Early diversification and complex evolutionary history of the p53 tumor suppressor gene family

The p53 tumor suppressor plays the leading role in malignancy and in maintaining the genome's integrity and stability. p53 belongs to a gene family that in vertebrates includes two additional members, p63 and p73. Although similar in sequence, gene structure, and expression potential, the three p53 members differ in domain organization (in addition to the transactivation, DNA-binding, and tetramerization domains, p63 and p73 encode a sterile alpha motif, SAM, domain) and functional roles (with p63 and p73 assuming additional key roles in development). It is interesting to note that outside vertebrates, p53-like sequences have only been found as single genes, of either the p53 or the p63/p73 type (i.e., without or with a SAM domain, respectively). In this paper, we report that the diversification of this family is not restricted to the vertebrate lineage, as both a p53- and a p63/p73-type sequence are present in the unicellular choanoflagellate, Monosiga brevicollis. Furthermore, multiple independent duplication events involving p53-type sequences took place in several other animal lineages (cnidarians, flat worms, insects). These findings argue that selective factors other than those associated with the evolution of vertebrates are also relevant to the diversification of this family. Understanding the selective pressures associated with the multiple independent duplication events that took place in the p53 family and the roles of p53-like proteins outside vertebrates will provide further insight into the evolution of this very important family. In addition, the presence of both a p53 and a p63/73 copy in the unicellular M. brevicollis argues for its suitability as a model system for elucidating the functions of the p53 members and the mechanisms associated with their functional diversification.     

Neurodevelopment on route p63

All known members of the p53 gene family, including the two homologs p73 and p63, have multiple biological functions. In neurons, p53 and p73 are known to regulate cell death in the developing and adult nervous system. A report by Jacobs et al. in this issue of Neuron shows that the more ancestral member of this gene family, p63, is an essential proapoptotic protein during neuronal development.     

Role of the newer p53 family proteins in malignancy

The most recently identified members of the p53 family, p63 and p73, share certain structural and functional similarities with p53. Both p63 and p73 can bind to canonical p53-DNA-binding sites, transactivate the promoters of known p53 target genes and induce apoptosis. Despite these similarities there are many important differences. In contrast to p53, p63 and p73 give rise to multiple distinct protein isoforms that have different functional properties. Upstream signaling pathways involved in the activation of p63 and p73 differ from those involved in p53 activation. Only a subset of the DNA damaging agents that induce p53 can induce p73. Cellular and viral oncoproteins can discriminate between p53 and the newer family members. In addition, the levels of p63 and p73 are affected by certain states of cellular differentiation. Finally, it is becoming clear that the newest members of the p53 family are not classical tumor suppressor genes. In contrast to the high prevalence of p53 mutations in human cancers, p63 and p73 mutations are rare. Indeed, levels of p73 increase during malignant progression. In addition, unlike p53-/- mice, mice lacking p63 and p73 do not develop tumors, but instead have significant developmental abnormalities. Mutations in p63 have also been detected in humans with the ectodermal dysplastic syndrome EEC. Further studies are required to determine whether qualitative or quantitative differences in the expression of p63 and p73 isoforms are important in the development of human cancers.     

Roles for p53 and p73 during oligodendrocyte development

Oligodendrocytes make myelin in the vertebrate central nervous system (CNS). They develop from oligodendrocyte precursor cells (OPCs), most of which divide a limited number of times before they stop and differentiate. OPCs can be purified from the developing rat optic nerve and stimulated to proliferate in serum-free culture by PDGF. They can be induced to differentiate in vitro by either thyroid hormone (TH) or PDGF withdrawal. It was shown previously that a dominant-negative form of p53 could inhibit OPC differentiation induced by TH but not by PDGF withdrawal, suggesting that the p53 family of proteins might play a part in TH-induced differentiation. As the dominant-negative p53 used inhibited all three known p53 family members - p53, p63 and p73 - it was uncertain which family members are important for this process. Here, we provide evidence that both p53 and p73, but not p63, are involved in TH-induced OPC differentiation and that p73 also plays a crucial part in PDGF-withdrawal-induced differentiation. This is the first evidence for a role of p73 in the differentiation of a normal mammalian cell.     

p73 and p63 are homotetramers capable of weak heterotypic interactions with each other but not with p53.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human cancers. Recent identification of two human homologues of p53 has raised the prospect of functional interactions between family members via a conserved oligomerization domain. Here we report in vitro and in vivo analysis of homo- and hetero-oligomerization of p53 and its homologues, p63 and p73. The oligomerization domains of p63 and p73 can independently fold into stable homotetramers, as previously observed for p53. However, the oligomerization domain of p53 does not associate with that of either p73 or p63, even when p53 is in 15-fold excess. On the other hand, the oligomerization domains of p63 and p73 are able to weakly associate with one another in vitro. In vivo co-transfection assays of the ability of p53 and its homologues to activate reporter genes showed that a DNA-binding mutant of p53 was not able to act in a dominant negative manner over wild-type p73 or p63 but that a p73 mutant could inhibit the activity of wild-type p63. These data suggest that mutant p53 in cancer cells will not interact with endogenous or exogenous p63 or p73 via their respective oligomerization domains. It also establishes that the multiple isoforms of p63 as well as those of p73 are capable of interacting via their common oligomerization domain.     

The p53 family in nervous system development and disease

The p53 family, consisting of the tumor suppressors p53, p63 and p73, play a vital role as regulators of survival and apoptosis in the developing, adult and injured nervous system. These proteins function as key survival and apoptosis checkpoints in neurons, acting as either rheostats or sensors responsible for integrating multiple pro-apoptotic and survival cues. A dramatic example of this checkpoint function is observed in developing sympathetic neurons, where a pro-survival and truncated form of p73 antagonizes the apoptotic functions of p53 and p63. Thus the levels and activities of the different p53 family members may ultimately determine whether neurons either live or die during nervous system development and disease.     

Interrelations between p73 and p53: a model, neuroblastoma

Homologies in sequence and gene organization of p53 and their relatives, p73 and p63, suggest similar biological functions. However differences exist between the p53 family members. Indeed in human tumors p53 is often mutated while p63 and p73 are very rarely mutated. In addition, in contrast to p53 which is transcribed in a unique mRNA species spanning all gene exons, each homologue expresses two types of isoforms: some with transactivation domain (TAD) showing tumor suppressive properties, the others deprived of TAD, with oncogenic properties. If p53 responds to immediate genotoxic stress, its homologues participate to the cell homeostasis of specific tissues along their development and differentiation, neuronal tissue for p73, epithelial for p63. However a collaboration between the three p53 family members has been shown to occur in response to cell genotoxic damages. Neuroblastic tumors characterized by a large spectrum of neuronal differentiation constitute a good model to study relationship between p73 and p53 as well as the regulation of their respective expression.     

Evaluation of p63 and p73 antibodies for cross-reactivity

The tumor suppressor p53 is commonly mutated in human cancers. However, two homologs of p53, p63 and p73, are frequently over-expressed in tumors and are associated with tumor subtypes, clinical outcomes, and responses to therapy. There are many isoforms of p53, p63, and p73 (the p53 family). Proper detection of and discrimination between the members of this tumor suppressor family in human tissues is of critical importance to cancer research and clinical care. In this study, we assessed the specificity of several commercially available and newly generated p73 antibodies, focusing on antibodies that distinguish between the TAp73 and ?Np73 isoforms by Western analysis, immunohistochemistry, and immunofluorescence. In addition, we found that the pan-p63 and pan-p73 antibodies tested cross-react with p73 and p63 respectively. The results of this study have important implications for analysis of p63 and p73 expression and co-expression in human tumors, and for potential use of these reagents in molecular diagnostics and therapeutic decision-making.