Characterization of spontaneous malignant lymphomas in Japanese macaques (Macaca fuscata).

Lymphomas are common spontaneous tumors in nonhuman primates but remain poorly characterized in Japanese macaques (Macaca fuscata). This study examined 5 cases of spontaneous malignant lymphoma in Japanese macaques, focusing on the immunophenotypes and presence of simian lymphocryptoviruses, which are Epstein-Barr virus-related herpesviruses in nonhuman primates. The macaques with lymphoma were 5 to 28 years old, indicating that lymphomas develop over a wide age range. The common macroscopic findings were splenomegaly and enlargement of lymph nodes. Histologic and immunohistochemical analyses revealed that all cases were non-Hodgkin type and exhibited a T-cell phenotype, positive for CD3 but negative for CD20 and CD79α. The lymphomas exhibited diverse cellular morphologies and were subdivided into 3 types according to the World Health Organization classification. These included 3 cases of peripheral T-cell lymphoma, not otherwise specified; 1 case of T-cell prolymphocytic leukemia; and 1 case of an unclassifiable T-cell lymphoma. Positive signals were detected by in situ hybridization in 2 of the 4 examined cases using probes for the Epstein-Barr virus-encoded small RNA (EBER). Furthermore, the presence of M. fuscata lymphocryptovirus 2, a macaque homolog of Epstein-Barr virus, was demonstrated in EBER-positive cases by polymerase chain reaction amplification followed by direct sequencing. Immunohistochemistry using antibody to the Epstein-Barr virus-encoded nuclear antigen 2 was negative, even in the EBER-positive cases. The present study suggests that T-cell lymphoma is more common than B-cell lymphoma in Japanese macaques and that M. fuscata lymphocryptovirus 2 is present in some cases.

Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma.

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B cells: 21 chronic lymphocytic leukemia (B-CLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients (B-ALL, B-CLL or AML, but not MM) was positive for EBV and contained <100 ebv copies/100 ng dna. ebv loads were occasionally high (>500 copies/100 ng DNA) in B-ALL (2/16) and in B-CLL (2/21) samples. The fractions of samples positive for HHV-8 and HHV-6A, less than 10% for MM patients, were high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV-6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+). B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of multiple HHVs, including EBV and CMV at high loads, is not rare in B-ALL and B-CLL cell samples. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.

Human DBI (endozepine): relationship to a homologous membrane associated protein (MA-DBI).

Human endozepine, an 86 amino acid polypeptide, was originally isolated from human brain tissue as a putative ligand of the benzodiazepine receptor. Complete amino acid sequencing of the human and bovine proteins revealed significant homology with the partial sequence of diazepam binding inhibitor (DBI), a protein from rat brain. Both endozepine and DBI have been shown to elicit behavioral effects, suggesting that they function as pharmacologically-active ligands of the GABA (gamma-aminobutyric acid) receptor complex. Subsequent cDNA cloning of human and bovine endozepine, rat DBI and human DBI has shown that these proteins are encoded by the same gene. A related cDNA, encoding a transmembrane protein of 533 amino acids with a domain homologous to DBI, has also been cloned from bovine brain.

Nucleotide sequence and genomic structure of duck acyl-CoA binding protein/diazepam-binding inhibitor: co-localization with S-acyl fatty acid synthase thioesterase.

A computer-aided homology search of the GenBank nucleotide database using the amino acid sequence of human acyl CoA-binding protein (ACBP)/diazepam-binding inhibitor (DBI)-endozepine as a probe revealed that a genomic fragment containing the gene encoding the mallard duck (Anas platyrhynchos) S-acyl fatty acid synthase thioesterase also contains sequences which encode the duck homolog of ACBP/DBI. The duck ACBP/DBI gene is positioned downstream of the thioesterase gene in a tail-to-tail orientation separated from the 3' end of the thioesterase gene by only several hundred nucleotides. Three exons were identified that have strong homology to the published cDNA sequences of human and bovine ACBP/DBI. These exons define all of the coding region except for the amino-terminal domain, which was subsequently cloned by polymerase chain reaction (PCR) amplification. The encoded amino acid sequence of the duck ACBP/DBI is 62-68% homologous to mammalian ACBP/DBI sequences. While the mammalian ACBP/DBI is expressed mainly in the liver, with smaller amounts in the brain and heart, mRNA transcripts of duck ACBP/DBI were detected only in the brain with no evidence for expression in the liver or heart. The close proximity of the genes for ACBP/DBI and S-acyl fatty acid synthase thioesterase raises the possibility of co-regulation of expression.

Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta).

Multifocal submucosal stromal tumors were diagnosed in a 5.5-year-old rhesus macaque (Macaca mulatta) experimentally infected with simian immunodeficiency virus, strain SIVsmE660, and CD4+ T cell depleted. The animal was negative for simian retroviruses, SRV-1, -2, and -5. Polymerase chain reaction analysis of DNA from tumor and spleen tissue revealed abundant, preferential presence of retroperitoneal fibromatosis herpesvirus, the macaque homologue of the Kaposi sarcoma-associated herpesvirus (human herpesvirus-8), in the tumors. This was corroborated by demonstration of viral latent nuclear antigen-1 in the nuclei of a majority of the spindeloid tumor cells. Low levels of an additional macaque herpesvirus, rhesus rhadinovirus, were also detected in the spleen and tumor tissues. The spindeloid cells labeled positively for vimentin and CD117 but were negative for CD31, CD68, desmin, and smooth muscle cell actin. Collectively, these findings suggest a relation to but not absolute identity with simian mesenchymoproliferative disorders (MPD) or typical gastrointestinal stromal tumors (GISTs).

Disruption of LT-antigen/p53 complex by heat treatment correlates with inhibition of DNA synthesis during transforming infection with SV40.

Transforming infection of Go/G1-arrested primary mouse kidney cell cultures with simian virus 40 (SV40) induces cells to re-enter the S-phase of the cell cycle. In Go-arrested cells, no p53 is detected, whereas in cells induced to proliferate by infection, a gradual accumulation of p53 complexed to SV40 large T-antigen is observed in the nucleus. Heat treatment of actively proliferating SV40-infected cells leads to inhibition of DNA synthesis and growth arrest. To determine the fate of p53 after heat treatment, proliferating infected cells were exposed to mild heat (42.5 degrees C) for increasing lengths of time. The results presented here show that after ninety minutes of treatment, the arrest of DNA synthesis by heat correlates with the disruption of the p53/LT-antigen complex. Longer treatments induce, in addition, a reduction in the solubility of p53, which was recovered tightly associated with the nuclear fraction. This contrasted with large T-antigen, whose solubility remained unaffected by heat treatment. Although the total amount of p53 in the nucleus remained constant, as shown by immunoblot analyses, p53 was no longer detectable after immunoprecipitation or by immunofluorescent staining techniques. These results suggest that heat treatment had either induced conformational changes in its antigenic sites, or had sequestered the sites through aggregation or binding to insoluble nuclear components.

Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6.

Oncostatin M (OSM), a glycoprotein of Mr approximately 28,000 produced by activated monocyte and T-lymphocyte cell lines, was previously identified by its ability to inhibit the growth of cells from melanoma and other solid tumors. We have detected significant similarities in the primary amino acid sequences and predicted secondary structures of OSM, leukemia-inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). Analysis of the genes encoding these proteins revealed a shared exon organization, suggesting evolutionary descent from a common ancestral gene. Using a panel of DNAs from somatic cell hybrids, we have shown that OSM, like LIF, is located on human chromosome 22. We have also demonstrated that OSM has the ability to inhibit the proliferation of murine M1 myeloid leukemic cells and can induce their differentiation into macrophage-like cells, a function shared by LIF, G-CSF, and IL-6. We propose that OSM, LIF, G-CSF, and IL-6 are structurally related members of a cytokine family that have in common the ability to modulate differentiation of a variety of cell types.

Characterization of the surface proteins of SV40-transformed mouse and human cells: absence of SV40-specific proteins.

The proteins of a number of SV40- and spontaneously transformed mouse and human cell lines were compared in an effort to identify a surface protein which would correspond to the SV40 tumor-specific transplantation antigen (TSTA). Analysis of the one- and two-dimensional electrophoretic patterns of 35S-methionine-labelled total proteins and 125I-labelled surface proteins of several of these cell lines failed to reveal the presence of proteins specific to transformation by SV40. Antisera were prepared against SV40- and spontaneously transformed mouse cells in syngeneic mice. In serological assays, these antisera reacted with surface antigens common to both SV40- and spontaneously transformed mouse cell lines. Electrophoretic analysis of the 125I-surface-labelled proteins which these antisera immunoprecipitated from extracts of SV40- and spontaneously transformed mouse and human cells identified a set of common surface proteins with apparent molecular weights of 15, 46, 50, 72, 77, 105, 150 and 230kdal. No SV40-specific surface proteins were detected. Two of the transformed cell surface proteins (105 and 150kdal) were present as well in membrane fractions of 35S-methionine-labelled primary mouse kidney cultures. The proteins of the primary cultures could not be iodinated by lactoperoxidase suggesting that these proteins were present at a "cryptic" location at the surface of normal cells. We were not able to obtain serological or immunochemical evidence for the presence of SV40 large T-antigen at the surface of any of the SV40-transformed cell lines tested using either hamster anti-SV40 tumor sera, a rabbit antiserum against SDS-denatured gel-purified large T-antigen or antisera against SV40-transformed mouse cells. In conjunction with the report that large T-antigen released from disrupted SV40-transformed cells will bind to cell surfaces (Lange-Mutschler and Henning, 1982), we consider the possibility that the specific rejection of SV40-induced tumors by sensitized animals is the result of immunological reactions against both common transformation-related surface antigens and SV40 T-antigen from disrupted cells that has bound to the surface of other tumor cells.

A 105 000 dalton antigen of transformed mouse cells is a stress protein.

Antisera prepared in mice against syngeneic spontaneously transformed AL/N cells (anti-TAL/N serum) identified a number of protein antigens synthesized by simian virus 40 (SV40) transformed cells, among which was a protein with a molecular mass of 105 000 daltons (p105). Of these transformed cell antigens which were immunogenic in a syngeneic system, only p105 was detected in primary mouse kidney cell cultures. p105 isolated from normal and transformed mouse cells was demonstrated to be identical by two-dimensional gel analysis. Relatively small amounts of p105 were synthesized in quiescent primary cultures, while the protein was actively synthesized in SV40-infected as well as in proliferating mouse kidney cells, and its synthesis in quiescent cells could be induced by subjecting the cultures to glucose starvation or heat-shock treatment. Immunofluorescent staining and cellular fractionation showed that p105 is normally localized to cytoplasmic structures. The results suggest that the expression of p105 is intimately associated with the metabolic state of the cell.

Molecular cloning and chromosomal localization of a pseudogene related to the human acyl-CoA binding protein/diazepam binding inhibitor.

The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5' ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-related sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1.

Repression of a G0-associated 65-kilodalton protein in actively proliferating and SV40-transformed mouse kidney cells.

The pattern of [35S]methionine-labeled proteins from primary cultures of mouse kidney epithelial cells arrested in G0 phase was analyzed by two-dimensional gel electrophoresis and compared with that observed from cultures of actively proliferating and SV40-transformed mouse kidney cells. A major polypeptide (p65) migrating with a molecular mass of 65,000 daltons and a pI of 5.8 was detected in quiescent cultures of cells which had exhausted their finite division potential. Under the experimental conditions used, these cells had lost sensitivity to growth factors and were irreversibly blocked in G0 phase of the cell cycle. In cultures of actively proliferating mouse kidney cells, the expression of p65 was not observed until just prior to arrest. Moreover, proliferating cultures of immortalized mouse kidney cells that had been reactivated from their quiescent state by infection with SV40 did not express p65. Subcellular localization studies suggest that p65 is associated with the crude nuclear fraction. In addition, p65 is glycosylated and binds the lectin concanavalin A. Pulse-chase experiments demonstrated that p65 was short lived with an estimated half life of 10 min. Thus, p65 appears to be a growth-arrest specific gene product whose expression is repressed during the proliferative state of mitotically active mouse kidney cells.

Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.

Molecular cloning of the gene for the yeast homolog (ACB) of diazepam binding inhibitor/endozepine/acyl-CoA-binding protein.

Diazepam binding inhibitor (DBI)/endozepine (EP)/acyl-CoA-binding protein (ACBP) is a small, highly conserved protein which has been independently isolated and characterized from different species using several different biological systems. To further investigate the structural and functional properties of this protein, we have cloned the homologous gene for DBI/EP/ACBP from the budding yeast Saccharomyces cerevisiae. The yeast gene contains no introns and encodes a polypeptide of 87 amino acids (including the initiating methionine), identical in length to the human gene product with 48% conservation of amino acid residues. The most highly conserved domain consists of 7 contiguous residues which are identical in all known protein species from yeast, birds, and mammals. This domain has previously been shown to constitute the hydrophobic binding site on DBI/EP/ACBP for acyl-CoA esters and is located within the second helical region of the molecule. Major and minor mRNA species of approximately 520 and 740 nucleotides, respectively, were detected in exponentially growing yeast. Sequences similar to those implicated in the regulation of fatty acid synthesis and beta-oxidation in yeast were detected in the promoter region of the gene. The presence of a highly conserved DBI/EP/ACBP gene in a primitive organism such as yeast provides support for the basic biological role of DBI/EP/ACBP as an acyl-CoA-binding protein and suggests that many of the biological functions attributed to it in higher organisms may result from its ability to interact with acyl-CoA. Hence, we have designated the yeast gene as ACB, for acyl-CoA-binding protein.

Oncostatin M.

Oncostatin M (OSM) was initially identified as a polypeptide cytokine which inhibited the in vitro growth of cells from melanoma and other solid tumors. OSM shows significant similarities in primary amino acid sequence and predicted secondary structure to leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), granulocyte colony-stimulating factor (G-CSF), interleukin 6 (IL-6), and interleukin 11 (IL-11). Analysis of the genes encoding these proteins reveals a shared exon organization suggesting evolutionary descent from a common ancestral gene. Recent data indicates that OSM also shares a number of in vitro activities with other members of this cytokine family. The overlapping biological effects appear to be explained by the sharing of receptor subunits.

Induction and yield of reaginic antibodies in mouse serum and ascitic fluid.

Reaginic antibody in ascitic fluid was isolated from mice immunized with ovalbumin and TAl(OH)3 or with avalbumin and pertussis adjuvant. Partial purification of this reagin by ammonium sulphate precipitation, gel filtration, and ion-exchange column chromatography yielded a gammaglobulin fraction highly active in the passive cutaneous anaphylaxis reaction. This was identical in purity and specific activity to a fraction of partially purified reagin isolated from mouse antiserum. The recovery of reaginic activity after purification from ascitic fluid was 19 per cent compared to the 7-8 per cent recovery from anti-serum. These studies demonstrate that ascitic fluid in mice immunized with methods known to produce reaginic antibody provides an excellent source of mouse IgE.

Oncostatin M (OSM) inhibits the differentiation of pluripotent embryonic stem cells in vitro.

Oncostatin M (OSM) is a cytokine which shares a common gene structure and amino acid sequence similarity with leukemia inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6), suggesting evolution from a common ancestral gene. These four cytokines share several biological activities including the ability to induce the differentiation of the murine M1 myeloid leukemic cell line. To further define the functional similarities within this family, we have investigated whether OSM can substitute for LIF in the maintenance in vitro of the undifferentiated state of pluripotent embryonic stem (ES) cells. In this study, we demonstrate that human recombinant OSM is similar to LIF in its ability to inhibit the differentiation of MBL-5 murine ES cells cultured in vitro. The level of differentiation was determined by morphological criteria and by the continued expression of the embryonic stem cell-specific surface antigen defined by the ECMA-7 monoclonal antibody. Competition binding studies demonstrate that OSM binds to the LIF receptor on MBL-5 ES cells. Our results implicate OSM as a developmental regulatory factor for embryonic stem cells in vivo.

Structural and sequence analysis of TGF-beta 2 cDNA clones predicts two different precursor proteins produced by alternative mRNA splicing.

Analysis of cDNA clones coding for human and simian transforming growth factor-beta 2 (TGF-beta 2) revealed the existence of two types of TGF-beta 2 precursor proteins of 414 amino acids (TGF-beta 2,414) and 442 amino acids (TGF-beta 2,442) in length. TGF-beta 2,442 contains a 29-amino-acid insertion in the amino terminus of the precursor region that replaces an Asn residue located at position 116 in TGF-beta 2,414. Of these 29 amino acids, three are cysteines, suggesting a more extensive disulfide-bond mediated secondary structure for TGF-beta 2,442 than for TGF-beta 2,414. Northern blot analysis using probes specific for the insert in TGF-beta 2,442 indicated that this protein is encoded by a minor 5.1-kb mRNA species present in human and simian cells. Since the DNA sequences flanking the insert are identical between clones coding for the two precursor protein, we suggest mRNAs coding for these proteins arise via differential splicing. Evidence is also presented that indicates that additional TGF-beta 2 mRNA heterogeneity is due to alternate polyadenylation. We propose that the 414-amino-acid precursor be referred to as TGF-beta 2a and the 442-amino-acid precursor be referred to as TGF-beta 2b.

Cloning and sequence analysis of simian transforming growth factor-beta cDNA.

We have isolated a cDNA clone coding for the simian TGF-beta precursor from a cDNA library made from an African green monkey cell line, BSC-40. The deduced amino acid sequence of the mature simian TGF-beta shows 100% homology with that of the human TGF-beta: Strong sequence homology was found between the precursor regions of the human and simian proteins with only five amino acid changes out of 278. The simian (and murine) precursor sequence was found to code for one less amino acid residue than the human. The implications of these results with respect to the isolation of a growth inhibitor from these cells functionally related to the TGF-beta molecule are discussed.

Structural and functional characterization of the human FMR1 promoter reveals similarities with the hnRNP-A2 promoter region.

Fragile X mental retardation syndrome is associated with an expansion of a CGG repeat within the 5'UTR of the first exon of the FMR1 gene, abnormal methylation of the CpG island in the promoter region, and a transcriptional silencing of this gene. We studied transcriptional regulation of the FMR1 gene using protein footprint analysis of the active and inactive gene in vivo . We identified four footprints within the FMR1 promoter region which correspond to consensus binding sites of known transcription factors, alpha-PAL/NRF1, Sp1, H4TF1/Sp1-like and c-myc. These footprints were present in normal cells with a transcriptionally active FMR1 gene. The same footprints were present in different cell types: primary fibroblasts, lymphoblastoid cells and peripheral lymphocytes. However, for the 1.1 kb region analyzed, no footprints were detected in a variety of cell types derived from patients with fragile X syndrome which have a transcriptionally inactive FMR1 gene. A BLAST nucleotide search identified sequence similarities between the region of the FMR1 gene containing the footprints and an analogous region within the promoter region of the gene for the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a member of a family of ribonucleoproteins implicated in mRNA processing and nuclear-cytoplasm transport. The nucleotide sequences identified in the hnRNP-A2 promoter region correspond to the same consensus binding sites showing DNA-protein interactions in the FMR1 gene. Our previous functional studies and the studies of others demonstrate that FMR proteins, like hnRNP-A2, are also ribonucleoproteins which appear to be involved in mRNA transport. The results from our footprint studies suggest that the expression of the FMR1 gene is regulated by the binding of specific transcription factors to sequence elements in the 5' region of the gene and that this expression may be regulated by elements in common with the hnRNP-A2 gene. Common regulation of these two genes might play an important role in the cooperative processing and transport of mRNA from the nucleus to the translation machinery.