The protein p53 is the most frequently mutated tumour suppressor to be identified so far in human cancers. The ability of p53 to inhibit cell growth is due, at least in part, to its ability to bind to specific DNA sequences and activate the transcription of target genes such as that encoding the cell-cycle inhibitor p21Waf1/Cip1 . A gene has recently been identified that is predicted to encode a protein with significant amino-acid sequence similarity to p53. In particular, each of the p53 amino-acid residues implicated in direct sequence-specific DNA binding is conserved in this protein. This gene, called p73, maps to the short arm of chromosome 1, and is found in a region that is frequently deleted in neuroblastomas. Here we show that p73 can, at least when overproduced, activate the transcription of p53-responsive genes and inhibit cell growth in a p53-like manner by inducing apoptosis (programmed cell death).

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The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.

The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73alpha and p73beta stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.p53 is the most frequently mutated human tumor suppressor gene identified to date. In model systems, restoration of p53 function in tumor cells leads either to a cell cycle block or to apoptosis. These activities have been linked to the ability of p53 to bind to specific DNA sequences and activate transcription. Among the genes that are activated by p53 are the cyclindependent kinase inhibitor p21 and a family of genes involved in oxidative stress (10,36).Recently, Kaghad et al. serendipitously identified a cDNA encoding a novel p53-like protein (22). The protein, called p73, is approximately 60% identical to p53 in the region corresponding to the p53 DNA-binding domain. Furthermore, all of the residues that contact DNA in the p53-DNA crystal structure are conserved in p73 (7). In addition, the N terminus of p73 is similar (ϳ25% identity) to the N-terminal transactivation domain of p53, and the C terminus of p73 contains a region which is comparably similar to a C-terminal oligomerization domain within p53 (34,44,48). Finally, the genomic organizations of p53 and p73 are highly similar, suggesting that they have a common ancestry (22). In keeping with this high degree of similarity, p73 can, at least when overproduced, activate transcription from p53-responsive promoters and induce tumor cell apoptosis (20,22).The p73 gene maps to chromosome 1p36, a region that is frequently deleted in a variety of human cancers (22). Nonetheless, to date no mutations have been identified in the remaining p73 allele in such tumors. Thus, p73, unlike many tumor suppressor gene products such as the retinoblastoma protein (pRB) and p53, does not appear to conform to a two-hit model of tumorigenesis. It has been suggested that this may be due to epigenetic silencing of the p73 allele retained in tumors (22). Alternatively, it is possible that p73 is not a tumor suppressor gene product and is not the relevant target of 1p36 deletions.A number of unrelated DNA tumor viruses can inactivate pRB. Inactivation of pRB leads to derepression of E2F-responsive promoters, which, in turn, allows quiescent cells to enter S phase. In model...

We have isolated a cDNA clone encoding the major protein-tyrosine-phosphatase (protein-tyrosine-phos- tTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. M31724). 2735The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

E2F DNA-binding activity in vivo is due to heterodimer formation between members of the E2F and DP transcription factor families. The ability of these heterodimers to serve as transcriptional regulators is modulated by complex formation with additional proteins such as the products of the retinoblastoma gene and the adenovirus E4 ORF 6/7. Each of the E2F family members cloned to date contains a highly conserved region of unknown function, termed the marked box, which lies between their DNA binding and transactivation domains. Mutational analysis showed that the marked box contributed to the recognition of E2F family members by the E4 ORF 6/7 protein in vitro and in vivo.

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