From the Department of Pathology, State University of New York at
Stony Brook, Stony Brook, New York 11794-8691 and
Dana-Farber Cancer Institute, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, June 29, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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The identification of upstream pathways that
signal to TP73 is crucial for understanding the
biological role of this gene. Since some evidence suggests that
TP73 might play a role in tumorigenesis, we asked whether
oncogenes can induce and activate endogenous TP73. Here, we
show that endogenous p73 TP53 is a crucial tumor suppressor for preventing the
malignant transformation of cells. Surprisingly, despite
TP53's central role in carcinogenesis, no related genes
were known for 20 years. In 1997, two novel family members were
identified and termed TP73 and TP63 (1-4). p73
shares 63% identity with the DNA-binding region of p53 including the
conservation of all DNA contact residues, 38% identity with the
tetramerization domain, and 29% identity with the transactivation
domain. In contrast to TP53, human TP73 produces
six C-terminal splice variants (p73 Despite this experimental evidence, the role of TP73 in
tumorigenesis is as yet unclear. Current genetic data have ruled out that TP73 is a Knudson-type tumor suppressor. Although
TP73 maps to chromosome 1p36.3, which undergoes frequent
loss of heterozygosity in breast cancer, neuroblastoma, and
several other cancers (12), mutations in the TP73 gene are
extremely rare in human tumors. Initially, imprinting of the
TP73 locus was thought to be an explanation to satisfy the
two-hit hypothesis in tumors with loss of heterozygosity but no
mutations. However, imprinting is highly variable from patient to
patient and tissue to tissue (6, 13-15). In fact, in lung, esophageal,
gastric, and renal carcinoma, the second TP73 allele is
specifically activated in the tumor compared with the normal tissue of
origin (loss of imprinting) (16-19). Furthermore, p73-deficient mice
lack a spontaneous tumor phenotype but have neurological and
immunological defects (7).
Both differences and similarities to p53 are found with respect to p73
inactivation by viral oncoproteins. SV40 T antigen, adenovirus
E1B 55-kDa protein, and HPV E6, which all target and inactivate p53
during host cell transformation, do not target the p73 protein
physically or functionally (20-22). Indeed, ectopic p73, but not p53,
induces apoptosis in E6-transformed cells, highlighting TP73's potential for gene therapy in HPV-mediated cancers
(23). However, the adenovirus E4orf6 oncoprotein specifically represses p73 but not p53 transactivation in some experimental systems (22, 24),
indicating that adenoviral transformation targets both p53 and p73 via
different viral products. Stable transfectants of a human rhabdoid
tumor cell line expressing E1A or adenovirus 5 large E1B showed
increased p73 levels, although functional activation of the protein was
not tested (24). The Tax protein of the human T-cell leukemia virus
type 1 represses the transactivation function of p73 In all normal human tissues studied, p73 is expressed at very low
levels (13, 30). In contrast, multiple primary tumor types and tumor
cell lines overexpress p73. Work by us and others showed that the most
common cancer-specific alteration is an overexpression of p73 rather
than a loss. To date, p73 overexpression has been found in tumors of
breast, neuroblastoma, lung, esophagus, stomach, colon, bladder, ovary,
ependymoma, hepatocellular carcinoma, and myeloid leukemia (CML blast
crisis and acute myeloid leukemia) (6, 13, 14, 17-19, 31-36). For
example, using quantitative reverse transcription-polymerase chain
reaction, we found overexpression (5-25-fold) of TP73
mRNA in 38% of 77 invasive breast cancers and in 5 of 7 breast
cancer cell lines (13-73-fold) but not in normal breast (6). Likewise,
we found that TP73 mRNA is overexpressed in a subset of
neuroblastoma and related embryonal tumors (8-80-fold) and in 12 of 14 neuroblastoma cell lines (8-90-fold) (13). The cause of p73
overexpression is unknown.
Unlike p53 protein, which becomes stabilized and activated in response
to a very broad spectrum of cellular stresses, little is known about
the upstream signals that induce a p73 response. p73 is not activated
by UV, actinomycin D, doxorubicin, and mitomycin C (1, 37), all of
which stabilize and activate p53. However, recently it has been shown
that endogenous p73 is activated for apoptosis in response to cisplatin
and Cell Culture and Reagents--
The human lung carcinoma line
H1299 and the human osteosarcoma line SaOs-2 each carry a homozygous
deletion for TP53 and were used for transfection. Other cell
lines were SK-N-AS (human neuroblastoma), MRC5 (human diploid
fibroblasts), and COS (monkey kidney cells). All cells were cultured in
Dulbecco's modified Eagle's medium plus 10% fetal calf serum.
Cisplatin was purchased from Sigma.
Plasmids--
The following mammalian expression plasmids were
used: pE1A, expressing E1A 12 S protein driven by the natural
adenovirus E1A enhancer/promoter (41); pCMXM45 E2F1, expressing human
E2F1 (42); pmc-Myc, expressing mouse c-Myc protein (gift from M. Cole,
Princeton University); pC53-C1N3 expressing human wild type p53; and
pcDNA3-p73
pcDNA3-p73DD and pcDNA3-mtp73DD were gifts of Dr. William
Kaelin and are described in detail in Ref. 43. Briefly,
pcDNA3-p73DD expresses T7-tagged amino acids 327-636 of human p73
Antibodies and Immunoblots--
Cell lysates were prepared as
described (45), subjected to SDS-polyacrylamide gel electrophoresis,
and transferred to nylon membranes. Immunoblots were visualized by ECL
(Pierce). Antibodies to p73 were the monoclonal GC15 (AB-3 from
Oncogene Science; recognizes amino acids 380-499 of human p73 Transfections--
H1299 cells were plated in 60-mm dishes and
grown overnight to 80% confluence. For transient transfections, 2 µg
of expression plasmid or empty vector was cotransfected with 200 ng of
green fluorescent protein-encoding plasmid using the LipofectAMINE Plus reagent (Life Technologies, Inc.). Cells were collected after 24 h. Stable transformants were seeded into P100 plates (1 × 107 cells) and selected for 21 days in medium containing
0.5 mg/ml G418 (Life Technologies), ring-cloned, and expanded into
single cell clones. For the SaOs-2 experiments (see Fig.
5D), transformants were pooled after 3 weeks of G418
selection and transiently transfected with c-Myc prior to parallel
TUNEL assays and immunofluorescence staining 24 h later. For
luciferase assays, cells were seeded into 24-well plates and
transfected with an expression vector or empty vector (400 ng) together
with the p53-responsive PG13-Luc from firefly (80 ng) and pRL-TK
Renilla luciferase cDNA (8 ng). To test the effect of
the dominant negative inhibitor and its mutant, each well was
transfected with 100 ng of expression vector plus 300 ng of empty
vector, with 100 ng of expression vector plus 300 ng of p73DD, or with
100 ng of expression vector plus 300 ng of mtp73DD, together with the
reporter constructs as above. Luciferase activity was measured after
24 h by the dual luciferase reporter assay (Promega), and
transfection efficiency was standardized against Renilla luciferase.
For apoptosis, SaOs-2 cells were seeded in duplicates into eight-well
chambers 48 h prior to transfection. At about 70% confluence, cells in duplicate wells were transfected with expression plasmid (150 ng) plus empty vector (350 ng) or expression plasmid (150 ng) plus
p73DD (350 ng) or expression plasmid (150 ng) plus mtp73DD (350 ng).
After 24 h, cells were fixed and processed in parallel for the
TUNEL assay (Roche Molecular Biochemicals) and for immunofluorescence using the appropriate antibodies against p73, E2F1, c-Myc, or E1A.
Expression was reproducibly about 30%, similar among all constructs
and evenly distributed throughout the wells. For each construct,
TUNEL-positive cells (494 fields at 40×) and transfected cells (30 fields at 40×; around 200 cells) from duplicate chambers were counted,
and the percentage of apoptosis of transfected cells was determined
after correction for background with vector alone (500 ng per well).
Experiments were performed 3-6 times, depending on the construct.
Endogenous p73
Next we tested whether viral and cellular oncogenes, which are major
upstream signals for TP53 activation, are also
physiologically relevant for triggering the induction of endogenous
TP73. To this end, H1299 cells were transiently transfected with
various oncogene-encoding plasmids. Their expression was verified by
immunoblotting with the respective antibodies (Fig.
2A). Both p73 Stable H1299 Clones Overexpressing c-Myc Recapitulate the
Up-regulation of p73 Proteins--
Since transient overexpression of
oncogenes induces the accumulation of p73 Oncogene-mediated Up-regulation of Endogenous p73 Protein Leads to
p73 Transcriptional Activation--
We then tested whether the
oncogene-mediated up-regulation of endogenous p73 translates into
activation of p73 transcriptional function. To this end, we carried out
luciferase reporter assays in transiently transfected H1299 cells using
the p53/p73-responsive PG13-Luc reporter. As shown in Fig.
4A, all three oncogenes were able to activate p73 reporter activity. E2F1 exhibited a 16.5-fold, c-Myc a 10.3-fold, and E1A a 13.9-fold induction of the p73-responsive reporter compared with vector controls. These data indicate that oncogenes induce the transcriptional activation of endogenous p73.
Oncogene-mediated Up-regulation of Endogenous p73 Leads to
Activation of TP73 Response Genes--
TP73 shares many
response genes with TP53 in vivo. This has been
shown in several cell systems using transient or inducible expression
of ectopic p73 (1, 8, 9, 38). To further support our previous results,
we tested whether oncogene-mediated accumulation of endogenous p73
leads to the induction of TP73 target gene products. When
p53-deficient H1299 cells were transiently transfected with expression
plasmids for E2F1, c-Myc, and E1A, endogenous p73 Oncogene-mediated Activation of Endogenous p73 Induces Apoptosis in
p53-deficient Tumor Cells; Conversely, Inactivation of p73 Inhibits
Oncogene-induced Apoptosis--
The activation of the p73
transcription function by oncogenes suggested that these upstream
signals might also induce the activation of the apoptotic function of
p73. To test this prediction, we performed apoptosis assays on
transiently transfected SaOs-2 cells using the in situ TUNEL assay.
To confirm that the oncogene-induced apoptotic activity is mediated
through TP73, we tested the effect of a coexpressed dominant negative inhibitor of p73 (p73DD) (43). p73DD is modeled after the
dominant negative p53 inhibitor (p53DD) (48) and encodes amino acids
327-636 of human p73
Prior to doing apoptosis assays, we needed to demonstrate that
oncogenes induce endogenous p73 in p53-deficient SaOs-2. As seen in
Fig. 5B, expression of E2F1, c-Myc, and E1A in these cells markedly induced p73 levels, analogous to what we already saw in H1299
cells (see Fig. 2). Importantly, the induced p73 protein was
functionally active (Fig. 5C). After transient transfection, all three oncogenes induced apoptosis in SaOs-2 cells, which resembled the one seen after transfecting p73 Our results show that during short term exposure, overexpression
of three different oncogenes induce the endogenous TP73 gene and activate it for transcriptional and apoptotic function in p53-deficient cells. We demonstrate that disruption of p73 function inhibits oncogene-induced apoptosis in p53-deficient human tumor cells.
Collectively, our findings identify an important novel afferent
signaling pathway to p73 that appears to operate in vivo. This conclusion is in agreement with evidence from several other experimental systems. For example, Myc-induced apoptosis in
p53 While this work was ongoing, W. G. Kaelin's group obtained similar
data on the relationship of E2F1 and p73 (43). They also found that p73
mediates E2F1-induced apoptosis in SaOs-2 cells and
p53 In contrast to transcriptional activation of TP73 by
oncogenes, cisplatin activates endogenous p73 by a posttranscriptional mechanism (38). Cisplatin increases protein stability from 45 min to
2 h without altering TP73 transcript levels.
Protein stability of endogenous and exogenous p73 We also find that stable deregulation of c-Myc in H1299 subclones
recapitulates the p73 overexpression seen with transient deregulation
of c-Myc. One might ask how stable clones can be generated, given that
in transiently transfected tumor cells deregulated c-Myc was able to
activate the transcriptional and apoptotic activity of p73. One
possibility is that clonal outgrowth of cells with stable
overexpression of oncogenes selects for loss of p73 transactivation function. In keeping with this idea, a preliminary analysis on three
clones with the highest p73 Most human tumors harbor deregulated oncogenes, including the Myc gene
(56). In particular, many human tumors have suffered a deregulation of
the E2F1 activity through mutations that inactivate the Rb pathway
(57-59), thus derepressing E2F1-responsive genes. Our finding could
provide a framework for the fact that p73 is frequently overexpressed
in human tumors. Our stable clones will be a helpful tool in future
studies aimed at determining the functional relevance of constitutive
p73 overexpression in tumors.
and
proteins are up-regulated in
p53-deficient tumor cells in response to overexpressed E2F1, c-Myc, and E1A. E2F1, c-Myc, and E1A-mediated p73 up-regulation leads to activation of the p73 transcription function, as shown by
p73-responsive reporter activity and by induction of known endogenous
p73 target gene products such as p21 and HDM2. Importantly, E2F1-,
c-Myc-, and E1A-mediated activation of endogenous p73 induces apoptosis
in SaOs-2 cells. Conversely, inactivation of p73 by a dominant
negative p73 inhibitor (p73DD), but not by a mutant p73DD, inhibits
oncogene-induced apoptosis. These data show that oncogenes can signal
to TP73 in vivo. Moreover, in the absence of
p53, oncogenes may enlist p73 to induce apoptosis in tumor cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
) (1, 5, 6). For example,
TP73
encodes all 14 exons, while TP73
lacks exon 13. In addition, mouse TP73 has an alternative
promoter in intron 3, which encodes a p73 protein that lacks the
transactivation domain (
N p73) and acts as a dominant
negative suppressor of p73
(7). When ectopically overexpressed in
cell culture, p73
and
closely mimic p53 activities. Ectopic p73
, and to a lesser extent p73
, transactivate many p53-responsive
promoters, although relative efficiency differences on a given promoter
are observed (8-10). Like p53, p73 forms a complex with p300/CBP, which mediates transcription by p73 (11). Ectopic p73 also promotes apoptosis irrespective of the p53 status (1), and overexpression of p73
,
, and
suppresses focus formation, while p73
does not
(5, 8). The suppressor activities of isoforms
and
have not been determined.
and
via a
p300-dependent mechanism (25). Moreover, analogous to p53,
Mdm2 suppresses p73 transactivation function via a negative
feedback loop (26-28). However, in contrast to p53, cellular
Mdm2 and the HPV E6 protein do not mediate degradation of
exogenous p73 (26, 27, 29), suggesting that the regulation of p73
degradation might be distinct from the one regulating p53.
-ionizing irradiation in a pathway that depends on the
nonreceptor tyrosine kinase c-Abl (38-40). This
DNA-damage-dependent up-regulation of p73 by c-Abl may be partly responsible for p53-independent apoptosis. What is
already evident is that TP73, at least qualitatively,
utilizes the same or very similar effector pathways as TP53.
Complete identification of all upstream signals of TP73 that
operate physiologically will be very important in elucidating its
normal function and role, if any, in tumorigenesis. We therefore asked
whether deregulated oncogenes, which are a preeminent signal for
triggering p53-dependent transactivation and apoptosis,
also induce and activate p73 function. We report that overexpression of
cellular and viral oncogenes do up-regulate endogenous p73 proteins and
activate their transactivation function. Moreover, E2F1-, c-Myc-, and
E1A-mediated activation of endogenous p73 induces apoptosis in
p53-deficient tumor cells. Disruption of p73 function by a dominant
negative p73 inhibitor (p73DD), but not by a mutant version thereof,
inhibited oncogene-induced apoptosis. These data show that
oncogenes can signal to TP73 in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and pcDNA3-p73
expressing
HA1-tagged human p73
and
(5).
and acts as a dominant negative p73 in vivo. The
corresponding loss-of-function mutant named mtp73DD, which contains a
L371P point mutation, is inactive as inhibitor. Green fluorescent
protein expression plasmid (CLONTECH) was
cotransfected in transient transfections to verify relative
transfection efficiency. The p53/p73-responsive reporter construct
PG13-Luc and its mutant counterpart MG15-Luc (gift of B. Vogelstein)
were used for luciferase assays. p73
exhibited 80% of the activity
of p53 (on a molar basis) in transactivating the PG13-Luc reporter and
no activity with the MG15-Luc reporter (data not shown).
),
the polyclonal p73N (raised in rabbit against the N-terminal peptide
FHLEGMTTSVMAQF), and the polyclonal p73
(raised in rabbit
against a C-terminal
-specific peptide; gift of K. Vousden).
Oncogene expression was confirmed with antibodies against E1A (clone
13S-5; Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), E2F-1 (clones
C20 and KH95; Santa Cruz Biotechnology), and c-Myc (Ab-3; Oncogene
Science). Antibodies against p53 (clone DO-1), HDM2 (clone IF2), and
p21 (Ab-1) were from Oncogene Science. For normalization of protein
loading, blots were reprobed with antibodies specific for vimentin (BioGenex).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
Proteins Are Induced in Response to
E2F1, c-Myc, and E1A--
The great majority of functional and
regulatory p73 studies to date have used ectopically expressed p73
proteins. To reliably detect endogenous p73 proteins, we used three
different p73-specific antibodies. They comprised a p73
-specific
monoclonal (GC15), a p73
-specific polyclonal raised against a
C-terminal peptide (poly-p73
) and a pan-p73 polyclonal raised
against an N-terminal peptide (poly-p73N). The antibodies detected
endogenous full-length p73
and
in several tumor cell lines
including the p53-deficient human H1299 line (Fig.
1). H1299 cells express a basal level of p73
(lane 1) and
(lane
9). Simian COS cells express the highest level of p73
(lane 4) and p73
(lanes
5 and 8). In contrast, SK-N-AS cells express no
detectable p73
(lane 3) or p73
(lane 6), consistent with previous reports
(20).
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Fig. 1.
Endogenous p73 and
proteins are detectable by various
p73-specific antibodies. Immunoblots of cell lysates from
p53-deficient H1299 cells (H), neuroblastoma SK-N-AS cells
(AS), and SV40 T antigen-transformed simian COS cells are
shown. Polyclonal p73N was raised against an N-terminal peptide,
polyclonal p73
against a C-terminal
-specific peptide, and GC15
against amino acids 380-499 of human p73
. Transiently transfected
HA-tagged p73
and
serve as positive control. HA-tagged
exogenous p73
shows a retarded migration (lane
2), while HA-tagged exogenous p73
does not
(lane 7). Each lane contains 30 µg of total
cell extract. Detection was by horseradish peroxidase-conjugated
secondary antibodies and enhanced chemiluminescence.
and
proteins were markedly induced after expression of E2F1, c-Myc, and
adenoviral E1A when compared with empty vector. A representative
experiment is shown in Fig. 2, B and C. By
molecular weight standards and reactivity with the polyclonal
p73N antibody, only full-length proteins were observed, with p73
migrating at about 83 kDa and p73
at about 75 kDa. The equal
loading of immunoblots was confirmed by reblotting the membranes with
an antibody specific for vimentin. Since transfection efficiency in
these transient assays ranged between 30 and 40% (judged by
coexpressed green fluorescent protein), the actual degree of induction
is higher than the one detected here by immunoblots, since this assay
is subject to dilution by untransfected cells. Significant induction of
endogenous TP73 was also seen after oncogene transfection in
SaOs-2 cells (see Fig. 5B).
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Fig. 2.
p73 and
proteins are induced in response to cellular and
viral oncogenes. A, immunoblots of H1299 cells after
transient transfection with empty vector or expression vectors for
E2F1, c-Myc and E1A. Blots were developed with the indicated
antibodies. Immunoblots (IB) are shown of H1299 cells probed
with polyclonal p73
antibody (B) or with p73
antibody (GC15) (C) after transient transfection with empty
vector (vect) or expression vectors for E2F1, c-Myc, and
E1A. HA-tagged p73
and p73
transfected into H1299 cells serve
as positive controls, respectively. Exogenous p73
shows a retarded
migration. SK-N-AS cells (AS) are used as negative control.
Membranes were reblotted for vimentin to ensure equal loading.
and
(Fig. 2), we next
asked if stable oncogene overexpression similarly would lead to long
term up-regulation of p73. To this end, vector control and
c-Myc-transfected H1299 cells were selected in G418 for 3 weeks. Of the
surviving c-Myc foci, seven were randomly picked, ring-cloned, and
successfully expanded into stable sublines. As shown in Fig.
3, all seven clones overexpressed c-Myc,
albeit to various degrees compared with vector control. Clones 1, 2, and 4 showed the highest c-Myc expression. Cell extracts were then
probed for p73 protein levels. As already seen with transient c-Myc
transfections, p73
and
were found to be induced above base line
in all seven subclones (Fig. 3). p73
induction appeared
proportional to the level of c-Myc expression in the individual clones,
with clones 1, 2, and 4 showing the highest p73
accumulation.
Interestingly, for reasons that remain to be elucidated, p73
reproducibly behaved inversely to
(i.e. whenever
was
high,
was low, and vice versa). Taken together, this
result indicates that stable deregulation of the c-Myc oncogene recapitulates the p73 overexpression already seen with transient deregulation of c-Myc.
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Fig. 3.
Stable H1299 clones overexpressing c-Myc
recapitulate the overexpression of p73 proteins. Upper
panel, Myc immunoblot of all seven H1299 subclones
overexpressing c-Myc and an empty vector clone. The first and last
lanes show H1299 cells transiently transfected with p53 and c-Myc,
respectively. Thirty µg of total protein per lane were loaded.
Lower panels, immunoblots of the seven subclones
and the empty vector clone were probed for p73 and
.
Control lane 9 shows H1299 cells
transiently transfected with p73
expression plasmids. The p73
blot was reprobed for vimentin to ensure equal loading (30 µg/lane).
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Fig. 4.
Oncogene-mediated induction of endogenous
TP73 leads to functional activation of p73.
A, oncogene-mediated activation of the p73-responsive
reporter PG13-Luc. H1299 cells were transiently transfected with empty
vector or the indicated expression plasmids (400 ng each) together with
80 ng of PG13-Luc and 8 ng of Renilla luciferase. Luciferase
activity was measured 24 h later and standardized for
Renilla activity. E2F1 exhibited a 16.5-fold, c-Myc a
10.3-fold, and E1A a 13.9-fold induction compared with vector controls.
Results are the average ± S.D. of three independent
experiments. B, H1299 cells were transiently transfected
with empty vector (vect) or the same amount of the indicated
expression plasmids (2 µg). Total cell extracts were immunoblotted
for p73 , Waf1p21, and HDM2. Membranes were reblotted for vimentin
to ensure equal loading (30 µg/lane). H1299 cells directly
transfected with 2 µg of HA-p73
and
expression plasmids,
respectively, are shown as positive controls (lanes
5 and 6). Four independent experiments gave
similar results.
protein was again
up-regulated (Fig. 4B, top panel,
lanes 2-4, compare with empty vector in
lane 1). Oncogene expression was confirmed by
immunoblots (data not shown). The p73 up-regulation was accompanied by
the induction of the TP73 response gene products Waf1p21 and
HDM2 (Fig. 4B, middle panels, lanes 2-4; compare with empty vector in
lane 1). Lanes 5 and
6 are positive controls after direct transfection of p73
and
expression plasmids. Although the induction is modest, it could be due to the fact that we are relying on endogenous rather than ectopic p73. Furthermore, since transfection efficiency was only between 30 and 40%, the actual induction is probably higher than the
one detected here by immunoblots, due to the dilutional effect by the
untransfected majority of cells. E2F1 reproducibly caused a stronger
transactivation of the p21 and HDM2 genes than c-Myc and E1A. Previous
studies have shown that Waf1p21 and HDM2 are direct in vivo
targets of ectopic p73, as demonstrated by detecting their products in
response to inducibly expressed p73
and
in EJ (mutant p53) and
p53-deficient H1299 cells (9, 38). In p53-expressing cells,
transactivation of HDM2 in response to a broad spectrum of
overexpressed oncogenes including the panel used here has been shown to
be indirect and strictly dependent on p53 (for a review, see Ref. 47).
Transactivation of p21 by c-Myc and E1A is also
p53-dependent, although E2F1, in addition to activating the
p21 promoter through p53, can transactivate p21 directly (47). Overall,
these data strongly suggest that with the partial exception of E2F1,
the induction of p21 and HDM2 in response to oncogenes is mediated
through p73 in H1299 cells. Together with the reporter assays, these
results are consistent with the idea that in the absence of p53,
oncogene-induced endogenous p73 is capable of activating its target genes.
. p73 DD acts as a specific inhibitor of p73
and
-dependent transactivation (Fig.
5A) but not of
p53-dependent transactivation (data not shown). Moreover, p73DD binds to p73
and
proteins in vitro and
in vivo but not to p53 protein (43). When coexpressed with
p73
, p73DD suppressed PG13-Luc reporter activity by 98%, making it
an efficient specific inhibitor (Fig. 5A). In contrast, an
inactive point mutant called mtp73DD, carrying a L371P exchange, does
not bind p73
and
and does not block p73
- and
-dependent transactivation (43). mtp73DD was completely
incapable of suppressing the reporter activity of p73
(only 4%
inhibition compared with p73
alone) (Fig. 5A). Together,
these results indicate the specificity of these two reagents.
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Fig. 5.
Oncogene-mediated activation of endogenous
p73 induces apoptosis in p53-deficient tumor cells.
Inactivation of p73 inhibits oncogene-induced apoptosis.
A, inhibition of p73 -de- pendent transactivation by the dominant negative inhibitor
p73DD but not by the inactive mtp73DD (L371P) mutant. H1299 cells were
transiently transfected with either p73
(100 ng) plus empty vector
(300 ng), p73
(100 ng) plus p73DD inhibitor (300 ng), or p73
(100 ng) plus mtp73DD mutant (300 ng), together with 80 ng of PG13-Luc
and 8 ng of Renilla luciferase. Luciferase activity was
measured 24 h later and standardized for Renilla
activity. Results are the average ± S.D. of three independent
experiments. B, immunoblot of SaOs-2 cells after transient
transfection with empty vector or expression vectors for E2F1, c-Myc,
and E1A. The blot was developed with a mixture of GC15 and vimentin
antibodies. C, SaOs-2 cells in duplicate wells were
transfected with either expression plasmid (150 ng) plus empty vector
(350 ng), expression plasmid (150 ng) plus p73DD inhibitor (350 ng), or
expression plasmid (150 ng) plus mtp73DD mutant (350 ng). After 24 h, cells were processed in parallel for TUNEL and for
immunofluorescence to determine expression. The percentage of apoptosis
of transfected cells is shown after correction for background with
vector alone (500 ng/well). The results represent the average ± S.D. of three independent experiments. D, SaOs-2 cells were
transfected with empty vector or expression vectors for p73DD or
mtp73DD. Cells were selected for 3 weeks in G418 (550 µg/ml) prior to
transfection with c-Myc or empty vector. Cells were seeded into
duplicate eight-well chamber slides. After 24 h, cells were
processed in parallel for TUNEL and for immunofluorescence to determine
expression. The percentage of apoptosis of transfected cells is shown
after correction for background with vector alone (500 ng/well).
directly (light grey
columns). Moreover, the apoptotic activity of each oncogene was
greatly suppressed or abrogated when oncogenes were coexpressed with
the dominant negative inhibitor p73DD, with 84% suppression for E2F1, 96% for c-Myc, and 72% for E1A (black columns) (Fig.
5C). The suppression of oncogene-mediated apoptosis by a
p73-specific inhibitor strongly suggests that E2F1, c-Myc, and E1A
mediate their apoptotic effects through p73. This conclusion is further
confirmed by the lack of significant suppression when p73DD is
exchanged for the functionally inactive inhibitor mtp73DD (dark
grey columns) (Fig. 5C). Furthermore, the dominant
negative p73DD inhibitor also prevented apoptosis when it was stably
expressed in SaOs-2 cells, while its mutant counterpart failed to
prevent apoptosis (Fig. 5D). Also, E2F1- and c-Myc- induced
apoptosis was found to be partially mediated through p73 in transiently
transfected HeLa cells (data not shown; E1A not tested). Taken
together, these data show that E2F1-, c-Myc-, and E1A-mediated
apoptosis in p53-deficient tumor cells largely depends on p73.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mouse embryo fibroblasts is only
attenuated but not abrogated compared with wild type p53 cells,
supporting the idea that part of the apoptotic response is
p53-independent (49). Based on our results, this p53-independent
apoptosis may be due, at least in part, to p73. The demonstration that
endogenous p73 is induced and activated by oncogenes is in line with
the known p73 activation by cisplatin and
-ionizing irradiation
(38-40). Together, these data suggest that TP73 can be a
component of a tumor surveillance pathway, which in the absence of p53
might respond to different types of incoming signals in a
p53-compensatory fashion.
/
mouse embryo fibroblasts. Moreover,
they identified the mechanism of this interaction to be directly
transcriptional; i.e. E2F1 induces transcription of
full-length p73
and
via E2F1 binding sites in the P1 promoter
of TP73 (43). A functional interaction between E2F1 and p73
was also recently shown in T-cell receptor activation-induced cell
death (44). A direct transcriptional mechanism of TP73
induction by E2F1 is in complete agreement with two predicted E2F1
binding sites at positions
284 and
1862 of the TP73
promoter (50). Moreover, the c-Myc-mediated up-regulation of
TP73 that we observed most likely also operates via E2F
(51-53). E2F1 (as well as E2F2 and E2F3a) is induced by c-Myc via a
group of E-box elements in its promoter conferring positive Myc
responsiveness. The TP73 promoter itself has not been
reported to contain Myc binding sites. In cells with functional Rb such
as in H1299 cells, E1A-mediated up-regulation of TP73 might
again be mediated through E2F1, albeit indirectly via inactivation of
Rb by E1A (54). SaOs-2 cells, which were also used in our study, are
deficient for Rb, indicating an additional Rb-independent mechanism of
TP73 induction by E1A. Taken together, our paper
independently confirms the E2F1-TP73 signaling pathway and
extends it to additional oncogenes, thus broadening the concept of
TP73 activation by oncogenes.
has been
shown to be regulated in part through proteasome-dependent
degradation, since cells accumulate p73
after treatment with
proteasome inhibitors (27, 29, 55). Furthermore,
-ionizing
irradiation activates p73 through c-Abl-mediated tyrosine
phosphorylation without protein stabilization (39, 40). Taken together,
it appears that the regulation of p73 activity occurs both on a
transcriptional and posttranscriptional level and might depend on the
specific activating stimulus.
overexpression suggested that this
might be the case. In contrast to transient Myc transfection, Myc
clones 1, 2, and 4 (Fig. 3) failed to show evidence of p21 and HDM2
induction compared with vector. The same clones also failed to show
transactivation activity in a p73-responsive reporter assay.2 Moreover, in a
previous study on five human breast cancer cell lines with p73
overexpression (four of the five lines were mutant for p53), we found
no correlation with p21 mRNA and protein levels (6). While it is
tempting to speculate, it is important to point out that more extensive
analyses need to be done before definitive statements about the
functional consequence of constitutive p73 overexpression in tumor
cells can be made. Nevertheless, the sustained p73 overexpression in
stable Myc clones is highly reminiscent of the fact that multiple
primary human tumor types and tumor cell lines overexpress p73. This
includes tumors of breast, neuroblastoma, esophagus, stomach, lung,
colon, bladder, ovary, ependymoma, hepatocellular carcinoma, and
myeloid leukemia (6, 13, 14, 17-19, 31-36).
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ACKNOWLEDGEMENT |
---|
We thank William G. Kaelin for sharing previously unpublished reagents and data relevant to this work and for useful comments.
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Note Added in Proof |
---|
SaOs-2 cells do not express p63 (Ratovitski, E. A., Pattarajan, M., Hibi, K., Trink, B., Yamaguchi, K., Sidransky, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 1817-1822)
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FOOTNOTES |
---|
* This work was supported by the United States Army Medical Research Command United States Army Grant BC971469 and a grant from the Carol Baldwin Breast Cancer Research Fund.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.
§ To whom correspondence should be addressed. E-mail: umoll@ path.som.sunysb.edu.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M005737200
2 A. Zaika and U. Moll, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: HA, hemagglutinin; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
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