Snyder-Theilen Feline Sarcoma Virus Research Articles

Viral gene mutation as determinant of low grade malignancy of lesions induced by perinatal retrovirus infection

Of 108 Wistar/Ma rats perinatally injected with Snyder-Theilen feline sarcoma virus, 75 developed tumors within 60 days (group A), and 25 after more than 117 days (group B). Metastases were detected in 59.1% and 11.8% in group A and B respectively. Antibody to v-fes gene product was found to occur before tumor development in group B and after tumor development in group A. v-Ses was found to be down-regulated and extensively mutated in tumors of group B. These results suggest viral gene mutation as a determinant of low grade malignancy of lesions induced.

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.

Differentiation of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus (ST:FeSV)

We have previously reported the conversion of human fibroblasts (HF) to tissue macrophages (TM) by transduction with the Snyder-Theilen feline sarcoma virus [ST:FeSV(FeLV)]. Here, we have demonstrated macrophage mediated tumor cytotoxicity (MTC) of human teratocarcinoma cells (NTERA-2) by ST:FeSV-induced TM. Inclusion of the NTERA-2 specific antibody (SSEA-4), substantially increased antibody-dependent cellular cytotoxicity (ADCC) of the tumor cells by ST:FeSV-induced TM. Tumor cell lysis by ST:FeSV-induced TM through either MTC or ADCC was especially effective at low serum concentrations (1%). The presence of cytokines during co-incubation, M-CSF in particular, enhanced the lysis of NTERA-2 cells by ST:FeSV-induced TM. Whereas pretreatment of the NTEBA-2 cells with all-trans retinoic acid (AtRA) enhanced MTC-induced lysis, it abrogated ADCC-induced lysis of the tumor cells by ST:FeSV-induced TM. Significantly, the co-incubation of NTERA-2 cells with ST:FeSV-induced TM in agar, resulted in a considerable stimulation of growth of untreated and TPA-pretreated NTERA-2 cells. In contrast, the growth of AtRA-pretreated NTERA-2 cells in agar was completely inhibited by ST:FeSV-induced TM. These results indicate that ST:FeSV-induced TM can produce potent growth-modulating cytokines, and that AtRA can modify their antitumor activity in monolayer cultures and in agar. The effects by AtRA, serum, and growth conditions (whether monolayers or agar) point to several levels of complexity regarding the interaction of tissue macrophages with tumor cells.

Differentiation of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus is accompanied by nuclear accumulation of the tumor suppressor p53

Snyder-Theilen feline sarcoma virus (ST:FeSV)-transduced human fibroblasts differentiate into tissue macrophages with many of the properties of normal macrophages. These cells express high levels of the gag-fes tyrosine kinase fusion protein, p85(v-fes), and exhibit an elevated level of tyrosine phosphorylation. Expression of the macrophage phenotype is accompanied by increased levels of DNA-binding activity and nuclear accumulation of wild-type p53. The DNA-binding activity of the transcription factors Egr-1, CREB and Sp1, which are known to be involved in cell differentiation, is also increased in ST:FeSV-induced macrophages. These observations suggest that v-fes can activate signal transduction pathways normally involved in macrophage differentiation, and that transcription factors such as p53, further facilitate v-fes-induced terminal differentiation.

Conversion of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus (ST:FeSV(FeLV)): productive infection by Leishmania major

The decisive role of macrophages in T-cell differentiation is best exemplified by cutaneous leishmaniasis. During infection, Leishmania attach to macrophages, the only site of replication for the parasite. We have recently demonstrated the conversion of human fibroblasts to tissue macrophages (TM) by transduction with the Snyder-Theilen feline sarcoma virus (ST:FeSV-(FeLV)). Since Leishmania have tropism only for macrophages, we have used the parasite to ascertain the functional phenotype of the ST:FeSV-induced TM. Here, we have demonstrated the productive infection of the ST:FeSV-induced TM by L. major. These results point to the utility of ST:FeSV-induced TM in studies that concern the role of human macrophages in T-cell differentiation during the course of infection by Leishmania.

Structural analysis of a variant clone of Snyder-Theilen feline sarcoma virus

A variant clone of Snyder-Theilen feline sarcoma virus (ST-FeSV) encoding a polyprotein with a molecular weight of approximately 104 kDa (P104) was compared to the P85 encoding prototype clone of ST-FeSV. Analysis of chimeric genes constructed with the viral oncogenes of the two clones indicated that the variant clone coded for a larger polyprotein than the prototype clone because of genetic differences in its 3' portion. Comparative DNA sequence analysis revealed that one nucleotide just upstream of the termination codon TGA in the prototype proviral DNA was deleted from the variant clone resulting in a 468-bp larger open reading frame. Furthermore, it appeared that the U3 regions of the long terminal repeats (LTRs) of the variant clone contained an insertion of 71 bp as compared to the LTRs of the prototype clone. In addition, both clones differed also from each other with respect to genetic sequences deleted from their env gene regions.

Monoclonal antibodies to the v-fes product and to feline leukemia: Virus P27 interspecies-specific determinants encoded by feline sarcoma viruses

Monoclonal antibodies to p27 gag and v- fes specific determinants on the gag-onc polyprotein encoded by Snyder-Theilen feline sarcoma virus (ST-FeSV) were prepared. In order to obtain hybridoma clones specific to the antigenic determinants encoded by the FeSV genome, Lou rats were immunized with ST-FeSV-transformed, virus-nonproducing syngeneic cells, and boosted with either the same cells or affinity-purified feline leukemia virus (FeLV) p27. Three distinct clones reactive to both FeLV p27 and p85 gag-fes , and one clone specific for a p85 fes determinant were established. The anti-p27 monoclonal antibodies also reacted with the polyproteins p959 gag-fes and p839 gag-fgr from Gardner-Arnstein (GA) and Theilen-Pedersen (TP1) FeSV, respectively. The anti-p27 monoclonal antibodies reacted with MuLV p30 and RD114 p28 but not with RSV, MMTV, or BLV. These results indicated that the part of the p27 gag gene that is preserved in ST-, GA, and TPI-FeSV encodes interspecies-specific p27 determinants.

Expression of the Mammalian c-fes Protein in Hematopoietic Cells and Identification of a Distinct fes-Related Protein

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

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.

Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins.

Two monoclonal antibodies have been obtained that recognize antigenic determinants within the C-terminal fps-encoded region of P140gag-fps, the transforming protein of Fujinami avian sarcoma virus (FSV). The hybridomas which secrete these antibodies (termed 88AG and p26C) were isolated after the fusion of NS-1 mouse myeloma cells with B lymphocytes from Fischer rats that had been immunized with FSV-transformed rat-1 cells. FSV P140gag-fps immunoprecipitated by either antibody is active as a tyrosine-specific kinase and is able to autophosphorylate and to phosphorylate enolase in vitro. The fps-encoded proteins of all FSV variants, including the gag- p91fps protein of F36 virus, are recognized by both monoclonal antibodies. However, the product of the avian cellular c-fps gene. NCP98, and the transforming proteins of the recently isolated fps-containing avian sarcoma viruses 16L and UR1 are recognized only by the p26C antibody. The 88AG antibody therefore defines an epitope specific for FSV fps, whereas the epitope for p26C is conserved between cellular and viral fps proteins. The P105gag-fps protein of the PRCII virus is not precipitated by p26C (nor by 88AG), presumably as a consequence of the deletion of N-terminal fps sequences. These data indicate that the fps-encoded peptide sequences of 16L P142gag-fps and UR1 P150gag-fps are more closely related to NCP98 than that of FSV P140gag-fps. This supports the view that 16L and UR1 viruses represent recent retroviral acquisitions of the c-fps oncogene. The P85gag-fes transforming protein of Snyder-Theilen feline sarcoma virus is not precipitated by either monoclonal antibody but is recognized by some antisera from FSV tumor-bearing rats, demonstrating that fps-specific antigenic determinants are conserved in fes-encoded proteins.

Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus.

When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells.

In vitro transformation of murine pre-B lymphoid cells by Snyder-Theilen feline sarcoma virus.

Snyder-Theilen feline sarcoma virus (ST-FeSV) codes for a protein kinase with specificity for tyrosine residues (Barbacid et al., Proc. Natl. Acad. Sci. U.S.A. 77:5158-5163, 1980), properties analogous to those of the transforming gene product of Abelson murine leukemia virus (Witte et al., Nature (London) 283:826-831, 1980). In the present report, ST-FeSV was demonstrated to transform murine hematopoietic cells under in vitro assay conditions which detect lymphoid cell transformation by Abelson murine leukemia virus. Bone marrow colony formation was shown to require ST-FeSV, follow single-hit kinetics, and require the presence of mercaptoethanol in the agar medium. ST-FeSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The hematopoietic blast cells transformed by ST-FeSV in culture appeared to be at an early stage of B cell differentiation. They possessed Lyb 2 surface antigens, were dependent on mercaptoethanol for growth, and contained only low levels of terminal deoxynucleotidyl transferase. Moreover, a large fraction of the lines synthesized immunoglobulin mu chain in the absence of light chains. Thus, the phenotype of ST-FeSV hematopoietic transformants was indistinguishable from that of the pre-B lymphoblast transformants induced by Abelson murine leukemia virus. These findings indicate that the in vitro functional similarities in the onc gene products of ST-FeSV and Abelson murine leukemia virus may reflect a common pathway by which they exert their oncogenic potential.

Regulation of viral and cellular oncogene expression by cytosine methylation

Mink cells morphologically transformed by either Snyder-Theilen feline sarcoma virus (ST-FeSV) or Abelson murine leukemia virus (Abelson-MuLV) exhibit relatively high rates of reversion to the nontransformed phenotype. The proviral DNAs are conserved within the revertant lines and have not undergone changes in integration sites due to translocations or other genomic rearrangements. In contrast, expression of well-defined viral-encoded transforming proteins is blocked and elevated levels of phosphotyrosine characteristic of the parental transformed cells are reduced to control levels. Loss of the transformed phenotype is associated with increased cytosine methylation of proviral DNA sequences while levels of methylation resume control levels upon spontaneous retransformation of revertant clones. Following molecular cloning, and transfection to Rat-2 cells, ST-FeSV proviral DNAs from revertant and transformed cells induced similar numbers of transformed foci. Cytosine methylation sites involved in regulation of expression of the major ST-FeSV encoded transforming protein have been localized within the proviral DNA itself rather than in adjacent cellular flanking sequences. In contrast to the v- fes proviral DNA, c- fes, the cellular homolog of the ST-FeSV acquired transforming sequences, is highly methylated in cytosine residues in both transformed and revertant clones. These findings demonstrate regulation of viral oncogene-mediated transformation by cytosine methylation and suggest that expression of cellular homologs of viral oncogenes, such as c- fes, are also subject to regulation at this level.

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.

Isolation and characterization of a human locus homologous to the transforming gene (v-fes) of feline sarcoma virus.

A single locus (designated c-fes) in the human genome which exhibits homology to the transformation-specific onc gene (v-fes) of Snyder-Theilen feline sarcoma virus was identified by the Southern blot technique. Recombinant clones containing 16- to 18-kilobase inserts of human DNA including the c-fes locus were constructed. Restriction endonuclease mapping of these clones verified their identity with native human c-fes and demonstrated the presence of at least two sequences in human c-fes interrupting v-fes-homologous regions. The v-fes-homologous locus in the human genome spans about 4 kilobases. The 5'-3' orientation of the c-fes clones with respect to feline sarcoma virus proviral DNA was determined. The region of the human genome that is homologous to v-fes is proximal to the highly reiterated human Alu sequence but not to the highly reiterated human alphoid sequence.

Isolation of human oncogene sequences (v-fes homolog) from a cosmid library.

To define the human homolog (or homologs) of transforming sequences (v-fes gene) common to Gardner (GA) and Snyder Theilen (ST) isolates of feline sarcoma virus (FeSV), a representative library of human lung carcinoma DNA in a cosmid vector system was constructed. Three cosmid clones were isolated containing GA/ST FeSV v-fes homologous cellular sequences, within 32- to 42-kilobase cellular inserts representing 56 kilobases of contiguous human cellular DNA. Sequences both homologous to, and colinear with, GA or ST FeSV v-fes are distributed discontinuously over a region of up to 9.5 kilobases and contain a minimum of three regions of nonhomology representing probable introns. A thymidine kinase selection system was used to show that, upon transfection to RAT-2 cells, the human c-fes sequence lacked detectable transforming activity.

Transformation with subgenomic fragments of feline sarcoma virus proviral DNA

Murine fibroblasts can be transformed by subgenomic fragments of feline sarcoma virus (FeSV) proviral DNA generated by BamH-I digestion of DNA of Snyder-Theilen FeSV and FeLV-infected mink cells. The efficiency of transformation of NIH 3T3 cells by BamH-I-treated FeSV proviral DNA was the same as that of untreated FeSV DNA. Focus-forming virus could not be rescued from the BamH-I-digested FeSV DNA-transformed cells. However, DNA from these transformed cells was able to induce transformation in secondary transfection experiments. Analysis of the DNAs from two BamH-I-digested FeSV DNA-transformed clones showed that the transformants contained only the 5′ two-thirds of the FeSV provirus. Cells transformed by subgenomic fragments of FeSV DNA produce the 85,000-dalton gag-x protein, but do not express FOCMA. These experiments support the notion that the gag-x protein plays an active role in maintenance of the transformed phenotype.