Snyder-Theilen Strain Research Articles

V-onc product expression in early phase post perinatal retrovirus infection predictive of malignancy of induced lesions

Sixteen sarcomas developed in rats within 40 days post perinatal infection with feline sarcoma virus of Snyder-Theilen strain were examined to elucidate the association of the viral oncogene product (P85) expression with malignancy of developed sarcomas. Of these, 6 grew up to index-finger-tip-size, consisted of spindle shaped sarcoma cells and had no metastasis (group A), and 10 grew over thumb-tip-size, consisted of polygonal shaped sarcoma cells and had frequent metastasis (group B). One group A tumor and 8 group B tumors expressed P85. The v-onc product expression was thus found to be predictive of malignancy of induced tumors.

Differentiation of primary human fibroblasts into tissue macrophages by the Snyder-Theilen strain of the feline sarcoma virus is associated with endoreduplication, a 1,9 translocation, phenotypic stability, and apoptosis

We have previously reported the conversion of human fibroblasts to tissue macrophages by transduction with the Snyder-Theilen feline sarcoma virus [ST:FeSV (FeLV)]. The ST:FeSV-induced macrophages were largely configured as spherical cells, each with an extensive network of microvilli. Nuclear alterations included the appearance of three sub-populations within these mononucleated macrophages consisting of diploid (19%), tetraploid (43%), and octaploid (38%) cells. A karyotypic analysis revealed a 1,9 translocation in a small, dividing, subpopulation within the diploid fraction of the ST:FeSV-induced macrophages. Clonotypic assays demonstrated the conversion of all individually expanded, cloned fibroblasts, to macrophages following transduction with ST:FeSV. None of the converted macrophages reverted to the fibroblastic cell phenotype as determined by co-expression of v-fes and the receptor for the macrophage colony stimulating factor (CSF-IR). The ST:FeSV-induced macrophages did not form colonies in agar, nor did they form tumors in athymic mice. They appeared to undergo apoptosis after 4-6 weeks in culture. The findings pertain to the heterogeneous nature of tissue macrophages and their recruitment process during normal conditions, and in response to inflammatory stimuli.

Development and characterization of a panel of monoclonal antibodies against the catalytic domain of the human fes proto-oncogene product.

In developing monoclonal antibodies (Moabs) against the human fes proto-oncogene product, recombinant DNA technology was used to target reactivity of the Moabs towards the catalytic domain of it. Therefore, sequences of human fes exons 15-19 encoding amino acid residues 612 to 822 which harbor the catalytic domain except the presumed ATP-binding region, were fused in phase to the bacterial trp E gene which encodes anthranilate synthase. After partial purification of it, the bacterially produced hybrid product of this trp E-delta fes fusion gene was used as immunogen. A series of twelve mouse Moabs was obtained which recognized the human p92fes protein and the viral oncogene product p85gag-fes encoded by the Snyder-Theilen strain of feline sarcoma virus. Reactivity appeared to be directed towards the catalytic domain of the human fes proto-oncogene product. This was demonstrated by in vitro transcription and translation experiments using human fes coding sequences from exons 16-19. Upon testing their functional activity in divers immunological techniques, the whole panel of Moabs appeared to be useful in immunoprecipitation, Western blot and immunohistochemical analysis. Immunocytochemical analysis indicated that p85gag-fes is predominantly a cytoplasmic protein.

Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.

Purification and characterization of the feline sarcoma virus tyrosine-specific kinase PP85 gag-fes

The transforming phosphoprotein pp85 gag-fes of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) was purified in a form which exhibits tryosine-specific kinase activity. Cell lysates of ST-FeSV-transformed mink nonproducer cells were applied to a column of DEAE-Sephacel and eluted with a linear gradient of NaCl in phosphate buffer. Kinase activity was found uncomplexed (pp85 gag-fes ) in the flow-through and was eluted with 200 m M NaCl in a complex with pp50 and pp90. The flow-through material was further fractionated by chromatography on hydroxylapatite, followed by phosphocellulose and a final gel filtration step using Sephacryl S200. The final preparation was specifically enriched 1400-fold, and tyrosine-specific kinase activity was increased by about 300-fold as determined by autophosphorylation or phosphorylation of casein. Pp85 gag-fes kinase activity was inhibited by nicotinamide adenosine dinucleotide (NAD) and slightly inhibited by NADH, while quercetin, a strong inhibitor for pp60 src -associated tyrosine kinase activity, had no inhibiting effect.

Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.

A beta-type transforming growth factor, present in conditioned cell culture medium independent of cell transformation, may derive from serum.

An alpha-type transforming growth factor (TGF alpha) is produced at high levels by rat embryo cells transformed by the Snyder-Theilen strain of feline sarcoma virus (FeSV). Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF-dependent growth factor of the TGF beta type (TGF beta) in this conditioned medium. This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography. This extracellular type of TGF beta activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation. Moreover, the presence of an EGF-dependent, 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests serum as an origin for the B-type transforming growth factor initially observed in conditioned medium of Snyder-Theilen FeSV transformed cells. This does not, however, preclude the possibility that TGF beta is also secreted by the transformed rat embryo cells themselves.

Addendum

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of [3H]myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chrotomagraphy to be [3H]myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid (A. Schultz and S. Oroszlan, J. Virol46, 355–361). It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins (L. E. Henderson, H. C. Krutzsch, and S. Oroszlan, Proc. Natl. Acad. Sci. USA80, 339–343). The possible role of myristylation of transforming proteins is discussed.

Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of [ 3H]myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29 gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chrotomagraphy to be [ 3H]myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid (A. Schultz and S. Oroszlan, J. Virol 46, 355–361). It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins (L. E. Henderson, H. C. Krutzsch, and S. Oroszlan, Proc. Natl. Acad. Sci. USA 80, 339–343). The possible role of myristylation of transforming proteins is discussed.

Biological behavior of tumors and associated retroviremia in cats inoculated with Snyder-Theilen fibrosarcoma virus and the phenomenon of tumor recurrence after primary regression.

The fate of tumors and associated retroviremia was studied in 111 cats infected with the Snyder-Theilen strain of feline sarcoma virus (FeSV). Tumors appeared at the site of inoculation within 7 to 10 days. A retroviremia, due mainly to the associated feline leukemia virus helper virus (FeLV-helper), developed at the same time as tumors. Of the cats, 44 developed progressively growing tumors and therefore had to be killed, and 67 developed tumors that regressed. There was a strong correlation between the persistence of the accompanying retroviremia and the growth of the tumors. The 44 cats with progressively growing fibrosarcomas remained retroviremic until death. Conversely, 53 of the 67 cats with solitary, regressing tumors were only transiently retroviremic. Tumor regression in these cats paralleled the disappearance of retrovirus from the blood. The fate of tumors and retroviremia was not always the same, however. Twelve cats remained persistently retroviremic after all signs of gross tumors disappeared. Two other kittens became nonviremic within 20 days after inoculation, yet tumors continued to grow and even metastasize for another 3 to 5 weeks before regressing. Fibrosarcomas recurred 3 weeks to 8 months later in 8 of 12 persistently retroviremic cats with regressed tumors. Although the blood and bone marrow from these cats contained predominantly FeLV-helper, tumor cells yielded both FeSV and FeLV-helper. Of 53 animals, 3 developed recurrent fibrosarcomas 5 weeks to 8 months after all signs of tumors and retroviremia had disappeared. Cells cultured from these tumors appeared initially like normal fibroblasts and were virus nonproducers. After one to three passages in culture, however, cells became malignantly transformed and replicated both FeSV and FeLV-helper. Cultures of the bone marrow from these and other nonviremic cats with regressed tumors yielded only FeLV-helper.

Chromosomal sublocalization of human c-myb and c-fes cellular onc genes.

The transforming genes of acutely transforming retroviruses are derived from conserved cellular genes (c-onc genes) which are believed to be important in normal cell growth and differentiation. Recent studies indicate that altered expression of c-onc genes, for example, by insertion of viral genomes, gene amplification or chromosomal translocation, can lead to development of malignant diseases in man and animals. c-myb and c-fes are homologues of the transforming genes of avian myeloblastosis virus and feline sarcoma virus (Gardner and Snyder-Theilen strains), respectively. c-myb is transcribed preferentially in immature haematopoietic cells and probably codes for a protein important in differentiation of these cells. The viral fes gene, like several other viral onc genes, encodes a tyrosine-specific protein kinase. However, c-fes transcripts have not been detected in the types of human cells examined so far. c-myb and c-fes have been assigned to human chromosomes 6 and 15, respectively. Specific aberrations involving these chromosomes have been observed at high frequency in several human neoplasms. We have now sublocalized c-myb to 6q22-24 and c-fes to 15q25-26 by in situ hybridization of the human c-onc probes to human mitotic chromosome preparations. These chromosomal segments are indeed involved in nonrandom translocations in several human tumours. The results encourage further investigation into the role of onc genes in the pathogenesis of specific neoplasms.

Antibodies of predetermined specificity detect two retroviral oncogene products and inhibit their kinase activities.

Oligopeptides predicted from the nucleotide sequence of the oncogene v-fes of feline sarcoma virus (FeSV) were synthesized chemically and used to generate specific antibodies. Antisera against a 12-amino-acid-long oligopeptide (12-mer) located 42 residues from the carboxyl terminus of the v-fes coding sequence efficiently recognized the transforming proteins encoded by Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of FeSV. This 12-mer also contains 10 amino acid residues homologous in order and position to those predicted from the nucleotide sequence of the oncogene v-fps of avian Fujinami sarcoma virus (FSV). The anti-12-mer immunoprecipitated the FSV-specific transforming protein molecules from FSV-transformed cells. Binding of these antipeptide antibody molecules to the v-fes and the v-fps gene products inhibited their associated tyrosine-specific protein kinase (EC 2.7.1.37) activities. The ability to generate such site-specific antisera to the products of related oncogenes will be valuable in the molecular characterization of retroviral transforming proteins and their normal cellular homologs.

Association of the transforming proteins of the ST and GA strains of feline sarcoma virus and their in vitro associated protein kinase activities with cellular membranes.

The translation products of the Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV), termed gag-fes proteins, are high molecular weight polyproteins containing different amounts of the amino terminus of the feline leukemia virus (FeLV) gag gene-coded precursor protein linked to a similar sarcoma virus-specific polypeptide. Both polyproteins are phosphoproteins with indistinguishable in vitro associated tyrosine-specific protein kinase activities. The polyproteins are extremely hydrophobic proteins which are intimately associated with the plasma membrane fraction of transformed cells. Approximately 10% of the proteins are modified by glycosylation and expressed on the cell surface where they are accessible to lactoperoxidase-mediated radio-iodination and trypsinization. Cell surface localization of the polyproteins does not appear to be necessary for transformation. However, preliminary evidence suggests that the amount of FeLV p30 sequences at the amino end of the proteins may have some effect on the intracellular distribution of the gag-fes polyproteins and on the phenotype of the transformed cell.

Similar transforming growth factors (TGFs) produced by cells transformed by different isolates of feline sarcoma virus

Fisher rat embryo cells transformed by each of three independent isolates of feline sarcoma virus (FeSV) are shown to release transforming growth factors (TGFs) into cell culture medium. These acid- and heat-stable peptides compete for binding to, and stimulate phosphorylation of, EGF membrane receptors and promote anchorage-independent cell growth. Cells transformed by the Gardner and Snyder-Theilen strains of FeSV produce high titers of TGF (60-200 ng eq EGF/liter) while cells transformed by McDonough FeSV produce TGF at only low levels (<10 ng eq EGF/liter). Growth factors produced by cells transformed by each of the three FeSV isolates functionally and biochemically resemble each other, mouse sarcoma growth factor (SGF), and TGFs produced by human tumor cells.

Tyrosine phosphorylation sites common to transforming proteins encoded by Gardner and Snyder-Theilen FeSV

The Gardner and Snyder-Theilen strains of feline sarcoma virus (FeSV) encode 110,000 molecular weight ( M r ) polyproteins as their primary translational products. Cells transformed by a variant of Snyder-Theilen FeSV express an 85,000 M r polyprotein. Single tyrosine phosphorylation sites identified within each of these viral gene products are shown to represent major in vitro acceptors phosphorylated by the polyprotein associated protein kinases. By two-dimensional tryptic peptide analysis, these acceptor sites are highly related although exhibit minor differences in map position, while by analytical high-performance liquid chromatography, all three elute at approximately the same acetonitrile concentration (21–23%). The tyrosine acceptor sites in these FeSV-encoded polyproteins have been localized by sequential Edman degradation at a position seven amino acid residues distal to their trypsin cleavage site. The major tyrosine acceptors, two serine phosphorylation acceptors common to the p12 structural components of all three polyproteins, a single major phosphothreonine site, and several minor less well-resolved acceptor sites are shown to be phosphorylated in vivo. In addition to the major tyrosine acceptor site initially phosphorylated in vitro, phosphorylation of secondary tyrosine acceptors is shown to occur to proportionately greater extents when in vitro phosphotransferase reactions are carried out for longer times or at higher temperatures.

Human gene (c-fes) related to the onc sequences of Snyder-Theilen feline sarcoma virus.

The onc gene (v-fes) of the acutely transforming feline sarcoma virus (Snyder-Theilen strain) has homologous cellular sequences (c-fes) in all vertebrate species, including humans. We isolated from a human DNA library recombinant phages containing overlapping c-fes sequences. The human c-fes locus spans a region of 3.4 kilobases and contains 1.4 kilobases of DNA homologous to the viral onc sequence interspersed with three intervening sequences.

Human gene (c-fes) related to the onc sequences of Snyder-Theilen feline sarcoma virus.

The onc gene (v-fes) of the acutely transforming feline sarcoma virus (Snyder-Theilen strain) has homologous cellular sequences (c-fes) in all vertebrate species, including humans. We isolated from a human DNA library recombinant phages containing overlapping c-fes sequences. The human c-fes locus spans a region of 3.4 kilobases and contains 1.4 kilobases of DNA homologous to the viral onc sequence interspersed with three intervening sequences.

Molecular cloning of the Fujinami sarcoma virus genome and its comparison with sequences of other related transforming viruses.

Full-length proviral DNA of Fujinami sarcoma virus (FSV) of chickens was molecularly cloned and characterized. An analysis of FSV DNA integrated in mammalian cells showed that restriction endonuclease SacI has a single cleavage site on FSV DNA. Unintegrated closed circular FSV DNA obtained from newly infected cells was linearized by digestion with SacI and cloned into lambdagtWES.lambdaB. The following three different molecules were isolated: FSV-1 (4.4 kilobases [kb]) and FSV-2 (4.7 kb), which appeared to be full-length FSV DNA molecules containing either one or two copies of the long terminal repeat structure, and FSV-3 (6 kb), which consisted of part FSV DNA and part DNA of unknown origin. An analysis of the structure of cloned FSV-1 and FSV-2 DNA molecules by restriction endonuclease mapping and hybridization with appropriate probes showed that about 2.6 kb of the FSV-unique sequence called FSV-fps is located in the middle of the FSV genome and is flanked by helper virus-derived sequences of about 1.3 kb at the 5' end and 0.5 kb at the 3' end. The long terminal repeats of FSV were found to have no cleavage site for either EcoRI or PvuI. Upon transfection, both FSV-1 DNA and FSV-2 DNA were able to transform mammalian fibroblasts. Four (32)P-labeled DNA fragments derived from different portions of the FSV-fps sequence were used for hybridization to viral RNAs. We found that sequences within the 3' half of the FSV-fps gene are homologous to RNAs of PRCII avian sarcoma virus and the Snyder-Theilen strain of feline sarcoma virus, both of which were previously shown to contain transforming genes related to FSV-fps. These results suggest that the 3' portion of the FSV-fps sequence may be crucial for the transforming activity of fps-related oncogenic sequences.

Origin and biological properties of a new feline sarcoma virus

A new strain of feline sarcoma virus (GR-FeSV) was isolated from a spontaneous sarcoma of an 8-year-old domestic house cat. The virus induced sarcomas at high incidence after a short latent period in fetal and newborn kittens and transformed cat embryo fibroblasts in vitro after 5 days. Compared to Gardner-Arnstein and the Snyder-Theilen strains of FeSV, GR-FeSV induced more pleomorphic sarcomas and larger, more rounded and discrete foci of cell transformation. GR-FeSV was shown to be defective for replication, and nonproducer transformed clones from several species were obtained at limiting GR-FeSV dilution. The defective sarcoma virus could be rescued from such transformants by superinfection with replication competent type-C viruses. The primary translational product of the GR-FeSV genome is a 70,000-dalton polyprotein that contains the amino-terminal domain of the FeLV gag gene precursor protein and a sarcoma virus-specific polypeptide. These results differentiate GR-FeSV from previously isolated FeSV strains, and establish it as an independent spontaneously occurring sarcomavirus isolate of the domestic cat.

Three distinct genes in human DNA related to the transforming genes of mammalian sarcoma retroviruses.

Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.