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Advances in Brief |
Np73
Departments of Cell Biology [O. I., C. K., K. E., M. O., S. I.], Respiratory Oncology and Molecular Medicine [O. I., T. N.], Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan
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ABSTRACT |
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Np73, which is capable of suppressing p53- and p73-dependent transactivation. We speculate that this suppression is achieved by competing for the DNA binding site in the case of p53 and by direct association in the case of TAp73. Expression of Np73 in cancer cell lines also inhibited suppressive activity of p53 and TAp73 in colony formation, implying possible involvement of Np73 in oncogenesis by inhibiting the tumor-suppressive function of p53 and TAp73. Also reported is the identification of TAp73
, a new member of the COOH-terminal truncated isoform of p73 and tissue-specific expression of these isoforms, along with other previously identified p73 isoforms. |
Introduction |
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This p53 has long been a lone gene with no related genes until the recent successive identification of the new family members, p73 and p51/p63 (also known as p40, p73L, CUSP, or KET; Refs. 2, 3, 4, 5 ). In addition to a structural similarity to p53, they both resemble this gene functionally, at least in in vitro overexpression systems. They are capable of transactivating p53 target genes, inducing apoptosis, and suppressing cell growth to various extents, depending on the individual isoform (Refs. 2, 3, 4, 5, 6, 7 ; see below). Despite this resemblance, they were shown to exhibit distinct biological functions; p51-targeted mice suffered from severe defects in the development of skin and extremities; p51 mutations were shown to play a causative role in human hereditary diseases such as EEC (ectrodactyly, ectodermal dysplasia, and facial clefts), dactylaplasia, and Hay-Wells syndromes (2) ; and p73-targeted mice exhibited defects in neuronal development and inflammatory responses (8) . Furthermore, p73 and p51 was transcribed as numerous splicing variants, which are expressed in a tissue specific manner (4, 5, 6, 7, 8) .
In an attempt to delineate this complex pattern of expression, our first effort was devoted to determine the genomic structures for these p53 family genes. Careful genomic analysis of human p73 and p51 enabled us to predict the presence of an alternative promoter and exon of p73 similar to those of p51. This exon encoded NH2-terminal truncated isoforms of p73 corresponding to the p51 isoforms termed Np51/p63 (5)
, which was shown to have characteristic potential to suppress transcriptional activity of both p53 and TAp51/p63. During analyses of the isoform, the discovery and functional evaluation of murine Np73 were described (8
, 9)
. We report here the first identification of human Np73. We show that it has the potential to suppress p53 or TAp73, implying that Np73 may function as an oncogenic isoform. Along with the analyses of Np73, we also describe the discovery of a COOH-terminal truncated variant of p73, TAp73, and analyses of the tissue-specific expression pattern of p73 isoforms in detail.
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Materials and Methods |
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Construction of a Panel of cDNA Libraries and Semiquantitative PCR.
We constructed a human cDNA library panel using 33 human poly(A)+ RNAs purchased from Clontech (Palo Alto, CA) using the SuperScript Lambda System for cDNA synthesis and 
cloning (Life Technologies, Inc., Rockville, MD), essentially as suggested by the supplier. Expression of the NH2-terminal splice variants of the p73;TA, N', and N was detected by semiquantitative PCR. TA and N' was detected by use of 5'-GGAATAATGAGGTGGTGGGC-3' designed from exon 3 and 5'-GCCTGTTTACAAGAAAGCGG-3' designed from exon 5. N was detected by use of 5'-GCGAAAATGCCAACAAACGG-3' designed from exon 3' and the above exon 5 primer. PCR for the COOH-terminal variants of p73 
, ß, 
, 
, 
, and 
was performed using the sense primer designed from exon 10 (5'-GAAGCTGAAAGAGAGCCTGG-3') and the antisense primer designed from exon 14 (5'-GATGGTCATGCGGTACTGC-3'). The expected length of the amplified fragments for the , ß, , , , and was 517, 423, 367, 190, 273, and 230 bp, respectively. To detect , the sense primer designed from exon 11 (5'-CCTTCTCTTCCTTGCTCTCG-3') and the antisense primer designed from exon 13 (5'-TTCTCGCCCATGAACAAGG-3') were used. PCR reaction was done by use of Taq DNA polymerase (Life Technologies, Inc., Rockville, MD) and anti-Taq start antibody (Clontech) as suggested by the suppliers.
Cloning of Np73/ß and TAp73.
A DNA fragment including p73 exon 3' was PCR amplified using the above exon 3 primer and an antisense primer designed from exon 6 (5'-GTGATGATGATGAGGATGGG-3') and then cloned into pRcCMV (Invitrogen, Carlsbad, CA) to obtain Np73/ß expression plasmids. HA3
-tagged and FLAG-tagged constructs were made by addition of synthetic oligonucleotides encoding the HA epitope of MYPYDVPDYA and the FLAG epitope of MDYKDDDDK to the NH2-terminal of TAp73/Np73. A DNA fragment containing the TAp73 isoform amplified by the 3'-RACE method was combined with TAp73/pRcCMV to create an expression construct.
Immunoprecipitation and Immunoblot Analysis.
Cells were transiently transfected with 10 µg of DNA per 10-cm dish and harvested after 48 h. Cell lysate preparation and immunoprecipitation were carried out as described either using anti-p53 (Oncogene Research Products, Cambridge, MA), anti-HA (Boehringer Mannheim, Tokyo, Japan), or anti-FLAG (Sigma Chemical Co.-Aldrich, Tokyo, Japan) antibody (10)
. The immunoprecipitants were resolved by SDS-polyacrylamide gels and immunoblotted with the corresponding antibodies.
Colony Formation and Cell Proliferation Assay.
The indicated expression plasmids were cotransfected with pBABEpuro, a puromycin-resistance plasmid (a gift from J. Morgenstern) into H1299 cells using Lipofectamine (Life Technologies, Inc., Rockville, MD) as described (4)
. H1299 stable transfectants were isolated by transfecting Np73 or pRcCMV using Lipofectamine with pBABEpuro as a selection plasmid. The recombinant adenovirus expressing human TAp73 cDNA (Ad-TAp73) and LacZ (Ad-LacZ) were constructed as described (11)
. Cell viability was assayed by alamarBlue (Alamar Biosciences, Sacramento, CA).
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Results |
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Np73 and TAp73.Np73 isoform, corresponding to Np51. Naturally, our next attempt was to isolate unidentified isoforms by an efficient RACE method (to be described elsewhere), which enabled us to isolate Np73 corresponding to Np51 lacking an NH2-terminal transactivation domain. Comparison of the Np73 cDNA to its genomic sequence revealed the existence of exon 3' in intron 3 of p73 gene. The length of the p73 intron 3' (between exon 3' and exon 4) was approximately 15 kb, which was similar to that of the p51 gene. Another interesting feature is the two-way usage of this exon 3', which might also be operating in the case of p51, considering the striking resemblance (Fig. 1A)
. One usage is the exon 3' transcribed by an alternative promoter similar to that of Np51. The 5'-RACE method showed that p73 has 276 bases of exon 3', which is homologous to that of murine p73 and similar to that of p51 (8)
. The other usage is that the exon 3' is being transcribed along with exon 3 by an alternative splicing event. This was determined by PCR screening of human cDNA libraries using various primers. This latter transcript, N'p73 mRNA, was shown to retain all of the other exons 1 through 14 with 198 base-exon 3' insertion. Both Np73 and N'p73 mRNA encoded an identical protein with a truncated NH2-terminal structure lacking the putative transactivation domain of TAp73 proteins (Fig. 1B)
. In addition to this 5'-RACE method, the 3'-RACE method enabled us to obtain a new COOH-terminal truncated isoform of p73, termed TAp73. Other than the two originally identified isoforms of p73, p73 and p73ß (generated by exon 13 skipping), four splicing variants of p73 have since been reported: p73, , , and generated by skipping the exon of 11, exons of 11–13, exons 11 and 12, and exons 11 and 13, respectively. Contrasting with these isoforms, p73 is generated by alternative termination (Fig. 1A)
. p73 mRNA has a long exon 13 (853 bp) containing a stop codon, encoding a 571-amino acid protein (Fig. 1C)
. As shown in Fig. 1A
, there are 11 different p73 mRNAs: TAp73, TAp73ß, TAp73, TAp73, TAp73, TAp73, TAp73, Np73, Np73ß, N'p73, and N'p73ß.
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N'p73 transcripts were distinguishable by the product length, 642 and 444 bp, respectively. The expression of the latter transcript was solely detected in the pancreas, whereas many tissues were positive for TAp73 expression. The Np73 transcript was only detected in fetal lung and corpus callosum by conducting PCR in separate reactions amplifying the 606-bp product (Fig. 1D)
. p73 was expressed in a wide variety of human tissues, whereas p73ß was expressed in lymph nodes, spinal cord, cerebellum, and substantia nigra; p73 was expressed in liver, small intestine, fetal lung, cerebellum, and thalamus (Fig. 1D)
. Only one 200-bp product was amplified in the hippocampus, which could be either p73, , , or a mixture of these (Fig. 1D)
. The p73 transcript was only detected in the lymph node (data not shown). Although not as potent, TAp73 showed significant transactivation potential comparable with that of p51A/TAp63, TAp73, and TAp73 against mdm2-P2 (Fig. 2A)
, BAX, p21waf1, 14-3-3, and RGC promoters (data not shown).
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Np73.N isoforms in inhibiting p53 or TAp73 transactivation activity. A designated amount of Np73 plasmid was cotransfected into SaOS2 cells with a constant amount of either p53 or TAp73 expression plasmid. FLAG tagged Np73 and ß almost completely suppressed the transactivation of mdm2-P2 promoter by TAp73ß and TAp73 (Fig. 2B
and similar data not shown). Similarly, transactivation of p53 was suppressed by the FLAG-Np73 and ß, although a four times larger amount was required to suppress the transactivation of p53 effectively (Fig. 2C)
. The expression constructs without the FLAG tag were capable of suppressing p53 and TAp73 transactivation to the same extent (data not shown).
Determination of Interaction between Np73 with Either p53 or TAp73.
To determine the mechanism of the above suppression, we investigated the ability of the Np73 proteins to bind to either p53 or TAp73. Lysates prepared from HA-TAp73 ( or ß) and FLAG-Np73 ( or ß) cotransfectants were subjected to immunoprecipitation by an anti-FLAG monoclonal antibody and then immunoblot detection by an anti-HA monoclonal antibody (Fig. 3, A and B)
. The experiment revealed readily detectable interaction of HA-TAp73 ( or ß) and FLAG-Np73 ( or ß). The reciprocal experiment using tagged epitopes and antibodies vice versa resulted in essentially identical results (data not shown). On the other hand, p53 was not coimmunoprecipitated with FLAG-Np73 or ß (Fig. 3C)
under the same conditions used, indicating that p53 does not stably interact with p73. We also experienced reduction of FLAG-Np73 in the presence of p53 (Fig. 3C)
. This is speculated to be caused by the general inhibitory effect of p53 on promoter activity (13)
or by enhanced Np73 degradation mediated by p53, as was observed in the case of Np63 (14)
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Np73 on Cell Growth.Np73, we next examined the effect of Np73 expression on cell growth- inhibitory activity of p53 and TAp73 by colony formation assay. Cotransfection of Np73 with either TAp73 or with p53 resulted in significant or dramatic increment in the number of colonies compared with transfection of either alone (Fig. 4A)
. Although suppression of colony formation by TAp73 expression had a marginal effect with no significant difference from pRcCMV alone, cotransfection of Np73 plasmid apparently increased the number of colonies. Thus, the assay revealed the potential of Np73, which can be interpreted as either anti-growth suppressing or growth promoting.
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Np73 (H1299-Np73) and stably integrating pRcCMV (H1299-RcCMV). H1299-Np73 cells, expressing exogenous Np73, exhibited more rapid growth profile than did H1299-RcCMV cells, when infected with Ad-TAp73 at the MOI of 100. In contrast, the growth rates of these two clones when infected with Ad-LacZ at the similar MOI resulted in almost identical profiles (Fig. 4B)
. Independent experiments using 20 and 50 MOI of Ad-TAp73 or Ad-LacZ confirmed the resistance of H1299-Np73 cells (data not shown). These data suggest that the cells expressing Np73 are more resistant to the growth-inhibitory effect of TAp73 and p53. Nevertheless, the results regarding TAp73 were somewhat weak because of the fact that TAp73 did not suppress cell growth to the extent reported previously by others (6
, 7)
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, , , , and p63ß; (c) both possess an alternative promoter producing transcript encoding N isoforms; and (d) p73 encoded the isoform TAp73 by alternative termination, and p51 encoded the isoform p51A/p63 by alternative exon (Fig. 1A)
. As a result of this complex genomic structure common to these two genes, they exhibited a much more complex pattern of expression than that of p53. Furthermore, we speculate the existence of different regulations among different species. Contrasting with predominant expression of Np73 in the murine brain as reported (8)
, we only detected human Np73 expression in the corpus callosum among eight subregions of the brain. In addition, neither TA nor N isoform of human p73 was expressed in hippocampus, whereas p73-deficient mice were demonstrated to suffer from hippocampal dysgenesis (8)
.
In addition to mdm2 oncoprotein, a well-known negative regulator of p53 and p73, we discovered a novel mechanism of regulation of p73 and possibly p53 by N isoforms. It is of note that the amount of Np73 comparable with that of TAp73 was fully capable of suppressing the transactivation by TAp73, whereas a 4-fold larger amount was necessary to suppress p53 to the same extent (Fig. 2, B and C)
. Np73 bound to TAp73 but was incapable of binding to p53 in vivo (Fig. 3)
, which was consistent with the data showing that wild-type p53 cannot associate with TAp73 in mammalian cells (15, 16, 17)
. Thus, we speculate that the suppression of TAp73 transactivation was achieved by direct association, whereas the suppression of p53 was achieved by competition for the DNA binding element.
One reason that the p53 mutations are the most frequently encountered in human tumors is attributable to the nature common to the vast majority of p53 mutant proteins, i.e., they are capable of inhibiting the remaining wild-type p53 allele in a dominant-negative manner (1)
. In contrast, the mutation of the p73 gene in human tumor is reported to be rare, despite the structural and functional similarity to p53 (18
, 19)
. Nevertheless, our analyses gave rise to the possibility that the overexpression of Np73 may contribute to the genesis of tumors by negating the tumor-suppressive activity of p53 or TAp73. In fact, overexpression of p73 in some tumors of the lung, bladder, and liver has been described (18)
. Although the overexpressed isoform of p73 has not been determined for most of these cases, it is highly likely that they are the Np73 isoform. Furthermore, one of the N isoforms of the p51 gene, p40, is reported to be oncogenic (20)
. It can readily be interpreted that the overexpression of the N isoform is much more easily actualized than acquisition of point mutations as in the case of p53. Further investigation is under way, and we are currently searching for human tumors with Np73 overexpression.
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FOOTNOTES |
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1 This study was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Otsuka Pharmaceutical Co., Ltd. 
2 To whom requests for reprints should be addressed, at Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8487; Fax: 81-22-717-8488; E-mail: sikawa{at}idac.tohoku.ac.jp
3 The abbreviations used are: HA, hemagglutinin antigen; RACE, rapid amplification of cDNA ends; MOI, multiplicity of infection.
Received 8/23/01. Accepted 12/14/01.
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and , with different transcriptional activity. J. Exp. Med., 188: 1763-1768, 1998.Np63 into a protein degradation pathway. Proc. Natl. Acad. Sci. USA, 98: 1817-1822, 2001.This article has been cited by other articles:
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  A. Kurihara, H. Nagoshi, M. Yabuki, R. Okuyama, M. Obinata, and S. Ikawa Ser46 phosphorylation of p53 is not always sufficient to induce apoptosis: multiple mechanisms of regulation of p53-dependent apoptosis Genes Cells, July 1, 2007; 12(7): 853 - 861. [Abstract] [Full Text] [PDF]
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U. Nyman, A. Sobczak-Pluta, P. Vlachos, T. Perlmann, B. Zhivotovsky, and B. Joseph Full-length p73{alpha} Represses Drug-induced Apoptosis in Small Cell Lung Carcinoma Cells J. Biol. Chem., October 7, 2005; 280(40): 34159 - 34169. [Abstract] [Full Text] [PDF]
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I. Dulloo and K. Sabapathy Transactivation-dependent and -independent Regulation of p73 Stability J. Biol. Chem., August 5, 2005; 280(31): 28203 - 28214. [Abstract] [Full Text] [PDF]
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T. Hanamoto, T. Ozaki, K. Furuya, M. Hosoda, S. Hayashi, M. Nakanishi, H. Yamamoto, H. Kikuchi, S. Todo, and A. Nakagawara Identification of Protein Kinase A Catalytic Subunit {beta} as a Novel Binding Partner of p73 and Regulation of p73 Function J. Biol. Chem., April 29, 2005; 280(17): 16665 - 16675. [Abstract] [Full Text] [PDF]
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A Oniscu, N Sphyris, R G Morris, S Bader, and D J Harrison p73{alpha} is a candidate effector in the p53 independent apoptosis pathway of cisplatin damaged primary murine colonocytes J. Clin. Pathol., May 1, 2004; 57(5): 492 - 498. [Abstract] [Full Text] [PDF]
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G. Melino, F. Bernassola, M. Ranalli, K. Yee, W. X. Zong, M. Corazzari, R. A. Knight, D. R. Green, C. Thompson, and K. H. Vousden p73 Induces Apoptosis via PUMA Transactivation and Bax Mitochondrial Translocation J. Biol. Chem., February 27, 2004; 279(9): 8076 - 8083. [Abstract] [Full Text] [PDF]
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T. Stiewe, J. Stanelle, C. C. Theseling, B. Pollmeier, M. Beitzinger, and B. M. Putzer Inactivation of Retinoblastoma (RB) Tumor Suppressor by Oncogenic Isoforms of the p53 Family Member p73 J. Biol. Chem., April 11, 2003; 278(16): 14230 - 14236. [Abstract] [Full Text] [PDF]
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 A. I. Zaika, N. Slade, S. H. Erster, C. Sansome, T. W. Joseph, M. Pearl, E. Chalas, and U. M. Moll {Delta}Np73, A Dominant-Negative Inhibitor of Wild-type p53 and TAp73, Is Up-regulated in Human Tumors J. Exp. Med., September 16, 2002; 196(6): 765 - 780. [Abstract] [Full Text] [PDF]
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T. Stiewe, S. Zimmermann, A. Frilling, H. Esche, and B. M. Putzer Transactivation-deficient {Delta}TA-p73 Acts as an Oncogene Cancer Res., July 1, 2002; 62(13): 3598 - 3602. [Abstract] [Full Text] [PDF]
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, 
) or transfected with empty pRcCMV vector (H1299-RcCMV; 
, 
) were selected. These cells were plated 1 day before adenoviral infection. Days were counted starting from day 0 when Ad-TAp73