Search

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., (2011 Elsevier Ltd. All rights reserved.)

Numerous reports have raised the level of national concern that chemicals found in the environment may have adverse effects on the health of humans and wildlife. Environmental exposure to pollutants, such as dioxin, has been implicated in gonadal tumor formation in Maine softshell clams (Mya arenaria). Prevalence of these tumors is as high as 40% in some populations. Although their etiology is still unknown, investigations into the mechanisms of tumor formation have revolved around a hypothesis of dioxin-induced toxicity. The aryl hydrocarbon receptor (AHR) was initially investigated, but was later determined to not bind the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suggesting that dioxin toxicity is mediated through an AHR-independent pathway. An alternative mechanism of tumor formation has been investigated, involving a protein with significant sequence similarity to mammalian E6AP, a HECT (homologous to E6AP carboxy terminus) E3 ubiquitin-protein ligase. E6AP, in association with the high-risk human papillomavirus (HPV) E6 protein, is involved in the abnormal degradation of the p53 tumor suppressor protein in human cervical cancer. Tumorigenic clam reproductive tissue revealed higher M. arenaria E3 (MaE3) protein levels concomitant with lower M. arenaria p53 (Map53) levels. While the function of MaE3 as a HECT E3 was verified, results from three methods agree that MaE3 does not associate with Map53. However, alteration in Map53 levels may still play a role in clam gonadal tumorigenesis. Due to upregulation of MaE3 in neoplastic reproductive tissue, further investigations will focus on determining the proteolytic targets of MaE3. In conjunction with our previous findings that dioxin toxicity in the softshell clam is not mediated by AHR, the results from our current investigation suggest a complex etiology for the clam germinomas.

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.

Homologues for human p53 (Hsp53) and p73 (Hsp73) genes were cloned and expression patterns for their corresponding proteins analysed in tissues from normal and leukemic softshell clams (Mya arenaria). These are the first structural and functional data for p53 and p73 cDNAs and gene products in a naturally occurring, non-mammalian disease model. Core sequence of the predicted clam p53 (Map53) and p73 (Map73) proteins is virtually identical and includes the following highly conserved regions: the transcriptional activation domain (TAD), MDM2 binding site, ATM phosphorylation site, proline rich domain, DNA binding domains (DBDs) II-V, nuclear import and export signals and the tetramerization domain. The core sequence is a structural mosaic of the corresponding human proteins, with the TAD and DBDs resembling Hsp53 and Hsp73, respectively. This suggests that Map53 and Map73 proteins may function similarly to human proteins. Clam proteins have either a short (Map53) or long (Map73) C-terminal extension. These features suggest that Map53 and Map73 may be alternate splice variants of a p63/p73-like ancestral gene. Map73 is significantly upregulated in hemocytes and adductor muscle from leukemic clams. In leukemic hemocytes, both proteins are absent from the nucleus and sequestered in the cytoplasm. This observation suggests that a non-mutational p53/p73-dependent mechanism may be involved in the clam disease. Further studies of these gene products in clams may reveal p53/p73-related molecular mechanisms that are held in common with Burkitt's lymphoma or other human cancers.

Softshell clams (Mya arenaria) were exposed to dioxin in controlled laboratory experiments in order to study their molecular response to dioxin exposure. A complementary DNA (cDNA) fragment with sequence similarity to E3 ubiquitin-protein ligase appeared to be upregulated in dioxin-exposed clams compared to controls. E3 covalently ligates ubiquitin onto a protein, targeting it for degradation. Our findings suggest that the ubiquitin-mediated proteolytic pathway in the softshell clam may be activated by dioxin exposure. Because the clam E3-predicted amino acid sequence is most similar to a specific vertebrate E3 protein (E6-AP), we hypothesize that dioxin may stimulate ubiquitin-mediated degradation of cell-cycle regulatory proteins, such as the tumor suppressor p53, which promotes cell proliferation. This pathway has been observed in human cervical cancer. Partial cDNA sequence of the clam E3 has been identified using the differential display polymerase chain reaction (ddPCR) and RACE (Rapid Amplification of cDNA Ends) PCR; the full-length sequence is currently being determined. Discovering the molecular mechanism(s) stimulated by dioxin exposure in this invertebrate model may contribute to a better understanding of the effects of dioxin on marine organisms.

A full-length clone with sequence similarity to genes in the cytochrome P450 superfamily was isolated from a cDNA library prepared from female Mercenaria mercenaria gonadal tissue. This clone was isolated while screening an expression library with an antibody prepared against a peptide sequence within the ligand-binding region of the murine Ah receptor. Comparison of the predicted amino acid sequence of this clone to those of other cytochrome P450 genes indicated that the closest overall sequence similarity (38%) was to proteins predicted from genes in the CYP3 family. Northern blots indicated the presence of a transcript of the appropriate size (3.0 kb) with homology to the clam cytochrome P450. In vitro translation of the cDNA clone produced a 50.7-kDa protein as determined by SDS-polyacrylamide gel electrophoresis. The in vitro translated protein was not recognized on Western blots by two polyclonal antibodies specific for members of the CYP3 family. Since the degree of similarity to existing cytochrome P450 families was below the 40% level required for membership, and the expressed protein was not recognized by CYP3-specific antibodies, this clam cytochrome P450 cDNA has been placed in a new family, cytochrome P450 30 (CYP30).

Ribosomal proteins contribute to the regulation and activity of ribosomes, and hence, the translational activity of the cell. Aberrant expression of ribosomal proteins has been linked to certain pathological conditions such as neoplasms. We have isolated and characterized a cDNA for the ribosomal protein (rp) S19 from a marine bivalve, the soft-shell clam (Mya arenaria), and we have examined its pattern of mRNA expression in the ovary and testis. The S19 cDNA contains a 450 nucleotide (nt) open reading frame (ORF), flanked by 89 nt and 26 nt of 5' and 3' untranslated regions, respectively. Probes synthesized from the S19 cDNA recognize a single transcript of approximately 550 nt in four different tissues. The predicted amino acid sequence from the ORF exhibits 58% identity with human and rat S19. Southern analysis of genomic DNA suggests that M. arenaria may have multiple copies of S19, a feature that is more similar to vertebrate than invertebrate rp genes. Expression of S19 mRNA in both ovary and testis was elevated throughout gametogenesis until after spawning, when a decrease in S19 message was observed. A comparison of S19 mRNA levels in post-spawn animals revealed a trend of elevated expression in ovaries and testes affected by a gonadal neoplasm, indicating that S19 may be a useful molecular marker for the pathological condition.

Expression of a clam p53 homologue was examined in tissues of the soft-shell clam, Mya arenaria, from Beal's Island, Maine. Southern analysis reveals that p53, in this population, is a single copy gene. A 1.7 to 1.9-kb p53 mRNA was detected at very low levels in normal adult gonadal tissue. This transcript is similar in size to that of vertebrate p53 genes. RNAs were harvested from several tissues, including individual clam gonads during gametogenesis. These were hybridized in ribonuclease (RNase) protection assays to a p53 antisense probe designed from the clam p53 cDNA sequence. RNase protection profiles indicate that p53 mRNA is expressed in adductor muscle, gill, and gonads of both sexes. Although p53 mRNA is expressed throughout gametogenesis in mature male and female gonads, ovaries have significantly higher levels of expression. The significance of our findings to the study of normal clam gametogenesis and to etiology of gonadal tumors is discussed.

Sequences of the internal transcribed spacer (ITS-1) ribosomal DNA region were compared among 88 soft-shell clams (Mya arenaria) from 12 sites (within three general areas) along the New England coast to determine whether populations were genetically heterogeneous. Two sequence variants were observed, with type 1 having a 3-nucleotide insertion and one point mutation relative to type 2. Allele-specific polymerase chain reaction (PCR); using primers specific to each sequence type, was performed to determine the distribution of individuals who had both allelic forms. DNA from soft-shell clams collected from three areas (Cobscook Bay, Maine; Gulf of Maine; and southern New England) were compared chi 2 analyses of allele-specific PCR results revealed no significant heterogeneity among the three population distributions.

Earlier studies have reported an unusually high incidence of gonadal tumors in marine bivalves in areas of potentially high exposure to herbicides including 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophe-noxyacetic acid. Some herbicides can be contaminated by halogenated aromatic compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Most of the effects of planar halogenated aromatic compounds, including carcinogenic effects in vertebrates, appear to be mediated through binding to the aryl hydrocarbon receptor. The current study investigated whether halogenated aromatic hydrocarbon-binding proteins are present in the marine bivalve, Mercenaria mercenaria. We used the TCDD photoaffinity analog 2-azido-3-[125I]-iodo-7, 8-dibromodibenzo-p-dioxin to detect the presence of two proteins (28 and 39 kDa) in cytosols prepared from M. mercenaria that specifically bound this ligand. Expression of these proteins is tissue-dependent with the highest concentrations being observed in gill and gonadal tissue. Gonadal tissue also exhibited gender-specific expression with female clams exhibiting higher levels of the 39-kDa protein. Gender-and tissue-specific expression are consistent with the hypothesis that these proteins might be involved in the carcinogenic response observed in clams exposed to herbicides.

An increase in both the numbers and types of tumors found in finfish and shellfish has been noted in the past several decades. In many cases, while the increase in tumor incidence can be correlated with increases in aquatic toxicant levels, causality cannot be definitively proven. One recent epidemiologic investigation identified the prevalence of gonadal cancers as high as 40% in softshell clams (Mya arenaria) in Maine and 60% in hardshell clams (Mercenaria spp.) from Florida. A second study of these same geographic areas identified human mortality rates due to ovarian cancer as significantly greater than the national average. The rise in mortality rates in humans correlated with the increased use of herbicides in these areas as well as with the appearance of significant numbers of gonadal tumors in the clams. Studies were initiated in our laboratory to examine the molecular basis of these neoplasms in bivalves. NIH3T3 transfection assays were used to examine DNA isolated from these molluscan tumors for the presence of activated oncogenes. DNAs isolated from advanced tumors in both species were able to transform NIH3T3 cells and induce tumors in athymic mice. Studies are now underway to identify the gene(s) detected by these assays and also to examine the molecular mechanisms of toxic response of herbicide-exposed clams.

Studies were initiated on oncogene activation in two bivalve species with high frequencies of histologically identifiable gonadal neoplasms. Pathological assessments identified epizootic seminomas and dysgerminomas in softshell clams (Mya arenaria) from three Maine estuarine sites contaminated by herbicides and in hardshell clams (Mercenaria) from the Indian River in Florida, an area of potential citrus agrochemical exposure. NIH3T3 transfection assays were used to examine DNA isolated from these molluscan tumors for the presence of activated oncogenes. DNAs isolated from advanced tumors in both species were able to transform NIH3T3 cells in a standard focus assay. These same cells were also able to form colonies in low concentrations of serum and induce tumors in athymic mice. Cells expanded from isolated foci demonstrated anchorage-independent growth in soft agar. The results of these studies indicate that DNA from the clam tumors is able to transform mouse fibroblasts, which suggests that a transforming gene is present in these tumor cells. Studies are under way to identify the gene(s) detected by these assays.