From the Department of Microbiology and Cell Biology,
Indian Institute of Science, Bangalore 560 012, India and the
¶ Howard Hughes Medical Institute, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Received for publication, November 18, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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p73 is a p53 homolog, as they are similar
structurally and functionally. Unlike p53, p73 is not inactivated by
the products of viral oncogenes such as SV40 T antigen and human
papilloma virus E6. Here we show that the product of adenoviral
oncogene E1A inhibits the transcriptional activation by both p73 The product of the tumor suppressor gene p53, which is often
described as the "guardian of the genome," is a transcription factor involved in maintaining genomic integrity by controlling cell
cycle progression and cell survival (1). Consistent with this view,
mutations in p53 are the most frequently seen genetic alterations in
human cancers (2, 3). Similarly, p53 knockout mice as well as
transgenic mice carrying mutant p53 alleles are highly prone to
developing spontaneous and carcinogen-induced tumors (4, 5).
Several p53 family members have been identified recently. They are p73,
p51, p63, and p40 (6-9). All of them have considerable homology to
p53, in particular to the conserved domains of p53. p73 produces two
major alternatively spliced isoforms, termed p73 Transforming proteins encoded by DNA tumor viruses target p53. In the
case of adenovirus, the E1A, E1B (55 kDa, but not 19 kDa), and
E4orf6 proteins specifically target and inhibit p53 (15-18).
Similarly, SV40 T antigen and papilloma virus E6 oncoproteins bind and
inactivate p53 (19, 20). However, p73 seems to be differentially
regulated by viral oncoproteins. Adenovirus E1B (55 kDa), SV40 T
antigen, and HPV16 E6 oncoproteins do not seem to inactivate p73 (21,
22).
In this work, we have studied the regulation of p73 by E1A. We found
that E1A could inhibit p73-mediated transcription very efficiently.
While this work was under progress, similar results were independently
reported (23, 24). We have further studied the mechanism of inhibition
of p73-mediated transcriptional activation by E1A; the structural
requirements within each protein for this inhibition; and more
importantly, the physiological relevance of this inhibition during
p73-mediated growth arrest with respect to transformation.
Plasmids--
pG13-Luc, WWP-Luc, pCEP4-p53, pcDNA3-p73 Cell Lines, Transfections, and Reporter Assays--
The mutant
p53-expressing human colon cancer cell line SW480 was maintained in
culture as described previously (16). The human lung carcinoma cell
line H1299 carries a homozygous deletion of p53 and was kindly provided
by Dr. Tapas Kumar Kundu (Jawaharlal Nehru Centre for Advanced
Scientific Research, Bangalore, India). Transfections were carried out
using Lipofectin (Invitrogen) or Escort (Sigma) following the
manufacturers' recommendations or by the calcium phosphate method as
described (26). In all transfections, 1 µg of pCMV-LacZ DNA was added
to normalize the transfection efficiency variation between samples.
Luciferase, Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was carried out essentially as described
previously (16). A p53-binding site (5'-AGCTTAGGCATGTCTAGGCATGTCTA-3') from the human p21 promoter was used as probe (28). The following reaction conditions were used: 25 mM Tris, pH 7.5, 20%
glycerol, 50 mM KCl, 50 mM dithiothreitol, 1 mg/ml bovine serum albumin, and 0.1% Triton X-100. The sequence of the
nonspecific competitor was as follows: 5'-GATCCGAATTCGGTACC-3'. The p73
proteins were made by in vitro coupled
transcription/translation (Promega), and E1A was bacterially
synthesized and purified as a GST fusion protein (27).
Adenoviral Reagents and Infections--
The adenoviruses used in
this study were as described previously (16, 29, 30). Ad-E1A/WT refers
to Ad5 Western and Immunohistochemical Analyses--
Western analysis
and immunohistochemical staining were performed as described previously
(16) with mouse anti-human p21WAF1/CIP1 antibody
(Ab-1, Oncogene), mouse anti-Ad2 E1A monoclonal antibody (M73,
Ab-1, Oncogene), mouse anti-human p73 monoclonal antibody (Ab-2,
Oncogene), mouse anti-human Rb monoclonal antibody (clone LM95.1, Ab-5, Oncogene), and goat anti-human actin polyclonal antibody
(I-19, Santa Cruz Biotechnology). Cells were harvested or fixed 24 h after viral infection and subjected to analysis. Under conditions in
which two viral infections were performed, cells were infected with the
second virus 6 h after the first viral infection; and 24 h
after the second viral infection, the experiment was terminated.
DNA Synthesis Inhibition--
BrdUrd incorporation (31) and
[3H]thymidine incorporation (16) were carried out as
described. BrdUrd (20 µM) or [3H]thymidine
(5 µCi) was added after 20 h of viral infection. The experiment
was terminated 4 h after addition of these compounds, and DNA
synthesis was measured either using anti-BrdUrd antibody (Ab-3,
Oncogene) or by measuring the radioactivity with a scintillation counter.
Cell Cycle Analysis--
SW480 cells were infected with Ad-LacZ,
Ad-E1A/WT, Ad-E1A/1101, Ad-p73, or the indicated combinations of
viruses at m.o.i. = 10. BrdUrd (20 µM) was added to the
cells during the last 4 h of the time points at which they were
harvested. The cells were washed with PBS twice and harvested by
trypsinization. The cells were washed again with PBS and fixed with
cold 70% ethanol for 1 h. Subsequently, the cells were
centrifuged, and the pellet was resuspended in 2 N HCl and
incubated at room temperature for 30 min. The cells were washed three
times with PBS and incubated with 1 µg of anti-BrdUrd antibody for 30 min. The cells were washed again twice with PBS and incubated with 2 µg of fluorescein isothiocyanate-conjugated goat anti-mouse antibody
(Oncogene) for 30 min at room temperature. The cells were washed with
PBS once and then incubated with 4 µg of ribonuclease A (Roche
Applied Science) for 30 min at room temperature. Propidium iodide was
added to the cell suspension at a final concentration of 20 µg/ml and
incubated for 30 min. The cells were then analyzed by flow cytometry
using a FACScan (BD Biosciences). The results were quantified using the
software WinList (Verity Software House Inc.).
E1A Inhibits p73-mediated Transcriptional Activation--
E1A has
been shown to inhibit p53-mediated transcriptional activation (15, 16).
To determine the effect of E1A on p73-mediated transcriptional
activation, we studied the ability of p73 to activate transcription in
the presence of E1A. We used the p53-specific reporter pG13-Luc to
assay p73-mediated transcription. pG13-Luc contains a synthetic
promoter with 13 copies of p53-binding sequence cloned in tandem
upstream of a basal polyoma promoter (25); transcription from this
promoter is dependent upon the presence of the wild-type p53 protein
(32). p73 activity can be studied by cotransfecting this reporter along
with the p73 expression vector into a p53 null or mutant cell line.
Neither p73 E1A Requires Its p300/CBP-binding Region to Inhibit
p73-mediated Transcription--
E1A is believed to transform cells by
binding and abrogating the activities of two sets of cellular proteins:
the p300/CBP family of coactivators and the pRb family of proteins
(33). Inhibition of p53-mediated transcription by E1A requires its
p300/CBP-binding region (16, 34-36). To determine the domain of E1A
required for the inhibition of p73 function, we tested the ability of
two deletion mutants of E1A to inhibit p73 function. The N-terminal
deletion mutant E1A( E1A Does Not Inhibit Sequence-specific DNA Binding by p73--
p73
binds to a sequence similar to p53-binding sequence and activates
transcription (21). We investigated the effect of E1A on the
sequence-specific DNA binding by p73 proteins, as it is essential for
transactivation (10). p73 E1A Targets Activation Domains of p73--
p73 has two activation
domains: one in the N-terminal region, which has ~25% similarity to
the N terminus of p53, and the other in the C-terminal region (37).
Whereas the N-terminal activation domain is identical in p73 E1A Inhibits p73-mediated Induction of
p21WAF1/CIP1--
Overexpression of p73 by transient
transfection leads to induction of p21WAF1/CIP1 and growth
arrest (10). To study the importance of inhibition of p73 function by
E1A, we studied the ability of p73 to transactivate p21WAF1/CIP1 in the presence of E1A. We used a
replication-deficient recombinant adenovirus carrying p73 E1A Inhibits p73-mediated Cell Cycle Arrest--
To correlate the
immunohistochemical and Western blot data with changes in cell cycle
phases, we studied the cell cycle profile under the same conditions. We
first monitored cellular DNA synthesis by measuring BrdUrd and
[3H]thymidine incorporation. The ability of p73 to
inhibit DNA synthesis in the absence or presence of either WT E1A or
its N-terminal deletion mutant was analyzed. Infection of H1299 cells
with Ad-p73 resulted in a drastic reduction in the percentage of cells
incorporating BrdUrd in comparison with Ad-LacZ-infected cells (Fig.
6a, compare panels
B and D), suggesting inhibition of cellular DNA
synthesis upon overexpression of p73. However, prior expression of E1A
by infecting cells with Ad-E1A/WT before Ad-p73 infection abrogated the
p73-mediated DNA synthesis inhibition (Fig. 6a, compare
panels D and F). The results obtained in Fig.
6a were quantified, and percent BrdUrd incorporation under
different conditions is shown in Fig. 6b. Cellular DNA
synthesis was also measured by [3H]thymidine
incorporation under similar conditions. Infection of cells with Ad-p73
resulted in a drastic reduction of [3H]thymidine
incorporation, which is indicative of DNA synthesis inhibition (Fig.
6c, compare bars 1 and 2). However,
expression of E1A before Ad-p73 infection resulted in the abrogation of
DNA synthesis inhibition as seen by increased
[3H]thymidine incorporation (Fig. 6c, compare
bars 2 and 3). In both of the above experiments,
the N-terminal deletion mutant of E1A failed to abrogate the DNA
synthesis inhibition by p73 (Fig. 6, a, compare
panels D and H; b, compare bars
2 and 4; and c, compare bars 2 and 4). The phosphorylation status of Rb correlated with the
extent of cellular DNA synthesis (Fig. 5c) under the conditions used in the above experiments. The hypophosphorylated form
of Rb was seen in Ad-p73-infected cells (Fig. 5c, lane
2). In addition, Ad-p73-induced hypophosphorylated Rb was seen
in cells expressing the N-terminal deletion mutant of E1A, but not WT
E1A (Fig. 5c, lanes 4 and 6). Thus, it
is evident from these results that abrogation of p73-mediated
inhibition of DNA synthesis correlates with the ability of E1A to
inhibit p73-mediated transcriptional activation, particularly that of
p21WAF1/CIP1. Moreover, the N terminus of E1A, which binds
to the p300/CBP family of coactivators, is essential for E1A to mediate
this function.
Exogenous overexpression of p73 also induces apoptosis in addition to
growth arrest (10). To distinguish between growth arrest and
apoptosis in Ad-p73-infected cells as well as to confirm the above
results, we analyzed the cell cycle profile by FACS. We first carried
out a control experiment in which we obtained FACS profiles of either
Ad-LacZ- or Ad-p73-infected cells at different time points after
infection (Fig. 7a).
Ad-p53-infected cells were used as a positive control. Upon Ad-p73
infection, cells with less than 2N DNA, representing apoptotic cells,
appeared only after 48 h (34.9% versus 4.2%) in
comparison with Ad-LacZ-infected cells. However, the proportion of
cells actively replicating DNA, representing S phase cells, decreased
by ~50% as early as 24 h (8.8% versus 16.6%) and
to negligible levels by 48 h (0.9% versus 19.3%) in
the Ad-p73-infected sample in comparison with the Ad-LacZ-infected sample. Thus, it is apparent that growth arrest as seen by a decrease in cells undergoing DNA synthesis occurs much earlier than apoptosis. Next, we checked the ability of WT E1A to abrogate p73-mediated growth
arrest by FACS (Fig. 7b). Ad-p73 infection led to a
significant decrease in S phase cells in comparison with Ad-LacZ
infection (26.2 to 5.2%) with a concomitant increase in cells
containing 2N DNA (58.8 to 81.3%), suggesting a potent G1
phase growth arrest mediated by p73. However, prior infection of
Ad-p73-infected cells with Ad-E1A/WT, but not with Ad-E1A/1101,
abrogated the decrease in S phase cells. Thus, in concurrence with our
earlier data (Fig. 6), WT E1A inhibits p73-mediated growth arrest
through its p300/CBP-binding region.
Stabilization of Endogenous p73 by E1A--
Expression of
wild-type E1A leads to stabilization of the p53 protein in rat embryo
fibroblast cells (38). p53 stabilization by wild-type E1A does not
require either the p300/CBP- or Rb-binding region of E1A (16). To study
the relation between inhibition of p73 function and transformation by
E1A, we studied the ability of E1A to stabilize endogenous p73.
Infection of H1299 cells with Ad-E1A/WT resulted in the stabilization
of p73 In this study, we found that E1A inhibits p73-mediated
transcription, confirming earlier observations by others. In addition, we have shown that the sequence-specific DNA binding by p73 is not
affected by E1A, whereas the transactivation domains of p73 are
targeted by E1A. A mutant of E1A carrying a deletion in the N terminus
failed to inhibit p73-mediated transcription. Furthermore, we have
shown that E1A abrogated p73-mediated growth arrest, which correlated
with its ability to inhibit p73-mediated activation of
p21WAF1/CIP1. E1A also stabilized endogenous p73 in a
time-dependent fashion. The N-terminal region of E1A, which
is needed for binding to the p300/CBP family of coactivators, was
required for inhibition of p73 function, but not for stabilization of
endogenous p73 by E1A.
E1A has been shown to inhibit p53-mediated transcription through its
p300/CBP-binding region (15, 16). Subsequently, it was shown that
p300/CBP molecules act as coactivators for p53 and that E1A binds to
p300/CBP coactivators, thereby inhibiting p53-mediated transcription
(34-36). In our study, we found that wild-type E1A, but not an
N-terminal deletion mutant of E1A, inhibited p73-mediated
transcription. Because the N-terminal deletion mutant does not bind to
the p300/CBP family of coactivators (33), it is evident that E1A
inhibits p73-mediated transcription by binding to the p300/CBP family
of proteins. In fact, p300/CBP has been shown to bind to p73 and to act
as a coactivator (39).
Our results also show that E1A targets the transactivation domains of
p73 and not the sequence-specific DNA binding by p73. p300/CBP has been
shown to interact with the N terminus of p73 and to coactivate
p73-mediated transcription (39). In good correlation with this,
wild-type E1A, but not the N-terminal deletion mutant, inhibited
transcription mediated by the N-terminal activation domain of p73. E1A
also inhibited transcription mediated by the C-terminal activation
domain of p73. Although we do not know the actual mechanism of this
inhibition, one possibility is that there may be an additional site for
p300/CBP binding on p73, as the N-terminal deletion mutant of E1A
failed to inhibit transcription from the C-terminal activation domain
of p73. Another possibility is that another protein, p400, which is
known to bind to the N terminus of E1A, could also be involved in the
inhibition of the C-terminal activation domain of p73 by E1A (40). p400
is related to SWI/SNF factors, and binding of E1A to p400 could recruit
SWI/SNF complexes to chromatin, leading to either transcriptional
activation or repression (40).
The level of p73 protein is increased in response to expression of
certain cellular and viral oncogenes. Cellular oncogene E2F is a direct
transcriptional activator of p73 by binding to several E2F1-responsive
elements within the P1 promoter of p73 (41, 42). The level of p73
protein, similar to p53, is increased upon adenoviral infection
(23, 43). Adenoviral E1A and E1B proteins are responsible for this p73
stabilization. Upon activation, p73 induces p21WAF1/CIP1
and mounts a strong G1 arrest by inhibiting DNA synthesis,
which would prevent transformation. Our study has also shown that
endogenous p73 was stabilized by the expression of WT E1A. In addition,
we have shown that the N-terminal region of E1A, which is essential for
binding to the p300/CBP family of proteins, was not needed for p73
stabilization (Fig. 8). However, p73 stabilized by WT E1A was unable to
activate p21WAF1/CIP1, whereas p73 stabilized by the
N-terminal deletion mutant of E1A was able to activate
p21WAF1/CIP1. These results suggest a mechanism whereby E1A
could bring about a successful transformation by inhibiting the
transcriptional activation of p21WAF1/CIP1 by endogenous
p73 induced in response to expression of WT E1A. The N-terminal
deletion mutant, which did not inhibit p73-mediated transcription, was
also able to stabilize endogenous p73, but with a concomitant
activation of p21WAF1/CIP1, thus explaining the fact that
this N-terminal deletion mutant is unable to transform cells successfully.
Our study suggests that E1A bypasses important cell cycle checkpoints
by inhibiting transcriptional activation by p73. The benefits from
inhibition of p73-mediated p21WAF1/CIP1 induction are
2-fold. First, inhibition of p73-mediated induction of
p21WAF1/CIP1 would relieve a block at the G1/S
boundary because complexes between cyclin and
cyclin-dependent kinase associated with G1/S progression would remain activated. Second, it would also relieve a
block during DNA synthesis, as it has been shown that
p21WAF1/CIP1 interacts with proliferating cell nuclear
antigen and affects processive DNA synthesis (44).
Increases in p53 levels have been observed frequently in primary
tumors. However, p53 is inactivated by mutational inactivation in the
majority of these cases. p53 is also inactivated by the ARF-MDM2-p53
pathway (45). This pathway includes amplification of MDM2, homozygous
deletion of ARF, or methylation silencing of ARF expression. The levels
of p73 in normal human tissues are generally very low (46, 47). In
contrast, overexpression of p73 has been shown in variety of primary
tumors and tumor cell lines (48-54). Unlike p53, p73 is very rarely
mutated in primary tumors, suggesting the existence of other mechanisms
that functionally inactivate stabilized p73. Binding of MDM2 and MDMX
proteins to p73 inhibits the transcriptional activation by p73 and
affects the subcellular localization of p73 (55-57). It remains to be
seen whether ARF regulates the function of p73. Thus, it is evident that there are cellular mechanisms of inactivation of p73 function during transformation similar to transcriptional inactivation of p73 by E1A.
p73 and p63 have been shown to regulate p53 function, in particular
p53-dependent apoptosis in response to DNA damage (58). p53
also utilizes the p300/CBP family of proteins as coactivators (34-36),
similar to p73. This raises the question of whether the inhibitory
effect of E1A on p73 function is mediated through a mechanism involving
p53. Because our experiments were carried out in cells in which p53 is
either mutated (SW480) or deleted (H1299), the inhibition of
p73-mediated transcription by E1A is p53-independent.
In summary, we conclude that adenoviral oncogene E1A inhibits
p73-mediated transcription in a manner similar to inhibition of p53
activity by E1A. However, inhibition of p73 function by E1A is
independent of p53. Our findings suggest that inhibition of
p73-mediated growth arrest by E1A could be an additional mechanism for
a successful transformation. In the p53 family, few other members, such
as p63 and p51, in addition to p73, have been identified. It will be
interesting to determine whether these members are up-regulated in
response to oncogene expression and whether E1A inhibits the function
of these p53 homologs as well.
and
p73
. Electrophoretic mobility shift assays revealed that E1A does
not inhibit the sequence-specific DNA binding by p73. Transcriptional activation by a fusion protein containing the Gal4 DNA-binding domain
and either of the activation domains of p73 was inhibited by wild-type
(WT) E1A, but not by the N-terminal deletion mutant E1A(
2-36). E1A(
2-36), which does not bind to the
p300/CBP family of coactivators, failed to inhibit p73-mediated
transcription, whereas E1A(
CR2), a deletion mutant that does not
bind to the pRb family of proteins, inhibited p73-mediated
transcription as efficiently as WT E1A. Consistent with these
observations, growth arrest induced by p73 expressed from a recombinant
adenovirus was abrogated by WT E1A, which correlated with inhibition of
p73-mediated induction of p21WAF1/CIP1 by E1A.
However, p73 was able to induce p21WAF1/CIP1 and to mediate
growth arrest in the presence of E1A(
2-36). Furthermore, the
expression of either wild-type E1A or E1A(
2-36) resulted in the
stabilization of endogenous p73. However, p73 stabilized in response to
the expression of E1A(
2-36), but not WT E1A, was able to activate
the expression of p21WAF1/CIP1. These results suggest that
the transcriptional activation function of p73 is specifically targeted
by E1A through a mechanism involving p300/CBP proteins during the
process of transformation and that p73 may have a role to play as a
tumor suppressor.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and p73
, which
differ at their C terminus (6). The homology between both p73 isoforms
and p53 is very extensive within the most conserved domains. p73
isoforms are also similar functionally to p53 in that they are able to
inhibit cancer cell growth by activating p21WAF1/CIP1 and
induce apoptosis when exogenously expressed (10). However, p73 is also
different from p53 in many different ways. Unlike p53, p73 is
monoallelically expressed and is not activated by either DNA damage or
ultraviolet irradiation (6). Moreover, mutations in the p73 gene have
not yet been found in several cancers (11), despite the fact that p73
is located in 1p36.3, which is one of the most frequently deleted
regions in neuroblastomas and other cancers (12-14).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
pcDNA3-p73
, WT1 E1A,
E1A(
2-36), E1A(
CR2), GST-E1A, G5E1BCAT, and pCMV-LacZ were described previously (16, 22, 25-27). WT E1A refers to 12 S form of
E1A, and all of the mutant forms of E1A were derived from it. The
fusion protein constructs consisting of the Gal4 DNA-binding domain and
either of the activation domains of p73 were made as follows. For the
N-terminal activation domain, the region between amino acids 1-112 of
p73
was amplified as a BamHI/XbaI fragment fused to the Gal4 DNA-binding domain by cloning the fragment into the
BamHI and XbaI sites of the pM plasmid
(Clontech). For the C-terminal activation domain,
the region between amino acids 380 and 513 of p73
was amplified as a
BamHI/XbaI fragment and fused to the Gal4
DNA-binding domain by cloning the fragment into the BamHI
and XbaI sites of the pM plasmid. Because the SV40 promoter, which drives the expression of Gal4 fusions in pM, is inhibited by E1A,
we transferred the entire fragment carrying Gal4-activation domain
fusions as BglII/XbaI fragments into the
BamHI and XbaI sites of pcDNA3 (Invitrogen).
-galactosidase, and chloramphenicol acetyltransferase
assays were performed as described previously (16, 26).
520E1B
, which lacks E1B, but expresses wild-type
12 S E1A only. Ad-E1A/1101 refers to Ad5
520E1B
/1101,
which lacks E1B, but expresses 12 S E1A lacking amino acids 4-25.
Ad-LacZ lacks both E1A and E1B, but carries
-galactosidase (25).
Ad-p73 lacks both E1A and E1B, but carries simian p73
. (The details
of the construction and characterization of Ad-p73 will be
published elsewhere.) Adenoviral infections were carried out as
described (16).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
nor p73
activated transcription in the presence of WT
E1A (Fig. 1, b and c). Moreover, the inhibition by WT E1A was
dose-dependent (Fig. 1d). When activated, p53
has been shown to induce p21WAF1/CIP1, which is the major
mediator of p53-mediated growth arrest (1). Because p73 also induces
p21WAF1/CIP1 upon overexpression, p21WAF1/CIP1
may play an important role in p73-mediated growth arrest. WT E1A
inhibited the transcriptional activation of the
p21WAF1/CIP1 promoter by p73
(Fig. 1e). These
results suggest that adenovirus E1A inhibits p73-mediated transcription
very effectively.
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Fig. 1.
Effect of E1A on p73-mediated
transactivation. SW480 colon cancer cells were transfected with 1 µg of pG13-Luc (a-d) or WWP-Luc (e) and, where
indicated (+), with wild-type p53 expression plasmid (1 µg),
wild-type p73 expression plasmid (1 µg), wild-type p73
expression plasmid (1 µg), or wild-type 12 S E1A expression plasmid
(1 µg). Transfections were carried out either by the calcium
phosphate method (a-d) or using Escort transfection reagent
(e). Lysates were prepared and analyzed for luciferase
reporter activity 48 h post-transfection as described under
"Experimental Procedures." pUC18 plasmid DNA was added to keep the
total amount of DNA per transfection at 7 µg. In d,
increasing amounts of wild-type 12 S E1A expression plasmid DNA (0.25, 0.5, 0.75, and 1 µg) were used (bars 3-6).
2-36), which does not bind to the p300/CBP
family of coactivators, failed to inhibit p73-mediated transcription (Fig. 2, a, bar 4;
and b, bar 4). However, the deletion mutant E1A(
CR2), which does not bind to the pRb family of proteins, inhibited p73 function very efficiently (Fig. 2, a,
bar 5; and b, bar 5). Both WT E1A and
its deletion mutants were detected in transfected cells (Fig.
2c, lanes 2-4). These results suggest that an
intact p300/CBP-binding region is required for E1A to inhibit p73
function.
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Fig. 2.
Mapping of the E1A domain responsible for
inhibiting p73-mediated transactivation. a and
b, SW480 colon cancer cells were transfected with 1 µg of
pG13-Luc and, where indicated (+), with wild-type p73 expression
plasmid (1 µg), wild-type p73
expression plasmid (1 µg),
wild-type 12 S E1A expression plasmid (1 µg), E1A(
2-36; 1 µg),
or E1A(
CR2; 1 µg). pUC18 plasmid DNA was added to keep the total
amount of DNA per transfection at 7 µg. Transfections were carried
out by the calcium phosphate method. Lysates were prepared and analyzed
for luciferase reporter activity 48 h post-transfection as
described under "Experimental Procedures." c, SW480 cell
lysates (a, bars 2-5) were immunoblotted for the
E1A protein.
and p73
proteins synthesized in
vitro using coupled transcription/translation system were used in
this assay. p73 proteins bound to the p53-binding site (Fig.
3, a, lane 3; and
c, lane 4). The binding was specific, as it was
competed efficiently by an unlabeled p53 oligonucleotide (Fig. 3,
a, lanes 4-6; and c, lanes
5-7), but not by a nonspecific oligonucleotide (a,
lanes 7-9; and c, lanes 8-10). E1A
did not inhibit the sequence-specific DNA binding by p73
(Fig.
3b, lane 5) and p73
(c, lane
12). These results suggest that the sequence-specific DNA binding
by p73 is not targeted by E1A during its inhibition of p73
function.
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Fig. 3.
Effect of E1A on the sequence-specific
DNA-binding activity of p73. Electrophoretic mobility shift assay
was performed using in vitro translated p73 (a
and b) and p73
(c) as described under
"Experimental Procedures." Competitions were performed by adding
increasing amounts of specific or nonspecific competitor
(com.; 0.03, 0.3, and 3 pmol) as indicated. Elution
buffer refers to the reduced glutathione buffer used for eluting
GST-E1A from glutathione beads. oligo,
oligonucleotide.
and
p73
, the C-terminal activation domain, which extends from amino
acids 380 to 513 in p73
, is terminated at amino acid 495 in p73
due to differential splicing (37). Because the sequence-specific DNA
binding by p73 was not affected by E1A, we thought that E1A could
inhibit p73-dependent transcription by targeting the
activation domains. To examine this possibility, we studied the effect
of E1A on the transcriptional activation by a fusion protein comprising
the Gal4 DNA-binding domain and either of the activation domains of p73. E1A inhibited transcription from the N-terminal activation domain
(Fig. 4, a, compare
lanes 2 and 3; and c, compare
bars 1 and 2) as well as from the C-terminal
activation domain (b, compare lane 1 and
2; and d, compare bars 1 and
2) of p73
. The inhibition of Gal4 fusion proteins by WT
E1A was specific because of the following reasons. Transcription by the
Gal4-VP16 fusion protein was not inhibited by WT E1A (16). The
N-terminal deletion mutant E1A(
2-36) failed to inhibit
transcription from either the N-terminal activation domain (Fig. 4,
a, compare lanes 2 and 4; and
c, compare bars 1 and 3) or the
C-terminal activation domain (b, compare lanes 1 and 3; and d, compare bars 1 and
3) of p73. These experiments suggest that E1A specifically
inhibits the activation domains of p73 and that the p300/CBP-binding
region of E1A is required for this inhibition.
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Fig. 4.
Effect of E1A on transactivation domains of
p73. SW480 colon cancer cells were transfected with 5 µg of
G5E1BCAT and, where indicated (+), with Gal4-p73 N-terminal activation
domain fusion protein (GAL4/N-ter; 2.5 µg)
(a), Gal4-p73 C-terminal activation domain fusion protein
(GAL4/C-ter; 2.5 µg) (b), WT E1A
(2.5 µg) (a and b), or E1A( 2-36; 2.5 µg)
(a and b). Transfections were carried out using
Lipofectin transfection reagent. pUC18 plasmid DNA was added to keep
the total amount of DNA per transfection at 11 µg. Lysates were
prepared and analyzed for chloramphenicol acetyltransferase reporter
activity 24 h post-transfection as described under "Experimental
Procedures." All experiments were performed in duplicate.
Percent chloramphenicol acetyltransferase (CAT) conversions
were quantified for samples from a and b, and the
values are shown in c and d, respectively.
Chloramphenicol acetyltransferase assays were redone for a
(lanes 2-4) after diluting the cell lysates appropriately
so that the percent chloramphenicol acetyltransferase conversion was
<30%, and the values are shown in c (bars 1-3,
respectively). Percent chloramphenicol acetyltransferase conversions
for b (lanes 1-3) were quantified, and the
values are shown in d (bars 1-3,
respectively).
(Ad-p73)
for this purpose. We first checked the ability of p73 expressed from
Ad-p73 to induce p21WAF1/CIP1 in the presence of E1A. We
used the human lung carcinoma cell line H1299 for this experiment
because it carries a homozygous deletion of p53. H1299 cells infected
with a control virus (Ad-LacZ) showed very little or no expression of
p73, E1A, and p21 proteins (Fig.
5a, panels A,
E, and I, respectively). Infection of H1299 cells
with Ad-p73 led to accumulation of high levels of p73 and p21 (Fig.
5a, panels B and J, respectively).
However, when Ad-p73 infection was preceded by Ad-E1A/WT infection,
cells expressed high levels of p73 and E1A, but not
p21WAF1/CIP1 protein (Fig. 5a, panels
C, G, and K, respectively). Results from
reporter assay experiments suggested that an intact p300/CBP-binding region is required for E1A to inhibit p73 function (Fig. 2,
a and b). To confirm this observation, we checked
the effect of an N-terminal deletion mutant of E1A on p73-mediated
induction of p21WAF1/CIP1. We used Ad-E1A/1101 for this
purpose because it expresses an E1A protein with a deletion of amino
acids 24 and 25, which makes it deficient for binding to p300/CBP
proteins (29, 30). Unlike WT E1A, the N-terminal deletion mutant failed
to inhibit p73-mediated induction of p21WAF1/CIP1 (Fig.
5a, panel L). We also analyzed the p73 and
p21WAF1/CIP1 levels by Western blotting in a similar
experiment (Fig. 5b), the results of which were comparable
to those of the immunohistochemical staining experiment. The presence
of WT E1A, but not its N-terminal deletion mutant, inhibited the
induction of p21WAF1/CIP1 by p73 (Fig. 5b,
compare lanes 5 and 6). These results suggest that E1A inhibits p73-mediated induction of p21WAF1/CIP1
efficiently through its p300/CBP-binding region.
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Fig. 5.
Effect of E1A on p73-mediated activation
of p21WAF1/CIP1. H1299 cells were infected with
Ad-LacZ, Ad-p73, Ad-E1A/WT, and Ad-E1A/1101 alone and in
combinations at m.o.i. = 10 as indicated in a-c.
a, immunohistochemical staining for p73 (panels
A-D), E1A (panels E-H), and p21WAF1/CIP1
(panels I-L); b, Western blot analysis for p73,
E1A, p21WAF1/CIP1, and actin proteins; c,
Western blot analysis for Rb.
View larger version (44K):
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Fig. 6.
Effect of E1A on p73-mediated DNA synthesis
inhibition. Cellular DNA synthesis was measured by BrdUrd
incorporation (a and b) or by
[3H]thymidine incorporation (c). H1299 cells
were infected with Ad-LacZ, Ad-p73, Ad-E1A/WT + Ad-p73, or Ad-E1A/1101 + Ad-p73 at m.o.i. = 10 as indicated in a-c.
BrdUrd-positive cells were identified by staining using anti-BrdUrd
antibody and Texas Red-conjugated anti-mouse secondary antibody
(a, panels B, D, F, and
H). Panels A, C, E, and
G show the results from 4,6-diamidino-2-phenylindole
staining of the cells in panels B, D,
F, and H, respectively. The percentage of
BrdUrd-positive cells was calculated as percent BrdUrd
(Brdu) incorporation from a and is shown in
b. Percent BrdUrd incorporation was calculated by dividing
the number of cells showing BrdUrd incorporation by the total number of
cells counted. The level of [3H]thymidine incorporation
in Ad-LacZ-infected cells was considered as 100% (c).
View larger version (52K):
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Fig. 7.
Flow cytometry analysis of Ad-p73-infected
cells. a, SW480 cells were infected with either Ad-LacZ
or Ad-p73 at m.o.i. = 10. The cells were harvested 12, 24, 36, and 48 h after infection and subjected to flow cytometry analysis
as described under "Experimental Procedures." The cells were
allowed to incorporate BrdUrd for the last 4 h of the time points
at which they were collected. S indicates cells undergoing
DNA synthesis (S phase), and A indicates cells containing
less than 2N DNA (apoptotic cells). The quantified values are
indicated. b, SW480 cells were infected with Ad-LacZ,
Ad-E1A/WT, or Ad-E1A/1101 at m.o.i. = 10. After 6 h of infection,
the cells were again infected with either Ad-LacZ or Ad-p73 as
indicated. 20 h after the second viral infection, BrdUrd
incorporation was carried out for 4 h, and the cells were
harvested and subjected to flow cytometry analysis as described under
"Experimental Procedures." G1 indicates cells containing
2N DNA, and G2/M indicates cells containing 4N
DNA. The quantified values are indicated.
in a time-dependent fashion (Fig.
8, lanes 2 and 5).
However, endogenous p73 stabilized in response to WT E1A expression was
unable to activate p21WAF1/CIP1 very efficiently (Fig. 8,
lanes 2 and 5). Expression of an N-terminal deletion mutant of E1A (Ad-E1A/1101) also resulted in the stabilization of endogenous p73
, but the difference was that p73 stabilized by the
N-terminal deletion mutant was able to activate
p21WAF1/CIP1 very efficiently (Fig. 8, lanes 3 and 6). p73
expressed from an exogenous source by
infection of H1299 cells with Ad-p73 resulted in the activation of
p21WAF1/CIP1 (Fig. 8, lane 7). These results
suggest at least two important points. E1A also inhibits activation of
p21WAF1/CIP1 by endogenously stabilized p73
in a
p300/CBP-dependent manner, and the p300/CBP-binding region
of E1A is not required for stabilization of endogenous p73, whereas it
is required for inhibition of p73-mediated transcription. This
observation is very important with respect to transformation by E1A
because p73 stabilized by E1A would otherwise suppress DNA synthesis
and induce growth arrest by activating p21WAF1/CIP1.
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Fig. 8.
Effect of E1A on endogenous p73. H1299
cells were infected with Ad-LacZ, Ad-E1A/WT, Ad-E1A/1101, or Ad-p73 at
m.o.i. = 10. The cells were harvested after 36 h (left
panels) or 48 h (right panels), and the levels of
p73 , E1A, and p21WAF1/CIP1 were analyzed by Western
blotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by grants from the Council for Scientific and Industrial Research and the Defense Research and Development Organization, Government of India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from the Council for Scientific and Industrial Research, Government of India.
To whom correspondence should be addressed. Tel.:
91-80-394-2973; Fax: 91-80-360-2697; E-mail:
skumar@mcbl.iisc.ernet.in.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M211704200
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ABBREVIATIONS |
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The abbreviations used are: WT, wild-type; GST, glutathione S-transferase; Ad, adenovirus; BrdUrd, bromodeoxyuridine; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; CBP, cAMP-responsive element-binding protein-binding protein; FACS, fluorescence-activated cell sorting; ARF, alternate reading frame; Rb, retinoblastoma protein.
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REFERENCES |
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