal antibodies raised against full-length human p21 and cyclin A
were described previously (8). The CDK2 antibody was raised against the
12 amino acid residues at the carboxyl-terminal as described (7). The
rabbit polyclonal antibody against human p27 was raised against a
fusion protein (GST-p27), between glutathione S-transferase
and human p27 using the protocols as described previously (7, 25). All
the antibodies were gifts from Dr. David Beach.
Control or irradiated cells were harvested and immunoprecipitated in a
buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.4, 100 mM sodium fluoride, and 10 µg/ml each of the following protease inhibitors: trypsin inhibitor,
aprotinin, and leupeptin, as well as 1 mM benzamidine for
3 h at 4 °C (25). Western blot analysis of various proteins was
conducted using the ECL method (Amersham) as described before (25). The
kinase assay using histone H1 as the in vitro substrate was
conducted as described (8).
Adenovirus Purification and Infection--
The construction of
adenoviruses encoding lacZ, p21, and p53 at the E1 locus under the
cytomegalovirus promoter control has been described previously (16, 26,
27). They were amplified using 293 cells as the host. The viruses were
purified through two rounds of CsCl density gradient centrifugation
according to Graham and Prevec (28). For infection, 5 × 105 cells were grown to log phase and infected with
adenoviruses at 10 plaque forming units/cell. Twenty-four or 48 h
post-infection, the cells were harvested and analyzed by flow cytometry
analysis using propidium iodide staining as described (29). The protein level and cleavage of p21 in the adenovirus-infected cells were analyzed using immunoprecipitation and Western blot analyses as described above.
Extract Preparation and Assays for p21
Cleavage--
Non-irradiated and irradiated ML-1 cells were collected
6 h post-irradiation. They were suspended in a buffer containing
10 M Hepes, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 10 µg/ml each of the following
protease inhibitors: trypsin inhibitor, aprotinin, leupeptin, as well
as 1 mM benzamidine (30). Cells were homogenized with a
Dounce homogenizer with a loose-type pestle. The cytosolic fraction was
obtained after spinning at 100,000 × g and was stored
at
80 °C until use. For in vitro translation, the
cloned p21 cDNA in Bluescript-II SK- (Stratagene) was transcribed and translated with T3 RNA polymerase in the T3-coupled TNT rabbit reticulocyte lysate system (Promega) in the presence of
[35S]methionine. Five microliters of the in
vitro translated p21 protein was incubated with 50-µl extracts
(40 µg of total protein) or caspase-3 (2 µg) for 30 min at
30 °C. The protein products were isolated by immunoprecipitation.
The GST-caspase-3 DNA construct was kindly provided by Dr. Emad
Alnemeri (Thomas Jefferson University). It was expressed in
Escherichia coli (BL21 strain) and affinity purified by
glutathione-Sepharose beads (Pharmacia Biotech Inc.). The caspase
inhibitor, AC-DEVD-CHO, was obtained from Bachem and used at 100 nM. The site-directed mutagenesis for the p21 mutants was
conducted using the method as described before (8).
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RESULTS |
Association of p14 with Cyclin A/CDK2 and p21 Complexes in
-Irradiated Cells--
We used the ML-1 cells, a human myeloblastic
leukemia cell that contains a functional p53, to investigate the DNA
damage response (31).
-Irradiation of ML-1 cells induced cell cycle
arrest (31). Microscopic examination revealed that a substantial
fraction of the cells underwent nuclear fragmentation and cell membrane blebbing, morphological changes typically associated with apoptotic cells (Fig. 1A). The apoptotic
response was damage dose-dependent and occurred in a time
dependent fashion (Figs. 1B and 3A). After a
latent period, apoptotic cells, about 30-50% of the total cell population, appeared at 5-6 h post-irradiation (Fig. 1B).
Further incubation up to 12 h or more, however, did not
significantly increase the fraction of the apoptotic cells. To monitor
the molecular events that may be associated with the cyclin·CDK and
p21 complexes in the irradiated ML-1 cells, the non-irradiated and
irradiated cells were labeled with [35S]methionine. The
cyclin·CDK and p21 complexes were then isolated by
immunoprecipitation with their respective antibodies. The proteins that
associated with cyclin·CDK and p21 complexes were resolved in an
SDS-polyacrylamide gel and compared between DNA damage treated and
non-treated samples. Strikingly, a 14-kDa protein was found to
specifically associate with the cyclin A, CDK2, and p21 complexes after
irradiation (Fig. 2A). The
specific association of this protein was confirmed by its disappearance
from the CDK2 complexes if a competing CDK2 antigenic peptide was
included during immunoprecipitation (Fig. 2A). Because of
these interesting features, we have further characterized p14.

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Fig. 1.
-Irradiation induces apoptosis in ML-1
cells. A, log-phase growing ML-1 cells were irradiated at 8 gray with a 137Cs irradiator. After irradiation, the cells
were cultured in RPMI 1640 medium for 8 h before being fixed with
4% formaldehyde and stained with a DNA dye, Hoechst 33248. Left, control cells without irradiation; right,
irradiated cells. The apoptotic cells show nuclear and cytoplasmic
blebbing. B, time course for the appearance of apoptosis.
The cells were irradiated (8 gray) and scored for apoptotic phenotypes
at various time points under a microscope. Vertical:
percentage of apoptotic cells. Horizontal: time (hours)
after irradiation.
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Fig. 2.
Association of p14 with cyclin A·CDK2 and
p21 complexes in response to DNA damage. A, ML-1 cells were
irradiated (8 gray) and labeled with [35S]methionine for
8 h. The cyclin A, CDK2, and p21 complexes were isolated from
non-irradiated control (0 h) or irradiated cells (8 h) by
immunoprecipitation with respective antibodies as indicated. The CDK2
immunoprecipitation was conducted either in the absence ( ) or
presence of an antigenic CDK2 peptide (+). The molecular weight
standards (in kDa) are shown on the left. B,
pulse and chase analysis. Irradiated ML-1 cells were labeled with
[35S]methionine for 2 h (pulse). The labeling medium
was then removed and cells were cultured in the regular medium in the
presence of 1 mM methionine (chase). The cells were
harvested at various time points during the chasing period as
indicated. The CDK2 complexes were isolated by immunoprecipitation.
Similar results were obtained with p21 and cyclin A
immunoprecipitation.
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To determine the identity of p14, we tested whether p14 is from a
pre-existing protein or a new protein that is induced by DNA damage. To
distinguish these possibilities, a pulse and chase experiment was
performed. Both control and irradiated cells were pulse labeled for
2 h with [35S]methionine. After removal of the
[35S]methionine-containing medium, the cells were
re-cultured in regular medium and harvested at different time points
(chase). The labeled proteins that associated with cyclin A, CDK2, and p21 complexes were isolated by immunoprecipitation and analyzed for
their kinetics of disappearance or appearance.
Comparison of the patterns of the proteins associated with the
CDK2·p21 complexes revealed that p14 was not present during the
initial pulse labeling period (first 2 h) after irradiation. However, although the labeled p21 and other proteins were disappearing during the chasing period, p14, in contrast, started to appear in the
cyclin A·CDK2 and p21 complexes during subsequent hours of chasing
(Fig. 2B). As observed before, the association of p14 occurred only in the irradiated cell samples, suggesting that p14 might
come from a protein that is labeled during the initial pulse labeling
period in irradiated cells.
p14 Is a Cleavage Product of p21 after DNA Damage--
To identify
the source of p14, we blotted the immunoprecipitated p21 and cyclin
A·CDK2 complexes with p21, CDK2, and cyclin A-specific antibodies. As
expected, p21 was rapidly induced after irradiation (Fig.
3A). In addition, the p21
antibody could detect the induction of an additional protein of 14 kDa
in the irradiated cell samples. p14 became detectable between 2 and
3 h after
-irradiation, which paralleled with the p21 induction
profile (Fig. 3A). To determine whether the presence of p14
is associated with the apoptotic process rather than cell cycle arrest,
the p21 complexes from WI38 human fibroblasts and ML-1 cells were
compared after
-irradiation. WI38 cells respond to DNA damage by
cell cycle arrest but not apoptosis (32). Although we could clearly
detect the induction of p21 in WI38 cells after irradiation, p14 was
present only in irradiated ML-1 cells (Fig. 3A). To further
examine whether p14 is associated with apoptosis, we tested its
potential presence in apoptotic cells induced by other means. ML-1
cells undergo apoptotic death once they reach high cell density.
p14 was also detectable in the ML-1 high density-induced apoptotic
cells but not in the actively growing cells (Fig. 3A). These
studies suggest that p14 might be a proteolytic product of p21 in cells
that are prone to apoptotic death. Since p14 is induced much earlier
than any detectable apoptotic phenotypes such as nuclear blebbing, the
cleavage of p21 appears to be an early event during the DNA damage-induced apoptosis. Interestingly, we found that the cleavage of
p21 is quite selective, since other cell cycle regulators such as CDK2,
CDK4, CDC2, cyclin A, and p27, were not detectably cleaved after
irradiation (Fig. 3B, and data not shown).

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Fig. 3.
p14 is a cleavage product of p21 in cells
that undergo apoptosis. A, left panel,
ML-1 cells were -irradiated (8 gray) and harvested at various time
points as indicated; middle panel, WI38 and ML-1 were
irradiated with various doses of -irradiation as indicated and
harvested at 6 h post-damage treatment. Right panel,
ML-1 cells were grown under log-phase (log) or saturation
(sat) conditions. p21 complexes were isolated by
immunoprecipitation and detected by Western blot analysis with the
anti-p21 antibody. B, p27 and CDK2 were isolated from
irradiated cells at various time points by anti-p27 or CDK2
immunoprecipitation and examined by Western blot analyses. The kinase
activity of CDK2 from the control and irradiated cells was also assayed
using the histone H1 as the substrate.
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We have examined the kinase activity of CDK2 complexes after
irradiation using the histone H1 as the in vitro substrate.
The kinase activity of CDK2 became inhibited 3 h after
-irradiation (Fig. 3B). This might be due to the fact
that only a fraction of p21 is cleaved and there is substantial
increase in total amounts of p21 after
-irradiation (Fig.
3A). In addition, we found that the transcription of cyclin
A and cyclin B became inhibited 6-8 h after the DNA damage treatment
(data not shown).
Induction of p21 Cleavage by p53-induced Apoptosis--
Since ML-1
is a p53 positive cell line (31) and p53 induces both cell cycle arrest
and apoptosis, we tried to determine whether the expression of p53
alone can induce p21 cleavage. To deliver and express p53 efficiently,
we used a recombinant adenovirus that encodes p53. We also used
adenoviruses that contain lacZ or p21 genes as the control.
Two well characterized human colon carcinoma cell lines, HCT116 and
DLD1, were used in these experiments. HCT116 cells contain a functional
p53 which have been shown to respond to p53 expression by the cell
cycle arrest (33). DLD1 cells are deficient in p53 activity and respond
to p53 expression by apoptotic death (33). While infection of HCT116 or
DLD1 cells with adenovirus encoding lacZ or p21 did not
affect cell morphology, expression of p53 in DLD1 cells caused a
fraction of these cells to become rounded up and detached from the
plate within 24-48 h post-infection. Such an apoptotic cell death was
confirmed by flow cytometry analysis (Fig.
4A). As reported before (33), p53 expression in HCT116 cells only caused the G1 cell
cycle arrest (Fig. 4A). Analysis of p21 from these
adenovirus-infected HCT116 and DLD1 cells revealed that while both p21-
and p53-containing adenoviruses induced similar high level expression
of p21, p14 was present only in the p53 adenovirus-infected DLD1 cells
(Fig. 4B). These data are consistent with our previous
findings in WI38 and ML-1 cells that p14 is likely to associate with
the p53-mediated apoptotic response during the DNA damage treatment.
The elevation of the p21 level alone is not sufficient to induce p14.

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Fig. 4.
p14 is induced during the p53-mediated
apoptosis. A, DLD1 and HCT116 cells were infected with
adenoviruses encoding lacZ (Ad-lacZ), p21 (Ad-p21), or p53 (Ad-p53) at
10 plaque forming units/cell. Cells were harvested and analyzed by flow
cytometry analysis at 24 (top) or 48 h
(bottom) post-infection. Sample order: Ad-lacZ,
front; Ad-p21, middle; and Ad-p53,
back. B, DLD1 and HCT116 cells were infected with
adenoviruses as described in A. Forty eight hours after
infection, p21 complexes were isolated by anti-p21 immunoprecipitation.
The immunoprecipitated proteins were blotted with the p21
antibody.
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p21 Is Cleaved by a Caspase Like Activity after
-Irradiation--
To determine the protease that cleaves p21 after
-irradiation, we prepared cell extracts from the control and
-irradiated ML-1 cells. To assay for p21 cleavage activity, in
vitro translated and [35S]methionine-labeled p21 was
incubated with the cell extracts. The reaction products were recovered
by immunoprecipitation with p21 antibodies and analyzed by protein gel
electrophoresis. In these experiments, the cleavage of p21 could be
recapitulated in the
-irradiated extract but not in the control
extract (Fig. 5A). As shown in
Fig. 5B, the in vitro cleaved p14 product
migrated precisely as that of p14 isolated from the in vivo
irradiated samples. Since one prominent event during apoptosis is the
activation of caspase proteases (30), we tested whether p21 could be
cleaved by known caspases such as caspase-3, a close homologue of
CED-3. A recombinant fusion protein between the glutathione
S-transferase and caspase-3 (formerly CPP32) (GST-caspase-3)
was expressed in bacteria and isolated using glutathione-Sepharose
beads. Incubation of in vitro translated p21 with caspase-3
produced a p14 which is identical to the one obtained from either
in vivo cell samples after
-irradiation or from the
irradiated cell extract (Fig. 5, A and B).
Partial V8 protease mapping of p14 obtained from various sources
confirmed that they were identical (Fig. 5C). In addition,
we also found that p21 cleavage in the irradiated cell extract could be
inhibited by the caspase-3 inhibitor, AC-DEVD-CHO (30) (Fig.
5A), suggesting a caspase-like activity in the
-irradiated cells is responsible for the p21 cleavage.

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Fig. 5.
p21 is cleaved by a caspase like activity.
A, in vitro translated p21 (p21 input) was
incubated with either ML-1 extracts prepared from non-irradiated
(control extract) or irradiated cells (IR extract). It was
also treated with recombinant GST S-transferase or
GST-caspase-3 (GST-Cas-3) as indicated. The effect of the caspase
inhibitor, AC-DEVD-CHO, was also examined ( /+ inhibitor). The
reaction products were isolated by immunoprecipitation with p21
antibodies and separated in a protein gel. The proteins were visualized
by autoradiography. B, comparison of p14 proteins from
in vivo and in vitro samples. The p21 complexes
were isolated from non-irradiated (control) and irradiated ML-1 (IR)
cells after labeling with [35S]methionine for 5 h.
The mobility of p14 was compared with p14 obtained either from the
in vitro irradiated cell extract (IR extract) or
from the caspase-3 cleavage reaction as indicated. C, V8
partial protease mapping. p14 obtained from either IR extract,
caspase-treated, or in vivo irradiated ML-1 cell samples
were compared by the partial V8 protease mapping method as indicated.
The p15 protein from the in vivo p21 immunoprecipitation was
used as a control (Fig. 2A).
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p21 Cleavage Affects Its Binding to PCNA in Vitro--
To examine
the biological effect of the p21 cleavage, we tried to determine the
precise location of the caspase cleavage site. Inspection of the human
p21 protein sequence revealed that it contained a potential caspase-3
cleavage motif between aspartic acids 109 and 112 (AEED*HVD*LSL, the
first D* is aspartic acid residue 109 and the second is 112) (20). To
test whether these aspartic acids are the recognition and cleavage
sites of caspase-3, we converted aspartic acid 109 or 112 individually
into alanine (D109A or D112A) by site-directed mutagenesis (8). Using
the in vitro cleavage assays, our data indicated that both
D109A and D112A mutants abolished the p21 cleavage by caspase-3 (Fig.
6A). To further analyze the
caspase cleavage site, we generated truncated p21 mutants that have
deleted the regions after aspartic acid 109 (Asp109) or 112 (Asp112). These truncated p21 derivatives were in
vitro translated and their electrophoretic mobilities were
compared with that of p14. Our results indicated that aspartic acid 112 is the caspase cleavage site (Fig. 6A). Since it has been
shown that caspase-3 preferentially cleaves at an aspartic acid if a
negatively charged amino acid residue is present near its
amino-terminal region (20), the effect of D109A mutant on p21 cleavage
is likely caused by the alteration of caspase recognition site on
p21.

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Fig. 6.
Cleavage of p21 by caspase abolishes its
binding to PCNA. A, mutational analysis.
Left, p21 and p21 mutants, containing either the conversion
of aspartic acid 109 or 112 to alanine (D109A or D112A), were in
vitro translated. They were treated with recombinant caspase-3 and
the cleavage products were analyzed. Right, p21 and p21
truncation mutants, containing a translational stop codon at the end of
either aspartic acid 109 or 112 (109Z or 112Z), were in
vitro translated. They were treated with caspase-3 and the
products were resolved in a protein gel. B, PCNA and CDK2
were in vitro translated in the presence of
[35S]methionine. They were incubated with 2 µg of
purified GST, GST-p21, and GST-p21/112Z proteins at 30 °C for 30 min. CDK2 or PCNA that associated with the GST-p21 or p21 mutant was
isolated by glutathione-Sepharose beads. The proteins were separated in
a protein gel and visualized by autoradiography. C,
recombinant cyclin A·CDK2 complexes were incubated in the presence of
increasing amounts of GST-p21 or GST-p21/112Z (0, 0.5, 1.5, and 4.5 µg) as indicated. The inhibition of the kinase activity was assayed
using the histone H1 as the substrate.
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p21 can bind to both CDK and PCNA independently. Such interactions have
been shown to reside on two separate regions of p21 (34). The CDK
recognition domain has been mapped to the amino-terminal half of p21
while the PCNA-binding domain is at the carboxyl-terminal region. To
determine the potential significance of p21 cleavage, we examined
whether such a cleavage affects its binding to either PCNA or CDK2. p21
and the p21-112Z mutant derivative were fused in-frame with the
glutathione S-transferase (GST-p21 or GST-p21-112Z, respectively) and expressed in bacteria. The fusion proteins were purified by glutathione-Sepharose beads. The purified GST-p21, GST-p21-112Z, and the control GST proteins were incubated with PCNA or
CDK2 that was obtained from in vitro translation. The PCNA
and CDK2 proteins that bound to the GST p21 proteins were then
recovered from the reactions by glutathione-Sepharose beads. Our
studies indicated that while GST-p21 could bind to both PCNA and CDK2
in these assays, the GST-p21-112Z protein could only bind to CDK2 and
failed to associate with PCNA (Fig. 6B). This is consistent
with the mapped position of p21 cleavage, which is located at the
carboxyl region of p21. In addition, we have also analyzed the effects
of GST-p21 and GST-p21/112Z on the cyclin A/CDK2 activity. Both the
wild type p21 and p21/112Z mutant could inhibit the in vitro
kinase activity of cyclin A/CDK2 (Fig. 6C). These data
suggest that one direct consequence of p21 cleavage is the loss of its
interaction with PCNA, although we cannot rule out the possibility that
in vivo the cleavage of p21 by the caspase-like activity may
potentially affect p21 stability or localization during the DNA damage
response.
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DISCUSSION |
DNA damage induces both cell cycle arrest and apoptosis (1). Many
lines of evidence indicate that failure to express p21 can lead to the
apoptotic cell fate in response to genotoxic stress (12, 15, 33). It
has been postulated that the imbalance of the cell cycle signals or
failure to arrest the cell cycle may trigger the apoptotic program (1).
Indeed, ectopic expression of positive cell cycle regulators such as
E2F, CDC25, Myc, or viral oncoprotein E1A causes many cells to die
through apoptosis (14, 35). Our finding that p21 is induced in
DNA-damaged cells but selectively cleaved by a caspase-like activity
prior to the appearance of apoptosis is consistent with the previous
observations that the insufficient expression of p21 during DNA damage
response may cause apoptosis. Although we show that a direct effect of p21 cleavage is to abolish its interaction with PCNA, the truncation of
p21 during apoptosis may cause other effects such as changes in the p21
stability or cellular/nuclear localization. The cleavage of p21 may
thus alter its effective inhibitory level in the cell, leading to a
failure in the cell cycle arrest in response to DNA damage.
Cells respond to DNA damage by arresting the cell cycle at
G1 or G2 phase, a process that allows the time
for repairing the DNA lesions (31). It has been shown that p21
associates with PCNA and such an association affects PCNA replication
function. Consistently, p21 is normally absent from the replicating
nucleus (9, 11). However, p21 does not appear to inhibit the repair activity of PCNA (10). In fact, it has been reported that p21 becomes
co-localized with PCNA in the nucleus at the repair foci during the DNA
damage response, suggesting p21 may perform a function during the DNA
repair process (11). Interestingly, it has been shown that
p21-associated cyclin·CDK complexes can exist in both active and
inactive states (8). One possibility is that p21 may bring its
associated active cyclin·CDK complexes to the repair sites through
its interaction with PCNA. If this is the case, the cleavage of p21 by
caspase-like activity may interfere with DNA repair, causing prolonged
presence of DNA lesions that may trigger apoptosis.
Our data, together with previous reports (12, 13, 33), suggest that p21
is a critical checkpoint target protein for both cell cycle arrest and
apoptosis in response to DNA damage. Manipulation of the p21 levels
during DNA damage response may thus provide a novel strategy to the
cancer therapy.
We thank Dr. Emad Alnemeri (Thomas Jefferson
University) for the GST-caspase-3 DNA construct.