Previous Article | Next Article 
Molecular and Cellular Biology, April 1999, p. 2746-2753, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
New Insights into the Mechanism of Inhibition of
p53 by Simian Virus 40 Large T Antigen
Hilary M.
Sheppard,
Siska I.
Corneillie,
Christine
Espiritu,
Andrea
Gatti, and
Xuan
Liu*
Department of Biochemistry, University of
California, Riverside, California 92521
Received 2 November 1998/Returned for modification 9 December
1998/Accepted 19 December 1998
 |
ABSTRACT |
Simian virus 40 (SV40) large tumor antigen (T antigen) has been
shown to inhibit p53-dependent transcription by preventing p53 from
binding to its cognate cis element. Data presented in this
report provide the first direct functional evidence that T antigen,
under certain conditions, may also repress p53-dependent transcription
by a mechanism in which the transactivation domain of p53 is abrogated
while DNA binding is unaffected. Specifically, p53 purified as a
complex with T antigen from mouse cells was found to bind DNA as a
transcriptionally inactive intact complex, while that purified from
human cells was found to bind DNA independently of T antigen and could
activate p53-dependent transcription. This difference in activity may
be dependent on a different interaction of T antigen with mouse and
human p53 and, in addition, on the presence of super T, which is found
only in transformed rodent cells. These results suggest that subtle yet
important differences exist between the inhibition of p53 by T antigen
in mouse and human cells. The implications of this finding with respect
to SV40-associated malignancies are discussed.
| |
INTRODUCTION |
p53 is an important tumor suppressor
gene, found to be mutated or absent in over 50% of all cancers studied
(23). It functions as a sequence-specific DNA-binding
transcription factor (21, 24). In response to
double-stranded DNA breaks, p53 is converted from a latent to an active
form (17). This results in increased expression of
p53-responsive proteins such as p21 which are required for growth
arrest at the G1-to-S phase transition (12). It
also mediates apoptosis via the increased expression of proteins such as Bax (30). Inactivation of p53, therefore, results in the loss of a cell cycle checkpoint required for repair of damaged DNA and
prevents apoptosis in response to severe DNA damage. In the absence of
these responses, oncogenic mutations which may result in tumor
progression can accumulate. From the above, it is clear that the
transcriptional activation function of p53 is critical to its role as a
tumor suppressor.
A number of proteins bind to p53 and negatively affect its
transcriptional activity. The cellular oncoprotein MDM2 has been shown
to inhibit p53 via three different mechanisms. First, when bound to
p53, MDM2 conceals the activation domain of p53 from the transcription
machinery, thereby indirectly repressing p53-dependent transcription
(32). Second, it has been found to promote the rapid
degradation of p53 via a ubiquitin-proteosome pathway, resulting in
decreased levels of p53 available to activate transcription (15,
22). Finally, MDM2 may itself function as an active repressor of
transcription, which, via its interaction with p53, represses p53-responsive genes (39).
Proteins encoded by DNA tumor viruses also inhibit p53 activity in
similar ways. The human papillomavirus type 16 E6 protein forms a
complex with p53, thus promoting its polyubiquitination and
subsequent degradation (35, 38). The adenovirus early 1B
(E1B) 55K protein, a transcriptional repressor, binds to p53 and is
thereby targeted to p53-responsive genes (41). The large tumor antigen (T antigen) of simian virus 40 (SV40) also forms with p53
a complex that inhibits p53 function in SV40-infected and
SV40-transformed cells. Experiments performed with
baculovirus-expressed human p53 and T antigen led Bargonetti et al.
(1) to propose that T antigen inhibits p53 function by
preventing it from binding to its cognate cis element.
Furthermore, Segawa et al. (36) reported similar results
when they examined the DNA-binding activity of p53 in crude nuclear
extracts isolated from a human p53 null cell line transiently
transfected with plasmids expressing p53 and T antigen; in addition,
when baculovirus-expressed T antigen was added to a mouse cell lysate
containing wild-type p53, DNA binding was abolished. Taken together,
data derived from these experiments support the model in which T
antigen inhibits p53 function by preventing it from binding to DNA.
T antigen is often found as a 90-kDa protein in the nuclei of
SV40-infected and/or -transformed cells. However,
higher-molecular-weight forms of T antigen, designated super T, have
also been detected in SV40-transformed rodent cell lines
(37). Forms of super T are reported to arise from internal
in-phase duplications in the coding region of the T-antigen gene
(28, 29) or, as is the case for a commonly occurring 100-kDa
form, by differential splicing between two integrated partial copies of
the T-antigen gene (25). The duplication which forms the
100-kDa protein includes the first exon, the intron, and part of the
second exon upstream of the complete coding sequence for the T-antigen
gene. It is proposed that transcription starts at the upstream copy of
T antigen and continues through the host DNA and into the full-length
copy of the gene. The long primary transcript is spliced, but a short region of extra RNA, possibly from the first copy of the duplicated control region, is retained and encodes the extra amino acids present
in the 100-kDa super-T protein (25). The presence of super T
was found to correlate with anchorage-independent growth in mouse cell
lines (2, 6). Despite the compelling effect of super T in
transformation, the molecular mechanism by which super T functions to
inhibit p53-dependent transcription remains to be elucidated.
In the present study, we show that p53 immunopurified from a human cell
line and a monkey cell line copurifies with T antigen, while that
purified from two mouse cell lines copurifies with a 100-kDa form of
super T. When purified from mouse cells, the p53-T antigen complex can
bind specifically to DNA in electrophoretic mobility shift analysis
(EMSA). However, despite this DNA-binding activity, the complex is not
capable of activating p53-dependent transcription in vitro. Therefore,
our data suggest that in mouse cell lines, T antigen/super T abrogates
the transactivation domain of p53 and does not affect DNA binding. When
purified from either a human or a monkey cell line, the p53-T antigen
complex also binds specifically to DNA in EMSA, but surprisingly T
antigen is not present in the resulting shifted complex and hence this complex can support p53-dependent transcription in vitro. This finding
suggests that in human and monkey cells, p53 may not be inhibited by T
antigen. The different activities of the p53-T antigen complex purified
from mouse cell lines and from human and monkey cell lines were found
to be dependent on a different interaction of T antigen with mouse and
human p53 and, in addition, possibly on the presence of super T. These
results suggest for the first time the existence of subtle differences
between the inhibition of p53 by T antigen in human and mouse cells
that may have physiologically significant consequences.
| |
MATERIALS AND METHODS |
Protein purification.
p53-T antigen complex was
immunopurified from nuclear extracts prepared by the method of Dignam
et al. (11). One milliliter of nuclear extract (7 mg of
protein/ml) was incubated for 3 h at 4°C with 100 µl of packed
protein A-Sepharose beads to which Pab 421, a monoclonal antibody
specific for p53, was covalently linked. Beads were washed twice with
0.5 M KCl D buffer (20 mM HEPES [pH 7.9], 20% glycerol, 0.2 mM EDTA,
1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and once with
0.1 M KCl D buffer. p53 was eluted from the washed beads with 100 µl
of 421 epitope oligopeptide (KKGQSTSRHKK) at 1 mg/ml in 0.1 M KCl D buffer. p53-T antigen complex was also purified by
using beads to which Pab 108, a monoclonal antibody specific for T
antigen, was covalently linked. In this case the complex was eluted
with EG (ethylene glycol) buffer (50% EG, 0.5 M NaCl, 10% glycerol,
20 mM Tris HCl [pH 8.5], 1 mM EDTA) and dialyzed overnight against
0.1 M KCl D buffer. To purify p53 in the absence of T antigen, Pab 421 anti-p53 beads were incubated with nuclear extract and washed as
described above. T antigen was eluted from the bound p53 by two 10-min
washes in 0.1 ml of 2 M urea in 0.1 M KCl D buffer. The
T-antigen-containing supernatant was dialyzed overnight against 0.1 M
KCl D buffer. The remaining beads were washed overnight with 0.1 M KCl
D buffer, after which p53 was eluted as described above. To
immunodeplete the Pab 421-purified complex, a volume of covalently
linked Pab 108 beads equal to one-fifth of the protein sample volume
was added, and incubation was carried out for 3 h at 4°C with
gentle rotation. The supernatant was retained and incubated with fresh Pab 108 beads for a further 3 h, after which the supernatant was collected. A control human p53 (no T antigen present) was prepared from
HeLa cells infected with recombinant vaccinia virus expressing p53 as
described previously (26) and purified with Pab 421 anti-p53 beads as described above. Recombinant T antigen was prepared from Spodoptera frugiperda SF21 insect cells infected with
recombinant baculovirus (a gift from C. Prives, Columbia University) as
described by Bargonetti et al. (1). Proteins were analyzed
by electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide
gels which were subjected to Western blotting or were silver stained to
visualize bands.
EMSA.
The sequence of the oligonucleotide probe containing
the ribosomal gene cluster (RGC) p53-binding site is
5'-AGCTTGCCTCGAGCTTGCCTGGACTTGCCTGGTCGACGC-3'; the sequence
of that containing the p53-binding site from the p21 promoter is
5'-AGCTTAATTCTCGAGGAACATGTCCCAACATGTTGCTCGAGG-3'. Probes
were labeled with the Klenow fragment of Escherichia coli DNA polymerase. When required, preincubation reactions were performed for 20 min at 4°C prior to EMSA. Binding reaction mixtures contained 60 mM KCl, 12% glycerol, 5 mM MgCl2, 1 mM EDTA, 0.1 µg
of bovine serum albumin, 0.5 µg of poly(dG-dC), 200 pg of
32P-labeled probe, proteins and antibodies as indicated,
and water in a total volume of 12.5 µl. Antibody N-19 (Santa Cruz
Biotechnology Inc.), recognizing an amino-terminal epitope mapping to
within residues 2 to 20, was used against p53. Pab 108, recognizing an amino-terminal epitope mapping to within residues 1 to 82, was used
against T antigen. Reaction mixtures were incubated for 30 min at
30°C and then analyzed on a 3% polyacrylamide gel containing 0.5×
TBE (0.045 mM Tris-borate, 0.045 mM sodium borate, 0.001 mM EDTA [pH
8.0]). Electrophoresis was carried out in 0.5× TBE. The gel was
dried, and DNA-protein complexes were visualized with a PhosphorImager
using Adobe Photoshop software. Densitometry was performed with
ImageQuant software.
In vitro transcription.
Reactions were performed as
described previously (26). Briefly, 70 µg of HeLa cell
nuclear extract (7 mg of protein/ml) and 150 ng of a synthetic target
promoter containing five p53-responsive sites immediately upstream of
the adenovirus E4 TATA box and chloramphenicol acetyltransferase (CAT)
reporter gene (5RGCE4CAT) were mixed in a final volume of 50 µl with
60 mM KCl, 12 mM HEPES (pH 7.9), 12% glycerol, 6 mM MgCl2,
0.4 mM ribonucleoside triphosphates, 7 mM 
-mercaptoethanol, p53, and
T antigen as indicated and incubated at 30°C for 60 min. Control
reactions were performed with 150 ng of synthetic promoter containing
five GAL4-binding sites immediately upstream of the adenovirus E4 TATA
box fused to CAT (5GAL4E4CAT) (5) in the presence of 100 ng
of bacterially expressed GAL4-VP16 protein. Reactions were stopped by
the addition of 50 µl of stop buffer (2% SDS, 200 mM NaCl, 20 mM
EDTA, 20 µg of tRNA per ml, 100 µg of proteinase K per ml) followed
by incubation at 39°C for 10 min. After phenol-chloroform extraction,
the RNA was ethanol precipitated. Primer extension was performed by
resuspending the RNA pellet in 10 µl of annealing buffer (125 mM KCl,
25 mM Tris-HCl [pH 8.3], 1,000 cpm of radiolabeled primer) and
incubating it at 70°C for 10 min and then at 39°C for 30 min.
Twenty-four microliters of extension buffer (5 mM MgCl2, 50 mM KCl, 20 mM Tris HCl [pH 8.3], 0.3 mM deoxynucleoside
triphosphates, 10 mM dithiothreitol, 10 U of Moloney murine leukemia
virus reverse transcriptase) was then added, and the mixture was
incubated for 30 min at 39°C. The reaction was stopped by the
addition of 35 µl of stop buffer; primer extension products were
ethanol precipitated and resuspended in 4 µl of formamide sequencing
dye prior to electrophoresis on a 10% acrylamide-urea gel. The gel was
dried, and primer extension products were visualized with a
PhosphorImager using Adobe Photoshop software. Densitometry was
performed with ImageQuant software.
| |
RESULTS |
p53-T antigen complex can specifically bind to DNA.
To define
how T antigen inhibits p53-mediated transcription, p53 was purified
from nuclear extracts prepared from two mouse (SCID and SVT2), one
monkey (COS-7), and one human (WI38 VA13) cell line, all of which are
stably transformed with SV40, using anti-p53 antibody Pab 421 (Fig.
1A). From the mouse cell lines, two
proteins with apparent molecular masses of 90 and 100 kDa copurified
with p53; from the human and monkey cell lines, only one protein of 90 kDa was copurified. All of the copurified proteins were identified as
SV40 T antigen by Western blot analysis using anti-T-antigen antibody
Pab 108 (Fig. 1B). From the SCID mouse cell line, the 100-kDa T antigen
copurified in stoichiometric amounts with the 90-kDa form; from the
SV-T2 mouse cell line, the 100-kDa protein was the major form of
purified T antigen. Two lines of evidence suggest that the 100-kDa
protein is a previously identified form of super T. First, the
identification of this band as T antigen was further confirmed by
sequence analysis of peptides resulting from the digestion of this
protein (data not shown). Second, a form of super T with the same
molecular mass as the 100-kDa T antigen that we observed has been
identified in SV-T2 cells (40).
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 1.
p53 immunopurified from SV40-transformed cells
copurifies with T antigen. (A) Silver-stained SDS-polyacrylamide gel of
the p53-T antigen complex purified from four SV40-transformed cell
lines derived from mouse (SCID and SV-T2), human (WI38 VA13), and
monkey (COS-7) cells. (B) Western blot of the p53-T antigen complex,
using anti-T antigen antibody Pab 108, indicating that two forms of T
antigen (regular T antigen and super T) are present in the mouse cell
lines.
|
|
As stated above, T antigen interacts with p53 and is thought to inhibit
its binding to DNA. Therefore, when the p53 samples
shown in Fig.
1A
were used in EMSA with a probe containing the
p53
cis
element identified in the RGC, the results were unexpected
(Fig.
2A). Vaccinia virus-expressed human p53
(vhp53) purified
from HeLa cells was used as a T-antigen-minus control.
It produced
a retarded p53-DNA complex (lane 2) which was supershifted
by
the addition of anti-p53 antibody (lane 3). p53 purified from
the
monkey cell line COS-7 and the human cell line WI38 VA13 formed
complexes similar in mobility to those formed by vhp53 (compare
lane 2 to lanes 7 and 13). These complexes could be supershifted
by the
addition of anti-p53 antibody but not by the addition of
anti-T-antigen
antibody (cf. lanes 8, 9, 14, and 15). Western
blot analysis was
performed to ensure that equivalent amounts
of p53 were present in all
samples (data not shown). This result
suggested that although p53 was
purified in a complex with T antigen,
it could bind DNA and that when
bound to DNA, the p53 was no longer
complexed with T antigen. p53
purified from the mouse cell lines
SCID and SVT2, however, formed a
complex with the DNA probe that
migrated more slowly than the control
p53-DNA complex (compare
lane 2 to lanes 4 and 10). The addition of
both anti-p53 antibody
and anti-T-antigen antibody supershifted this
complex (lanes 5,
6, 11, and 12), suggesting that the mouse p53-T
antigen complex
remained intact during the gel electrophoresis. This
result indicated
that the DNA-binding ability of mouse p53 was also
unaffected
by T antigen, but when purified from mouse cells, the p53-T
antigen
complex remained intact when bound to DNA.
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 2.
p53 copurified with T antigen can bind to a DNA probe in
EMSA. (A) A 200-pg aliquot of radiolabeled probe containing the
p53-binding site from RGC was incubated with approximately 50 ng of
vhp53 purified from HeLa cells or 50 ng of each of the protein samples
shown in Fig. 1A and 100 ng of antibody as indicated for 30 min at
30°C prior to electrophoresis on a 3% polyacrylamide gel. Arrows
indicate positions of retarded complexes: 1, DNA-p53; 2, DNA-p53-N19
( p53); 3, DNA-p53-T antigen; 4, DNA-p53-T antigen-N19 or Pab 108 (T antigen). (B) Like panel A but with a probe containing the
p53-binding site identified in the p21 promoter. The gels shown in
panels A and B were subjected to electrophoresis for 3 and 4 h,
respectively, which accounts for the more advanced migration of the
bands in panel B. (C) Like panel A but with increasing amounts (50 and
100 ng) or equivalent volumes of p53-T antigen complex purified from
COS-7 cells with Pab 421 either before (lanes 1 and 2) or after one
(lanes 3 and 4) or two (lanes 5 and 6) rounds of immunodepletion with
anti-T-antigen antibody Pab 108.
|
|
To determine if these results were peculiar to the
cis
element identified in the RGC, further EMSA was performed with a probe
containing the p53 element identified in the p21 promoter. Similar
results were obtained (Fig.
2B), again demonstrating that p53-T
antigen
complexes purified from the mouse cell lines SCID and
SV-T2 can bind
DNA in the presence of T antigen (lanes 2, 3, 6,
and 7) while those
purified from the monkey and human cell lines
bind DNA in the absence
of T antigen (lanes 4, 5, 8, and 9). It
was possible that excess p53
not bound to T antigen present in
the p53-T antigen preparation from
the human and monkey cell lines
was responsible for the p53-DNA shift
observed in EMSA, even though
visualization by silver staining
indicated that the ratio of p53
to T antigen was approximately 1:1
(Fig.
1A). To test this possibility,
Pab 421-purified p53-T antigen
complex from COS-7 cells was immunodepleted
with anti-T-antigen
antibody. Analysis by SDS-PAGE followed by
silver staining indicated
that immunodepletion resulted in the
equal loss of both p53 and T
antigen from the sample (data not
shown). Importantly, EMSA revealed no
DNA binding with the twice-immunodepleted
sample (Fig.
2C; compare
lanes 1 and 2 to lanes 5 and 6). This
result strongly argues that the
p53 DNA binding observed was not
due to the presence of excess unbound
p53. Taken together, these
results suggest the existence of alternative
mechanisms for the
inhibition of p53-dependent transcription by T
antigen. In addition,
they suggest that the mechanism for inhibition of
p53 may differ
between mouse cells and human and monkey
cells.
The p53-T antigen complex purified from mouse cells is
transcriptionally inactive.
We next wanted to determine whether
the mouse p53-T antigen complex, which could bind to DNA, could also
support p53-dependent transcription. The complex was immunopurified
from SCID cells; as a control, SCID p53 was also purified in the
presence of 2 M urea in order to dissociate T antigen. The resulting
p53 samples are shown in Fig. 3A. When
tested by EMSA, the p53-T antigen complex resulted in the slowly
migrating band in which both p53 and T antigen were present (Fig. 3B,
lanes 4 to 8). In contrast, the p53 protein purified in the presence of
2 M urea produced a retarded complex that was similar in mobility to
that produced by the control vhp53 (compare lanes 9 to 12 to lanes 2 and 3) and did not react with the anti-T-antigen antibody Pab 108 (lane
13).
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 3.
Mouse p53 binds to DNA as a complex with T antigen in
EMSA. (A) Silver-stained SDS-polyacrylamide gel of p53 purified from
SCID cells with (+ T) or without ( T) T antigen. p53 was purified in
the absence of T antigen by washing the p53-T antigen complex when
bound to Pab 421 antibody linked to protein A-Sepharose beads with a 2 M urea solution. Sizes are indicated in kilodaltons. (B) Approximately
50 ng of vhp53 or increasing amounts (25, 50, and 75 ng) of the
proteins used for panel A were incubated for 30 min at 30°C with 200 pg of radiolabeled probe containing the p53-binding site from RGC with
100 ng of antibody as indicated prior to electrophoresis on a 3%
polyacrylamide gel. Arrows indicate positions of retarded complexes: 1, DNA-p53; 2, DNA-p53-N19 (p53); 3, DNA-p53-T antigen; 4, DNA-p53-T
antigen-N19 (p53) or Pab 108 (T antigen).
|
|
The ability of these p53 samples to stimulate transcription was next
tested in an in vitro transcription assay as described
previously
(
26). Unfractionated HeLa cell nuclear extract and
a
promoter template containing five p53 DNA-binding sites positioned
immediately upstream of the adenovirus E4 TATA box (Fig.
4A) were
incubated in the absence and presence of increasing amounts of
p53. In
the absence of exogenously added p53, the nuclear extract
supported a
low level of basal transcription (Fig.
4B, lane 1).
The addition of
vhp53 resulted in a threefold stimulation of transcription
above this
basal level (lane 6). By comparison to the control
vhp53, addition of
the mouse p53-T antigen complex isolated from
SCID cells did not
enhance transcription over basal levels (compare
lane 6 to lanes 2 and
3) even though it bound DNA in EMSA. When
the T-antigen proteins were
removed, however, mouse p53 activated
transcription 2.6-fold over basal
levels (compare lane 1 to lanes
4 and 5). This indicates that loss of
p53 activity was due to
inhibition by T antigen and that the p53 was
fully active on its
own. These results were reproducible and therefore
strongly suggest
that in mouse cells, T antigen may inhibit
p53-dependent transcription
by blocking the transactivation domain of
p53 and not by preventing
it from binding to its cognate
cis element.
Interestingly, COS-7 p53, which also purified in a complex with T
antigen (Fig.
1) but bound DNA independently of T antigen
(Fig.
2),
retained the ability to activate p53-dependent transcription
2.8-fold
over basal levels (Fig.
4C; compare lane 1 to lanes 2
to 4). This
surprising result suggests that in monkey cells, T
antigen may not
necessarily inhibit p53 function despite its association
with p53. T
antigen can itself function as an activator of transcription
from a
simple promoter consisting of certain TATA elements and
one upstream
transcription factor-binding site (
13,
33). Therefore,
we
performed assays in the presence of baculovirus-expressed purified
T
antigen to determine if T antigen alone could activate transcription
in
this system (Fig.
4D). p53-dependent
transcription was inhibited
when a fourfold excess of
baculovirus-expressed T antigen was
added to vhp53 (cf. lanes 2 and 4).
The effect of T antigen on
basal transcription was tested in assays
using the amount of baculovirus
T antigen sufficient to inhibit
p53-dependent transcription. Transcription
was not affected by the
addition of T antigen alone (cf. lanes
7 and 8), suggesting that the
transcription observed in Fig.
3C
is p53 dependent and not T antigen
dependent. Finally, the effect
of T antigen on activated transcription
driven by bacterially
expressed GAL4-VP16 was also tested in assays
using template DNA
containing five upstream GAL4-binding sites fused to
the adenovirus
TATA box. The addition of GAL4-VP16 to these reactions
resulted
in a 12-fold increase in transcription levels. This
transactivation
was minimally affected by the addition of baculovirus T
antigen
(compare lane 10 to lanes 11 and 12). Therefore, unbound T
antigen
does not affect transcription in these assays.
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 4.
Mouse p53-T antigen complex is transcriptionally
inactive, while human p53-T antigen complex retains activity. (A)
Schematic diagram of template DNA used in in vitro transcription
reactions. (B) In vitro transcription reaction using HeLa nuclear
extract (lane 1) with increasing amounts (300 and 600 ng) of SCID p53
purified with (lanes 2 and 3) or without (lanes 4 and 5) T antigen.
vhp53 (200 ng) was used as a positive control (lane 6). Transcription
(txn) products were subjected to electrophoresis on a 10%
acrylamide-urea gel and visualized with a PhosphoImager using Adobe
Photoshop software. (C) Like panel B but with increasing amounts (250, 500, and 750 ng) of COS-7 p53-T antigen complex (lanes 2 to 4). (D)
Like panel B but with 100 ng of vhp53 (lanes 2 to 4 and 6) and
increasing amounts (100 [lanes 3 and 7] and 400 [lanes 4 and 8] ng)
of baculovirus-expressed T antigen (Bacl. T). In lanes 9 to 12, in
vitro transcription was performed with a promoter similar to that used
for panel A but with the RGC elements replaced with GAL4-binding sites.
GAL4-VP16 (100 ng) was added to activate transcription (lanes 10 to 12)
in the presence of increasing amounts (100 and 400 ng) of
baculovirus-expressed T antigen (lanes 11 and 12).
|
|
DNA binding by a p53-T antigen complex is species dependent.
Next we wanted to determine what contributed to the different
activities of the p53-T antigen complexes purified from either the
mouse or the human and monkey cell lines. The difference may depend on
the p53 present in these cell lines. At the amino acid level, monkey
p53 is 96% identical to human p53, while mouse p53 is only 79%
identical to human p53. Therefore, differences at the amino acid level
or in protein modification may account for distinct interactions of
mouse and human or monkey p53 with T antigen, with subsequent effects
on p53-T antigen complex activity. Alternatively, the difference may be
due to the presence of super T in the mouse cell lines but not in the
human and monkey cell lines.
To address this issue, we performed EMSA with vhp53 and mouse p53
purified from SCID cells in the absence of T antigen. These
samples
were incubated with either a baculovirus-expressed T antigen
(a 90-kDa
protein) or T antigen purified from mouse SCID cells
(90- and 100-kDa
proteins [Fig.
5A]). Ideally, a
preparation of
the 100-kDa form of super T alone would have been used
in these
experiments, but to our knowledge no clone that only makes
super
T is available. Results are shown in Fig.
5B and C. The addition
of T antigen/super T purified from SCID cells to mouse p53 resulted
in
the generation of a supershifted complex (Fig.
5B, lane 6).
This
complex was abolished by the addition of anti-T-antigen antibody
(lane
7). T antigen/super T alone did not bind to DNA (lane 8).
The addition
of the same amount of T antigen/super T to human
p53 also resulted in
the generation of a supershifted complex
(lane 3). However, we
consistently observed that less human p53
than mouse p53 was
supershifted under these conditions (cf. lanes
3 and 6), suggesting
that T antigen/super T can form a DNA-binding
complex more efficiently
with mouse p53 than with human p53.
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 5.
Both mouse p53 and mouse T/super T contribute to the
formation of a p53-T antigen complex that can bind to DNA. (A)
Silver-stained SDS-polyacrylamide gel of baculovirus-expressed T
antigen purified with anti-T-antigen antibody Pab 108 from SF21 insect
cells (Bacl. T) and T antigen purified from stably transformed mouse
SCID cells in the absence of p53 as described in Materials and Methods
(SCID T). Sizes are indicated in kilodaltons. (B) Approximately 50 ng
of vhp53 or 100 ng of SCID p53 (no T antigen present; mp53) was
incubated for 30 min at 30°C with 200 pg of radiolabeled probe
containing the p53 binding site from RGC, 50 ng of T antigen purified
from SCID cells, and 100 ng of antibody (T) as indicated prior to
electrophoresis on a 3% polyacrylamide gel. DNA-protein complexes were
visualized with a PhosphoImager using Adobe Photoshop software. Arrows
indicate positions of retarded complexes: 1, DNA-p53; 2, DNA-p53-T
antigen. (C) Like panel B but with 100 ng of SCID p53-T antigen complex
(lane 2), 100 ng of vhp53, or 100 ng of SCID p53 (no T antigen present;
mp53) incubated with 50 or 100 ng of baculovirus-expressed T antigen as
indicated. Arrows indicate positions of retarded complexes: 1, DNA-p53;
2, DNA-p53-T antigen.
|
|
When baculovirus T antigen was used in these experiments, a
supershifted complex was formed only with mouse p53 (Fig.
5C;
compare
lane 6 to lanes 7 and 8). Two slowly migrating complexes,
possibly due
to incomplete renaturation of the mouse p53 after
the harsh conditions
of the purification procedure, were observed.
The slower-migrating
complex migrated at a position similar to
that of the complex formed by
the mouse p53-T antigen complex
immunopurified from the SCID cell line
(lane 2), and addition
of both anti-p53 antibody and anti-T-antigen
antibody supershifted
this slower-migrating complex (data not shown).
In comparison
to mouse p53, DNA binding of human p53 was inhibited by
baculovirus
T antigen (compare lane 3 to lanes 4 and 5 [1.4- and
1.9-fold
reduction in binding, respectively]). Baculovirus T antigen
alone
did not bind specifically to DNA (lane 9). Therefore, in these
in
vitro assays, the DNA-binding ability of human p53 was inhibited
by the
addition of baculovirus-expressed T antigen, in accordance
with
previous observations (
1). This result concurs with the
data
from in vitro transcription assays using vhp53, where transcription
was
inhibited by the addition of baculovirus-expressed T antigen
(Fig.
4D,
lanes 2 to 4). The fact that transcription and DNA binding
are observed
with p53-T antigen complex purified from COS-7 monkey
cells suggests
that there may be an important difference between
this in vivo complex
and that prepared in vitro by using virally
expressed proteins. In
addition, the molar ratio of T antigen
to p53 may be higher in the in
vitro complex, therefore affecting
its
activity.
The EMSA results presented in Fig.
5 suggest that mouse p53 is
different from human p53 in its ability to interact with T
antigen.
Indeed, this difference was also noted when we attempted
to purify
p53-T antigen complex from monkey and mouse cells by
using
anti-T-antigen antibody. The complex remained intact when
purified
under stringent conditions from the SCID mouse cell line
but
dissociated when purified from the monkey COS-7 cell line
(data not
shown), suggesting that T antigen binds strongly to
mouse p53 and
weakly to human p53. Therefore, mouse p53, in addition
to mouse T/super
T, may contribute to the formation of a DNA-binding
p53-T antigen
complex.
An active conformation of p53 is required for p53-T antigen complex
DNA binding.
Wild-type p53 can be converted from an inactive or
latent state to an active state that binds DNA (17). The
latent state is thought to be dependent on a C-terminal negative
regulatory domain within p53 that is proposed to interact with a motif
in the core of the p53 tetramer, thereby forming a conformationally inactive complex (18). A number of conditions that modify
the C terminus of p53 which convert p53 from a latent to an active state have been described. These include anti-p53 antibody Pab 421 (17, 18), phosphorylation (16, 17), acetylation
(14), short single strands of DNA (19), and the
redox/repair protein Ref-1 (20). The biological relevance of
this model of activation is demonstrated by the fact that UV-induced
activation of the transcriptional function of p53 does not require an
increase in p53 protein levels (18).
The p53-T antigen complexes used in the previous experiments were
purified by using Pab 421 antibody. This antibody recognizes
an epitope
in the C terminus of p53 and is thought to convert
p53 from its latent
to its active state, thereby significantly
increasing its DNA-binding
activity (
18,
31). Consequently,
we wanted to determine if
this method of purification affected
the DNA-binding ability of the
complex. First, the mouse p53-T
antigen complexes eluted from Pab 421 beads by using a 50% EG
solution and 421 peptide were compared. In
both cases, similar
binding affinities were observed in EMSA (data not
shown), indicating
that binding was not dependent on the presence of
421 peptide.
Next, we attempted to purify the complex by using
p53-specific
antibodies D0-1 and Pab 1801 (Santa Cruz), both of which
recognize
amino-terminus epitopes in human p53. However, in each case
control
experiments indicated that the stringent conditions required to
elute p53 from these antibodies resulted in DNA-binding-deficient
protein. Therefore, the complex was purified by using anti-T-antigen
antibody Pab 108, which is not known to activate p53, and eluted
with
50% EG solution, which does not affect the DNA-binding ability
of p53
(
36a). This method of purification resulted in a complex
similar to that purified with Pab 421 when analyzed by SDS-PAGE
followed by silver staining (Fig.
6A).
However, the complex purified
with Pab 108 had a significantly reduced
affinity for DNA compared
to that purified with Pab 421 (Fig.
6B;
compare lanes 2 to 4 to
lanes 5 to 7), suggesting that an active
conformation of p53 may
be required for p53-T antigen complex
DNA-binding ability. To
test if this was the case, we incubated the Pab
108-purified complex
with Pab 421 prior to EMSA and then observed DNA
binding (Fig.
6C; cf. lanes 3 and 4). The resulting shifted band
migrated parallel
with that produced by the Pab 421-purified complex
(cf. lanes
4 and 6) and could be supershifted by the addition of
anti-p53
and anti-T-antigen antibodies (data not shown). The activation
of binding was not observed following incubation with an unrelated
antibody (lane 5) or with bovine serum albumin (data not shown).
These
results strongly argue that the DNA-binding ability of the
complex is
dependent on the active conformation of p53.
View larger version (52K):
[in this window]
[in a new window]
|
FIG. 6.
Purification of p53-T antigen complex by Pab 421 is
required for DNA-binding ability. (A) Silver-stained SDS-polyacrylamide
gel of p53-T antigen complex purified from mouse SCID cells by using
Pab 108 (anti-T antigen) or Pab 421 (anti-p53). Sizes are indicated in
kilodaltons. (B) vhp53 (50 ng), increasing amounts (20, 40, and 60 ng)
of Pab 421-purified SCID p53-T antigen complex, or increasing amounts
(50, 75, and 100 ng) of Pab 108-purified complex were incubated for 30 min at 30°C with 200 pg of radiolabeled probe containing the
p53-binding site from RGC prior to electrophoresis on a 3%
polyacrylamide gel. Arrows indicate positions of retarded complexes: 1, DNA-p53; 2, DNA-p53-T antigen. (C) Like panel B but with 50 ng of
vhp53, 100 ng of Pab 108-purified SCID p53-T antigen complex, or 50 ng
of Pab 421-purified complex as indicated. In lanes 4 and 5, Pab
108-purified complex (108 comp) was preincubated for 20 min at 4°C
with 5 µg of Pab 421 or 5 µg of antibody 12CA5 in 0.1 M D buffer,
as indicated.
|
|
| |
DISCUSSION |
In this paper we present biochemical evidence that p53 isolated in
a complex with T antigen can bind to DNA, provided that p53 is in an
active conformation. We also demonstrate that p53 exhibits
species-specific interactions with T antigen. In vitro, T antigen
apparently dissociates from human p53 in the presence of DNA, resulting
in a transcriptionally active form of p53 bound to DNA. In contrast,
mouse p53 binds DNA in a complex with T antigen, resulting in
transcriptionally inactive p53. Our results indicate that this
difference may be attributable to a difference between mouse and human
p53 and, in addition, possibly to the presence of super T in the mouse
cell lines. On the basis of our results, we conclude that T antigen,
under certain conditions, may repress p53-dependent transcription by a
mechanism in which the transactivation domain of p53 is inhibited while
DNA binding is unaffected. This mechanism is different from the
previous proposed mechanism in which T antigen inhibits p53 by
preventing it from binding DNA (1, 36). Therefore, similar
to the inhibition of p53 by MDM2, it seems that T antigen may inhibit
p53 by more than one mechanism.
Long et al. (27) have shown, using nuclear extracts in EMSA,
that a small fraction of p53 from SV40-transformed monkey and rat cells
specifically binds to DNA as a complex with T antigen. Our results,
however, differ from theirs in that we observe significant DNA binding
by p53-T antigen complex purified from mouse cells. The reason for this
discrepancy may be that we used purified p53 activated by Pab 421 antibody as opposed to crude nuclear extracts. However, we did not
observe DNA binding by p53-T antigen complex when using crude nuclear
extracts in EMSA, either in the presence or in the absence of Pab 421 (data not shown). This may have been because the concentration of p53
in the crude extract was too low for Pab 421 activation to occur or for
DNA binding to be observed.
The observation that the transactivation domain of mouse p53 may be
inhibited by T antigen while DNA binding is unaffected has several
implications. First, the surface of p53 required for DNA binding must
be accessible in the mouse p53-T antigen complex. The region of p53
that is required for T-antigen binding has been mapped to residues 126 to 218, a region within the core domain required for DNA binding
(34). However, resolution of the crystal structure of a
human p53 core domain-DNA complex has localized the region of p53
directly contacted by DNA to three structural elements which include
the H2 helix (amino acids [aa] 278 to 286), the L1 loop (aa 112 to
124), and the L3 loop (aa 236 to 251) (7). None of these
elements overlap with the region required by p53 to interact with T
antigen. Therefore, it is possible that p53 can concurrently form a
complex with T antigen and bind DNA. Second, interaction of T antigen
with the DNA-binding domain of p53 must either alter or block the
activation domain of p53. Thus, it appears that the alteration of one
domain on p53 may affect the function of the others.
Our results suggest that T antigen can form a transcriptionally
inactive DNA-binding complex with mouse p53. The adenovirus E1B 55K
protein also forms an inhibitory complex with p53 without displacing
p53 from its cognate cis element (41).
p53-mediated transcription is prevented because E1B 55K is a repressor
of transcription which, via its interaction with p53, is targeted to
p53-responsive genes. The mechanism of repression adopted by T antigen,
however, is unlikely to be identical to that adopted by E1B 55K, as T
antigen can function as a transcriptional activator (13,
33). T antigen is thought to activate transcription through
direct interactions with both the basal transcription complex and
upstream-bound transcription factors and may act like a component of
TFIID by augmenting, or replacing, a function of TAFII250
(9). Additionally, T antigen has been shown to enhance
formation of TATA-binding protein-TFIIA complex on certain TATA
elements (10). The fact that T antigen does not activate
transcription when tethered to promoter DNA by p53 indicates that its
activation function, as previously suggested (42), may be
conformation dependent and that binding to p53 may alter its
conformation. Consistent with this view, GAL4-T antigen fusion protein
has been shown to be transcriptionally inactive when brought to a
target promoter bearing GAL4 DNA-binding sites (13). When
bound as a complex with p53 to DNA, T antigen may inhibit p53-dependent
transcription by steric hindrance of the activation domain of p53 or by
deleteriously affecting the assembly or conformation of the basal
transcription machinery. In addition, T antigen may interfere with the
interaction of p53 with coactivators of transcription such as p300
(14).
Mouse cells are more readily transformed with SV40 than are human
cells. Using the data presented in this paper, we propose a model which
may explain the molecular mechanism of this difference (Fig.
7). In mouse cell lines, latent p53 and T
antigen/super T form a complex that cannot bind DNA. Upon activation of
p53, a conformational change in p53 may alter the p53-T antigen-super T
complex such that DNA binding occurs. p53-responsive promoters would
therefore be blocked by the transcriptionally inactive complex, and the
tumor suppressor function of p53 would be reduced or lost. In human
cell lines, latent p53 and T antigen also form a complex that is unable
to bind to DNA. Again, upon activation of p53, a conformational change
in p53 may alter the p53-T antigen complex such that DNA binding
occurs. However, because human p53 appears to bind to DNA independently
of T antigen and to retain the ability to activate transcription, p53
function would not be completely lost. Therefore, p53 target genes that
are required for growth arrest and apoptosis would be activated in
human cells containing T antigen and an active form of p53. This model
relies on the fact that p53 must adopt an active conformation.
Significantly, cellular factors that may activate p53 similarly to Pab
421 antibody, including protein kinases (16, 17),
coactivator p300 (14), and the redox/repair protein Ref-1
(20), are found in the cells.
It is tempting to speculate that our model may also explain why SV40 is
known to cause tumors in rodents (3, 8) but has not proven
to do so in humans. Although recently this virus has been linked to
some human cancers, a cause-and-effect relationship has not been
established (4). Finally, it will be of interest to
determine whether the differences reported here between
species-specific forms of p53 are also applicable to interactions with
other viral and cellular proteins. If so, such a distinction between
rodent and primate p53 could have significant ramifications for the use of rodent models of transformation.
| |
ACKNOWLEDGMENTS |
We thank Arnold Berk and Carol Prives for providing cells and
viruses, and we thank Francisco Renteria for help with cell culture. We
also thank Frances Sladek, Noelle L'Etoile, Renee Yew, and members of
the Liu laboratory for many helpful discussions and valuable comments
on the manuscript.
This work was supported by grants CA75180-01 (X.L.) from the National
Cancer Institute and DAMD17-96-1-6076 (X.L.) from U.S. Army Breast
Cancer Research Program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of California, Riverside, CA 92521. Phone:
(909) 787-4350. Fax: (909) 787-4434. E-mail:
xuan.liu{at}ucr.edu.
| |
REFERENCES |
| 1.
|
Bargonetti, J.,
I. Reynisdottir,
P. Friedman, and C. Prives.
1992.
Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53.
Genes Dev.
6:1886-1898[Abstract/Free Full Text].
|
| 2.
|
Butel, J. S.,
C. Wong, and B. K. Evans.
1986.
Fluctuation of simian virus 40 (SV40) super T-antigen expression in tumors induced by SV40-transformed mouse mammary epithelial cells.
J. Virol.
60:817-821[Abstract/Free Full Text].
|
| 3.
|
Carbone, M.,
P. Rizzo, and H. I. Pass.
1997.
Simian virus 40, poliovaccines and human tumors: a review of recent developments.
Oncogene
15:1877-1888[Medline].
|
| 4.
|
Carbone, M.,
P. Rizzo,
P. M. Grimley,
A. Procopio,
D. J. Y. Mew,
V. Shridhar,
A. de Bartolomeis,
V. Esposito,
M. T. Giuliano,
S. M. Steinberg,
A. Levine,
A. Giordano, and H. I. Pass.
1997.
Simian virus-40 large T-antigen binds p53 in human mesotheliomas.
Nat. Med.
3:908-912[Medline].
|
| 5.
|
Carey, M.,
J. Leatherwood, and M. Ptashne.
1990.
A potent Gal4 derivative activates transcription at a distance in vitro.
Science
247:710-712[Abstract/Free Full Text].
|
| 6.
|
Chen, S.,
M. Verderame,
A. Lo, and R. Pollack.
1981.
Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.
Mol. Cell. Biol.
1:994-1006[Abstract/Free Full Text].
|
| 7.
|
Cho, Y.,
S. Gorina,
P. D. Jeffrey, and N. P. Pavletich.
1994.
Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations.
Science
265:346-355[Abstract/Free Full Text].
|
| 8.
|
Cicala, C.,
F. Pompetti, and M. Carbone.
1993.
SV40 induces mesotheliomas in hamsters.
Am. J. Pathol.
142:1524-1533[Abstract].
|
| 9.
|
Damania, B., and J. C. Alwine.
1996.
TAF-like function of SV40 large T antigen.
Genes Dev.
10:1369-1381[Abstract/Free Full Text].
|
| 10.
|
Damania, B.,
P. L. Lieberman, and J. C. Alwine.
1998.
Simian virus 40 large T antigen stabilizes the TATA-binding protein-TFIIA complex on the TATA element.
Mol. Cell. Biol.
18:3926-3935[Abstract/Free Full Text].
|
| 11.
|
Dignam, J. D.,
R. M. Lebovitz, and R. G. Roeder.
1983.
Accurate transcript initiation by RNA Pol II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:1475-1489[Abstract/Free Full Text].
|
| 12.
|
El-Diery, W. S.,
T. Tokino,
V. E. Velculescu,
D. B. Levy,
R. Parsons,
J. M. Trent,
D. Lin,
W. E. Mercer,
K. W. Kinzler, and B. Vogelstein.
1993.
WAF1, a potential mediator of p53 tumor suppression.
Cell
75:817-825[Medline].
|
| 13.
|
Gruda, M. C.,
J. M. Zabolotny,
J. H. Xiao,
I. Davidson, and J. C. Alwine.
1993.
Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.
Mol. Cell. Biol.
13:961-969[Abstract/Free Full Text].
|
| 14.
|
Gu, W., and R. G. Roeder.
1997.
Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.
Cell
90:595-606[Medline].
|
| 15.
|
Haupt, Y.,
R. Maya,
A. Kazaz, and M. Oren.
1997.
Mdm2 promotes the rapid degradation of p53.
Nature
387:296-299[Medline].
|
| 16.
|
Hupp, T. R., and D. P. Lane.
1994.
Regulation of the cryptic sequence-specific DNA-binding function of p53 by protein kinases.
Cold Spring Harbor Symp. Quant. Biol.
59:195-206[Abstract/Free Full Text].
|
| 17.
|
Hupp, T. R.,
D. W. Meek,
C. A. Midgley, and D. P. Lane.
1992.
Regulation of the specific DNA binding function of p53.
Cell
71:875-886[Medline].
|
| 18.
|
Hupp, T. R.,
A. Sparks, and D. P. Lane.
1995.
Small peptides activate the latent sequence-specific DNA binding function of p53.
Cell
83:237-245[Medline].
|
| 19.
|
Jayaraman, L., and C. Prives.
1995.
Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus.
Cell
81:1021-1029[Medline].
|
| 20.
|
Jayaraman, L.,
K. G. K. Murthy,
C. Zhu,
T. Curran,
S. Xanthoudakis, and C. Prives.
1997.
Identification of redox/repair protein Ref-1 as a potent activator of p53.
Genes Dev.
11:558-570[Abstract/Free Full Text].
|
| 21.
|
Ko, L. J., and C. Prives.
1996.
p53: puzzle and paradigm.
Genes Dev.
10:1054-1072[Free Full Text].
|
| 22.
|
Kubbutat, M. H. G.,
S. N. Jones, and K. H. Vousden.
1997.
Regulation of p53 stability by Mdm2.
Nature
387:299-303[Medline].
|
| 23.
|
Levine, A. J.,
J. Momand, and C. A. Finlay.
1991.
The p53 tumor suppressor gene.
Nature
351:453-456[Medline].
|
| 24.
|
Levine, A. J.
1997.
p53, the cellular gatekeeper for the growth and division.
Cell
88:323-331[Medline].
|
| 25.
|
Levitt, A.,
S. Chen,
G. Blanck,
D. George, and R. E. Pollack.
1985.
Two integrated partial repeats of simian virus 40 together code for a super-T antigen.
Mol. Cell. Biol.
5:742-750[Abstract/Free Full Text].
|
| 26.
|
Liu, X., and A. J. Berk.
1995.
Reversal of in vitro p53 squelching by both TFIIB and TFIID.
Mol. Cell. Biol.
15:6474-6478[Abstract].
|
| 27.
|
Long, S.-B.,
H.-Y. Ho,
C.-L. Chen, and M.-D. Lai.
1995.
Complex of simian virus large T antigen and p53 can bind DNA specifically.
Anticancer Res.
15:1375-1380[Medline].
|
| 28.
|
Lovett, M.,
C. E. Clayton,
D. Murphy,
P. W. J. Rigby,
A. E. Smith, and F. Chaudry.
1982.
Structure and synthesis of a simian virus 40 super-T antigen.
J. Virol.
44:963-973[Abstract/Free Full Text].
|
| 29.
|
May, E.,
M. Kress,
L. Daya-Grosjean,
R. Monier, and P. May.
1981.
Mapping of the viral mRNA encoding a super-T antigen of 115,000 daltons expressed in simian virus 40-transformed rat cell lines.
J. Virol.
37:24-35[Abstract/Free Full Text].
|
| 30.
|
Miyashita, T., and J. C. Reed.
1995.
Tumor suppressor p53 is a direct transcriptional activator of the human bax gene.
Cell
80:293-299[Medline].
|
| 31.
|
Mundt, M.,
T. Hupp,
M. Fritsche,
C. Merkle,
S. Hansen,
D. Lane, and B. Groner.
1997.
Protein interactions at the carboxyl terminus of p53 result in the induction of its in vitro transactivation potential.
Oncogene
15:237-244[Medline].
|
| 32.
|
Oliner, J. D.,
J. A. Pietenpol,
S. Thiagalingam,
J. Gyuris,
K. W. Kinzler, and B. Vogelstein.
1993.
Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53.
Nature
362:857-860[Medline].
|
| 33.
|
Rice, P. W., and C. N. Cole.
1993.
Efficient transcriptional activation of many simple modular promoters by simian virus 40 large T antigen.
J. Virol.
67:6689-6697[Abstract/Free Full Text].
|
| 34.
|
Ruppert, J. M., and B. Stillman.
1993.
Analysis of a protein-binding domain of p53.
Mol. Cell. Biol.
13:3811-3820[Abstract/Free Full Text].
|
| 35.
|
Scheffner, M.,
B. A. Werness,
J. M. Huibregtse,
A. J. Levine, and P. M. Howley.
1990.
The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53.
Cell
63:1129-1136[Medline].
|
| 36.
|
Segawa, K.,
A. Minowa,
K. Sugasawa,
T. Takano, and F. Hanaoka.
1992.
Abrogation of p53-mediated transactivation by SV40 large T antigen.
Oncogene
8:543-548.
|
| 36a.
| Sheppard, H. M., and X. Liu. Unpublished
results.
|
| 37.
|
Smith, A. F.,
R. Smith, and E. Pouche.
1979.
Characterization of different tumor antigens present in cells transformed by simian virus 40.
Cell
18:335-346[Medline].
|
| 38.
|
Storey, A.,
M. Thomas,
A. Kalita,
C. Harwood,
D. Gardiol,
F. Mantovani,
J. Breuer,
I. M. Leigh,
G. Matlashewski, and L. Banks.
1998.
Role of a p53 polymorphism in the development of human papillomavirus-associated cancer.
Nature
393:229-234[Medline].
|
| 39.
|
Thut, C. J.,
J. A. Goodrich, and R. Tijan.
1997.
Repression of p53-mediated transcription by MDM2: a dual mechanism.
Genes Dev.
11:1974-1986[Abstract/Free Full Text].
|
| 40.
|
Wilson, V. G., and R. L. Williams.
1985.
Origin binding by a 100,000-dalton super-T antigen from SVT2 cells.
J. Virol.
56:102-109[Abstract/Free Full Text].
|
| 41.
|
Yew, P. R.,
X. Liu, and A. J. Berk.
1994.
Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53.
Genes Dev.
8:190-202[Abstract/Free Full Text].
|
| 42.
|
Zhu, J.,
M. Abate,
P. W. Rice, and C. Cole.
1991.
The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind p53.
J. Virol.
65:6872-6880[Abstract/Free Full Text].
|
Molecular and Cellular Biology, April 1999, p. 2746-2753, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Kassam, S. N., Rainbow, A. J.
(2009). UV-inducible base excision repair of oxidative damaged DNA in human cells. Mutagenesis
24: 75-83
[Abstract]
[Full Text]
-
Bhana, S., Hewer, A., Phillips, D. H., Lloyd, D. R.
(2008). p53-dependent global nucleotide excision repair of cisplatin-induced intrastrand cross links in human cells. Mutagenesis
23: 131-136
[Abstract]
[Full Text]
-
Bocchetta, M., Eliasz, S., De Marco, M. A., Rudzinski, J., Zhang, L., Carbone, M.
(2008). The SV40 Large T Antigen-p53 Complexes Bind and Activate the Insulin-like Growth Factor-I Promoter Stimulating Cell Growth. Cancer Res.
68: 1022-1029
[Abstract]
[Full Text]
-
Bhana, S., Lloyd, D. R.
(2008). The role of p53 in DNA damage-mediated cytotoxicity overrides its ability to regulate nucleotide excision repair in human fibroblasts. Mutagenesis
23: 43-50
[Abstract]
[Full Text]
-
Amin, A. R. M. R., Thakur, V. S., Paul, R. K., Feng, G. S., Qu, C.-K., Mukhtar, H., Agarwal, M. L.
(2007). SHP-2 tyrosine phosphatase inhibits p73-dependent apoptosis and expression of a subset of p53 target genes induced by EGCG. Proc. Natl. Acad. Sci. USA
104: 5419-5424
[Abstract]
[Full Text]
-
Valls, E., Blanco-Garcia, N., Aquizu, N., Piedra, D., Estaras, C., de la Cruz, X., Martinez-Balbas, M. A.
(2007). Involvement of chromatin and histone deacetylation in SV40 T antigen transcription regulation. Nucleic Acids Res
35: 1958-1968
[Abstract]
[Full Text]
-
Deng, L., Nagano-Fujii, M., Tanaka, M., Nomura-Takigawa, Y., Ikeda, M., Kato, N., Sada, K., Hotta, H.
(2006). NS3 protein of Hepatitis C virus associates with the tumour suppressor p53 and inhibits its function in an NS3 sequence-dependent manner. J. Gen. Virol.
87: 1703-1713
[Abstract]
[Full Text]
-
Valls, E., de la Cruz, X., Martinez-Balbas, M. A.
(2003). The SV40 T antigen modulates CBP histone acetyltransferase activity. Nucleic Acids Res
31: 3114-3122
[Abstract]
[Full Text]
-
Yuan, L., Yu, W.-M., Yuan, Z., Haudenschild, C. C., Qu, C.-K.
(2003). Role of SHP-2 Tyrosine Phosphatase in the DNA Damage-induced Cell Death Response. J. Biol. Chem.
278: 15208-15216
[Abstract]
[Full Text]
-
Nghiem, P., Park, P. K., Kim, Y.-s., Desai, B. N., Schreiber, S. L.
(2002). ATR Is Not Required for p53 Activation but Synergizes with p53 in the Replication Checkpoint. J. Biol. Chem.
277: 4428-4434
[Abstract]
[Full Text]
-
Xing, J., Sheppard, H. M., Corneillie, S. I., Liu, X.
(2001). p53 Stimulates TFIID-TFIIA-Promoter Complex Assembly, and p53-T Antigen Complex Inhibits TATA Binding Protein-TATA Interaction. Mol. Cell. Biol.
21: 3652-3661
[Abstract]
[Full Text]
-
Nie, Y., Li, H.-H., Bula, C. M., Liu, X.
(2000). Stimulation of p53 DNA Binding by c-Abl Requires the p53 C Terminus and Tetramerization. Mol. Cell. Biol.
20: 741-748
[Abstract]
[Full Text]
-
Otsuka, M., Kato, N., Lan, K.-H., Yoshida, H., Kato, J., Goto, T., Shiratori, Y., Omata, M.
(2000). Hepatitis C Virus Core Protein Enhances p53 Function through Augmentation of DNA Binding Affinity and Transcriptional Ability. J. Biol. Chem.
275: 34122-34130
[Abstract]
[Full Text]
Top
Abstract
Introduction
