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Transactivation-deficient TA-p73 Acts as an Oncogene¹

Centre for Cancer Research and Cancer Therapy, Institute of Molecular Biology [T. S., S. Z., H. E., B. M. P.] and Department of General and Transplantation Surgery [A. F.], University of Essen, Medical School, D-45122 Essen, Germany

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
The recently discovered p53-family member p73 displays significant homology to p53, but data from primary tumors and knockout mice argue against p73 being a classical tumor suppressor. We report on the overexpression of NH₂-terminally truncated, transactivation-deficient p73 proteins (TA-p73) in human cancer cells. Moreover, we show that TA-p73 overexpression results in malignant transformation of NIH3T3 fibroblasts and tumor growth in nude mice, thereby providing the experimental evidence for an oncogenic function of TA-p73. Apparently, increased expression of NH₂-terminally truncated p73 isoforms conveys the TP73 gene with oncogenic activity that appears to be actively selected for during tumor development.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
The TP53 gene was the first tumor suppressor 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) . Although the recently discovered p53 family member p73 shows remarkable structural and functional similarities to p53, several pieces of evidence argue against p73 being a classical tumor suppressor. In contrast to mice lacking p53, p73-negative mice are not prone to tumor development (2) . 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 (3 , 4) . Instead, overexpression of wild-type p73 in various tumor types compared with normal tissues has been reported and shown to correlate with a poor patient prognosis (5, 6, 7, 8) . Together, these data raise the question about additional activities of p73 in cancer. In this study we report that p73 overexpression is associated with increased levels of NH₂-terminally truncated p73 proteins (TA-p73). Moreover, we show that overexpression of TA-p73 protein results in malignant transformation of NIH3T3 cells supporting an oncogenic function of TA-p73. Therefore, our data provide a possible explanation for the observed overexpression of p73 in human cancers.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Cell Culture.
Unless otherwise indicated, cell lines were obtained from American Type Culture Collection, Manassas, VA. HeLa, C-33A, H1299 (human bronchoalveolar carcinoma; obtained from B. Opalka, University of Essen, Essen, Germany), A549, AsPC-1, Capan-1, Capan-2, MZA (human pancreas carcinoma; obtained from D. I. Smith, Mayo Clinic, Rochester, MN), HT29, MCF-7, SK-OV-3, Saos-2, 293, NHDF cells (normal human diploid fibroblasts; obtained from M. Roggendorf, University of Essen), and NIH3T3 cells were maintained in DMEM supplemented with 10% FCS. K-562, and LAMA-84 chronic myeloid leukemia cell lines (obtained from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and TT cells were grown in RPMI 1640 plus 15% FCS. The 4-OHT³ inducible Saos-2 ER-E2F1 cells have been described previously (9) . CHX (Sigma, Taufkirchen, Germany) was used at a final concentration of 10 µg/ml. For genotoxic treatment, NIH3T3 cells were exposed to 10 µg/ml Adr, 20 µg/ml CP, 250 ng/ml Act, or 400 µM Eto for 3 h. For gene expression analysis, cells were harvested 9 h later. Transfection of NIH3T3 cells was performed using Polyfect (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.

HCC Tissue Samples.
Tissue samples from HCC tissue and adjacent normal liver were obtained from patients undergoing partial hepatectomy at the Department of General and Transplantation Surgery at the University of Essen, Essen, Germany. The tissues were frozen and stored in liquid nitrogen until RNA extraction. Histological analysis showed >75% tumor cells in all of the tumor sections.

Plasmids and Recombinant Retroviruses.
cDNAs encoding NH₂-terminally truncated p73 were amplified by PCR using HA-p73 as a template (kindly provided by G. Melino, University Tor Vergata, Rome, Italy), cloned into pcDNA3.1 and sequence-verified. TA-p73ß (aa72–499) was cloned into the retroviral vector pLXRN (Clontech, Palo Alto, CA). Retroviruses pseudotyped with the envelope glycoprotein from the vesicular stomatitis virus were produced as described (9) . The luciferase-reporter plasmids (pGL3-p53 and pGL3basic) have been described (9) .

IP and Western Blotting.
Endogenous p73 from 5 mg of radioimmunoprecipitation assay cell extract (50 mM Tris/HCL, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) was immunoprecipitated with 1 µg of anti-p73 (ER15) antibody. Immunoprecipitates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with various p73 antisera. Murine anti-p73 monoclonals ER15 and ER13 were obtained from Oncogene Science. Goat polyclonal anti-p73 (Ab-7) and rat monoclonal anti-H-ras (sc-35) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA.

Two-dimensional Electrophoresis.
For two-dimensional Western blot analysis, the anti-p73 immunoprecipitates were solubilized in 125 µl isoelectric focusing rehydration buffer [8 M urea, 1 mM DTT, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonic acid, 0.25% Bio-Lyte (pH 3–10), and 5 mM Pefabloc] and subjected to isoelectric focusing using IPG strips (pH 3–10; Bio-Rad, Munich, Germany) with a total of 20,000 Vh. IPG strips were equilibrated in SDS equilibration buffer [6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl (pH 8.8)] and subjected to second dimension separation on 8% SDS-PAGE.

RT-PCR, Southern Blotting, and Luciferase Assays.
Semiquantitative RT-PCR was carried out on total RNA prepared with the RNeasy Mini kit (Qiagen) essentially as described (9) . To obtain a semiquantitative result, we used the minimum number of cycles required to obtain a clear signal in the linear range and labeling of PCR products with -[³²P]dCTP for high-sensitivity detection. The amount of PCR product was quantitated on a phosphorimager. Primer sequences were as follows: p73: 5'-GACGGAATTCACCACCATCCT-3' and 5'-CCAGGCTCTCTTTCAGCTTCA-3'; TA-p73: 5'-GGCTGCGACGGCTGCAGAGC-3' and 5'-GCTCAGCAGATTGAACTGGGCCATG-3'; p73ex2 and p73ex2/3: 5'-GGCTGCGACGGCTGCAGGGA-3' and 5'-GGCTGCGACGGCTGC AGGCC-3' as the sense primers and 5'- CAGGCGCCGGCGACATGG-3' as the antisense primer; N'-p73: 5'-TCGACCTTCCCCAGTCAAGC-3' and 5'-TGGGACGAGGCATGGATCTG-3'; N-p73: 5'-CAAACGGCCCGCATGTTCCC-3' and 5'-TTGAACTGGGCCGTGGCGAG-3'; and p73–1/4: 5'-ACGCAGCGAAACCGGGGCCCG-3' and 5'-GCCGCGCGGCTGCTCATCTGG-3'. For Southern blotting, RT-PCR products were separated on 2% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech), and probed with the [³²P]dCTP end-labeled oligonucleotide p73-exon4 5'-TTCATGGTGCTGCTCAGCAGATTGAACTGGGC-3'. Luciferase assays were essentially performed as described (9) .

Real-Time RT-PCR.
MDM2 and p21 transcripts levels were quantitated on a LightCycler (Roche Diagnostics) using SYBR Green I. Primer sequences were as follows: MDM2: 5'-GGATCCTTTGCAAGCGCCAC-3' and 5'-TCAAAGGACAGGGACCTGCG-3'; and p21: 5'-TTTCAGCCACAGCGACCATG-3' and 5'-AAAGTTCCACCGTTCTCGGG-3'. Transcript levels were normalized to GAPDH expression and presented as the fold induction relative to untreated control cells.

Tumorigenicity Assays.
For soft agar analysis, 10⁴ cells in DMEM/10% FCS were mixed with an equal volume of 2% methylcellulose medium and poured onto a bed of 0.6% agarose. After 3 weeks, colonies were microscopically counted and measured. All of the experiments were performed in triplicate. Tumor growth in nude mice was assayed by injecting 5 x 10⁶ cells in 200 µl of DMEM s.c. into the flanks of nude mice. Tumor dimensions were measured in two perpendicular diameters. Care of experimental animals was in accordance with institutional animal care and use committee guidelines.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Identification of NH₂-terminally Truncated p73 Protein in Human Tumor Cells.
Analysis of p73 protein in a panel of human tumor cell lines showed various expression levels ranging from absence of p73 protein in MCF-7 cells to high-level expression in 293 cells (Fig. 1A) . p73 expression in 293 cells is consistent with p73 induction by the adenoviral E1A oncogene (10) . Interestingly, high-level p73 expression correlated with a complex isoform pattern in MZA, K562, and 293 cells. All of the detected bands share the epitope of the ER15 antibody in exon 11 (data not shown), which is only present in the p73 and ß-isoforms, but not in the other COOH-terminal variants described thus far, suggesting that the additional bands might be because of either novel COOH-terminal variants or NH₂-terminally truncated forms. These can be differentiated by the Ab-7 antibody, which was raised against amino acids 1–15 and detects only full-length p73 and p73ß (Fig. 1B) . A detailed analysis of p73 immunoprecipitates from 293 cells reveals four distinct isoforms (Fig. 1C) . In addition to the previously described full-length p73 proteins TA-p73 and TA-p73ß, we could identify two novel isoforms, which lack the NH₂-terminal epitope recognized by the Ab-7 antibody. Consistent with the lack of the acidic NH₂-terminal transactivation domain they have a more basic isoelectric point as determined by two-dimensional gel electrophoresis (Fig. 1D) . They appear as a series of spots differing in isoelectric point, potentially reflecting post-translational modifications. Because of the lack of the major transactivation domain, these two isoforms are termed TA-p73 and TA-p73ß. These results demonstrate that several human tumor cells express NH₂-terminally truncated p73 proteins.

View larger version (62K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Identification of NH2-terminally truncated p73 proteins in human cancer cells. A, IP-Western blot analysis of p73 in various cancer cell lines. The observed bands are labeled according to their reactivity with specific antibodies as determined in C. Lanes 1, HeLa; 2, C-33A; 3, H1299; 4, MZA; 5, MCF-7; 6, K-562; 7, 293; 8, HepG2; 9, NHDF (normal human diploid fibroblasts). B, Western blot analysis of in vitro translated, 35S-labeled NH2-terminal deletion mutants of p73. The upper panel shows an autoradiography, the lower panels immunoblots with three different p73 antibodies. C, IP-Western blot analysis of 293 cell extracts with the indicated p73 antibodies. The first and third lane show appropriate controls without the primary antibody. The bands are labeled according to their reactivity with the various antibodies. D, two-dimensional IP-Western blot analysis. Analysis of recombinant proteins TA-p73, TA-p73 (amino acids 49–637), and TA-p73 (amino acids 72–637) expressed in H1299 cells is shown in the upper right panel with brackets denoting the location of the individual proteins. Analyses of endogenous p73 proteins from 293 cells is shown in the other panels.

Transcripts Encoding TA-p73.

View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Transcripts encoding TA-p73. A, genomic organization of the TP73 locus. The splicing patterns generating COOH-terminal isoforms p73 , ß, , , , and , and the NH2-terminal isoforms p73ex2, p73ex2/3, and N'-p73 are shown. Transcriptional start sites are indicated by arrows. The N-p73 isoform is generated from a cryptic promoter within intron 3. Color coding: red, transactivation domain; orange, exon 3B-derived coding sequence; blue, DNA-binding domain; green, oligomerization domain; yellow, COOH terminus; white, noncoding sequences. B, alignment of p73 NH2-terminal isoform amino acid sequences. The underlined amino acids are unique to the N-p73 and N'-p73 isoforms, and are derived from the alternative exon 3B located in intron 3. C, semiquantitative RT-PCR analysis of p73 expression in various cancer cell lines. p73 transcripts were amplified with specific primer pairs and the radioactively labeled RT-PCT products visualized by autoradiography. NHDF, normal human diploid fibroblasts; -, no template control. The p53 status is indicated; *, inhibition of wild-type p53 by HPV E6 or adenoviral E1B-55K; ?, p53 status unknown; -, p53-deleted. D, Southern blot of RT-PCR products. NH2-terminally truncated p73 transcripts were simultaneously amplified with primers corresponding to sequences in exon 1 and 4, respectively. Probing of a Southern blot of the RT-PCR products with an oligonucleotide from exon 4 revealed distinct transcripts with similar expression levels depending on the cell line analyzed. Lanes are labeled as in C. E, induction of TA-p73 by E2F1. E2F1 activity was induced with 4-OHT in ER-E2F1-expressing Saos-2 cells for the time indicated. Total RNA was analyzed for the various TA-p73 encoding transcripts by semiquantitative RT-PCR. F, TA promoter-derived TA-p73 transcripts are direct targets of E2F1. E2F1 activity was induced in the absence or presence of CHX. Total RNA was analyzed by semiquantitative RT-PCR. CCNE1 is shown as a control for a direct, MMP16 for an indirect E2F1 target gene.

Induction of TA-p73 by E2F1.
Expression of p73 is regulated by E2F, which shows deregulated activity in most tumors (9 , 14) . Therefore, increased levels of p73 in cancer tissues might be attributable to increased transcriptional activation of the TP73 gene by E2F. To analyze the regulation of the various p73 isoforms by E2F, we used Saos-2 cells that constitutively express E2F1 fused to a modified, 4-OHT-responsive version of the ligand binding domain of the mouse ER as described previously (9) . Conditional activation of E2F1 in the presence of 4-OHT resulted not only in an increase of total p73 mRNA levels but also in a parallel induction of both full-length and NH₂-terminally truncated p73 isoforms (Fig. 2E) . Activation of the ER-E2F1 fusion protein in the presence of the protein synthesis inhibitor CHX allows us to discriminate a direct effect of E2F1 on p73 isoform expression from secondary effects because of E2F1-mediated cell cycle stimulation. All of the TA promoter-derived transcripts (TA-p73, p73ex2, p73ex2/3, and N'-p73) were strongly induced by E2F1 (23.7-fold) even in the presence of CHX, whereas the N-promoter-regulated N-p73 transcript showed only a modest induction (2.2-fold). Therefore, at least the TA promoter-derived TA-p73 isoforms are direct transcriptional targets of E2F1 (Fig. 2F) suggesting that increased expression levels of TA-p73 in tumor cells are also attributable to increased E2F activity. Depending on the cellular context E2F1 apparently exhibits a dual function as an oncogene and a tumor suppressor by stimulating both proliferation and apoptosis (15) . Therefore, activation of both proapoptotic full-length and transforming TA-p73 isoforms by E2F1 appears to be intriguingly similar.

Increased Expression Levels of TA-p73 in HCCs.
Importantly, the correlation between increased expression of p73 and TA-p73 is not only found in cell lines but also in primary tumor samples. As has been reported, p73 overexpression is a common finding in HCCs and is significantly correlated with a poor patient survival prognosis (7) . Consistent with their data, our analysis of the various NH₂-terminal p73-isoforms by RT-PCR clearly showed enhanced expression of p73 in most HCC samples compared with adjacent normal liver tissue (Fig. 3) . Of note, all of the samples with high level p73 overexpression (samples #1, #3, #5, and #6) demonstrated tumor-specific up-regulation of the TA promoter-derived alternatively spliced TA-p73 isoforms (p73ex2, p73ex2/3, and N'-p73), whereas the N-p73 transcript was barely detectable (data not shown). These data are consistent with findings by Sayan et al. (11) , who also failed to detect tumor-specific up-regulation of the N-p73 transcript. This underlines the importance of the TA promoter-derived alternatively spliced transcripts and questions the relevance of the N-promoter for hepatocarcinogenesis.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Increased expression levels of TA-p73 in HCCs. Expression of NH2-terminal p73 isoforms was analyzed by semiquantitative RT-PCR in six matched pairs of tumor (T) and adjacent normal (N) liver tissue. Amplification of 18S-rRNA demonstrates use of equal amounts of total RNA. -, no template control.

Overexpression of TA-p73 Results in Malignant Transformation of NIH3T3 Cells.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. TA-p73 acts as a transforming oncogene in NIH3T3 cells. A, luciferase assay demonstrating the dominant-negative function of TA-p73. Four hundred ng of a reporter plasmid together with 150 ng of the indicated TA-p73 plasmid were transfected into NIH3T3 cells and assayed for luciferase activity after 48 h. B, TA-p73 acts as a dominant-negative inhibitor of full-length p73ß. Transfections including 50 ng p73ß plasmid were performed as described in A. C, suppression of p53-dependent reporter activity by TA-p73ß following genotoxic damage induced by Adr. Mock or TA-p73ß-expressing NIH3T3 cells were transfected with 100 ng p53-reporter plasmid and treated 24 h later with 3 µM Adr as indicated for another 24 h before luciferase measurement. D, induction of endogenous p53 targets genes by various genotoxic agents (Adr, CP, Act, and Eto) in parental (light gray) or TA-p73ß-expressing (dark gray) NIH3T3 cells. Transcript levels for MDM2 and p21Waf1 were assessed by quantitative RT-PCR and normalized to GAPDH expression. Fold induction was calculated in relation to untreated control cells. Data are expressed as mean. *P < 0.05 (t test). E, Western blot of stably transduced NIH3T3 cells with antibodies against p21ras, p73, and actin. F, tumor growth kinetics of mock- (), TA-p73ß- (+), or H-rasV12- (x) transduced NIH3T3 cells inoculated s.c. into the flank of athymic nude mice. Shown is the average from three mice per cell population. G, anchorage-independent growth in semi-solid medium of TA-p73ß or H-rasV12 expressing NIH3T3 cells. Mock-infected cells did not form any colonies. Representative colonies from both cell populations are depicted in the upper panel. Photographs of nude mice 2 weeks after injection of TA-p73ß- or H-rasV12-infected NIH3T3 cells (middle panels). Corresponding formalin-fixed and paraffin-embedded tumor sections stained with H&E show a typical sarcomatous morphology (lower panels); bars, ± SD.

	ACKNOWLEDGMENTS

 
We thank O. Dirsch for preparation and analysis of tumor sections and S. Tuwe for support in real-time PCR analysis.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a grant of the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung (to B. M. P.).

² To whom requests for reprints should be addressed, at Centre for Cancer Research and Cancer Therapy Institute of Molecular Biology, University of Essen, Medical School, Hufelandstr. 55, D-45122 Essen, Germany. Phone: 49-201-723-3158; Fax: 49-201-723-5974; E-mail: brigitte.puetzer{at}uni-essen.de

³ The abbreviations used are: 4-OHT, 4-hydroxytamoxifen; CHX, cycloheximide; RT-PCR, reverse transcription-PCR; Adr, Adriamycin; IP, immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; Act, actinomycin D; CP, cisplatin; Eto, etoposide; ER, estrogen receptor.

Received 1/14/02. Accepted 5/ 7/02.

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