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INTRODUCTION |
The protein, p202, is an interferon
(IFN)1-inducible
phosphoprotein (52 kDa) whose ectopic expression in a variety of cell types retards proliferation (1-4). Interestingly, the reduced basal
levels of p202 (in consequence of the expression of antisense RNA to
202) in fibroblasts, under reduced serum condition, increase the susceptibility to apoptosis (5) and overexpression of p202 in Rat-1
cells inhibits c-myc-induced apoptosis (6). Together, these
observations support the notion that p202 participates in the
regulation of apoptosis. However, the molecular mechanism(s) remains to
be identified.
The p53 family genes include genes encoding p73, p63, and
p53 (7). The p53 tumor suppressor gene from the family
continues to hold distinction as the most frequently mutated gene in
human cancer (7-11). This has stimulated efforts to understand the
function of this gene in normal and neoplastic state. A large number of functions have been attributed to p53, including cell-cycle checkpoints and apoptosis (11). In human and rodent cells containing wild-type p53 genes, the p53 protein is induced by a variety of
stimuli, including chemotherapeutic agents, oxidative stress, hypoxia, nucleotide depletion, and oncogenic expression. Accumulated data suggest that the high frequency of p53 mutations in human cancer reflect the ability of this protein to induce programmed cell death or
apoptosis (7-11).
The underlying mechanism of tumor suppressor activity of p53 resides in
part in its ability to bind DNA in a sequence-specific manner (8, 10).
It has been reported that a substantial number of genes containing p53
binding site(s) are activated by p53 (8, 11). These include
mdm2, p21, and gadd45. p21
and gadd45 have been implicated in p53-mediated cell cycle
regulation. In addition to playing a role as a DNA
binding-dependent activator, p53 has also been reported to
negatively regulate the transcription of a number of genes (11),
including those for presenilin (12), topoisomerase II
(13, 14), map4
(15), hsp70 (16), and other viral and cellular promoters (17). In
contrast to the transcriptional activation by p53, no consensus
sequence has been found in the promoters that are repressed by p53. It
was initially reported that only the promoters containing a TATA box
are repressed by p53 (18). However, in at least two cases, it has been
reported that the specific DNA binding of p53 in the regulatory region of these genes results in transcriptional repression (19, 20).
Several studies have implicated a role for transcriptional repression
in p53-dependent apoptosis (9, 11). For example, proteins
such as BCL2 have been shown to inhibit p53-mediated transcriptional
repression, whereas transactivation and G1 arrest functions
remain unaffected (21). Additionally, deletion of the proline-rich
region (amino acids 60-90) in human p53, which contains five repeats
of the PXXP motif (22), renders it defective at apoptosis
induction and transrepression, but not transactivation (23-25).
The observation that p202 negatively regulates the transcriptional
activity of p53 (26), coupled with the fact that a number of proteins
that regulate p53 functions are in turn regulated by p53, led us to
test if p53 regulates the expression of p202. Here we report that the
expression of wild-type, but not mutant, p53 results in a decrease in
the 202 RNA and protein. We show that p53 binds to a p53
DNA-binding consensus sequence present in the 5'-regulatory region of
the 202 gene in gel mobility shift assays. Moreover, p53
represses the activity of a reporter gene, whose transcription is
driven by the 5'-regulatory region of the 202 gene.
Additionally, we demonstrate that overexpression of p202 significantly
delays p53-induced apoptosis, suggesting that the decrease in p202
levels by wild-type p53 may be important for p53-induced apoptosis.
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EXPERIMENTAL PROCEDURES |
Cell Cultures, Antibodies, and Reagents--
Murine AKR-2B cells
(originally a gift from Dr. H. L. Moses, Vanderbilt University,
Nashville, TN), murine fibroblasts (between passages 7 and 15) derived
from a p53-null mouse (generously provided by Dr. Gigi Lozano,
University of Texas M. D. Anderson Cancer Center, Houston, TX),
10.1 Val5 (27), and Vm10 cells (28) (generously provided by Dr. Arnold
Levine, Rockefeller University, New York, NY), human C-33A (from
American Type Culture Collection), a cervical carcinoma cell line with
a mutant p53, were grown in Dulbecco's modified Eagle's medium (with
high glucose) supplemented with 10% fetal bovine serum in an incubator
with 5% CO2. Val5 and Vm10 cells were maintained at
39 °C. If so indicated, recombinant IFN (Universal Type-1, Research
Diagnostic, Inc., Flanders, NJ) (1000 units/ml) was added to
subconfluent cultures as described previously (29).
Plasmids and Generation of Stable Cell Lines--
The
202 reporter plasmid (202-luc), containing the 5'-flanking
sequence (0.8 kilobase) from the 202 gene (30, 31), was constructed by ligating HindIII-PstI fragment
(nucleotides from 1 to 804 in Fig. 7 in Ref. 32) from plasmid pBA into
the pGL3 basic (without any enhancer and promoter sequences) vector
(from Promega, Madison, WI). Polymerase chain reaction (PCR)-based
mutagenesis was performed with a primer containing the desired point
mutations in p53CS1 sequence thus changing the sequence to
5'-CTAAATAACTTCTACCAATACTT-3' (altered bases underlined). The resulting PCR fragment containing the
5'-regulatory region was subloned into the pGL3 basic vector, resulting
in p53CS1mut-luc reporter plasmid. Similarly, a PCR-based approach was
used to generate deletions in the 5'-regulatory region of the
202 gene, resulting in deletions corresponding to the
nucleotides from
495 to
283 and
495 to
122 (from the 5' end of
the transcription initiation site in Fig. 7 of Ref. 32). The deletions
included the p53CS1 site alone (delp53CS1) or the p53CS1 and p53CS2
(delp53CS1 and p53CS2) sites in the 202 gene. The PCR
fragments were ligated into the pGL3 basic vector, resulting in the
reporter plasmid delp53CS1-luc and delp53CS1 and p53CS2-luc. The
reporter plasmid hdm2-luc was generously provided by Dr. Carol Prives
(Columbia University, New York, NY). pRL-TK reporter vector allowing
expression of Renilla luciferase was purchased from Promega
(Madison, WI). The plasmid pCMV53Val135, encoding the
temperature-sensitive mutant of murine p53 (33), was generously
provided by Dr. Moshe Oren (Weizmann Institute, Rehovot, Israel).
pCMH6K53 plasmid encoding wild-type murine p53 was provided by Dr.
Stanley Fields (University of Washington, Seattle, WA). pCMV53 plasmid
encoding wild-type human p53 was purchased from
CLONTECH Inc. (Palo Alto, CA). Bidirectional expression plasmids pBIRP175H and pBIRP248W, allowing coexpression of
both wild-type and the dominant negative mutants of p53 (34), were
generously provided by Dr. E. J. Stanbridge (University of California, Irvine, CA). Plasmid p53(
62-91) (25) was provided by
Dr. X. Chen (Medical College of Georgia, Augusta, GA).
To express the temperature-sensitive mutant of p53, murine AKR-2B
fibroblasts were cotransfected with plasmid pCMV53Val135 and pCMV (in
10:1 ratio) and the transfected cells were selected in G418 (500 µg/ml). After 2 weeks, the drug-resistant colonies (>70) were pooled
and maintained at 37 °C (favoring the mutant conformation of p53).
If so indicated, cells were shifted to 32 °C (favoring the wild-type
conformation of p53) for indicated times. If so indicated, AKR-2B
fibroblasts or fibroblasts derived from p53-null mouse were exposed to
UV-C light (using UV-Stratalinker) without any growth medium.
Vm10 cells (these cells express a temperature-sensitive mutant of p53
and c-myc) (28) were transfected with plasmid
pcDNA3.1-202 or empty vector (pcDNA3.1) and the transfected
cells were selected at 39 °C in medium containing Zeocin (100 µg/ml) for about 2 weeks. More than 100 Zeocin-resistant colonies
transfected with p202-encoding plasmid or empty vector were pooled for
further studies. To initiate apoptosis in the pooled transfected
cells, cells were cultured without Zeocin and were shifted to the
permissive temperature (32.5 °C) for the indicated times.
Flow Cytometric Analysis--
Flow cytometry was performed on
single cell suspensions on adherent (after trypsin and EDTA treatment)
as well as the floating cells after pooling them. Briefly, for cell
cycle analysis, cells were stained with propidium iodide (50 µg/ml,
Sigma) and subjected to flow cytometry using a Coulter Epics XL-MCL
flow cytometer as described previously (35). Apoptosis was measured by
the accumulation of cells with a sub-G1 DNA content.
Immunoblotting--
To detect p202 levels, cells were collected
from plates in phosphate-buffered saline, resuspended in a modified
radioimmune precipitation assay lysis buffer (50 mM
Tris-HCl, pH 8.0, 250 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors
(50 µg/ml leupeptin, 50 µg/ml pepstatin A, 1 mM
phenylmethylsulfonyl fluoride), and incubated at 4 °C for 30 min.
The cell lysates were sonicated briefly before centrifugation at 14,000 rpm in a microcentrifuge for 10 min. The supernatants were collected,
and equal amounts of proteins were processed for immunoblotting as
described previously (29). The p202 polyclonal antiserum has been
described previously (29). Antibodies to p21 (sc-6246) and p53
(sc-6243) were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). For gel-shift assays, anti-p53 monoclonal antibodies (clone 421)
and anti-E2F-1 antibodies (clone KH95) were purchased from Oncogene
Science (Boston, MA).
Northern Blotting--
Total cytoplasmic RNA was isolated from
cells and subjected to Northern blotting, followed by hybridization
with the 202-specific cDNA probe (HincII fragment).
DNA Fragmentation Assays--
Assays were performed on single
cell suspensions on adherent (after trypsin/EDTA treatment) as well as
the floating cells after pooling them. Cells (about 2-5 × 105) were lysed in 20 µl of lysis buffer (20 mM EDTA, 100 mM Tris-HCl, pH 8.0, 0.8% sodium
dodecyl sulfate). To lysates 10 µl of RNase A/T1 mixture mix (500 and
20,000 units/ml, respectively) was added, and the mixture was incubated
at 37 °C for 2 h. Proteinase K solution (10 µl of 20 mg/ml)
was added, and the samples were incubated overnight at 50 °C.
Samples were mixed with 6× DNA loading buffer (30% glycerol and
0.25% bromophenol blue) before their analysis on 1.5% agarose gel.
The agarose gel was run at 2 V/cm until the dye reached to the end of
gel. The gel was stained with ethidium bromide and photographed.
Gel Electrophoretic Mobility Shift Assays--
Nuclear extracts
were prepared from murine AKR-2B cells treated with UV-C (5 mJ/m2) for 24 h or untreated cells as described
previously (36). Equal amounts of nuclear proteins or the recombinant
purified p53 protein (250-500 ng; purchased from Santa Cruz
Biotechnology) were used for binding to labeled oligonucleotides
(labeled using T4 polynucleotide kinase and annealed into
double-stranded oligonucleotides) containing either the p53 DNA binding
consensus sequence (purchased from Santa Cruz Biotechnology) or the
202 gene p53 DNA binding sequence p53CS1
(5'-CTACATGACTTCTACCCATGCTT-3' and the complementary sequence) or
p53CS2 (CTAGTTTTAACACCTTGATCTGG-3' and the complementary sequence (see
Table I for sequence). For supershift assays, nuclear extract proteins
or the recombinant p53 were incubated with anti-p53 (clone 421)
antibodies (1 µg) for 20 min at room temperature. Binding reactions
were subjected to electrophoretic mobility shift assays as described
previously (36).
Luciferase Assays--
For reporter assays, subconfluent cells
in six-well plates were transfected with the reporter plasmids 202-luc
(5 µg) and pRL-TK (0.5 µg) along with indicated amounts of protein
expression plasmid using the calcium phosphate transfection system
(Life Technologies, Inc.), as suggested by the supplier. Cells were harvested 42-48 h after transfections and the firefly luciferase and
Renilla luciferase activities were determined using a
Dual-Luciferase reporter assay kit (from Promega) and TD-20/20
luminometer (from Turner Designs). The firefly luciferase activity was
normalized to the Renilla luciferase in order to control for
variation in transfection efficiencies. The luciferase activity in
control vector-transfected cells is shown as 1.
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RESULTS |
Expression of Wild-type p53 Results in a Decrease in p202
Levels--
To test if the expression of wild-type p53 regulates the
expression of p202, we generated a stable cell line of murine AKR-2B fibroblasts (we chose this cell line because the basal levels of p202
RNA and protein can be detected in this cell line) (29) constitutively
expressing the temperature-sensitive mutant of murine p53 (p53Val135)
(33). While incubation of these cells at 37 °C (the nonpermissive
temperature) favors mutant conformation of p53, upon shift of cells to
32 °C (the permissive temperature) favors a wild-type conformation
of p53 (33). Using this cell system, we found that the expression of
wild-type p53 significantly decreased the steady-state levels of the
202 RNA (Fig. 1A)
and the decrease was apparent 24 h after shift of cells to the
permissive temperature (compare lane 1 with
lane 2). Additionally, the decrease in the
202 RNA was accompanied by a decrease in the basal levels of
p202 (Fig. 1B, compare lane 1 with
lane 2) and also the interferon-induced levels of
p202 (Fig. 1B, compare lane 3 with
lane 4). Furthermore, the decrease in p202 levels
was accompanied by an increase in p21WAF1/CIP1
levels, a known transcriptional target of the p53 tumor suppressor (8,
10).

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Fig. 1.
Expression of wild-type p53 results in a
decrease in p202 levels. A, upper
panel, murine AKR-2B fibroblasts, stably transfected with
plasmid p53Val135, were incubated at 37 °C (lanes
1 and 3) or 32 °C (lanes
2 and 4) for 1 day (lanes 1 and 2) or 2 days (lanes 3 and
4). Total cytoplasmic RNA was isolated and analyzed by
northern hybridization using the 202-specific cDNA
probe. The arrow indicates the location of the
202-specific mRNA. Middle panel, the blot in
upper panel was stripped and reprobed with the
-actin-specific probe. The arrow indicates the location
of actin-specific RNA. Lower panel, the cytoplasmic RNA
applied on the agarose gel was visualized with ethidium bromide
staining to control for equal amounts of RNA loading. B,
AKR-2B cells, stably transfected with plasmid p53Val135, were incubated
at 37 °C (lanes 1 and 3) or
32 °C (lanes 2 and 4) for 2 days.
Total cell lysates prepared from untreated cells (lanes
1 and 2) or cells that had been treated with IFN
(lanes 3 and 4) were analyzed by
immunoblotting using an anti-p202 antiserum, anti-p53, and anti-p21.
p68 protein (29) served as a control for loading of equal amounts of
proteins. The location of a protein band is indicated by an
arrow. C, Val5 cells were incubated at 39 °C
(lanes 1 and 2) or 32.5 °C
(lanes 3 and 4) after they had been
treated with IFN (1000 units/ml for 24 h) (lanes
2 and 4) or left untreated (lanes
1 and 3). The total cell lysates prepared from
cells were analyzed by immunoblotting using anti-p53, anti-p21,
anti-p202, or anti- -actin antibodies. The location of a protein band
is indicated by an arrow.
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Because AKR-2B cells express wild-type p53, which could affect the
regulation by a transgene (p53Val135) by dimerization (9), we utilized
the Val5 cell line (in these cells both alleles of p53 are deleted)
(27), which also expresses the temperature-sensitive mutant of p53. As
shown in Fig. 1C, these cells had higher levels of p202 at
the nonpermissive temperature, which were comparable with the p202
levels in IFN-treated cells (compare lane 2 with lane 1), and shift of these cells to the
permissive temperature resulted in a decrease in p202 levels (compare
lane 3 with lane 1).
Moreover, the decrease in p202 levels was accompanied by an increase in
p21WAF1/CIP1 levels. Interestingly, as noted
above (Fig. 1B), the IFN treatment of these cells at the
permissive temperature resulted in only a moderate increase in p202
levels. Together, these observations provide support to the idea that
the expression of wild-type p53 negatively regulates the steady-state
levels of the 202 RNA and protein in these cell lines.
Wild-type p53 Binds Strongly to One of the Two p53 DNA Binding
Consensus Sequence Present in the 5'-Regulatory Region of the 202 Gene--
The 5'-regulatory region of the 202 gene, which
is immediately upstream to the transcription initiation sites, has been
characterized (30-32). It does not contain a TATA box (31, 32).
Therefore, to identify a potential mechanism(s) by which wild-type p53
regulates the expression of the 202 gene, we searched the
5'-regulatory region of the 202 gene for the presence of
other cis-regulatory elements that are shown to be involved
in p53-mediated transcriptional regulation of genes. Our search
revealed that the regulatory region of the 202 gene contains
several cis-acting elements (Fig.
2A), including two potential
p53 DNA binding sites (indicated as p53CS1 and p53CS2), shown to be
involved in the transcriptional regulation of genes by p53 (8, 11).
Interestingly, as shown in Table I, the
two potential p53 DNA binding sites p53CS1 and p53CS2 in the
5'-regulatory region of the 202 gene, like other known p53
DNA binding sites in other p53-responsive genes (37, 38), contain
C(A/T)(T/A)G at the core of the ten base palindrome, which is important
for the DNA binding activity of p53 (37, 38). However, as noted in
Table I, these two potential p53 DNA binding sites in the
202 gene contain variations (in the case of p53CS1, 5 variations out of 20 nucleotides, and, in the case of p53CS2, 4 out of
20 nucleotides) from the p53 DNA binding consensus sequence (38).

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Fig. 2.
Wild-type p53 binds to one of the potential
p53 DNA binding site present in the 5'-regulatory region of the
202 gene in gel mobility shift assays.
A, schematic of the locations of the cis-acting
elements within the HindIII and PstI fragment
(~0.8 kilobase) from the 5'-flanking sequence of the 202 gene, which is linked to luciferase reporter gene. These
cis-elements include potential binding sites for p53, CCAAT
enhancer-binding proteins, and one of the AP-1-like sequences. The
5'-flanking sequence of the 202 gene contains two potential
p53 DNA binding sites (indicated as p53CS1 and p53CS2). A
bold letter in the p53CS1 or p53CS2 sequence
indicates deviation from the p53 DNA binding consensus sequence. The
upstream and downstream end of the transcription initiation region in
the 202 gene are indicated by t and
t. B, subconfluent cultures of murine AKR-2B
cells were either treated with UV-C light (5 mJ/m2) or left
untreated for 24 h. The nuclear extracts were prepared as
described under "Experimental Procedures." The extracts containing
equal amounts of proteins were analyzed by gel mobility shift assays
using either a labeled oligonucleotide (probe) containing p53
DNA-binding consensus sequence (purchased from Santa Cruz Biotechnology
Inc.) (left panel) or the p53CS1 site in the
202 gene (right panel). Extracts from
untreated (lane 2) or UV-C-treated
(lanes 3 and 7) cells after incubation
with probe. Extracts from UV-C-treated cells were incubated with probe
plus a 20-fold mole excess of cold oligonucleotide (lanes
4 and 8), anti-p53 antibodies (clone 421)
(lanes 5 and 9), or anti-E2F-1
antibodies (clone KH95) (lanes 6 and
10). As a control, probe without an incubation with extract
was also run in lane 1. A black
arrowhead denotes p53 supershift (sup.), and a
white arrow indicates the p53-specific band. An
asterisk denotes a nonspecific band. C, nuclear
extracts containing equal amounts of protein were either incubated with
labeled oligonucleotide containing the p53 DNA binding consensus
sequence (lanes 1-3) or the p53CS1 sequence from
the 202 gene (lanes 4-6) or p53CS2
sequence from the 202 gene (lanes
7-9). Extracts were incubated with probe alone
(lanes 2, 5, and 8) or
probe plus antibodies to p53 (lanes 3,
6, and 9). As a control, oligonucleotide without
any incubation with extracts were also run (lanes
1, 4, and 7). A black
arrowhead denotes p53 supershift, and a white
arrow indicates the p53-specific band. An
asterisk denotes a nonspecific band. D,
recombinant purified p53 was incubated with an oligonucleotide
containing either the p53 DNA binding consensus sequence
(lanes 1-5) or the p53CS1 sequence from the
202 gene (lanes 6-10). p53 protein
either 250 ng (lanes 2, 4,
7, and 9) or 500 ng (lanes
3, 5, 8, and 10) was
incubated with labeled oligonucleotide alone (lanes
2, 3, 7, and 8) or with
oligonucleotide plus antibodies to p53 (clone 421) (lanes
4, 5, 9, and 10). As a
control, oligonucleotides without any incubation with extracts were
also run (lanes 1 and 6). A
black arrowhead denotes the p53 supershift.
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Table I
Comparison of the two potential p53 DNA binding site sequences present
in the regulatory region of the 202 gene with other known p53 DNA
binding sites
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Next, we tested if p53 could bind to the p53CS1 or p53CS2 sequence in
gel-mobility shift assays. For this purpose, we used nuclear extracts
from AKR-2B cells (these cells contain wild-type p53) untreated or
after exposure to low levels (5 mJ/m2) of UV-C treatment.
As shown in Fig. 2B (left panel), p53
in these nuclear extracts specifically bound to a p53 consensus
sequence in gel mobility shift assays and the complex was competed well with 20-fold excess of cold consensus sequence oligonucleotide (compare
lane 3 with lane 4). Furthermore, the
complex was supershifted after incubation with antibodies to p53
(antibody pAb 421) that are known to improve the binding of p53 to its
cognate site (39). However, no such supershift was seen after
incubation with an isogenic control antibody to E2F-1 (compare
lane 5 with lane 6). Interestingly, incubation of nuclear extracts with a labeled
oligonucleotide containing the p53CS1 sequence resulted in binding of
p53 (see Fig. 2B, right panel) and the
binding was also competed out well by 20-fold excess of cold
oligonucleotide containing the p53 DNA binding consensus sequence
(compare lane 8 with lane
7). Additionally, the p53 complex was supershifted after
incubation with antibodies to p53 (antibody pAb 421). However, no such
supershift was seen after incubation with an isogenic control antibody
to E2F-1 (compare lane 9 with lane
10). Thus, these experiments indicated that, in these
assays, p53 specifically bound to the 202 p53CS1 sequence and the p53 consensus sequence with a comparable affinity.
Because the p53CS2 sequence in the 5'-regulatory region of the
202 gene differs from the p53CS1 and the p53 consensus
sequence (see Table I), we tested its ability to bind p53 in gel-shift assays. As shown in Fig. 2C, oligonucleotide containing a
p53 DNA binding consensus sequence bound to p53 (lane
2) and incubation with anti-p53 antibodies resulted in
supershift of p53-oligonucleotide complex (lane
3). As expected from our above experiment (Fig. 2B), the 202 p53CS1 sequence also bound to p53 to
a comparable extent (compare lane 5 with
lane 2) and incubation with anti-p53 antibodies
resulted in a supershift of p53-oligonucleotide complex (compare
lane 6 with lane 3).
However, the p53CS2 sequence bound to p53 weakly (compare
lane 8 with lanes 2 and
5) and incubation with anti-p53 antibodies did not result in
a significant supershift of p53-oligonucleotide complex (compare
lane 9 with lanes 3 and 6). Thus, together, these experiments indicated that in
these assays p53 can specifically bind to one of the two (p53CS1) p53 consensus sequence present in the 5'-regulatory region of the 202 gene with an affinity comparable to that of p53 DNA
binding consensus sequence.
Because in nuclear extracts p53 is bound to other nuclear proteins,
which could facilitate binding of p53 to an oligonucleotide, we tested
if the recombinant purified p53 can bind to an oligonucleotide containing the p53CS1 sequence from the 202 gene. As shown
in Fig. 2D, purified recombinant p53 bound to an
oligonucleotide containing the p53 DNA binding consensus sequence
(lanes 2 and 3) and the
p53-oligonucleotide complex was supershifted upon incubation with
anti-p53 antibodies (lanes 4 and 5).
Incubation of recombinant p53 with an oligonucleotide containing the
p53CS1 sequence did not result in a significant binding to the
oligonucleotide (lanes 7 and 8).
However, incubation of p53 with anti-p53 antibody resulted in a
significant increase in DNA binding and supershift (lanes 9 and 10). These experiments, thus, clearly
demonstrated that in gel mobility shift assays one of the two p53 DNA
binding sites (p53CS1) present in the 5'-regulatory region of the
202 gene can specifically bind to the recombinant purified
p53 and p53 in nuclear extracts. Therefore, these experiments provide
further support to the idea that the specific DNA binding of p53 in the
5'-regulatory region of the 202 gene may contribute to the
transcriptional regulation of the 202 gene.
Expression of the Wild-type p53 Results in a Decrease in the
Activity of a Reporter Gene Whose Expression Is Driven by the
5'-Flanking Sequence of the 202 Gene--
To test if p53
transcriptionally regulates the 202 gene, we transfected
murine fibroblasts derived from a p53-null mouse or human C-33A cells
(these cells are null for p53) with the 202-luc reporter plasmid, a
luciferase reporter driven by a 800-base pair fragment from the
5'-flanking sequence of the 202 gene (Fig. 2A), together with varying amounts of pCMV-p53 plasmid encoding wild-type murine p53. As shown in Fig.
3A, the expression of the
wild-type p53 decreased the activity of luciferase reporter in a
dose-dependent manner. In several of these transient
transfection assays, we observed up to a 10-fold reduction in the
activity of luciferase reporter after the expression of wild-type p53.
These observations, thus, provide support to the idea that wild-type
p53 can negatively regulate the transcription of the 202 gene.

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Fig. 3.
Expression of the wild-type p53 in transient
transfection assays results in a decrease in the activity of the
202-luc reporter gene. A, murine fibroblasts (passages
7-15) derived from p53-null mouse or human C-33A cells were
transfected with the 202-luc reporter plasmid and indicated increasing
amounts of plasmid pCM6HK53 (encoding the wild-type murine p53) as
described under "Experimental Procedures." The activity of the
firefly luciferase was determined 42-44 h after transfections, and the
activity from cells transfected with vector alone is indicated as 1. The standard deviation is indicated by bars. B,
murine fibroblasts derived from p53-null mouse were transfected with
202-luc reporter plasmid and equal amounts (4 µg) of plasmid pCMV
(column 1), pCM6HK53 (encoding the wild-type
murine p53) (column 2), pCMV53 (encoding
wild-type human p53) (column 3), or p53mt135
(encoding a mutant human p53) (column 4) as
described under "Experimental Procedures." The activity of the
firefly luciferase was determined 42-44 h after transfections as
described in A. C, murine fibroblasts derived
from p53-null mouse were transfected with 202-luc reporter plasmid and
indicated amounts of plasmid either encoding wild-type p53 or a point
mutant of p53 (p53mt135) as described under "Experimental
Procedures." The activity of the firefly luciferase was determined
42-44 h after transfections as described in A. D, murine fibroblasts derived from p53-null mouse were
transfected with 202-luc reporter plasmid and equal amounts (4.5 µg)
of plasmid pCMV (first column), pCMV53 (encoding
wild-type human p53) (second column),
bidirectional plasmid pBIRP175H (encoding wild-type p53 and point
mutant 175H of p53) (third column), or pBIRP248W
(encoding wild-type p53 and point mutant 248W of p53)
(fourth column) as described under
"Experimental Procedures." The activity of the firefly luciferase
was determined 42-44 h after transfections as described in
A. E, murine fibroblasts derived from p53-null
mouse were transfected with either the 202-luc or hdm2-luc reporter
plasmid and equal amounts (4.5 µg) of plasmid pCMV (first
column), pCMV53 (encoding wild-type human p53)
(second column), or a plasmid encoding a
deletion mutant of p53 (p53( 62-91)) (third
column) as described under "Experimental Procedures."
The activity of the firefly luciferase was determined 42-44 h after
transfections as described in A. F, murine
fibroblasts derived from p53-null mouse were transfected with the
202-luc, p53CS1mut-luc, delp53CS1-luc or delp53CS1 and CS2-luc reporter
plasmid and equal amounts (100 ng) of plasmid pCMV (left
columns) or pCMV53 (encoding wild-type murine p53)
(right columns) as described under
"Experimental Procedures." The activity of the firefly luciferase
was determined 42-44 h after transfections as described in
A.
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Acetylation of p53 upon coexpression of cofactor p300, a histone
acetyltransferase, has been shown to potentiate the repression of
-fetoprotein gene (20). Therefore, we tested if coexpression of p300
with p53 affects p53-mediated transcriptional repression of the
202 gene. We found that the expression of p300 alone or its
coexpression with wild-type p53 did not affect the activity of the
202-luc reporter in p53-null fibroblasts (data not shown). Therefore,
it is unlikely that the acetylation of p53 by p300 contributes to the
transcriptional repression of the 202 gene.
We next tested if the expression of a mutant of human p53 (p53mt135),
which is defective in the specific DNA binding activity (40), affects
the 202-luc reporter activity. As shown in Fig. 3B,
transfection of a plasmid encoding wild-type murine or human p53
resulted in decreased activity of the 202-luc reporter (compare second and third columns with
first column). However, transfection of a plasmid
encoding p53mt135 only moderately decreased the activity of the 202-luc
reporter as compared with a plasmid encoding wild-type human p53
(compare fourth and third columns).
Curiously, no such decrease by the expression of p53mt135 was seen
under conditions in which transfection of nanogram amounts of a plasmid
encoding wild-type p53 still decreased the activity of the 202-luc (see Fig. 3C) (in this experiment, we used lesser amounts of the
expression plasmids to rule out repression because of excess amounts of
plasmid). We also tested how coexpression of two dominant negative
mutants of human p53 with wild-type p53 affects p53's ability to
repress the 202-luc reporter activity. For this purpose, we utilized
bi-directional plasmids (pBIRP) allowing coexpression of equal amounts
of wild-type and the dominant negative mutant of p53 (34). As shown in
Fig. 3D, coexpression of wild-type p53 together with the
dominant negative mutant 175H or 248W did not result in a decrease in
the activity of 202-luc (compare lane 1 with
lanes 3 or 4). Instead, coexpression of these mutants of p53 with wild-type p53 moderately increased the
activity of the 202-luc reporter. Thus, these observations indicated
that the specific DNA binding activity of p53 is required for
transcriptional repression of the 202 gene.
Because the transcriptional repression of the genes by p53 is shown to
involve the PXXP domain in p53 (23-25), we tested if this
domain in p53 is needed for the repression of the 202 gene. As shown in Fig. 3E, the expression of a deletion mutant of
human p53 (p53(
62-91)), which lacks the five PXXP
motifs, did not result in repression of the 202-luc reporter activity.
To rule out the possibility that the lack of repression of the 202-luc
reporter activity in the above experiments was due to the lack of
expression of mutant p53, we used hdm2-luc reporter plasmid, which is
shown to be weakly responsive to the expression of the above deletion mutant of p53 (25). Consistent with an earlier report (25), the
expression of this mutant of p53 weakly stimulated the activity of
hdm2-luc reporter. Thus, these experiments indicated that the PXXP motif in p53 is important for the transcriptional
repression of the 202-luc reporter activity.
Next, to examine the relative contribution of the two p53 DNA binding
sites (p53CS1 and p53CS2) in p53-mediated transcriptional repression of
the 202 gene, we deleted either both p53 DNA binding sites
(delp53CS1 and CS2) or only one site (delp53CS1) from the 5'-regulatory
region of the 202 gene and performed reporter assays. As
shown in Fig. 3F, these deletions in the 5'-regulatory
region of the 202 gene relieved p53-mediated repression of
the 202-luc activity. Because an oligonucleotide containing the p53CS1
sequence bound to p53 in gel-mobility shift assays (Fig. 2), we tested if mutations in the p53CS1 site could relieve p53-mediated
transcriptional repression of the 202-luc activity. As shown in Fig.
3F, site-directed mutagenesis of p53CS1 p53 DNA binding site
in the 202 gene, that has been shown to result in abrogation
of the specific DNA binding activity of p53 (37), relieved murine
p53-mediated repression of the 202-luc activity. Additionally, the
repression mediated by expression of human p53 was also diminished by
the CS1 mutation (data not shown). These observations, thus, raise the
possibility that p53 utilizes the p53CS1 site in the 202 gene to regulate the expression of the 202 gene.
Exposure of AKR-2B Cells to UV Light Results in a Decrease in p202
Levels--
To test if the physiological increases in p53 levels
affect the expression of p202, we transiently transfected murine AKR-2B cells (these cells harbor wild-type p53) or fibroblasts derived from
p53-null cells with the 202-luc reporter plasmid and exposed cells to
UV-C light. As shown in Fig.
4A, exposure of AKR-2B cells to UV light decreased the activity of 202 reporter in a
dose-dependent manner. However, in fibroblasts derived from
the p53-null mouse, only a moderate decrease in the 202-luc reporter
activity was seen. Therefore, these observations suggested that the
decrease in the activity of the 202-luc reporter in response to DNA
damage caused by UV-C light depends on the presence of wild-type
p53.

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Fig. 4.
Exposure of AKR-2B cells to UV light results
in a decrease in 202 expression. A, UV
treatment of murine AKR-2B fibroblasts represses the activity of the
202-luc reporter in a dose-dependent manner. Murine AKR-2B
fibroblasts or fibroblasts derived from p53-null mouse were transiently
transfected with the 202-luc reporter plasmid, and after 24 h of
transfections cells were exposed to indicated doses of UV-C light.
Cells were incubated for another 24 h, and the cell lysates were
analyzed for luciferase activity. The activity of the firefly
luciferase detected in unexposed control cells is indicated as 1. The
standard deviation is indicated by bars. B,
murine AKR-2B fibroblasts were incubated for indicated times after
treatment with UV-C (5 mJ/m2) (lanes
2 and 4) or without it (lanes
1 and 3). The total cell lysates prepared from
cells were analyzed by immunoblotting using anti-p53, anti-p21,
anti-p202, or anti- -actin antibodies. The location of a protein band
is indicated by an arrow.
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We next tested if the decrease in the expression of 202 gene
(following UV-exposure of cells) correlates with an increase in p53
levels. For this purpose, we exposed AKR-2B cells to low dose (5 mJ/m2) of UV light and analyzed the levels of p202. As
shown in Fig. 4B, UV-C treatment of cells resulted in a
decrease in p202 levels after 14 h of treatment. More importantly,
the decrease in p202 levels was accompanied by an increase in the
levels of p53 and p21WAF1/CIP1. These findings
raised the possibility that the physiological increases in the levels
of p53, after DNA damage caused by UV-C treatment of cells, results in
the transcriptional repression of the 202 gene.
Expression of p202 Decreases the Rate of p53-induced
Apoptosis--
To investigate how the decrease in the basal
levels of p202, after the physiological increases in the levels of p53,
affect p53-induced apoptosis, we overexpressed p202 in murine Vm10 cell line. These cells express a temperature-sensitive mutant of p53 (p53Val135) and c-myc (28). Upon shift of temperature to the permissive temperature (32.5 °C), these cells undergo p53-induced apoptosis in about 14 h (28). As shown in Fig.
5A, overexpression of p202 at
the permissive temperature in these cells significantly reduced number
of cells morphologically appearing apoptotic. Flow cytometry of these
cultures for the presence of sub-G1 cells revealed that
overexpression of p202 in these cells significantly (up to 10% after
19 h and 50% after 36 h) reduced the extent of
sub-G1 cells (Fig. 5B). Additionally, the
analysis of the genomic DNA content from both adherent and floating
cells in these cultures (transfected with vector) revealed that these
cells indeed exhibited a significant laddering of DNA, a hallmark of
apoptosis, at the permissive temperature (Fig. 5C, compare
lane 1 with lane 3), but
not at the nonpermissive temperature, and overexpression of p202
significantly (about 50%) reduce it (compare lane
3 with lane 4). Thus, these
experiments provide support to the idea that the transcriptional
repression of the 202 gene by the wild-type p53 may be
important for a rapid induction of p53-induced apoptosis.

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Fig. 5.
Overexpression of p202 delays p53-induced
apoptosis. A, murine Vm10 cells stably transfected with
vector (left four panels) or a
p202-encoding plasmid (right four
panels) were incubated for indicated times at 39 °C or
32.5 °C, and cells were subjected to flow cytometry analyses after
taking phase contrast photographs. Insets depict extent of
apoptosis in cells as determined by propidium iodide staining (% sub-G1 DNA content). B, percentage of apoptosis
in cells induced to undergo p53-induced apoptosis in the above
experiment was plotted against time in hours. C, cultures
undergoing p53-induced apoptosis shown in A were analyzed
for apoptosis by DNA ladder assays as described under "Experimental
Procedures." Approximately equal number of cells were lysed to
isolate DNA at the indicated times, and isolated DNA was subjected to
agarose gel (1.5%) electrophoresis. The gel was stained with ethidium
bromide and photographed.
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DISCUSSION |
In the present study, we demonstrate that wild-type p53 negatively
regulates the levels of p202 by repressing the transcription of the
202 gene. We used two approaches to investigate if the expression of wild-type p53 regulates the basal levels of p202. In the
first approach, we used two murine cell lines (AKR-2B and Val5)
constitutively expressing a temperature-sensitive mutant of p53. Upon
incubation of these cell lines at the permissive temperature, which
favors the wild-type conformation of p53, both the 202 RNA
and protein levels decreased significantly (Fig. 1). As expected, the
decrease in p202 levels was accompanied by an increase in the levels of
p21WAF1/CIP1, a known transcriptional target of
p53 (9, 10). In the second approach, we exposed murine AKR-2B cells to
UV-C light (which results in increase in p53 levels in these cells) to
test if the physiological increase in p53 levels correlate with
decrease in p202 levels. We found that UV treatment of cells, which
resulted in a decrease in the levels of p202 (Fig. 4), was correlated
with increases in the levels of p53 and
p21WAF1/CIP1 (Fig. 4). The decrease was also
evident at the 202 RNA levels (data not shown). Thus, these
two approaches revealed that increases in p53 levels are associated
with decreases in p202 protein and mRNA, thus suggesting that p53
may negatively regulate p202 expression.
Unlike the activation of gene expression, the mechanism of repression
of gene expression by wild-type p53 is not well defined and p53 has
been shown to repress transcription of genes by different mechanisms
(11, 41, 42). These mechanisms include: (i) the binding of p53 to the
TATA-binding proteins, which are bound to the TATA containing promoters
(11, 18); (ii) the recruitment of mSin3a and histone deacetylases by
p53 to the gene promoter (41); (iii) the sequence-specific DNA binding
of p53 to the consensus sequence present in the regulatory region of
genes (19, 20); and (iv) the interactions of p53 with other
cis-acting elements, which include SP-1 (16), CCAAT (43),
and AP-1 (44). Because the 5'-flanking sequence of the 202 gene does not contain a TATA box (30, 31), it is conceivable that the
mechanism(s) by which p53 represses the transcription of the
202 gene is independent of TATA-binding proteins (45-47).
Consistent with this prediction, we found that the expression of
adenovirus-encoded E1A protein, which is shown to relieve p53-mediated
transcriptional repression of genes by interacting with the
TATA-binding protein (46), did not relieve p53-mediated
transcriptional repression of the 202-luc reporter activity (data not shown).
The presence of two potential p53 DNA binding sites (37, 38) in the
5'-regulatory region of the 202 gene (Table I) prompted us
to test if p53 regulates the expression of the 202 gene
through the specific DNA binding. These experiments revealed that: (i) one of the two potential p53 DNA binding site (p53CS1, Fig.
2A) present in the 5'-regulatory region of the
202 gene specifically bound to p53 in gel-mobility shift
assays (Fig. 2, C and D); (ii) in transient
transfection assays, the expression of wild-type p53, but not various
mutants of p53 (defective in the specific DNA binding activity),
resulted in a decrease in the 202-luc reporter activity (Fig. 3); and
(iii) site-directed mutagenesis of the p53CS1 sequence in the
202 gene or its deletion relieved p53-mediated transcriptional repression of the 202-luc reporter activity (Fig. 3F). Thus, our observations are consistent with the
possibility that the sequence-specific DNA binding activity of p53
contributes to the transcriptional repression of the 202 gene. Therefore, the 202 gene joins the class of genes whose
transcriptional repression by p53 depends on the sequence-specific DNA
binding activity of p53 (19, 20).
Although our observation that point mutations in the p53CS1 site (or
its deletion) in the 202 gene relieve p53-mediated
transcriptional repression of the 202-luc activity provides support to
the idea that p53-mediated transcriptional repression of the
202 gene depends on the specific DNA binding activity of
p53, our observations do not completely rule out the possibility that
other mechanism(s), such as interactions of DNA-bound p53 with other
transcription factors, also contribute to the regulation of the
202 gene. Because in addition to the p53 DNA-binding sites
(p53CS1 and p53CS2), the 5'-flanking sequence of the 202 gene also contains other cis-acting elements, such as CCAAT
enhancer, SP-1, and an AP-1-like site, shown to be involved in
p53-mediated transcriptional repression of the genes (16, 43, 44),
further work will be needed to identify the relative contributions of
these cis-elements in p53-mediated transcriptional
repression of the 202 gene.
Curiously, the proline-rich region in human p53, which contains five
PXXP motifs (where P represents proline and X any
amino acid) (22), was found to be dispensable for transactivation and
cell cycle arrest mediated by p53 (23-25). However, the proline-rich region was found to be necessary for transrepression and apoptosis mediated by p53 (24, 25). Our observation that the expression of a
deletion mutant of p53, lacking the proline-rich region (amino acids
62-91), was not able to repress the transcription of the 202 gene (Fig. 3E) raises the possibility that
the mechanism(s) by which wild-type p53 negatively regulates the
transcription of the 202 gene depends on the presence of
PXXP motifs in p53. Similar to the 202 gene, the
requirement for the p53 proline-rich region was also demonstrated for
the transcriptional regulation of the PIG3 gene (24).
Our previous studies revealed that the decreased levels of p202 in
fibroblasts increase the propensity of cells to undergo apoptosis (5).
Interestingly, in a recent study overexpression of p202 was found to
inhibit c-myc-induced apoptosis in Rat-1 cells, which harbor
a wild-type p53 (6). Together, these observations support the notion
that p202 participates in the regulation of apoptosis. Consistent with
this notion, our observations provide additional evidence that the
increased levels of p202 in Vm10 cells significantly delay p53-induced
apoptosis (Fig. 5B). Therefore, it is likely that the
decrease in the basal endogenous levels of p202, after the
physiological increases in the levels of wild-type p53, contribute to
the potentiation of p53-induced apoptosis. Additionally, because p202
negatively regulates the transcriptional activity of p53 (26) and other
factors (for example, NF-
B and E2F-1) (49, 50), with which p53
cooperates to induce apoptosis (48, 51), the transcriptional repression
of the 202 gene by p53 may be part of an important cascade
of events by which p53 induces apoptosis. However, it remains to be
seen how the decrease in the levels of p202 following the expression of
wild-type p53 affect the activity of these other transcription factors.
p53-mediated biological effects in cultured cells, which include the
growth arrest and apoptosis, are shown to be dependent on the levels of
p53 expressed, the type (for example UV or ionizing radiation exposure)
and the extent of stress (different doses of UV), and the cell type
(11, 23). Therefore, it is conceivable that p53-mediated
transcriptional repression of the 202 gene depends on these
factors, and further work will be needed to examine these possibilities.
The identification of the 202 gene as a p53-repressible gene
in the present study provides a novel feedback mechanism by which p53
negatively regulates the expression of a negative regulator. Our
findings also raise the interesting possibility that the basal levels
of p202 are negatively regulated by other signaling pathways whose
activation ultimately results in an increase in the levels of p53 (7,
9, 11). Notably, these pathways include activation of p53 by oncogenes.
Consistent with this prediction, we note that p202 levels are
negatively regulated by the adenovirus-encoded E1A oncogene (data not
shown). Thus, our findings reported herein will serve as a basis to
study the regulation of the basal levels of p202 by these cell growth
regulatory pathways and to identify the functional role of p202 in
these cell growth-regulatory processes.