Received for publication, January 13, 2003, and in revised form, February 12, 2003
The p53 family includes three members that share
significant sequence homology, yet exhibit fundamentally different
functions in tumorigenesis. Whereas p53 displays all characteristics of a classical tumor suppressor, its homologues p63 and p73 do not. We
have previously shown, that NH2-terminally truncated
isoforms of p73 (
TA-p73), which act as dominant-negative inhibitors
of p53 are frequently overexpressed in cancer cells. Here we provide evidence that
TA-p73 isoforms also affect the retinoblastoma protein
(RB) tumor suppressor pathway independent of p53.
TA-p73 isoforms
inactivate RB by increased phosphorylation, resulting in enhanced E2F
activity and proliferation of fibroblasts. By inactivating the two
major tumor suppressor pathways in human cells they act functionally
analogous to several viral oncoproteins. These findings provide an
explanation for the fundamentally different functions of p53 and p73 in tumorigenesis.
 |
INTRODUCTION |
The TP53 gene was the first tumor supressor gene to be
identified and is still considered to be the prototype tumor
suppressor. In more than half of human tumors the TP53 gene
is directly inactivated by mutations and in many others, p53 is
functionally compromised epigenetically by various mechanisms (1, 2).
In fact, several transforming oncogenes have been shown to be potent
inhibitors of p53 (1). Loss of functional p53 therefore appears to be crucial for the development of most, if not all, cancers. While p53 was
long considered to be unique, recently two p53-related genes were
discovered (3-5). TP73 and TP63 encode proteins
with remarkable sequence homology to p53, suggesting that they are also
involved in the regulation of cell growth and apoptosis. Indeed, in
experimental systems, p73 showed many p53-like properties: it could
bind to p53 DNA binding sites, transactivate p53-responsive genes, and
induce cell cycle arrest or apoptosis (3, 6).
However, apart from structural and functional similarities between p53
and p73, several pieces of evidence argue against p73 being a classical
tumor suppressor. In contrast to p53, p73 is not inactivated by classic
viral oncoproteins to allow host cell transformation, indicating that
p73 may augment, rather than inhibit, viral and cellular transformation
(7). In contrast to mice lacking p53, p73-negative mice are not prone
to tumor development (8). Despite initial reports suggesting
tumor-associated deletion of p73, many subsequent studies failed to
demonstrate mutational inactivation of the TP73 gene in a
wide variety of tumors (9, 10). Instead, overexpression of p73 in its
wild-type form has been reported for tumor entities as different as
neuroblastomas, hepatocellular carcinomas, lung, prostate, colorectal,
gastric, breast, bladder, ovarian, and esophageal cancers (10-17). In
some cases overexpression of p73 could even be correlated with an
advanced tumor stage or poor prognostic parameters. In hepatocellular
carcinomas high p73 expression levels were revealed as an independent
marker of poor patient survival prognosis (17). Considering that p73 overexpression also correlates with poor prognostic parameters in other
tumor types and is observed in ~20-90% of all cancer patients,
overexpression likely contributes to the tumorigenic phenotype (9, 10,
17-19).
The molecular basis for the apparently different functions of p53 and
p73 in human tumors is presently unknown but might be related to the
differences in genomic organization of the TP53 and
TP73 genes. Whereas TP53 does not show much
splice variations, the TP73 gene encodes a complex number of
isoforms. We have recently reported that p73 overexpression is
accompanied by increased expression levels of
NH2-terminally truncated anti-apoptotic isoforms which lack
the transactivation domain and are therefore termed
TA-p73 (or
N-p73) (20). The origin of
TA-p73 proteins is still
controversially discussed (20-27). Some
TA-p73 transcripts are
generated by aberrant splicing (p73
ex2, p73
ex2/3,
N'-p73), and
others are derived from a second intronic promoter (
N-p73) (20).
Considering that full-length, transactivation-competent p73 (TA-p73) is
a proapoptotic protein (6), we focused our further analyses on the
possible function of the anti-apoptotic
TA-p73 isoforms in
tumorigenesis. In fact, ectopic expression of
TA-p73 results in
malignant transformtion of NIH3T3 cells, suggesting that
TA-p73
isoforms play the role of putative oncoproteins (20). Since
TA-p73 species lack the NH2-terminal transactivation
domain but retain an intact DNA binding domain, they act as
transdominant inhibitors of p53-mediated transactivation of specific
genes implicated in cell cycle control and apoptosis (21, 28). However,
many primary tumors harbor p53 mutations accompanied by
overexpression of p73 (15, 17, 29, 30). The common clonal expansion of
cells that harbor both p53 inactivation and p73 overexpression suggests
that the oncogenic properties of
TA-p73 extend beyond a
dominant-negative effect on p53 function.
Here, we report that overexpression of
TA-p73 induces proliferation
of serum-starved normal human diploid fibroblasts which can be
attributed to an increase in the activity of the cell cycle-promoting transcription factor E2F. Further we show, that activation of E2F is
accomplished by increased phosphorylation of the retinoblastoma tumor
suppressor retinoblastoma protein
(RB)1 resulting in a release
of active E2F. This proliferative function of
TA-p73 provides a
possible explanation for the selective advantage of p73-overexpressing
cells during human tumorigenesis, suggesting that p73 expression is not
simply a consequence of malignant transformation but rather actively
contributes to the development of the tumorigenic phenotype.
Importantly, this effect of
TA-p73 on RB function is independent of
its known p53 inhibitory activity and therefore represents a novel
function of p73.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Transfections--
SAOS-2, MCF7, and H1299 cell
lines were obtained from the ATCC. Cell lines and normal human diploid
fibroblasts (NHDFs) were maintained in Dulbecco's modified Eagle's
medium (Invitrogen) supplemented with 10% fetal calf serum and
1% penicillin G/streptomycin sulfate (Invitrogen). For serum
starvation experiments cells were washed twice with phosphate-buffered
saline and cultured in Dulbecco's modified Eagle's medium
supplemented with 0.1% bovine serum albumin fraction V (Sigma) for
48 h. After infection with recombinant adenoviruses the cells were
maintained under conditions of serum starvation for 6-48 h as
indicated. Transfections were performed with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol or by
electroporation as described in Ref. 31. For inhibition of CDK2 the
cell culture medium was supplemented with roscovitine (Calbiochem) at a
concentration of 50 µM.
Plasmids and Adenoviral Vectors--
Expression plasmids for the
various p73 isoforms (21), the p63 isoforms
N-p63
and TA-p63
(32), RB (33), the RB phosphorylation site mutant (34), cyclin E (35),
p107 (36), p130 (37), E2F1 (38), the inhibitors p53DD and p73DD (39),
and the p53 mutant R175H (40) have been described previously.
Adenoviral vectors expressing GFP or
TA-p73
have also been
described previously (21).
Luciferase Assays--
The pGL3-TATA-E2F plasmid used to measure
E2F activity was obtained from Ali Fattaey (Onyx
Pharmaceuticals) and contains six E2F binding sites in front of a TATA
element. In general, 200 ng of this reporter plasmid were used and
cotransfected with up to 600 ng of expression plasmids for p73 or p63
isoforms, p53-/p73 inhibitors, E2F1, RB family members, or inhibitors
of cyclin-dependent kinases (p21CDKN1A,
p16CDKN2A). Luciferase activity was measured 48 h
post-transfection using a premanufactured luciferase reporter assay
system (Promega) and normalized to the total protein concentration in
the cell extract. Error bars represent the S.D. between two
to three independent transfections.
Western Blot Analysis--
Cells were lysed in RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS), and total protein concentration
was quantitated by Bradford assay. Samples (100 µg per lane) were
separated by SDS-PAGE, transferred to nitrocellulose membranes
(Amersham Biosciences) and probed with p73 (ER15, Oncogene Science) or RB antibodies (C-19, Santa Cruz; phospho-specific RB
antibodies against serine residues Ser780,
Ser795 and Ser807/811 were purchased from Cell
Signaling Technology).
Immunofluorescnce--
NHDFs were grown on coverslips under
serum starvation for 48 h. Infection with recombinant adenoviruses
expressing
TA-p73
, or GFP was performed at an multiplicity of
infection of 100 under serum starvation. Positive control cells were
grown in Dulbecco's modified Eagle's medium containing 15% fetal
calf serum. 48 h after infection the cells were fixed with
ice-cold ethanol:acetic acid and stained with the Ki-67 antibody
(BioGenex). A secondary goat anti-rabbit antibody conjugated to Alexa
Fluor 546 (Molecular Probes) and 4',6-diamidino-2-phenylindole
(Molecular Probes) were used for visualization with a laser scanning microscope.
Flat Cell Assay--
1 × 107 SAOS-2 cells were
transfected by electroporation with plasmids encoding for RB (3 µg),
cyclin E (6 µg), or
TA-p73
(6 µg). A puromycin resistance
gene pIRESpuro2 (Becton Dickinson, 1 µg) was cotransfected. After
48 h cells were selected with 1 µg/ml puromycin for 10 days. The
number of flat cells was determined manually under the microscope.
 |
RESULTS |
TA-p73 Induces Proliferation in Serum-starved
Fibroblasts--
To further investigate the underlying mechanisms of
TA-p73 mediated oncogenicity, we analyzed the effects of
TA-p73
expression in primary non-transformed cells. Human diploid fibroblasts
(NHDFs) that were cultured under conditions of serum starvation for 2 days showed a complete growth arrest. To measure proliferative activity
we stained the cells with the Ki-67 monoclonal antibody, which
recognizes a nuclear antigen present in proliferating, but not resting
cells. To introduce the
TA-p73 cDNA into these growth-arrested primary cells, we used adenoviral vectors that are able to transduce resting fibroblasts with an efficiency of close to 100% (data not
shown). As shown in Fig. 1, both exposure
of the cells to fetal calf serum as well as infection with an
adenoviral vector expressing
TA-p73
induced a strong nuclear
Ki-67 staining, whereas cells infected with a GFP control virus only
showed a slight and predominantly cytoplasmic Ki-67 staining. Thus,
expression of
TA-p73
is sufficient to promote proliferation of
primary cells in the absence of growth factors.

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Fig. 1.
TA-p73 induces proliferation in
quiescent NHDFs. NHDFs were sterum-starved for 48 h,
infected with 100 multiplicity of infection of recombinant adenovirus
expressing TA-p73 , or GFP as a control and stained for Ki-67
expression 48 h post-infection. As a positive control,
cells were treated with 15% fetal calf serum (FCS).
Left, Ki-67 immunofluorescence staining (red);
middle, 4',6-diamidino-2-phenylindole labeling
(blue); right, merged images.
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TA-p73 Isoforms Activate E2F Independent of p53
Inhibition--
Entry of quiescent cells into proliferation is
primarily regulated at the G1 to S phase transition. A
major regulator of G1/S transition is the RB, which binds
to the E2F transcription factor, thereby converting E2F from a
transcriptional activator to a transcriptional repressor. Upon
transition into late G1, RB becomes hyperphosphorylated (inactivated) through the action of G1
cyclin-dependent kinases (Cdk) preventing RB from binding
and inactivating E2F. Using an E2F-regulated luciferase reporter, which
contains six E2F binding sites in front of a TATA element, we show that
TA-p73
or
TA-p73
expression induces a more than 10-fold
increase in E2F activity in MCF7 cells (Fig.
2A).

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Fig. 2.
TA-p73 activates E2F-regulated
transcription independent of its dominant-negative function.
A and B, increasing amounts of TA-p73 or
TA-p73 transactivate an E2F-responsive promoter in p53 wild-type
MCF7 (A) or p53-null H1299 (B) cells. 200 ng of
pGL3-TATA-E2F were cotransfected with 300-600 ng of TA-p73
expression plasmid. Luciferase activity was measured 48 h after
transfection. Shown is the average of two independent transfections.
Error bars denote the S.D. C, induction of E2F
activity is independent of the dominant-negative function of TA-p73
as shown by the absence of this activity for other known p53-/p73
inhibitory molecules (p53 mutant R175H, p53DD, and p73DD) (39, 41). 200 ng of reporter plasmid (pGL3-TATA-E2F) were cotransfected with 600 ng
of expression plasmid for the p53/p73 inhibitors.
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Previously,
TA-p73 has been shown to function as a dominant-negative
regulator of p53 (21-23, 27, 28). It is, therefore, possible that
TA-p73 induces cell cycle-promoting E2F activity by antagonizing
p53-induced expression of genes involved in cell cycle arrest such as
the Cdk inhibitor p21. We therefore analyzed the effect of
TA-p73
expression on E2F acitvity in p53-null H1299 cells. As shown in Fig.
2B, both
TA-p73
and
TA-p73
have a similar
stimulatory effect on E2F activity in these cells. In addition, this
effect of
TA-p73 is not seen with other inhibitors of the
p53-family. Neither the p53-specific inhibitor p53DD nor the p73/p63
inhibitor p73DD activated the E2F reporter (Fig. 2C) (39).
Not even the tumor-derived p53 mutant p53-R175H, which acts as a
combined inhibitor of p53, p63, and p73, scored positive in this assay
(41-43). The mechanism of E2F induction by
TA-p73, therefore,
appears to be distinct from its known dominant-negative activity.
As mentioned before,
TA-p73 is a heterogeneous class of p73 proteins
with different amino- and carboxyl-terminal sequences. p73
ex2,
p73
ex2/3, and
N'-p73 transcripts are generated from the
TA-promoter by alternative splicing, whereas the
N-p73 transcript is
generated from an alternative intronic promoter and therefore under a
different transcriptional regulation (20). Of note, the
N'-p73 and
N-p73 transcripts code for the same
N-p73 protein, which contains
a unique 13-amino acid epitope encoded by the alternative exon that is
not present in the full-length TA-p73 protein. We compared the
different NH2-terminal (p73
2, p73
2/3,
N-p73) and COOH-terminal (
,
,
, and
) variants (44) for induction of the E2F-regulated reporter. All of the different variants of
TA-p73 induced E2F activity with
TA-p73
being the most active and
TA-p73
being the weakest (Fig. 3,
A and B). These data clearly show that the gain
of the unique epitope present in the
N-p73 isoform is not required
for inducing E2F and that changes in the carboxyl-terminal region only
serve to modulate this activity. Therefore loss of the amino-terminal
transactivation domain is the major determinant of this novel p73
function.

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Fig. 3.
Activation of E2F by various
TA-p73 isoforms. A, various
carboxyl-terminal p73 isoforms of TA-p73 ( , , , and )
were analyzed for their E2F-activating function in H1299 cells by
luciferase assays using the pGL3-TATA-E2F plasmid as a reporter. 200 ng
of reporter plasmid were cotransfected with 600 ng of p73 expression
plasmid. Luciferase activity was measured 48 h after transfection.
Shown is the average of two independent transfections. Error
bars denote the S.D. B, comparative analysis of
full-length TA-p73 and three amino-terminal p73 isoforms (p73- 2 ,
p73- 2/3 , and N-p73 ) for their E2F-activating function as
described in A.
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Concerning the structural homology within the p53 family, p73 and p63
are more closely related to each other than to p53 (4, 5). In addition,
p63 and p73 share the characteristic gene structure with two
differently regulated promoters and multiple COOH-terminal splice
variants. We therefore compared the
N-p73
isoform with the
corresponding p63 isoform (
N-p63
). Surprisingly, only
N-p73
is able to activate the E2F reporter (Fig.
4), although
N-p63
has also been
shown to possess oncogenic activity (45, 46). Activation of E2F
activity therefore represents a novel and specific function of p73.

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Fig. 4.
Comparative analysis of p73 and p63.
Full-length, transactivation-competent isoforms (TA-p73 and
TA-p63 ) and NH2-terminally truncated,
transactivation-deficient isoforms ( N-p73 or N-p63 )
were analyzed for activation of the E2F-regulated reporter
pGL3-TATA-E2F by luciferase assay in the p53-null H1299 cell line.
Shown is the average of two independent experiments. The error
bars denote the S.D.
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TA-p73 Isoforms Induce Hyperphosphorylation of RB--
Since
E2F is regulated by binding to RB, we further investigated whether
TA-p73 influences E2F by interfering with RB function. When we
ectopically expressed RB in the RB-, p53-, and p73-negative SAOS-2 cell
line, we observed one single band after SDS-PAGE. When we coexpressed
RB with
TA-p73, another more slowly migrating band appeared (Fig.
5A). Considering that the
electrophoretic mobility of RB is strongly influenced by its
phosphorylation state, the faster migrating band most likely represents
hypophosphorylated RB as the active E2F-binding form of RB, whereas the
retarded band contains extensively hyperphosphorylated RB, which is the inactive form of RB.

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Fig. 5.
TA-p73 induces
hyperphosphorylation of RB. A, Western blot for RB
in RB-negative SAOS-2 cells transfected with a RB expression plasmid
alone or in combination with TA-p73 . RB-P indicates
hyperphosphorylated, and RB-OH indicates hypophosphorylated
RB. B, serum-starved NHDFs were infected with
recombinant adenoviruses expressing wild-type p53, full-length
TA-p73 , TA-p73 , or GFP as a negative control. Whole-cell
lysates were separated by SDS-PAGE and immunoblotted with antibodies to
total RB or RB phosphorylated on specified serine residues.
RB-P indicates hyperphosphorylated, RB-OH
indicates hypophosphorylated RB, and the asterisk indicates
an unspecific band. C, NHDFs were infected with recombinant
adenoviruses expressing TA-p73 or GFP and maintained under serum
starvation. At the indicated time points post-infection whole-cell
lysates were prepared and analyzed for phosphorylation of RB and p73
expression by Western blot.
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Similar changes in the electrophoretic mobility of endogenous RB were
observed in normal diploid fibroblasts (Fig. 5B).
Serum-starved fibroblasts arrested in G1 contain only low
levels of hyperphosphorylated (inactive) RB. Infection with adenoviral
vectors for p53 or TA-p73
reduces RB phosphorylation even further,
so that only hypophosphorylated RB can be detected. In contrast, we
observed a pronounced increase in retarded, hyperphosphorylated RB
species in
TA-p73-expressing NHDFs. Using a panel of
phosphorylation-specific RB antibodies, we could detect increased
phosphorylation at serine residues Ser780,
Ser795, and Ser807/811 (Fig. 5B).
When comparing different
TA-p73 isoforms, RB hyperphosphorylation was seen with both NH2-terminally truncated p73
and
p73
variants (data not shown).
The direct link between expression of
TA-p73 and
hyperphosphorylation of RB is further substantiated by the time course
experiment shown in Fig. 5C. When proliferating NHDFs were
serum-starved, RB became completely hypophosphorylated within 24 h
in the control cells. In cells infected with the Ad vector expressing
TA-p73, RB phosphorylation initially declined but started to
increase around 24 h.
TA-p73 expression could be observed as
early as 18 h post-infection therefore preceeding the
increase in RB phosphorylation.
Hyperphosphorylation of RB Is Required for Activation of E2F by
TA-p73--
Since the ability of RB to interact with E2F is
regulated by phosphorylation, the data implicate that
hyperphosphorylation of RB by
TA-p73 is responsible for the observed
increase in E2F activity. To test this, we performed additional
luciferase reporter assays. Activation of an E2F reporter by E2F1 in
RB-negative SAOS-2 cells was completely repressed by coexpression of RB
or a non-phosphorylatable RB mutant (PSM-RB) (34). Repression by
wild-type RB could be completely rescued by expression of
TA-p73,
whereas repression by the RB mutant was only partially rescued (Fig.
6A). Therefore hyperphosphorylation of RB is necessary for
TA-p73 to completely relieve RB-mediated transcriptional repression and induce E2F activity.
The residual activation by
TA-p73 seen with the pRB mutant is most
likely due to activation of E2F by phosphorylation of the other RB
family members, which are present in SAOS-2 cells and partially
compensate for the lack of RB. Consistent with this,
TA-p73
expression enhanced E2F activity in the absence of RB (Fig.
6A) and not only rescued repression by RB but also by p107 and p130 (Fig. 6B). Thus,
TA-p73 expression abrogates
transcriptional repression mediated by all three RB family members.

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Fig. 6.
Inactivation of RB by hyperphosphorylation
accounts for the increase in E2F activity. A, TA-p73
expression rescues RB-mediated repression of E2F activity in a
phosphorylation-dependent manner in SAOS-2 cells as
determined by luciferase assay. Repression by a RB mutated at most
phosphorylation sites (PSM-RB) was only partially rescued
(34). 1 µg of pGL3-TATA-E2F reporter was cotransfected with 1 µg of
pCMV-E2F1, 5 µg of RB or PSM-RB, and 5 µg of TA-p73 .
B, TA-p73 expression relieves transcriptional repression
by the other RB family members p107 and p130. 1 µg of pGL3-TATA-E2F
reporter was cotransfected with 1 µg of pCMV-E2F1, 5 µg of p107 or
p130, and 5 µg of TA-p73 . Error bars denote the
S.D.
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Role for Cdks in Hyperphosphorylation of RB--
According to the
current model pRB is sequentially phosphorylated at the G1
to S phase transition by the action of cyclin D-Cdk4/6 and
cyclin E-Cdk2 (47). The cyclin D-Cdk4/6 complex is
specifically inhibited by the Cdk inhibitor p16 (CDKN2A), whereas p21
(CDKN1A) is a ubiquitous inhibitor of both cyclin D- and
cyclin E-dependent kinases. A specific pharmacological
inhibitor of cyclin E-Cdk2 is roscovitine. To investigate which kinases
are responsible for phosphorylation of RB in response to
TA-p73
expression, we used these inhibitors in luciferase assays. As shown in
Fig. 7, all three kinase inhibitors
efficiently abrogate activation of E2F by
TA-p73, demonstrating that
both cyclin E-Cdk2 and cylin D-Cdk4/6 kinase acitivities
are required for
TA-p73 to phosphorylate RB and activate E2F.

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Fig. 7.
Repression of TA-p73
induced E2F activity by inhibitors of cyclin-dependent
kinases (p21CDKN1A,
p16CDKN2A, roscovitine). H1299 cells were
cotransfected with 1 µg of pGL3-TATA-E2F, 10 µg of TA-p73, and
2.5 µg of p21 or p16 expression plasmid. Roscovitine was used at a
final concentration of 50 µM. Luciferase activity was
measured 48 h after transfection. Shown is the average of two
experiments. Error bars denote the S.D.
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TA-p73 Inhibits the Function of RB in Differentiation
Control--
RB participates in dual tumor suppressor functions, one
linked to cell cycle progression and the other to differentiation control (33). RB mutations that destroy either function abolish RB
tumor suppressor activity (33). The ability to block cell cycle
progression is intimately linked to its ability to repress E2F-regulated transcription and consequently RB mutants deficient for
E2F-binding are unable to cause a G1/S block.
Phosphorylation of RB by expression of
TA-p73 inhibits RB mediated
transcriptional repression and therefore abrogates the ability of RB to
block cell cycle progression. The RB function in the control of
differentiation control, however, is rather linked to transcriptional
activation. Reintroduction of functional RB into SAOS-2 osteosarcoma
cells leads to acute growth arrest and in the long term induces a
senescent flat cell phenotype with expression of markers suggestive of
bone differentiation (33, 35). As shown in Table
I,
TA-p73 expression suppresses flat
cell formation by RB comparable with expression of cyclin E, which is
known to inhibit flat cell formation by induction of RB
hyperphosphorylation.
TA-p73 therefore inhibits both tumor
suppressor functions of RB, its ability to regulate proliferation and
its function in differentiation control.
 |
DISCUSSION |
Here we demonstrate that
TA-p73 inactivates the functions of
the RB tumor suppressor in cell cycle and differentiation control. Previously,
TA-p73 has been reported to act as a dominant-negative regulator of all transactivation-competent, proapoptotic p53 family members (20-23, 25, 27, 28, 48). Considering that inhibition of the
tumor suppressor function of p53 is a major step during tumor
development, overexpression of
TA-p73 could enhance the malignant
phenotype just like increased expression of other p53 inhibitors such
as the MDM2 or human papillomavirus E6 oncoproteins (1). In fact,
increased
TA-p73 expression levels were reported for various tumors
compared with the surrounding normal tissues (19, 20, 27, 49). Although
the causal link has not been established so far, the dominant-negative
activity of
TA-p73 is a suitable mechanism to explain the driving
force for selection of p73-overexpressing cells during tumor
development (19, 20, 27, 49).
However, if inhibition of p53 is the driving force for selection of
TA-p73 overexpressing cells, one would expect to find
TA-p73
overexpression only in p53 wild-type tumors but not in those harboring
p53 mutations. Yet, most studies that have addressed both the p53 and
p73 status in tumors have not been able to establish a correlation
between high p73 levels and wild-type p53 (15, 17, 29, 30). Therefore,
we assume that
TA-p73 contributes to tumor development by other
p53-independent mechanisms.
Here, we describe that
TA-p73 inactivates the RB tumor suppressor
leading to an increase in E2F activity and proliferation of normal
human diploid fibroblasts in the absence of exogenous growth factors.
Importantly, this possibly oncogenic effect of
TA-p73 is independent
of its known dominant-negative activity. It is also seen in the
p53-null cells (H1299 cell line) and cannot be observed with other
inhibitors of p53 function like the p53-specific inhibitor p53DD or the
dominant-negative p53 mutant p53R175H.
TA-p73 can therefore exert
oncogenic activity even in the absence of wild-type p53 providing a
possible explanation why overexpression of p73 and p53 mutations are
not mutually exclusive.
Entry of quiescent cells into the cell cycle is regulated at the
G1 to S phase transition by the RB family members. Whereas RB in association with E2F acts as a transcriptional repressor in
G1, sequential phosphorylation of RB in late G1
induces the release of active E2F, which serves to transactivate
multiple target genes to promote S-phase entry (50). We show that
TA-p73 obviously imitates this physiological process of RB
inactivation during normal cell cycle progression and inactivates RB by
inducing its hyperphosphorylation to induce E2F activity and promote
cell proliferation. Whereas all different NH2-terminally
truncated p73 isoforms induce E2F activity to a similar extent,
full-length p73 has the completely opposite effect consistent with its
p53-like function in cell cycle arrest. The transactivation domain in
the full-length TA-p73 appears to conceal a proliferative effect of p73, which becomes apparent only in the NH2-terminally
truncated isoforms. Variations in the carboxyl-terminal region of
TA-p73 only serve to modulate the effect on E2F activity so that
even the shortest p73 isoform (
TA-p73
), which lacks most of the
COOH-terminal sequences and is therefore most similar to p53, retains
the ability to induce E2F activity.
How
TA-p73 induces RB hyperphosphorylation is still unclear. Since
TA-p73 is competent for DNA binding but transactivation-deficient, a
possible mechanism might be the competition with other transactivating p53 family members for DNA-binding sites that has already been extensively analyzed (21-23,48). However, we failed to observe similar effects with other inhibitors of DNA binding such as p53DD, p73DD, or
N-p63. Active transcriptional repression of, for example, cell cycle inhibitors might be another possible mechanism.
Kartasheva et al. (23) have recently identified a
transcriptional repressor element in the carboxyl-terminal domain of
N-p73
, which is absent in
N-p73
. But similar induction of
E2F activity using both
N-p73
and
N-p73
argues against
transcriptional repression as the major underlying mechanism.
Interestingly only
N-p73, but not the closely related
N-p63, is
able to induce E2F activity. Possibly, slight differences in the DNA
binding specificity target
N-p73 to other promoters than
N-p63 to
cause this effect. Alternatively, DNA binding-independent mechanisms
such as direct protein-protein interactions also need to be considered
and analyzed to further delineate the underlying mechanism for
inhibition of RB function by
TA-p73.
Formation of RB-E2F complexes, and consequent repression of E2F
dependent promoters, likely contributes to RB-mediated tumor suppression. However, p107 and p130, the other two members of the RB
family, can likewise bind to E2F and repress E2F-dependent transcription, and yet only RB appears to be clinically important as a
tumor suppressor (33, 51, 52). RB itself is frequently inactivated in a
subset of human tumors, including osteosarcomas (53). In fact, RB acts
as an essential transcriptional coactivator to promote osteoblast
differentiation, which may contribute to the targeting of RB in
osteosarcomas (54). Typically, tumor-derived RB mutants have not only
lost their ability to repress E2F-dependent transcription,
but they have also lost this transactivation function, underscoring a
potential role in tumor suppression (54). A common model to measure the
function of RB in differentiation control is the induction of flat
cells in the SAOS-2 cell line. Reintroduction of RB into the
RB-negative SAOS-2 osteosarcoma cells induces a senescent-like
phenotype with expression of markers, suggestive of bone
differentiation (33). Here, we observed a significant reduction in the
number of RB-induced flat cells in the presence of either (TA-p73 or
cyclin E, which also induces hyperphosphorylation of RB. As flat cell
induction is intimately linked to the transactivation function of RB,
but not its ability to repress E2F-dependent transcription, this finding clearly demonstrates that hyperphosphorylation of RB in
the presence of
TA-p73 interferes with the function of RB in
differentiation control. Interestingly,
N-p73 is the predominant p73
species in the developing mouse (8), so that
N-p73 possibly functions as a negative regulator of RB during embryonic development.
In summary, our data show that
TA-p73 proteins are able to target
the two major tumor suppressor pathways in human cells, which are
consistently inactivated during malignant transformation. Through
inhibition of p53 and RB,
TA-p73 isoforms might act as oncoproteins
that convey the TP73 gene with oncogenic functions that can
be selected for during tumorigenesis, thereby explaining the frequently
observed overexpression of p73 in human tumors.
We thank W. G. Kaelin, L. Zhu, G. Vairo,
K. Helin, B. Vogelstein, R. Weinberg, J. Y. Wang, M. Senoo, and A. Fattaey for providing reagents.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M300357200
The abbreviations used are:
RB, retinoblastoma protein;
NHDF, normal human diploid fibroblast;
GFP, green fluorescent protein.
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