Inactivation of Retinoblastoma (RB) Tumor Suppressor by Oncogenic Isoforms of the p53 Family Member p73*

Thorsten StieweDagger §, Jens StanelleDagger , Carmen C. TheselingDagger , Barbara PollmeierDagger , Michaela Beitzinger§, and Brigitte M. PützerDagger

From the Dagger  Center for Cancer Research and Cancer Therapy, Institute of Molecular Biology, University of Essen, Medical School, D-45122 Essen, Germany and the § Rudolf-Virchow-Center for Experimental Biomedicine, University of Würzburg, D-97078 Würzburg, Germany

Received for publication, January 13, 2003, and in revised form, February 12, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta TA-p73), which act as dominant-negative inhibitors of p53 are frequently overexpressed in cancer cells. Here we provide evidence that Delta TA-p73 isoforms also affect the retinoblastoma protein (RB) tumor suppressor pathway independent of p53. Delta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Delta TA-p73 (or Delta N-p73) (20). The origin of Delta TA-p73 proteins is still controversially discussed (20-27). Some Delta TA-p73 transcripts are generated by aberrant splicing (p73Delta ex2, p73Delta ex2/3, Delta N'-p73), and others are derived from a second intronic promoter (Delta 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 Delta TA-p73 isoforms in tumorigenesis. In fact, ectopic expression of Delta TA-p73 results in malignant transformtion of NIH3T3 cells, suggesting that Delta TA-p73 isoforms play the role of putative oncoproteins (20). Since Delta 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 Delta TA-p73 extend beyond a dominant-negative effect on p53 function.

Here, we report that overexpression of Delta 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 Delta 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 Delta TA-p73 on RB function is independent of its known p53 inhibitory activity and therefore represents a novel function of p73.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Delta N-p63alpha and TA-p63gamma (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 Delta TA-p73beta 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 Delta TA-p73beta , 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 Delta TA-p73beta (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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Delta TA-p73 Induces Proliferation in Serum-starved Fibroblasts-- To further investigate the underlying mechanisms of Delta TA-p73 mediated oncogenicity, we analyzed the effects of Delta 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 Delta 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 Delta TA-p73beta 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 Delta TA-p73beta is sufficient to promote proliferation of primary cells in the absence of growth factors.


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Fig. 1.   Delta TA-p73 induces proliferation in quiescent NHDFs. NHDFs were sterum-starved for 48 h, infected with 100 multiplicity of infection of recombinant adenovirus expressing Delta TA-p73beta , 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.

Delta 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 Delta TA-p73alpha or Delta TA-p73beta expression induces a more than 10-fold increase in E2F activity in MCF7 cells (Fig. 2A).


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Fig. 2.   Delta TA-p73 activates E2F-regulated transcription independent of its dominant-negative function. A and B, increasing amounts of Delta TA-p73alpha or Delta TA-p73beta 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 Delta 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 Delta 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.

Previously, Delta TA-p73 has been shown to function as a dominant-negative regulator of p53 (21-23, 27, 28). It is, therefore, possible that Delta 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 Delta TA-p73 expression on E2F acitvity in p53-null H1299 cells. As shown in Fig. 2B, both Delta TA-p73alpha and Delta TA-p73beta have a similar stimulatory effect on E2F activity in these cells. In addition, this effect of Delta 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 Delta TA-p73, therefore, appears to be distinct from its known dominant-negative activity.

As mentioned before, Delta TA-p73 is a heterogeneous class of p73 proteins with different amino- and carboxyl-terminal sequences. p73Delta ex2, p73Delta ex2/3, and Delta N'-p73 transcripts are generated from the TA-promoter by alternative splicing, whereas the Delta N-p73 transcript is generated from an alternative intronic promoter and therefore under a different transcriptional regulation (20). Of note, the Delta N'-p73 and Delta N-p73 transcripts code for the same Delta 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 (p73Delta 2, p73Delta 2/3, Delta N-p73) and COOH-terminal (alpha , beta , gamma , and delta ) variants (44) for induction of the E2F-regulated reporter. All of the different variants of Delta TA-p73 induced E2F activity with Delta TA-p73alpha being the most active and Delta TA-p73gamma being the weakest (Fig. 3, A and B). These data clearly show that the gain of the unique epitope present in the Delta 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 Delta TA-p73 isoforms. A, various carboxyl-terminal p73 isoforms of Delta TA-p73 (alpha , beta , gamma , and delta ) 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-Delta 2alpha , p73-Delta 2/3alpha , and Delta N-p73alpha ) for their E2F-activating function as described in A.

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 Delta N-p73alpha isoform with the corresponding p63 isoform (Delta N-p63alpha ). Surprisingly, only Delta N-p73alpha is able to activate the E2F reporter (Fig. 4), although Delta N-p63alpha 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-p73beta and TA-p63gamma ) and NH2-terminally truncated, transactivation-deficient isoforms (Delta N-p73alpha or Delta N-p63alpha ) 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.

Delta TA-p73 Isoforms Induce Hyperphosphorylation of RB-- Since E2F is regulated by binding to RB, we further investigated whether Delta 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 Delta 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.   Delta 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 Delta TA-p73beta . 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-p73beta , Delta TA-p73beta , 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 Delta TA-p73beta 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.

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-p73beta 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 Delta 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 Delta TA-p73 isoforms, RB hyperphosphorylation was seen with both NH2-terminally truncated p73alpha and p73beta variants (data not shown).

The direct link between expression of Delta 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 Delta TA-p73, RB phosphorylation initially declined but started to increase around 24 h. Delta 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 Delta TA-p73-- Since the ability of RB to interact with E2F is regulated by phosphorylation, the data implicate that hyperphosphorylation of RB by Delta 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 Delta TA-p73, whereas repression by the RB mutant was only partially rescued (Fig. 6A). Therefore hyperphosphorylation of RB is necessary for Delta TA-p73 to completely relieve RB-mediated transcriptional repression and induce E2F activity. The residual activation by Delta 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, Delta 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, Delta 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, Delta 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 Delta TA-p73beta . B, Delta 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 Delta TA-p73beta . Error bars denote the S.D.

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 Delta 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 Delta TA-p73, demonstrating that both cyclin E-Cdk2 and cylin D-Cdk4/6 kinase acitivities are required for Delta TA-p73 to phosphorylate RB and activate E2F.


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Fig. 7.   Repression of Delta 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 Delta 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.

Delta 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 Delta 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, Delta 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. Delta TA-p73 therefore inhibits both tumor suppressor functions of RB, its ability to regulate proliferation and its function in differentiation control.


                              
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Table I
SAOS-2 flat cell assay


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we demonstrate that Delta TA-p73 inactivates the functions of the RB tumor suppressor in cell cycle and differentiation control. Previously, Delta 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 Delta 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 Delta 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 Delta 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 Delta TA-p73 overexpressing cells, one would expect to find Delta 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 Delta TA-p73 contributes to tumor development by other p53-independent mechanisms.

Here, we describe that Delta 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 Delta 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. Delta 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 Delta 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 Delta TA-p73 only serve to modulate the effect on E2F activity so that even the shortest p73 isoform (Delta TA-p73delta ), which lacks most of the COOH-terminal sequences and is therefore most similar to p53, retains the ability to induce E2F activity.

How Delta TA-p73 induces RB hyperphosphorylation is still unclear. Since Delta 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 Delta 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 Delta N-p73alpha , which is absent in Delta N-p73beta . But similar induction of E2F activity using both Delta N-p73alpha and Delta N-p73beta argues against transcriptional repression as the major underlying mechanism. Interestingly only Delta N-p73, but not the closely related Delta N-p63, is able to induce E2F activity. Possibly, slight differences in the DNA binding specificity target Delta N-p73 to other promoters than Delta 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 Delta 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 Delta TA-p73 interferes with the function of RB in differentiation control. Interestingly, Delta N-p73 is the predominant p73 species in the developing mouse (8), so that Delta N-p73 possibly functions as a negative regulator of RB during embryonic development.

In summary, our data show that Delta 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, Delta 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by a grant from the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung (to B. M. P.), and the Deutsche Forschungsgemeinschaft (to T. S.).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: Center for Cancer Reserach and Cancer Therapy, Inst. of Molecular Biology, University of Essen, Medical School, Hufelandstr. 55, D-45122 Essen, Germany. Tel.: 49-201-723-3687; Fax: 49-201-723-5974; E-mail: brigitte.puetzer@uni-essen.de.

Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M300357200

    ABBREVIATIONS

The abbreviations used are: RB, retinoblastoma protein; NHDF, normal human diploid fibroblast; GFP, green fluorescent protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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