Identification of DeltaN isoform and polyadenylation site choice variants in molluscan p63/p73 -like homologues

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Marine Biotechnology 9 (2007): 217-230, doi:10.1007/s10126-006-6045-1.The p53 family of transcription factors has been implicated in many vertebrate cancers. Altered p53 and p73 protein expression observed in leukemic cells of mollusks 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 mollusks Mytilus trossulus (bay mussel) and Mytilus edulis (blue mussel). These species, widely used for environmental assessment, develop a haemic neoplasia (leukemia) that is frequently fatal. The correlation between expression of p53 and its close relative p73 and onset of molluskan 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 trans-activation (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 over-expression serves as a robust marker for mammalian cancer. In addition, we report on the occurrence of alternate polyadenylation sites in the molluskan p63/p73: one proximal and one distal site, which differ by 1260 nt. We hypothesize that differential expression of various molluskan 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 molluskan 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 molluskan p53 gene family members have acquired an intronic promoter or splicing mechanism, either by invention that predates the evolutionary split of deuterostoms 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. This supports the notion that alternative splicing, intronic promoter usage and polyadenylation site choice may lead to expression of distinct isoforms originating from one common gene.A.F.M. was supported by the Greater Vancouver Regional District, BC, Canada and a Collaborative Research and Development Grant from the Natural Science and Engineering Research Council of Canada (#CRDP J 323120 –05). R.L.C. was supported by Environmental Protection Agency grant # R82935901 and National Institute of Health grant #1R21ES012273-01

A new invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls.

The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike clam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R(2) = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs

Identification and phylogenetic comparison of p53 in two distinct mussel species (Mytilus)

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B. V. for personal use, not for redistribution. The definitive version was published in Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 140 (2005): 237-250, doi:10.1016/j.cca.2005.02.011.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.AFM was supported by the Greater Vancouver Regional District, BC, Canada, and RLC was supported by STAR grant R82935901 from the Environmental Protection Agency (USA)

Diminished macrophage apoptosis and reactive oxygen species generation after phorbol ester stimulation in Crohn's disease.

BACKGROUND: Crohn's Disease (CD) is a chronic relapsing disorder characterized by granulomatous inflammation of the gastrointestinal tract. Although its pathogenesis is complex, we have recently shown that CD patients have a systemic defect in macrophage function, which results in the defective clearance of bacteria from inflammatory sites. METHODOLOGY/PRINCIPAL FINDINGS: Here we have identified a number of additional macrophage defects in CD following diacylglycerol (DAG) homolog phorbol-12-myristate-13-acetate (PMA) activation. We provide evidence for decreased DNA fragmentation, reduced mitochondrial membrane depolarization, impaired reactive oxygen species production, diminished cytochrome c release and increased IL-6 production compared to healthy subjects after PMA exposure. The observed macrophage defects in CD were stimulus-specific, as normal responses were observed following p53 activation and endoplasmic reticulum stress. CONCLUSION: These findings add to a growing body of evidence highlighting disordered macrophage function in CD and, given their pivotal role in orchestrating inflammatory responses, defective apoptosis could potentially contribute to the pathogenesis of CD

Suppressor cell regulation of immune response to tumors: Abrogation by adult thymectomy

The regulatory role of the adult thymus on the appearance of cytotoxic and suppressor T cells (thymus-derived lymphocytes) to allogeneic and autochthonous virus-induced tumors in mice was investigated. It was demonstrated that C57BL/6 mice challenged with allogeneic P815 mastocytoma cells and complete Freund's adjuvant failed to develop cytotoxic cells but instead developed suppressor T cells which inhibited cytotoxic T cell function. Further, adjuvant-induced suppressor cells prevented the primary in vitro induction of cytotoxic T cells to P815 mastocytoma cells. In contrast, adult thymectomized animals, when challenged with adjuvant and allogeneic cells, had a normal cytotoxic response in vivo and their cells could not inhibit the generation of cytotoxic T cells in vitro. These studies suggested that the intact adult thymus was necessary for the induction of suppressor cells. Moreover, suppressor cells regulated cytotoxic T cell activity both in vivo and in vitro. Further, it was shown that adjuvant could prevent the normal immune response to virus-induced tumors. BALB/c mice treated with murine sarcoma virus developed tumors which reached a maximal size by day 14 and then regressed. Sham thymectomized animals treated with virus and complete Freund's adjuvant to generate suppressor cells died from progressive tumor growth. In contrast, thymectomized animals similarly treated had normal regression of tumor and survived. These studies lead to the conclusion that the adult thymus may regulate immune responsiveness by the export of suppressor T cells which regulate other T cell responses to both allogeneic and tumor antigens

Detecting p53 family proteins in haemocytic leukemia cells of Mytilus edulis from Pictou Harbour, Nova Scotia, Canada

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