Article

p63/73 homologues in surf clam: Novel signaling motifs and implications for control of expression

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Abstract

To understand the role of p53 gene family members during invertebrate embryonic development, we used polymerase chain reaction (PCR) to identify p63/73 homologues in the marine mollusc Spisula solidissima. Here, we report the sequences of two distinct p63/73-like homologues, both cloned from Spisula embryos. The first, Ssp63/73alpha is 2699 nucleotide (nt); the second, Spp63/73beta is 3920 nt. The nucleotide sequences of the two variants are nearly identical up to their stop codons but diverge in their 3'-untranslated regions (UTRs). The deduced amino acid sequence of both Ssp63/73 variants is 597 amino acids, coding for a protein with predicted molecular weight of approximately 68 kDa. We conclude that the two unique transcripts, containing 3' UTRs of variable lengths, represent tandem alternate polyadenylation sites for the Ssp63/73 gene. While alternative splicing has been well documented in the p63/73 gene family, this is the first report of alternate polyadenylation site choice as a control point for p63/73 gene expression in any species. In order to identify specific post-transcriptional as well as post-translational signals potentially involved in regulation of p63/73-like expression, we compared Ssp63/p73 nucleotide and Ssp63/73 deduced amino acid sequences to corresponding regions of other mammalian and nonmammalian p63 and p73 homologues. Within the Spisula 3' UTRs we identified multiple AU-rich elements (AREs) which may control translation activation. Within the deduced amino acid sequence, we identified potential sites for sumoylation, a post-translational process that has been identified in mammalian p63 and p73 proteins. Identification of these novel signaling sites provides information about potential mechanisms controlling expression of multiple p63/73 isoforms during development.

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... Evidence for p53 family genes was either extracted from Ensembl database or manually verified using BLAST (for the most recent assemblies available à ) (Altschul et al. 1990;Hubbard et al. 2009;Sayers et al. 2009). Evidence for p53 family genes in clams comes from gene isolation by C. Walker and others (Kelley et al. 2001;Cox et al. 2003) à Several species, such as lancelet and sea squirts, also have tentative intronless paralogs; these could be pseudogenes or candidate p53 family genes that are unlikely predecessors for mammalian p53-family and are not shown in the tree. One possible scenario is that they arose through independent duplication events with intron loss (Mourier and Jeffares 2003) The Origins and Evolution ...
... Thanks to Charles Walker and his colleagues (Kelley et al. 2001;Jessen-Eller et al. 2002;Cox et al. 2003;Bottger et al. 2008;Holbrook et al. 2009;Siah et al. 2009), we know a great deal about the p53 gene in clams. Mya arenaria, a common clam species, has a single p63/p73 hybrid-like gene but can splice the transcripts from this gene into a p53-like sequence and a p63/p73-like sequence. ...
... It is also clear that a genera of clams, such as the common surf clam, has been exploring evolutionary expansions of splice variants of the p63/p73 gene, producing a p97 and a p120 set of isoforms with common DNA binding domains. Interestingly, the p120 isoform is expressed only in the embryonic stages of clam development and appears to be involved in neuronal functions (like p73 in mice) (Jessen-Eller et al. 2002;Cox et al. 2003). ...
Article
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A common ancestor to the three p53 family members of human genes p53, p63, and p73 is first detected in the evolution of modern-day sea anemones, in which both structurally and functionally it acts to protect the germ line from genomic instabilities in response to stresses. This p63/p73 common ancestor gene is found in almost all invertebrates and first duplicates to produce a p53 gene and a p63/p73 ancestor in cartilaginous fish. Bony fish contain all three genes, p53, p63, and p73, and the functions of these three transcription factors diversify in the higher vertebrates. Thus, this gene family has preserved its structural features and functional activities for over one billion years of evolution.
... ath pathway ( Urist et al . , 2004 ) . 8 9 In invertebrate animals , p53 - like proteins have been discovered , such as CEP - 1 in the worm 10 Caenorhabditis elegans ( Lu and Abrams , 2006 ) , DMP53 in the fly Drosophila melanogaster , and 11 various homologs in different molluscan models ( Schmale and Bamberger , 1997 ; Kelley et al . , 2001 ; 12 Cox et al . , 2003 ; Muttray et al . , 2005 ; Goodson et al . , 2006 ; Muttray et al . , 2007 ) . Molluscan family 13 members , more so than CEP - 1 or DMP53 , show high sequence similarity to their vertebrate 14 counterparts . Thus , molluscan p53 isoform transcription may afford a relevant but simple model to 15 describe core properties of this ancient ...
... suggested as a biomarker in human cancers ( Concin et 11 al . , 2004 ) . 12 13 The expression of p53 proteins during haemic neoplasia has been studied in Mytilus edulis and the 14 soft - shell clam Mya arenaria ( Barker et al . , 1997 ; Kelley et al . , 2001 ; McGladdery et al . , 2001 ; 15 Stephens et al . , 2001 ; Jessen - Eller et al . , 2002 ; Cox et al . , 2003 , St - Jean , 2005 ) . Using an antibody to 16 the central DNA binding domain of the M . arenaria p53 / p73 , it was found that p53 and / or p63 / 73 is 17 expressed in the cytoplasm of leukaemic animals instead of the nucleus where it is required as a 18 transcription factor ( Kelley et al . , 2001 ) . In addition , a protein band with ...
... p63 / 73 was present only in leukaemic haemocytes of M . arenaria , while a band corresponding to p53 20 was present at equal levels in normal and leukaemic haemocytes ( Kelley et al . , 2001 ; Stephens et al . , 21 2001 ) . Using a cross - reactive antibody specific to the homodimerization domain of the surf clam 22 Spisula solidissima p63 / 73 ( Cox et al . , 2003 ) , it was shown that p63 / 73 was expressed at higher levels 23 in leukaemic haemocytes when compared to normal haemocytes of Mytilus edulis ( St - Jean et al . , 24 ACCEPTED MANUSCRIPT 5 2005 ) . Since these initial studies found that some p53 - family members were upregulated in end - stage 1 haemic neoplasia cells , we wanted to inv ...
Article
Mussels of the genus Mytilus are widely used in environmental monitoring. They can develop a leukaemia-like disease, haemic neoplasia, which could be induced, in part, by environmental stressors. The molluscan p53 tumor suppressor gene family was previously shown to be involved in haemic neoplasia at the protein level. The purpose of this study was the quantification of molluscan p53-like isoforms at the mRNA level in mussels with haemic neoplasia compared to normal controls. The three isoforms monitored were a p53-like, a TAp63/73-like containing an intact transactivation (TA) domain, and an NH(2)-terminally truncated p63/73 isoform termed DeltaNp63/p73-like that lacks the full TA domain. Using a comprehensive data set of 62 individual Mytilus trossulus and reverse transcription real-time PCR, we found that both the p53 and the DeltaNp63/73 isoforms were up-regulated in neoplastic haemocytes compared to normal haemocytes (p<0.0001). In contrast, the mRNA levels of the non-truncated isoform TAp63/73 did not change significantly in mussels with the disease at alpha=0.01 (p=0.0141), in contrast to previous findings at the protein level. Correlations in mRNA levels between the truncated isoform and the full-length isoforms in normal haemocytes were lost in neoplastic haemocytes. The increase in mRNA concentration of the truncated DeltaNp63/73 isoform in molluscan haemic neoplasia is similar to observations in many human cancers and cell lines and underlines the phylogenetically ancient oncogenic role of this isoform.
... multiple isoforms of p63) and proteases involved in the destruction of proteins (e.g. caspase 3/7-1 and 3/7-2) [70][71][72][73]. Six isoforms of glutathione-S-transferase enzymes involved in the production of antioxidants were present in the upper 25% of M. galloprovincialis proteins with the most hydrogen bonds per amino acid [74]. ...
... M. galloprovincialis proteins that could exert a strong influence on cell fate during heat stress, including p63, DNA damage regulated protein (PDRP), and multiple caspase enzymes [72,73,83], had more hydrogen bonds and salt bridges per amino acid than M. trossulus proteins or other functional classes of M. galloprovincialis proteins. p63 is a member of the p53 family of transcription factors that serve as connection points for several stress response pathways and that govern the decision between cell survival and apoptosis by controlling the abundance of gene products that determine cell fate [70,71,73,84,85]. PDRP is a downstream target of p53, suggesting that this molecule also contributes to the balance between cell survival and death during environmental stress [73,86]. ...
Article
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Background: Temperature exerts a strong influence on protein evolution: species living in thermally distinct environments often exhibit adaptive differences in protein structure and function. However, previous research on protein temperature adaptation has focused on small numbers of proteins and on proteins adapted to extreme temperatures. Consequently, less is known about the types and quantity of evolutionary change that occurs to proteins when organisms adapt to small shifts in environmental temperature. In this study, these uncertainties were addressed by developing software that enabled comparison of structural changes associated with temperature adaptation (hydrogen bonding, salt bridge formation, and amino acid use) among large numbers of proteins from warm- and cold-adapted species of marine mussels, Mytilus galloprovincialis and Mytilus trossulus, respectively. Results: Small differences in habitat temperature that characterize the evolutionary history of Mytilus mussels were sufficient to cause protein structural changes consistent with temperature adaptation. Hydrogen bonds and salt bridges that increase stability and protect against heat-induced denaturation were more abundant in proteins from warm-adapted M. galloprovincialis compared with proteins from cold-adapted M. trossulus. These structural changes were related to deviations in the use of polar and charged amino acids that facilitate formation of hydrogen bonds and salt bridges within proteins, respectively. Enzymes, in particular those within antioxidant and cell death pathways, were over-represented among proteins with the most hydrogen bonds and salt bridges in warm-adapted M. galloprovincialis. Unlike extremophile proteins, temperature adaptation in Mytilus proteins did not involve substantial changes in the number of hydrophobic or large volume amino acids, nor in the content of glycine or proline. Conclusions: Small shifts in organism temperature tolerance, such as that needed to cope with climate warming, may result from structural and functional changes to a small percentage of the proteome. Proteins in which function is dependent on large conformational change, notably enzymes, may be particularly sensitive to temperature perturbation and represent foci for natural selection. Protein temperature adaptation can occur through different types and frequencies of structural change, and adaptive mechanisms used to cope with small shifts in habitat temperature appear different from mechanisms used to retain protein function at temperature extremes.
... Although the gene sequence is not resolved for Mytilus ssp., it appears that Mytilus can similarly produce p53-and p63/p73-like isoforms and in addition an N-terminally truncated deltaN p63/p73-like isoform through alternative splicing (Muttray et al. 2007). The surf clam S. solidissima has additional splice variants, p97 and p120 (Cox et al. 2003), and interestingly the p120 is only expressed in the embryonic stages of clam development in response to stress and appears to be involved in neuronal development (similar to vertebrate p73) (Jessen-Eller et al. 2002). ...
... Interestingly, concentrations of a p63/73-like protein were also increased in neoplastic hemocytes in Mya and Mytilus (Kelley et al. 2001;Stephens et al. 2001), which was consistent with an increase observed in deltaNp63-p73 (but not TAp63/ p73) mRNA levels in those species (Muttray et al. 2008(Muttray et al. , 2012. Similarly, an antibody to the p73 HOMO-domain of the surfclam S. solidissima (Cox et al. 2003) detected an increased amount of p63/73 in neoplastic Mytilus hemocytes . Thus, the increase in p63/73-like proteins observed in Mya and Mytilus neoplastic hemocytes may be correlated with the increase in the truncated deltaNp63/73 mRNA. ...
Chapter
Insights into the common mechanisms that likely exist in invertebrate and in vertebrate cancers will lead to a more coherent understanding of the fundamental processes that are altered during tumorigenesis and of tumor immunity. Invertebrates possess an innate immune system, primarily consisting of cellular and humoral defenses. In mollusks, hemocytes provide the first line of defense against foreign particles or organisms but lose their functionality when transformed into neoplastic cells. Neoplasia, or abnormal growth and proliferation of cells, has been described in many invertebrate species ranging from sponges to mollusks and arthropods. Occurrence of neoplasia in invertebrates appears to be by far not as common or as diverse in nature as it is in vertebrates, but some invertebrates even display cancers with metastatic potential, notably the bivalve mollusks. This chapter will focus on a cancer of the hemocytes in marine bivalves called disseminated neoplasia. Disseminated neoplasia in bivalves can be induced by a retrotransposon, appears to have contributing environmental factors, and is one of only four known cases of a transmissible cancers, not only between members of the same species but also between members of closely related species. This chapter will describe how disseminated neoplasia and the components of the innate immune system are linked by one of the central nodes in a wide signaling network that integrates DNA stability, apoptosis, and cell growth: the p53 tumor suppressor family.
... In addition, each member of the human p53 superfamily produces different protein isoforms that play critical roles in the regulation of biological processes and their abnormal expression contributes to tumorigenesis [78]. In mollusks, p53 superfamily coding sequences have been characterized for clams [79][80][81], mussels [82][83][84], cockles [85], oysters [86], and gastropods [87]. Interestingly, in some species, various isoforms have been detected, and these isoforms seem to arise from alternative splicing, as regions that overlap between different proteins from the same species are 100% identical [79,80,83,84]. ...
... In mollusks, p53 superfamily coding sequences have been characterized for clams [79][80][81], mussels [82][83][84], cockles [85], oysters [86], and gastropods [87]. Interestingly, in some species, various isoforms have been detected, and these isoforms seem to arise from alternative splicing, as regions that overlap between different proteins from the same species are 100% identical [79,80,83,84]. Examination of neoplastic hemocytes, using expression profiles and antibodies to p53 family members, has shown differential up-regulation of p53 gene in neoplastic cells compared with healthy ones [22,79,85,[88][89][90]. ...
Article
Full-text available
Neoplasia-the abnormal growth of cells-is associated mainly with higher vertebrates (e.g., humans and mammals). However, neoplastic processes or cancers are ubiquitous among living organisms, with a high incidence of reported neoplasms in mollusks. Both benign and malignant cancers have been described in mollusks, but just two malignant neoplasms have raised scientific and industry concerns: gonadal neoplasia and disseminated or leukemia-like neoplasia. These cancers have been reported in either wildlife or captive populations; and as in humans, different causes have been suggested including genetic alterations, virus, retrotransposons, and pollutants. In this review, I give a general overview on neoplasia in mollusks with a focus on genes and molecular pathways involved in gonadal and disseminated neoplasia. Subsequently, I highlight some similarities between disseminated neoplasia and human cancers, particularly with leukemia, as well as the advantages of using mollusks affected by this disease as a model system to better understand cancer in humans. Finally, I discuss the feasibility of using mollusks to investigate tumorigenesis. As the field of marine genomics advances, I predict that comparative oncology will gain more attention in the years to come.
... Genes coding for the P53 protein family have already been described in marine bivalves, e.g. Mya arenaria (Van Beneden et al., 1997) and Spisula solidissima (Cox et al., 2003). More recently, the identification of p53 genes in soft tissues of Mytilus spp. was reported (Ciocan and Rotchell, 2005;Muttray et al., 2005). ...
... Some further consideration on the newly identified mussel p53like gene should also be made. This gene is likely to belong to the p63/73 gene family that has already been described in bivalves (Cox et al., 2003), which in mammals is expressed several splicing variants. The full length gene variants-α and β include the p53-like transactivating (TA) domain that provides their function during proapoptosis, while ΔN genes lack the TA domain and instead behave as negative isoforms that dominate active variants (Moll et al., 2001). ...
Article
In this study we describe the design and implementation of a novel low-density oligonucleotide microarray (the "Mytox-chip"). It consists of 24 mussel genes involving both normalizing elements and stress response related genes, each represented on the array with one or two different 50 mer oligonucleotide-probe reporters spotted in replicated samples on glass-activated slides. Target genes were selected on the basis of their potential involvement in mechanisms of pollutant and xenobiotic response. They are implicated in both basic and stress related cellular processes such as shock response, biotransformation and excretion, cell-cycle regulation, immune defense, drug metabolism, etc. The microarray was tested on mussels exposed to sublethal concentrations of mercury or a crude North Sea oil mixture. RNA samples were extracted from digestive glands of control and treated mussels for the synthesis of fluorescence labeled cDNAs to be used in dual color hybridizations. Transcription rates of two metallothionein iso-genes (mt10 and mt20), a p53-like gene and actin were quantitatively estimated also by real-time PCR to confirm microarray data. Significant alterations in the gene transcription patterns were seen in response to both treatments.
... The regulation of p53 function is tightly controlled through several mechanisms including p53 transcription and translation, protein stability and post-translational modification, as well as p53 location in the cell nucleus or cytoplasm (O'Brate et al., 2003, and references therein). Several authors have used various monoclonal and polyclonal antibodies raised against human and clam (Spisula, Mya) p53 family members to study the expression of the p53 family and to distinguish leukemic cells from normal cells (Kelley et al., 2001; Stephens et al., 2001; Jessen-Eller et al., 2002; Cox et al., 2003). We isolated haemocytes from two species of bivalve mollusc, Mytilus spp. ...
... p53 sequences revealed that unique and highly conserved sequence sites occur in the 3' untranslated regions (3' UTRs). Our previous reports suggest that the occurrence of cis-acting signaling sites in the 3' UTRs of the p53 gene family members in clams may control gene expression (Cox et al., 2003). Columbia. ...
Article
The extent to which humans and wildlife are exposed to anthropogenic challenges is an important focus of environmental research. Potential use of p53 gene family marker(s) for aquatic environmental effects monitoring is the long-term goal of this research. The p53 gene is a tumor suppressor gene that is fundamental in cell cycle control and apoptosis. It is mutated or differentially expressed in about 50% of all human cancers and p53 family members are differentially expressed in leukemic clams. Here, we report the identification and characterization of the p53 gene in two species of Mytilus, Mytilus edulis and Mytilus trossulus, using RT-PCR with degenerate and specific primers to conserved regions of the gene. The Mytilus p53 proteins are 99.8% identical and closely related to clam (Mya) p53. In particular, the 3' untranslated regions were examined to gain understanding of potential post-transcriptional regulatory pathways of p53 expression. We found nuclear and cytoplasmic polyadenylation elements, adenylate/uridylate-rich elements, and a K-box motif previously identified in other, unrelated genes. We also identified a new motif in the p53 3'UTR which is highly conserved across vertebrate and invertebrate species. Differences between the p53 genes of the two Mytilus species may be part of genetic determinants underlying variation in leukemia prevalence and/or development, but this requires further investigation. In conclusion, the conserved regions in these p53 paralogues may represent potential control points in gene expression. This information provides a critical first step in the evaluation of p53 expression as a potential marker for environmental assessment.
... El gen supresor de tumores p53 y su expresión proteica han sido muy estudiados debido a sus múltiples funciones en la regulación del ciclo celular, apoptosis, mantenimiento de la estabilidad genética y su alta implicación detectada en cánceres humanos (Oren, 2003). Se ha secuenciado el gen p53 en M. arenaria (Barker et al, 1997;Kelley et al., 2001), Spisula solidissima (Cox et al., 2003), M. edulis (Ciocan y Rotchell, 2005;Muttray et al, 2007), M. trossulus (Ciocan et al., 2006;Muttray et al, 2007), C. gigas (Farcy et al., 2008), M. galloprovincialis (Stifanic et al., 2009) y C. edule (Ruiz et al., 2013b). En fase avanzada de la enfermedad se detecta la expresión de la proteína p53 mutada en almejas M. arenaria; secuenciando el gen de la proteína mutada se observó, en dos de cinco almejas estudiadas, una transversión de citosina a guanina al final del exón 6, produciendo un cambio del aminoácido de ese codón de prolina a alanina (Barker et al, 1997). ...
Thesis
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Cockle Cerastoderma edule is a species of great commercial interest in Galicia. It is one of the bivalve species with more annual catches and, therefore, has a high economic and ecological importance in this region. Pathological studies associated with cockle mortality events in Galicia detected high prevalence of a pathological condition known as disseminated neoplasia. Knowledge of cancer in molluscs is scarce. This study aims to deep in the knowledge about the epidemiology of this disease, its etiology and the biochemical and physiological changes it causes. A thorough knowledge of this disease is required to develop strategies to fight it.
... Moreover, human p53 and p73 sequences are not identical where they overlap, so that human p53 is more similar to mouse p53 than to human p73. In molluscs, several authors highlighted the putative existence of p53, p63 and p73 isoforms (Jessen-Eller et al., 2002; Stephens et al., 2001; Kelley at al, 2001; Cox et al., 2003; Muttray et al., 2007). Surprisingly, the data thus far (Mya sp. and Mytilus sp.) show that the central DNA binding domain is 100 percent conserved between all members of the p53 family within one species. ...
Article
Like other sessile filter-feeding molluscs, oysters may be exposed in the natural environment to a variety of contaminants. Long-term exposure to pollutants may be one factor affecting prevalence of cancerous-like disorders, such as neoplasia. Environmentally induced alterations in p53 protein expression, in relation to leukemia, have been reported in various mollusc species inhabiting polluted water, suggesting that p53 proteins can also be used as a marker for environmental research. This work reports the cloning and sequencing of a p53-like cDNA in the mollusc bivalve Crassostreagigas. The deduced amino acid sequences of p53 shared a high degree of homology with the homologues from other mollusc species, including typical eukaryotic p53 signature sequences. We examined the p53 transcription expression pattern during the annual cycle in oyster gills and whole soft tissues in four locations along the French coasts. Real-time PCR analysis suggested that strong variations at p53 mRNA level are probably synchronized with the seasonal cycle at the four locations investigated.
... It is interesting to note that outside vertebrates, p53-like sequences have only been found as single genes. Noteworthy, while some of the invertebrate p53-like sequences reported to date appear more similar in domain organization (i.e., lacking a SAM domain) to the vertebrate p53 (e.g., the p53-like sequences from the fly Drosophila melanogaster [Ollmann et al. 2000], the nematode Caenorhabditis elegans [Schumacher et al. 2001], and the unicellular amoeba Entamoeba histolytica [Mendoza et al. 2003]), others (i.e., the p53-like sequences from mollusks; Cox et al. 2003; Muttray et al. 2005) are more similar to the vertebrate p63/ p73 counterparts (i.e., they contain a SAM domain). Considering the phylogenetic distribution of p53-like sequences, the structural and functional differences between the p53 and p63/p73 paralogs, and the recently emerging picture of cooperative and internecine interactions among the p53 family members, several questions arise. ...
Article
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.
... arenaria, GenBank Accession Nos. AF253323 and AF253324) (23), three for surf clam (Spisula solidissima, AF285104, AY289767, and AY289768) (24,25), one for squid (Loligo forbesi, U43595), and one for oyster (Crassostrea rhizophorae, AY442309). ...
Article
Mussels are susceptible to a wide range of environmental toxicants, including carcinogens, and thus are often employed as bioindicator species. To elucidate the molecular aetiology of such neoplastic damage, we have cloned Mytilus edulis homologues of the vertebrate ras proto-oncogene, and p53 tumor suppressor gene. The M. edulis ras cDNA encodes a predicted protein of 184 amino acids. The DNA sequence analysis with vertebrate ras sequences demonstrates that the M. edulis ras cDNA is highly conserved in regions of functional importance, including mutational hot spots. The partial p53 sequence also demonstrates that M. edulis p53 is highly conserved in two regions of functional importance and that these regions also include four of the five mutational hot spots for this gene. In contrast, the M. edulis p53 sequence shows little similarity to the other published invertebrate p53-like sequences. The cancer gene sequences characterized herein will allow development of specific biomarkers of genotoxic damage.
... As already discussed by other authors (Goodson et al., 2006), this suggests we deal here with alternative sequences originating from the same gene, which is not surprising given that all p53 family genes are able to express alternative products (MurrayZmijewski et al., 2006). Protein alignments showed all the domains, motifs and conserved residues already discussed by other authors (Kelley et al., 2001; Jessen-Eller et al., 2002; Cox et al., 2003; Muttray et al., 2005; Goodson et al., 2006;). The facts that drew our attention were: ...
Article
Genes of the p53 family are known to be critical regulators of the cell cycle. They have already been established as possible biomarkers. Elaborate regulation mechanisms result in numerous cDNA and protein isoforms being expressed from each gene of the p53 family. Their similarity caused an often misleading nomenclature in non-vertebrate species. The aim of the present work is a clarification of the nomenclature of molluscan p53 family sequences, an essential prerequisite for reliable interpretation of gene expression and protein function studies. Here, we report five partial cDNA and one partial genomic p63 sequences, all originating from two Mytilus galloprovincialis individuals. DNA, deduced protein sequences, and the exon/intron architecture were analyzed and compared to p53, p63 and p73 sequences from other organisms. Along with our sequences, we analyzed all similar molluscan sequences found in the GenBank database. The analysis showed our cDNA sequences code for the TAp63gamma isoform of the p63 protein, and identified all other molluscan p53 family sequences as p63 genes or their expression isoforms. Our results also indicate p63 as the ancestral gene of the p53 family as well as the only gene of the family present in non-chordate metazoan species.
... Its sister proteins p63 and p73 have important functions in embryonic development (Yang et al. 2002). Homologous proteins have been isolated in a number of invertebrates (Schmale et al. 1997; Kelley et al. 2001; Cox et al. 2003; Muttray et al. 2005; Goodson et al. 2006; Muttray et al. 2007); functional domains exhibit a high degree of similarity to their vertebrate homologues. We have shown previously that p53 mRNA and an N-terminal truncated isoform, ΔNp63/73, were expressed during late-stage haemic neoplasia in M. trossulus at levels higher than those seen in normal cells (Muttray et al. 2008). ...
Article
The mussel Mytilus trossulus can develop a neoplasia of the haemolymph, which occurs with high frequency (up to 40%) in nature. Associated with this disease are pro-apoptotic tumor-suppressor protein p53 isoforms, which are highly conserved between molluscs and vertebrates. The vertebrate wildtype p53 protein is maintained at low levels by the MDM2 protein in non-stressed cells to prevent undesired apoptosis. Identification of a putative invertebrate MDM-like homolog suggests early evolution of this mechanism of p53 regulation. The M. trossulus MDM homolog consists of a conserved NH(2)-terminal p53 binding domain, an acidic domain with highly conserved phosphorylation sites, and a highly conserved C-terminal RING-finger Zn-binding domain. Although BLAST queries predict this homologue to be more similar to vertebrate MDM2 than to MDM4, phylogenetic analysis suggests that it may be an ancestral form to both vertebrate MDM genes. Using yeast-two-hybrid assays and pull-down assays, we show that this molluscan MDM is able to bind to its p53 counterpart. We also show that MDM expression levels are directly correlated with p53 expression levels in healthy and in neoplastic haemocytes, but not with other p53 isoforms or with the proto-oncogene RAS. The combination of expression levels of five gene transcripts (p53, mdm, ras, Np63/73, and TAp63/73) is significantly correlated with late-stage haemic neoplasia in M. trossulus.
... Some of these mutations have been shown to result in dysfunctional p53 proteins thus contributing to the deregulation of cell proliferation and the progression of cancer [20]. Deduced amino acid sequences and proteins homologous to mammalian p53, p63 and p73 have been identified in clams and mussels [21][22][23][24][25]. It has been suggested that there is only one gene in mollusks from which p53-like and p63/p73-like mRNA isoforms are expressed, although several conflicting papers have been published [26][27][28]. ...
Article
Several bivalve species, including mussels (Mytilus spp.) and clams (Mya spp.), are susceptible to a leukemia-like disease called haemic neoplasia that has been known to decimate whole populations. Previous studies of molecular processes associated with late stages of this disease have implicated analogs of the p53 tumour suppressor protein family in disease etiology. We detected synonymous single nucleotide polymorphisms (SNPs) in the coding region sequence of p53-like cDNA from Mytilus trossulus (bay mussel) that differ between normal and neoplastic haemolymph. SNPs were located at positions 182, 392 and 821 bp. Most (94%) of the late leukemic animals sampled from cages in Burrard Inlet (British Columbia, Canada) had the same p53-like genotype, C182T G392G C821T, whereas 75% of the healthy animals were homozygous at positions C182C and T821T, independent of the genotype at the 392 bp position. As well, we detected an increased number of allelic variants in the leukemic animals that may arise from separate somatic mutation events in haemocyte precursors or from additional p53-like gene copies in polyploidy. Therefore, detection of these SNPs may provide a useful genetic biomarker for efficient monitoring of mussel population health.
... p53 superfamily coding sequences have been published for six mollusc species, two in the soft-shell clam Mya arenaria (Kelley et al. 2001), five each for the mussels Mytilus edulis and Mytilus trossulus, including the only known deltaN isoforms in invertebrates (Muttray et al. 2005;Muttray et al. 2007), two in the Hawaiian bobtail squid Euprymna scolopes (Goodson et al. 2006), four in the surf clam Spisula solidissima (Jessen-Eller et al. 2002;Cox et al. 2003), and one in the Pacific oyster Crassostrea gigas (Farcy et al. 2008). Interestingly, whereas within some species a number of different proteins have been identified, it appears that all isoforms may arise from alternate splicing as regions that overlap between different proteins from the same species are 100% identical as seen in Mytilus trossulus and Mytilus edulis (Muttray et al. 2007), Mya arenaria (Kelley et al. 2001), Euprymna scolopes (Goodson et al. 2006), and possibly S. solidissima (Jessen-Eller et al. 2002). ...
Article
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The origin of the p53 superfamily predates animal evolution and first appears in unicellular Flagellates. Invertebrate p53 superfamily members appear to have a p63-like domain structure, which seems to be evolutionarily ancient. The radiation into p53, p63, and p73 proteins is a vertebrate invention. In invertebrate models amenable to genetic analysis p53 superfamily members mainly act in apoptosis regulation in response to genotoxic agents and do not have overt developmental functions. We summarize the literature on cnidarian and mollusc p53 superfamily members and focus on the function and regulation of Drosophila melanogaster and Caenorhabditis elegans p53 superfamily members in triggering apoptosis. Furthermore, we examine the emerging evidence showing that invertebrate p53 superfamily proteins also have functions unrelated to apoptosis, such as DNA repair, cell cycle checkpoint responses, compensatory proliferation, aging, autophagy, and innate immunity.
... Homologues for human p53 and the p53 family have been cloned in several bivalves, e.g. Mya arenaria (Kelley et al. 2001), Spisula solidissima (Cox et al. 2003), Crassostrea rhizohorae (GenBank accession no. AY442309), Crassostrea gigas (GenBank AM236465), Mytilus edulis (Ciocan & Rotchel 2005, Muttray et al. 2005 and Mytilus trossulus (Muttray et al. 2005), and have shown highly conserved regions. ...
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High prevalence of disseminated neoplasia has been found in cockles Cerastoderma edule of Galicia (NW Spain). Disseminated neoplasia has been associated with high mortalities of various bivalve species. In vertebrates, proteins such as p53 and heat shock proteins (HSPs) play important roles in carcinogenesis. The protein p53 has been detected in neoplastic cells of bivalve molluscs such as Mytilus edulis, Mytilus trossulus, Mya arenaria, Spisula solidissima, Crassostrea rhizophorae and Crassostrea gigas. In this study, western blotting analyses were used to test the expression of Hsp70, Hsp90 and mutant p53 proteins in the cells and plasma of the haemolymph of cockles showing various intensities of neoplasia. Disseminated neoplasia was previously diagnosed by examination of stained haemolymph monolayers with light microscopy. In the present study, mutant p53 was detected in haemolymph cells of cockles diagnosed as affected by moderate and heavy neoplasia intensity, whereas it was not detected in cockles with either no or light neoplasia. The higher the neoplasia intensity, the higher the levels of Hsp70 and Hsp90. These proteins were not found in plasma. The results reveal the possible association between p53 and HSPs in neoplastic cells of cockles, which could prevent p53 from carrying out its functions, as occurs in human cancers.
... p53 family in development p53, p63 and p73 are conserved during evolution between species. For example, p63 gene has been identified at least in mouse, human, Xenopus, zebrafish, chick and a marine mollusc (Lu et al. 2001; Lee and Kimelman 2002; Bakkers et al. 2002; Yasue et al. 2001; Cox et al. 2003). Mice engineered to have p53 deletions have multiple types of tumors (increased risk of tumorigenesis) (Donehower et al. 1992). ...
... 1.3 and 1.4). These residues are also present in p53 sequences of the Pacific oyster Crassostrea gigas (Farcy et al., 2008) and the surf clam Spisula solidissima (Cox et al., 2003). In molluscs, the putative Mdm2 binding site is identical for both p53-and p63/73-like proteins. ...
Article
The human p53 tumour suppressor protein is inactivated in many cancers and is also a major player in apoptotic responses to cellular stress. The p53 protein and the two other members of this protein family (p63, p73) are encoded by distinct genes and their functions have been extensively documented for humans and some other vertebrates. The structure and relative expression levels for members of the p53 superfamily have also been reported for most major invertebrate taxa. The functions of homologous proteins have been investigated for only a few invertebrates (specifically, p53 in flies, nematodes and recently a sea anemone). These studies of classical model organisms all suggest that the gene family originally evolved to mediate apoptosis of damaged germ cells or to protect germ cells from genotoxic stress. Here, we have correlated data from a number of molluscan and other invertebrate sequencing projects to provide a framework for understanding p53 signalling pathways in marine bivalve cancer and stress biology. These data suggest that (a) the two identified p53 and p63/73-like proteins in soft shell clam (Mya arenaria), blue mussel (Mytilus edulis) and Northern European squid (Loligo forbesi) have identical core sequences and may be splice variants of a single gene, while some molluscs and most other invertebrates have two or more distinct genes expressing different p53 family members; (b) transcriptional activation domains (TADs) in bivalve p53 and p63/73-like protein sequences are 67-69% conserved with human p53, while those in ecdysozoan, cnidarian, placozoan and choanozoan eukaryotes are ≤33% conserved; (c) the Mdm2 binding site in the transcriptional activation domain is 100% conserved in all sequenced bivalve p53 proteins (e.g. Mya, Mytilus, Crassostrea and Spisula) but is not present in other non-deuterostome invertebrates; (d) an Mdm2 homologue has been cloned for Mytilus trossulus; (e) homologues for both human p53 upstream regulatory and transcriptional target genes exist in molluscan genomes (missing are ARF, CIP1 and BH3 only proteins) and (f) p53 is demonstrably involved in bivalve haemocyte and germinoma cancers. We usually do not know enough about the molecular biology of marine invertebrates to address molecular mechanisms that characterize particular diseases. Understanding the molecular basis of naturally occurring diseases in marine bivalves is a virtually unexplored aspect of toxicoproteomics and genomics and related drug discovery. Additionally, increases in coastal development and concomitant increases in aquatic pollutants have driven interest in developing models appropriate for evaluating potential hazardous compounds or conditions found in the aquatic environment. Data reviewed in this study are coupled with recent developments in our understanding the molecular biology of the marine bivalve p53 superfamily. Taken together, they suggest that both structurally and functionally, bivalve p53 family proteins are the most highly conserved members of this gene superfamily so far identified outside of higher vertebrates and invertebrate chordates. Marine bivalves provide some of the most relevant and best understood models currently available for experimental studies by biomedical and marine environmental researchers.
... 1.3 and 1.4). These residues are also present in p53 sequences of the Pacific oyster Crassostrea gigas (Farcy et al., 2008) and the surf clam Spisula solidissima (Cox et al., 2003). In molluscs, the putative Mdm2 binding site is identical for both p53-and p63/73-like proteins. ...
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The physiological mechanisms that regulate adaptive plasticity of clonal organisms are key to their success in changing environments. Here, we review the mechanisms that regulate morphological plasticity of colonial hydrozoans. There is a heritable, genetic basis to colony form, but environmentally-induced plasticity and self-reinforcing developmental physiology explain much of total phenotypic variance. Morphological development of colonial hydrozoans emerges from interactions among (1) behaviors which drive gastrovascular transport, (2) architecture of the gastrovascular system that determines hydrodynamic characteristics of vascular flow, and, (3) gene products that vary in response to physiological signals provided by gastrovascular transport. Several morphogenetic signaling mechanisms have been identified, including, reactive oxygen species and nutrient concentrations in the hydroplasm, and hydromechanical forces associated with gastrovascular transport. We present a conceptual model of the interacting forces that drive hydrozoan morphological development. Several avenues for future research are suggested by the synthesis of information from prior studies of hydrozoans. Elucidating the morphogenetic signaling pathways responsive to metabolites or hydromechanical forces and the epigenetic effect of vascular architecture on colony form may give new insight into the self-maintenance of indeterminately growing and continuously developing vascular systems.
... The mouse monoclonal antibody Mab-1E10, previously described and characterized (Reinisch et al., 1983), was raised from existing hybridomas and affinity purified by CedarLane Laboratories (Burlington, ON); this new batch of 1E10 antibody was successfully compared to the previously produced 1E10. The rabbit polyclonal p63/73 homodomain antibody was previously raised against the Spisula solidissima p63/73 homodimerization domain and characterized by Cox et al. (2003). Antibodies were purchased from the following sources: mouse monoclonal anti-human p53 (SC-99) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-actin (A2066) (Sigma, Oakville, ON), tetramethylrhodamine goat anti-mouse (T2762) and anti-rabbit (T2769) secondary antibodies (Invitrogen, Burlington, ON), and goat anti-mouse (81-6522) and anti-rabbit (62-6122) secondary antibodies conjugated to alkaline phosphatase (Zymed Laboratories, San Francisco, CA). ...
Article
Intensive farming of potatoes in Prince Edward Island (PEI) relies on the repeated and widespread application of fertilizers and pesticides. In PEI the main potato farming areas are in close proximity and drain directly to estuaries. Runoff from high agricultural activity watersheds could impact benthic organism health in the depositional zone of downstream estuaries. The estuarine filter feeder Mya arenaria (soft-shell clam) could be particularly vulnerable to both particle-adsorbed and water soluble contaminants. M. arenaria is susceptible to haemocytic leukemia. In May 2009, we established that heavily proliferated leukemia (HPL) prevalence was generally higher in PEI estuaries located downstream of high intensity potato farming (Dunk and Wilmot estuaries) watersheds than in estuaries downstream of lower intensity areas. Using Mab-1E10 based immunocytochemistry we observed that leukemic haemocytes from the Dunk and Wilmot estuaries were 1E10 negative whereas those from the Ox/Sheep estuary (low potato farming intensity) were 1E10 positive. The expression of genes in the p53 tumour suppressor pathway enabled us to differentiate groups of leukemic and normal M. arenaria, validating our diagnoses. In October 2009, we confirmed that HPL prevalence was elevated in the Dunk and Wilmot estuaries compared to reference (Souris River). Moreover, leukemia prevalence declined with distance from the river mouths along transects through the Dunk and Wilmot estuaries. The pesticides ß-endosulfan and α-endosulfan were detected in surface sediments from the Dunk and Wilmot estuaries, but not in sediments from either the Souris River or several other lower intensity potato farming watersheds. Our study provides evidence of an association between intensity of potato farming and prevalence of clam leukemia at downstream estuaries in PEI.
... Alteration of P53 was also found in soft-shell clams in advanced stages of the disease (Barker et al., 1997). The use of a set of monoclonal and polyclonal antibodies raised against clam (Spicula solidissima, M. arenaria) P53 protein allowed to distinguish neoplastic cells from normal cells in M. arenaria (Cox et al., 2003;Kelley et al., 2001;Stephens et al., 2001). Up-regulation of P53 protein expression has been reported in M. edulis affected by DN . ...
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Disseminated neoplasia (DN) is a pathological condition reported for several species of marine bivalves throughout the world, but its aetiology has not yet been satisfactorily explained. It has been suggested that chemical contamination could be a factor contributing to neoplasia. The aim of the present study was to compare cell and tissue biomarkers and the transcription level of cancer-related genes in cockles (Cerastoderma edule) affected by DN with those of healthy cockles in relation to chemical contaminant burdens. For this, cockles were collected from a natural bed in Cambados (Ria de Arousa, Galicia) in May 2009. The prevalence of DN was 12.36% and 3 degrees of DN severity were distinguished. No significant differences in metal accumulation, non-specific inflammatory responses and parasites were observed between healthy and DN-affected cockles. Lysosomal membrane stability was significantly reduced in cockles affected by DN, which indicates a poorer health condition. Very low frequencies of micronuclei were recorded and no significant differences were detected between DN severity groups. Haemolymph analyses showed a higher frequency of mitotic figures and binucleated cells in cockles affected by moderate and heavy DN than in healthy ones. Neoplastic animals showed significantly higher transcription levels of p53 and ras than healthy cockles and mutational alterations in ras gene sequence were detected. Low concentrations of metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and phthalate esters were measured in cockles from Cambados. In conclusion, cockles affected by DN suffer a general stress situation and have altered patterns of cancer-related gene transcription. Further studies are in progress to elucidate mechanisms of carcinogenesis in this species.
... Furthermore, according to our previous and present studies, p63 is the only gene of the p53 family present in molluscs and vertebrate p63 nomenclature is fully applicable to all known molluscan p63 expression isoforms (Štifanić et al., 2009). Complete DNA coding sequences of different p63 isoforms (Kelley et al., 2001;Jessen-Eller et al., 2002;Cox et al., 2003;Muttray et al., 2005;Goodson et al., 2006;Muttray et al., 2007;Farcy et al., 2008;Estévez-Calvar et al., 2013) and only one complete genomic sequence (Mya arenaria p63/p73 and p53 gene; GenBank accession no. FJ041332) are at present publicly available in GenBank. ...
... The tumor-suppressor protein p53 regulates genes involved in progression through the cell cycle, DNA repair, senescence or apoptosis in response to cell stress; thus dysregulation or dysfunction of p53 can result in uncontrolled cellular proliferation ; inactivation of p53 occurs in almost all cancers (Oren, 2003 ). A p53-like gene has been characterized in a number of mollusk species, including M. arenaria (Barker et al., 1997; Kelley et al., 2001), Spisula solidissima (Cox et al., 2003), M. edulis (Ciocan and Rotchell, 2005; Muttray et al., 2007), M. trossulus (Ciocan et al., 2006; Muttray et al., 2007), M. galloprovincialis (Stifanic et al., 2009), and C. edule (Ruiz et al., 2013b ). The structure and phylogeny of p53 family in mollusks was reviewed by Walker et al. (2011); various isoforms have been detected, all seem to derive from the same gene through different splicing or other transformations . ...
... The tumor-suppressor protein p53 regulates genes involved in progression through the cell cycle, DNA repair, senescence or apoptosis in response to cell stress; thus dysregulation or dysfunction of p53 can result in uncontrolled cellular proliferation; inactivation of p53 occurs in almost all cancers (Oren, 2003). A p53-like gene has been characterized in a number of mollusk species, including M. arenaria (Barker et al., 1997;Kelley et al., 2001), Spisula solidissima (Cox et al., 2003), M. edulis (Ciocan and Rotchell, 2005;Muttray et al., 2007), M. trossulus (Ciocan et al., 2006;Muttray et al., 2007), M. galloprovincialis (Stifanic et al., 2009), and C. edule (Ruiz et al., 2013b). The structure and phylogeny of p53 family in mollusks was reviewed by Walker et al. (2011); various isoforms have been detected, all seem to derive from the same gene through different splicing or other transformations. ...
Article
Two types of prevalent neoplastic diseases have been described in marine bivalves of commercial interest: disseminated neoplasia (DN) and gonadal neoplasia. The first involves the excessive proliferation of abnormal cells with unknown origin (probably of hemic source in some cases/species), disseminating through the circulatory system and infiltrating the connective tissue of various organs; the second consists of an abnormal proliferation of undifferentiated germinal cells of the gonad. These two types of bivalve neoplasia fit the criteria of malignant tumors: pleomorphic and undifferentiated cells, rapid and invasive growth, abundance of mitotic figures, metastasis and progressive development often resulting in the death of the affected individual. Different causes have been suggested regarding etiology: genetic alterations, virus, retrotranspons, and contaminants, although it could depend on the mollusk species; evidence of horizontal transmission of clonal cancer cells as the cause of DN spreading in clam Mya arenaria populations has been recently reported. In some species and populations, the neoplastic disorders affect only a few individuals, but in others reach high prevalence. Among the diagnostic methods, DN has been detected by histology and cytologic examination of hemolymph, and with developed specific antibodies. Recently, flow cytometry has also been applied, allowing detecting DNA quantity alteration. Several studies reported many genes and pathways critically involved in neoplastic transformation in Mya arenaria, Mytilus spp. and Ostrea edulis. These genetic studies will allow the development of diagnosis by PCR which can be used in biomonitoring studies. Copyright © 2015. Published by Elsevier Inc.
... Las mutaciones del gen p53 son las más estudiadas debido a su función reguladora del ciclo celular y de la apoptosis y a su alta frecuencia de mutación en cánceres humanos. Se ha secuenciado el gen p53 en Mytilus edulis (MUTTRAY et al. 2005; CIOCAN y ROTCHEL 2005), Mytilus trossolus (MUTTRAY et al. 2005), Spisula solidissima (COX et al. 2003), Crassostrea rhizophorae (Genbank Accession Number AY442309) y Crassostrea gigas (Genbank Accession Number AM236465). En células neoplásicas de Mya arenaria se ha detectado una transversión de citosina a guanina en el final del exon 6 (BARKER et al. 1997). ...
... arenaria, GenBank Accession Nos. AF253323 and AF253324) (23), three for surf clam (Spisula solidissima, AF285104, AY289767, and AY289768) (24,25), one for squid (Loligo forbesi, U43595), and one for oyster (Crassostrea rhizophorae, AY442309). ...
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Mussels are susceptible to a wide range of environmental toxicants, including carcinogens, and thus are often employed as bioindicator species. To elucidate the molecular aetiology of such neoplastic damage, we have cloned Mytilus edulis homologues of the vertebrate ras proto-oncogene, and p53 tumor suppressor gene. The M. edulis ras cDNA encodes a predicted protein of 184 amino acids. The DNA sequence analysis with vertebrate ras sequences demonstrates that the M. edulis ras cDNA is highly conserved in regions of functional importance, including mutational hot spots. The partial p53 sequence also demonstrates that M. edulis p53 is highly conserved in two regions of functional importance and that these regions also include four of the five mutational hot spots for this gene. In contrast, the M. edulis p53 sequence shows little similarity to the other published invertebrate p53-like sequences. The cancer gene sequences characterized herein will allow development of specific biomarkers of genotoxic damage.
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Disseminated neoplasia (DN), an oyster disease resembling leukaemia, has been reported in a number of species of marine bivalve molluscs. The disease is characterised by a proliferation of abnormal circulating cells of unknown origin resulting in the invasion of tissues and organs, frequently with a fatal end of the affected individuals. To obtain a more comprehensive view of bivalve cancer processes, suppressive subtracted hybridisation (SSH) and quantitative RT-PCR (q-PCR) approaches were combined to investigate changes in the transcriptome of Ostrea edulis haemolymph cells associated to DN. Two SSH libraries were constructed and 587 expressed sequence tags (ESTs) were sequenced, obtaining 329 ESTs which showed expression changes in neoplastic process. Transcription expression analyses (q-PCR) were done for a total of 24 genes that could be relevant in neoplastic process, including genes with role in the regulation of cell cycle, apoptosis or chromosomal defects. Most of those genes had not been reported in association with cancer in non-vertebrate organisms. The over-expression and under-expression of some of those genes in DN-affected oysters was in agreement with observations in vertebrate cancer. The results herein reported contribute to cancer understanding in bivalve molluscs.
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Author Posting. © National Research Council Canada, 2005. This article is posted here by permission of National Research Council Canada for personal use, not for redistribution. The definitive version was published in Canadian Journal of Fisheries and Aquatic Sciences 62 (2005): 2055-2066, doi:10.1139/F05-119. Evaluating patterns of expression of p53-related proteins in cells is a novel approach in defining environmentally linked diseases. We have examined the induction of haemocytic leukemia in Mytilus edulis by municipal and industrial contaminants in Pictou Harbour, Nova Scotia, Canada. We used a murine monoclonal antibody, 1E10, as a diagnostic reagent to detect leukemic cells. We first characterized the reactivity of 1E10 with both normal and leukemic Mytilus haemocytes by confocal microscopy. We then compared p53 gene family expression (p53, p63–p73, and p97) in normal versus leukemic haemocytes using a panel of monoclonal and polyclonal antibodies to p53 family proteins. The immunochemical data demonstrate that haemocytic leukemia cells of M. edulis differentially express p63–p73 and p97–p120 proteins. We subsequently used 1E10 to diagnose haemocytic leukemia in 500 M. edulis previously deployed 6 months earlier in Pictou Harbour. In the field, Mytilus caged near untreated municipal wastewater and bleached kraft pulpmill effluents have a significantly greater chance of developing haemocytic leukemia than do mussels exposed to reference sites. This research was funded in part by the Pictou Harbour Biomonitoring Project, Fisheries and Oceans Canada, and Environment Canada.
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The purpose of this chapter is to review the neoplasms (or tumors) affecting different species of clams. We include as "clams" any bivalve mollusc other than oysters, mussels and scallops. Two types of neoplasm have been described in clams: disseminated neoplasms and germinoma (one histotype of gonadal neoplasms). Disseminated neoplasm, the type prevalent in bivalve molluscs, including clams, involves the extensive proliferation of circulating abnormal cells (neoplastic cells) of unknown origin, through the tissues of the organisms. Germinomas comprise the abnormal proliferation of altered immature germ cells (neoplastic cells). Some clam species and populations present epidemic levels of neoplasms (Mya arenaria, Mercenaria spp., Cerastoderma edule and Venerupis aurea), while others are less affected (Venerupis decussata (=Ruditapes decussatus), Ensis magnus (=E. arcuatus) and Ensis siliqua). The etiology of neoplasms is unknown, some studies suggesting the implication of an infectious agent and other factors in the case of disseminated neoplasms. By contrast, only stress factors seem to be involved in gonadal neoplasms. In recent years, many studies have been carried out focusing on the molecular basis of this disease.
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Within hours of hatching, the squid Euprymna scolopes forms a specific light organ symbiosis with the marine luminous bacterium Vibrio fischeri. Interactions with the symbiont result in the loss of a complex ciliated epithelium dedicated to promoting colonization of host tissue, and some or all of this loss is due to widespread, symbiont-induced apoptosis. Members of the p53 family, including p53, p63, and p73, are conserved across broad phyletic lines and p63 is thought to be the ancestral gene. These proteins have been shown to induce apoptosis and developmental morphogenesis. In this study, we characterized p63-like transcripts from mRNA isolated from the symbiotic tissues of E. scolopes and described their role in symbiont-induced morphogenesis. Using degenerate RT-PCR and RACE PCR, we identified two p63-like transcripts encoding proteins of 431 and 567 amino acids. These transcripts shared identical nucleotides where they overlapped, suggesting that they are splice variants of the same gene. Immunocytochemistry and Western blots using an antibody specific for E. scolopes suggested that the p53 family members are activated in cells of the symbiont-harvesting structures of the symbiotic light organ. We propose that once the symbiosis is initiated, a symbiont-induced signal activates p53 family members, inducing apoptosis and developmental morphogenesis of the light organ.
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The p53 family of transcription factors has been implicated in many vertebrate cancers. Altered p53 and p73 protein expression observed in leukemic cells of molluscs suggests that these transcription factors might be involved in invertebrate cancers as well. Here, we fully characterize the mRNA of four novel p53-like variants in the bivalve molluscs Mytilus trossulus (bay mussel) and Mytilus edulis (blue mussel). These species, widely used for environmental assessment, develop a hemic neoplasia (leukemia) that is frequently fatal. The correlation between expression of p53 and its close relative p73 and onset of molluscan leukemia was documented previously. We report the sequences of two distinct and novel p63/p73-like mRNAs, amplified by polymerase chain reaction (PCR) from both species. One of the p63/p73-like isoforms contains a 360 nt truncation in the 5' coding region. Based on this truncation and concomitant lack of a transactivation (TA) domain, we designate this variant as a DeltaNp63/p73-like isoform: the first to be reported in an invertebrate species. In mammalian species, DeltaNp73 potently inhibits the tumor-suppressive function of p73 and p53, and its overexpression serves as a robust marker for mammalian cancer. In addition, we report on the occurrence of alternate polyadenylation sites in the molluscan p63/p73: one proximal and one distal site, which differ by 1260 nt. We hypothesize that differential expression of various molluscan p63/p73-like isoforms, controlled in part by polyadenylation site choice variation, may help to interpret the apparently opposing roles of this gene in the development of cancer. Overall, this research further illustrates the utility of the molluscan model for studies involving the molecular mechanisms of oncogenesis in naturally occurring populations. The data presented here require a revisiting of hypotheses regarding evolution of the p53 gene family. Current hypotheses indicate that (1) the protostome gene family does not contain an intronic promoter for DeltaN expression and (2) p53 gene duplication did not occur in protostomes. Our characterization of DeltaN p63/73 in mussel suggests that molluscan p53 gene family members have acquired an intronic promoter or splicing mechanism, either by invention that predates the evolutionary split of deuterostomes from protostomes, or by parallel evolution. Our data also show that Mytilus p53, p63/p73, and DeltaNp63/p73 are identical in their core regions with variation limited to their C- and N-terminals, supporting the notion that alternative splicing, intronic promoter usage, and polyadenylation site choice may lead to expression of distinct isoforms originating from one common gene.
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The tumor suppressor p53 is mutated in approximately 50% of all human cancer cases worldwide. It is commonly assumed that the phylogenetic history of this important tumor suppressor has been thoroughly studied; however, few detailed studies of the entire extended p53 protein family have been reported, and none comprehensively and simultaneously consider functional, molecular, and phylogenetic data. Herein we examine a diverse collection of reported p53-like protein sequences, including representatives from the arthropods, nematodes, and protists, with the goal of answering several important questions. First, what evidence supports these highly divergent proteins being true homologues to the p53 family? Second, is the inferred overall family phylogeny concordant with known structures and functions? Third, does the extended p53 family possess recognizable conserved sites outside of the within-chordate, highly-conserved DNA-binding domain? Our study shows that the biochemical and functional evidence of p53 homology for nematodes, arthropods, and protists is inconsistent with their implied phylogenetic relationship within the overall family. Although these divergent sequences are always reported as functionally similar to human p53, our results confirm and extend the hypothesis that p63 is a far more appropriate protein for comparison. Within these divergent sequences, we find minimal conservation within the DNA-binding domain, and no conservation elsewhere. Taken together, our findings suggest that these sequences are not bona fide homologues of the extended p53 family and provide baseline criteria for the future identification and characterization of distant p53-family homologues.
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Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.
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The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.
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The cytoplasmic life of an mRNA revolves around the regulation of its localization, translation and stability. Interactions between the two ends of the mRNA may integrate translation and mRNA turnover. Regulatory elements in the region between the termination codon and poly(A) tail - the 3' untranslated region - have been identified in a wide variety of systems, as have been some of the key players with which these elements interact.
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We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.
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We describe the cloning of p63, a gene at chromosome 3q27-29 that bears strong homology to the tumor suppressor p53 and to the related gene, p73. p63 was detected in a variety of human and mouse tissues, including proliferating basal cells of epithelial layers in the epidermis, cervix, urothelium, and prostate. Unlike p53, the p63 gene encodes multiple isotypes with remarkably divergent abilities to transactivate p53 reporter genes and induce apoptosis. Importantly, the predominant p63 isotypes in many epithelial tissues lack an acidic N terminus corresponding to the transactivation domain of p53. We demonstrate that these truncated p63 variants can act as dominant-negative agents toward transactivation by p53 and p63, and we suggest the possibility of physiological interactions among members of the p53 family.
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p53 plays an essential pro-apoptotic role, a function thought to be shared with its family members p73 and p63. Here, we show that p73 is primarily present in developing neurons as a truncated isoform whose levels are dramatically decreased when sympathetic neurons apoptose after nerve growth factor (NGF) withdrawal. Increased expression of truncated p73 rescues these neurons from apoptosis induced by NGF withdrawal or p53 overexpression. In p73–/– mice, all isoforms of p73 are deleted and the apoptosis of developing sympathetic neurons is greatly enhanced. Thus, truncated p73 is an essential anti-apoptotic protein in neurons, serving to counteract the pro-apoptotic function of p53.
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Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that accumulate to toxic levels in the food chain. Using Spisula solidissima (surf clam) embryos as a developmental model, it was shown that Aroclor 1254 specifically targets two neuronal structures during embryonic development. Embryos were exposed to 1, 10), or 100 ppm Aroclor 1254 or an acetone vehicle control posthatching for 24, 48, and 72 h. Embryos labeled with a serotonin antibody or a neural antigen antibody and a rhodamine-conjugated secondary antibody were viewed by confocal microscopy. The cerebropleural ganglion showed a decrease both in serotonin production and in the size of the serotonin-synthesizing region upon exposure to 10 and 100 ppm Aroclor 1254. These decreases were detectable as early as 48 h postfertilization. When exposed to 100 ppm Aroclor 1254, the primitive neural plexus, which coordinates the movements of the mouth and velum, showed a delay in onset and cessation of expression of a molluscan-specific neural antigen. Exposure to Aroclor 1254 did not affect the overall growth and morphology of the embryos. In addition, analyses of total protein profiles and heat-shock protein 70 levels showed that exposure to Aroclor 1254 did not trigger protein degradation or cause a stress or shock response. These results show that exposure of Spisula embryos to Aroclor 1254 specifically targets neurogenesis while having no effect on the overall growth of the embryo.
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The discoveries of the p53 homologs, p63 and p73, have both fueled new insights and exposed enigmas in our understanding of the iconic p53 tumor suppressor. Although the pivotal role of p53 in cancer pathways remains unchallenged, because p63 and p73 are now implicated in stem cell identity, neurogenesis, natural immunity and homeostatic control. Despite their seemingly separate tasks, there are hints that the p53 family members both collaborate and interfere with one another. The question remains, therefore, as to whether these genes evolved to function independently or whether their familial ties still bind them in pathways of cell proliferation, death and tumorigenesis.
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p53 controls crucial stress responses that play a major role in preventing malignant transformation. Hence, inactivation of p53 is the single most common genetic defect in human cancer. With the recent discovery of two close structural homologs, p63 en p73, we are getting a broader view of a fascinating gene family that links developmental biology with tumor biology. While unique roles are apparent for each of these genes, intimate biochemical cross-talk among family members suggests a functional network that might influence many different aspects of individual gene action. The most interesting part of this family network derives from the fact that the p63 and p73 genes are based on the "two-genes-in-one" idea, encoding both agonist and antagonist in the same open reading frame. In this review, we attempt to present an overview of the current status of this fast moving field.
An anti-apoptotic role for the p53 family member p73 Towbin, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications
  • C D Pozniack
  • S Radinovic
  • A Yang
  • F Mckeon
  • D R Kaplan
  • F D H Miller
  • T Staehelin
  • J Gordon
Pozniack, C.D., Radinovic, S., Yang, A., McKeon, F., Kaplan, D.R., Miller, F.D., 2000. An anti-apoptotic role for the p53 family member p73 Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350 – 4354.
Spisula p63/p73 alpha variant; Ssp63/73h, Spisula 63/73 beta variant; SUMO, small ubiquitin-related modifier; TA, transactivation domain; TI, transactivation-inhibitory domain; UTR, untranslated region. * Corresponding author
  • Ssp
Ssp63/73a, Spisula p63/p73 alpha variant; Ssp63/73h, Spisula 63/73 beta variant; SUMO, small ubiquitin-related modifier; TA, transactivation domain; TI, transactivation-inhibitory domain; UTR, untranslated region. * Corresponding author. Tel.: +1-508-289-7517; fax: +1-508-540-6902.
On the shoulders of giants: p63, p73 and the rise of p53
  • Yang
p63 and p73 are required for p53-dependent apoptosis in response to DNA damage
  • Flores
Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Salinovich
Sequence determinants in human polyadenylation site selection
  • Legendre
An anti-apoptotic role for the p53 family member p73 during developmental neuronal cell death
  • Pozniack
p63 is essential for regenerative proliferation in limb, cranio-facial and epithelial development
  • Yang
p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage
  • Yuan