Carol L. Reinisch

Horizontal Transmission of Clonal Cancer Cells Causes Leukemia in Soft-Shell Clams

Outbreaks of fatal leukemia-like cancers of marine bivalves throughout the world have led to massive population loss. The cause of the disease is unknown. We recently identified a retrotransposon, Steamer, that is highly expressed and amplified to high copy number in neoplastic cells of soft-shell clams (Mya arenaria). Through analysis of Steamer integration sites, mitochondrial DNA single-nucleotide polymorphisms (SNPs), and polymorphic microsatellite alleles, we show that the genotypes of neoplastic cells do not match those of the host animal. Instead, neoplastic cells from dispersed locations in New York, Maine, and Prince Edward Island (PEI), Canada, all have nearly identical genotypes that differ from those of the host. These results indicate that the cancer is spreading between animals in the marine environment as a clonal transmissible cell derived from a single original clam. Our findings suggest that horizontal transmission of cancer cells is more widespread in nature than previously supposed.

Expression of homologues for p53 and p73 in the softshell clam ( Mya arenaria ), a naturally-occurring model for human cancer

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 Burkitts lymphoma or other human cancers.

Widespread transmission of independent cancer lineages within multiple bivalve species

Most cancers arise from oncogenic changes in the genomes of somatic cells, and while the cells may migrate by metastasis, they remain within that single individual. Natural transmission of cancer cells from one individual to another has been observed in two distinct cases in mammals (Tasmanian devils and dogs), but these are generally considered to be rare exceptions in nature. The discovery of transmissible cancer in soft-shell clams (Mya arenaria) suggested that this phenomenon might be more widespread. Here we analyse disseminated neoplasia in mussels (Mytilus trossulus), cockles (Cerastoderma edule), and golden carpet shell clams (Polititapes aureus) and find that neoplasias in all three species are attributable to independent transmissible cancer lineages. In mussels and cockles, the cancer lineages are derived from their respective host species; however, unexpectedly, cancer cells in P. aureus are all derived from Venerupis corrugata, a different species living in the same geographical area. No cases of disseminated neoplasia have thus far been found in V. corrugata from the same region. These findings show that transmission of cancer cells in the marine environment is common in multiple species, that it has originated many times, and that while most transmissible cancers are found spreading within the species of origin, cross-species transmission of cancer cells can occur.

Detection of micronuclei in hemocytes of Mya arenaria: association with leukemia and induction with an alkylating agent

Abstract Leukemia of the soft shell clam, Mya arenaria , is characterized by circulating tumor cells detected initially in the hemolymph and, as the disease progresses, in solid tissue. Because they are estuarine filter-feeders, clams are exposed to a wide variety of environmental pollutants. Genotoxic pollutants may react directly in vivo with DNA. Genotoxins may also act indirectly after cellular metabolism resulting in DNA adducts or lesions which then induce genetic changes. The micronucleus (MN) assay was used to detect genomic damage in clam hemocytes. We initially determined if the number of leukemia cells identified by a monoclonal antibody (MAb) correlated with micronucleus formation. Clams collected from a site heavily contaminated with PCBs (New Bedford Harbor, NBH, MA) showed a significant increase in MN formation when compared with clams from a relatively pristine site (Marthas Vineyard, MV, MA). Specifically, micronucleus frequency was 3 times higher in NBH clams than MV clams. Further, a positive correlation between each stage of leukemic disease (number of leukemic cells/ml hemolymph) and the corresponding MN frequency was found. When MV clams were exposed to the alkylating agent ethyl methanesulfonate (EMS: 50, 100 and 150 mg/l) in the laboratory (exposure time; 2–10 days), micronuclei were detected in normal hemocytes. The number of MN was directly related to the dose of EMS. After treatment with 150 mg/l EMS and an exposure time of 10 days, MN formation was 4 times higher than without treatment. Hemocyte reactivity with the MAb 1E 10 did not increase following treatment with EMS, but the percent MN formation showed a relationship with the percent leukemia cells within the clams from the field. Thus MN are generated either in response to an alkylating agent (EMS) or in response to environmental genotoxic materials.

Unique antigens on neoplastic cells of the soft shell clam Myaarenaria

Soft shell clams (Mya arenaria) are commonly found in coastal waters of both the eastern and western United States. These invertebrates, which have an open circulatory system, may develop neoplasms of the haemolymph which ultimately kill the host. In this study we have 1) recorded the prevalence of hematopoietic neoplasms (HN) in Mya arenaria within a 50 mile radius of Woods Hole, Massachusetts and 2) utilized cells from one HN bearing clam to generate a series of monoclonal antibodies. Our data show that determinants are expressed on HN cells which are not detected on normal clam haemocytes, suggesting separate ontogenetic pathways of cell differentiation.

Early development of the serotonergic and dopaminergic nervous system in Spisula solidissima (surf clam) larvae

We have defined the development of the serotonergic and dopaminergic components of the central nervous system in the early Spisula solidissima (surf clam) embryo using HPLC and immunocytochemistry. HPLC analysis reveals norepinephrine, dopamine, and serotonin are present at 24 h post-fertilization. Immunocytochemistry shows that the serotonergic nervous system emerges during the late trochophore stage with the development of a single serotonergic cell, C/A1, in the cerebral/apical ganglion. After 48 h, a second serotonergic cell forms, C/A2, which is connected to C/A1 by two serotonergic processes, and a single serotonergic cell emerges in the visceral ganglion, V1. At 72 h, a new serotonergic cell body develops in the cerebral/apical ganglion, C/A3. After 96 h, the cerebral/apical ganglion and visceral ganglion are connected by a serotonergic process. Expression of the dopamine receptor, D2, begins by 24 h with a generalized expression in the region of the developing gut. D2 expression in the gut ceases by 48 h. At 48 h, a network of fibers forms dorsolateral to the mouth. By 72 h, D2 expressing projections emerge from this network.

Activation of transcription and retrotransposition of a novel retroelement, Steamer, in neoplastic hemocytes of the mollusk Mya arenaria

Significance The soft shell clam in many areas of the North Atlantic is afflicted with a fatal leukemia-like disease of unknown origin. Leukemic cells from the diseased animals were found to release reverse transcriptase and to express high RNA levels of a previously unknown member of the gypsy family of retroelements, Steamer. The DNA copy number of the element was increased to enormously high levels in diseased cells, mediated by reverse transcription and integration into the host genome. The activation of Steamer expression and transposition may initiate or accelerate the course of leukemia and constitutes a potential diagnostic marker of the disease. Bivalve mollusks of the North Atlantic, most prominently the soft shell clam Mya arenaria, are afflicted with an epidemic transmissible disease of the circulatory system closely resembling leukemia. The disease is characterized by a dramatic expansion of blast-like cells in the hemolymph with high mitotic index. Examination of hemolymph of diseased clams revealed high levels of reverse transcriptase activity, the hallmark of retroviruses and retroelements. By deep sequencing of RNAs from hemolymph, we identified transcripts of a novel retroelement, here named Steamer. The DNA of the element is marked by long terminal repeats and encodes a single large protein with similarity to mammalian retroviral Gag-Pol proteins. Steamer mRNA levels were specifically elevated in diseased hemocytes, and high expression was correlated with disease status. DNA copy number per genome was present at enormously high levels in diseased hemocytes, indicative of extensive reverse transcription and retrotransposition. Steamer activation in M. arenaria is an example of a catastrophic induction of genetic instability that may initiate or advance the course of leukemia.

The Nerve Hemoglobin of the Bivalve Mollusc Spisula solidissima MOLECULAR CLONING, LIGAND BINDING STUDIES, AND PHYLOGENETIC ANALYSIS

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (μm). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mm), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P50 ∼0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.

Multiple protein differences distinguish clam leukemia cells from normal hemocytes: evidence for the involvement of p53 homologues

In coastal locations, marine invertebrates, primarily molluscs, develop fatal leukemias in their blood or hemolymph. In the clam Mya arenaria, non-adhesive, mitotic, spherical leukemia cells replace adhesive, motile, normal hemocytes as leukemia progresses. End-stage leukemia cells express a unique antigen, IE10, while normal cells express the 2A4 marker. The goals of this work were to further differentiate the normal and leukemia specific antigens relative to protein structure, determine if other protein distinctions exist, and examine p53 gene family expression in both cell types. Recognized by the monoclonal antibody 2A4, normal cells express a 185-kDa glycoprotein that may have multiple forms. Detected by the monoclonal antibody 1E10, leukemic cells express a very hydrophobic 252-kDa glycoprotein that is likely to be a transmembrane protein with spectrin/dystrophin-like characteristics. After normalization to the major cytoskeletal protein actin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals major distinguishing protein and glycoprotein differences between the two cell types. Most obvious is the near-absence of tubulin in the non-mitotic normal hemocytes. We have also characterized the expression of p53 gene family members in normal and end-stage leukemia cells, finding shifts in expression of the p53 gene homologues p73 and p97 coincident with leukemia-specific protein synthesis.

Indirect peroxidase staining using monoclonal antibodies specific for Mya arenaria neoplastic cells

Two cytological methods are primarily used to diagnose hematopoietic neoplasia (HN) in the soft shell clam, Mya arenaria. These methods involve (1) the examination of fresh hemocytes by using a hemocytometer or (2) the evaluation of fixed hemocyte preparations stained with Feulgen Picromethyl Blue. This paper presents a new method which uses indirect peroxidase (IP) staining of hemocyte preparations treated with murine monoclonal antibodies specific for HN cells. Application of the IP method is described, and a comparison is made between the three methods. The hemocytometer method is only 53% as accurate as either the IP or the Feulgen Picromethyl Blue methods. When compared to the Feulgen Picromethyl Blue method, both the IP and Hemocytometer methods are quicker to read (3 vs. 15 min). Additionally, the IP method is more specific than either the hemocytometer or the Feulgen Picromethyl Blue methods because questionable cells are identified as neoplastic or normal by the IP stain. Thus, the IP method is the best method of the three for the diagnosis of HN in soft shell clams.