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.     

5-Azacytidine inhibits adipocytic conversion of human skin fibroblasts by Kirsten murine sarcoma virus and dexamethasone

Transformation of human skin fibroblasts (SF) by the Kirsten murine sarcoma virus (KiMSV) is associated with their neodifferentiation into preadipose cells. Hydrocortisone or dexamethasone (DX) promote the transformation/neodifferentiation of preadipocytes to fully differentiated adipocytes. 5-Azacytidine (5-Aza-CR) by itself, or together with DX, or immediately after inoculation with KiMSV did not cause adipocytic conversion of human SF. Addition of 5-Aza-CR either before or immediately after exposure of human SF to KiMSV and DX reduced the total number of foci and significantly inhibited expression of adipocyte-containing foci. The results suggest that adipocytic conversion of human SF by KiMSV and DX is specific. This conversion apparently does not involve mechanisms directly related to the effects of 5-Aza-CR on the adipocytic conversion of murine embryo cells.     

Transformation-defective mutants of Snyder-Theilen feline sarcoma virus lack tyrosine-specific protein kinase activity.

Four phenotypically normal mink cell clones, each containing a transformation-defective provirus of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV), synthesized an 85,000-dalton viral polyprotein (P85) indistinguishable in size and antigenic complexity from that encoded by wild-type transforming ST-FeSV. An additional transformation-defective, ST-FeSV-containing flat cell clone produced a polyprotein of 88,000 daltons (P88). The viral polyproteins immunoprecipitated from cytoplasmic extracts of these cells lacked the tyrosine-specific protein kinase activity associated with the wild-type ST-FeSV gene product. In addition, the products encoded by representative transformation-defective ST-FeSV genomes were poorly phosphorylated in vivo and lacked detectable phosphotyrosine residues. Whereas proteins of ST-FeSV transformants contained elevated levels of phosphotyrosine, those of mink cells containing transformation-defective ST-FeSV exhibited phosphotyrosine levels no higher than those found in uninfected cells. These findings provide genetic evidence that the tyrosine-specific protein kinase activity associated with ST-FeSV P85 is required for virus-induced transformation.     

Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.     

Conversion of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus (ST:FeSV): tumoricidal potential in monolayers and in agar suspensions.

In earlier studies [1-3], we have demonstrated the conversion of human fibroblasts (HF) to tissue macrophages (TM) by the Snyder-Theilen feline sarcoma virus (ST:(FeSV)). The purpose of the present study is to determine the cytolytic potential of ST:FeSV(FeLV)-induced TM against tumorigenic target cells under defined conditions in vitro. The results show that ST:FeSV-induced TM, but not mock-infected HF, produced significant lysis of human colon adenocarcinoma cells (LS-180) after a 3-day preincubation period, followed by a 4-day coincubation period at an effector to target cell ratio of 5:1. The presence of IFN-gamma, or lipopolysaccharides (LPS), and especially of M-CSF, during the coincubation period generally yielded optimal lysis of the tumor cells. Addition of LS-180 specific antibody (NRCO-4) substantially increased the cytolytic potential of TM. Significantly, coincubation of TM with LS-180 tumor cells in an agar medium, where no direct contact between cells occurs, resulted in the inhibition of tumor cell proliferation. Addition of LPS has further accentuated this inhibition. The results indicate that ST:FeSV-induced macrophages are potent oncocytolytic agents of LS-180 tumor cells in the absence and in the presence of direct contact between effector and target cells.     

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.     

Snyder-Theilen feline sarcoma virus P85 contains a single phosphotyrosine acceptor site recognized by its associated protein kinase.

Cells nonproductively transformed by a variant of the Snyder-Theilen strain of feline sarcoma virus (FeSV) expressed an 85,000-dalton polyprotein (P85) with associated tyrosine-specific protein kinase activity. We identified within this polyprotein a single tyrosine acceptor site for its enzyme activity. This acceptor site, as well as two serine phosphorylation sites localized with the p12 structural component of Snyder-Theilen FeSv P85, was phosphorylated in cells nonproductively transformed by Snyder-Theilen FeSv. In contrast, infection by Snyder-Theilen FeSV transformation-defective mutants resulted in phosphorylation only of the two serine acceptor sites, indicating phosphorylation of the tyrosine acceptor site to be transformation specific. In addition, we describe in vitro labeling conditions, using unfractionated cell extracts, which resulted in preferential phosphorylation of the single Snyder-Theilen FeSV tyrosine-specific acceptor site.     

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.     

Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus.

The McDonough (SM), Gardner-Arnstein (GA), and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) code for high-molecular-weight polyproteins that contain varying amounts of the amino-terminal region of the FeLV gag gene-coded precursor protein and a polypeptide(s) of an as yet undetermined nature. The SM-FeSV primary translational product is a 180,000-dalton polyprotein which is immediately processed into a highly unstable 60,000-dalton molecule containing the p15-p12-p30 fragment of the FeLV gag gene-coded precursor protein and a 120,000-dalton FeSV-specific polypeptide. The GA- and ST-FeSV genomes code for polyproteins of 95,000 and 85,000 daltons, respectively, which in addition to the amino-terminal moiety (p15-12 and a portion of p30) of the FeLV gag gene-coded precursor protein also contain FeSV-specific polypeptides. However, the GA- and ST-FeSV polyproteins appear to be relatively stable molecules (half-lives of around 16 h) and are not significantly processed into smaller polypeptides. Immunological and biochemical analysis of each of the above FeSV translational products revealed that the sarcoma-specific regions of the GA- and ST-FeSV polyproteins are antigenically cross-reactive and exhibit common methionine-containing peptides. These findings favor the concept that these sarcoma-specific polypeptides are coded for by the similar subsets of cellular sequences incorporated into the GA- and ST-FeSV genomes during the generation of these transforming agents.     

Differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus.

Hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait, is associated with a predisposition to neoplasia. The present study describes the differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Primary cutaneous outgrowths were derived from normal appearing subepidermal biopsies of ACR phenotypes and appropriate controls. Exponentially growing cell cultures from ACR subjects and a portion of the clinically asymptomatic ACR progeny subjected to the viral probe were 100-1000 fold more susceptible to transformation than were normal skin fibroblast cultures. The virally transformed human skin fibroblasts showed a loss of anchorage dependency in carboxymethylcellulose suspension and formed tumors in athymic mice. The results suggest that skin fibroblasts obtained from individuals gine sarcoma virus.     

Hyaluronan uptake by adult human skin fibroblasts in vitro

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.     

Cellular transformation by subgenomic feline sarcoma virus DNA

The genome of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) is a 4.3-kilobase-pair (kbp) RNA molecule that contains a 1.5-kbp cellular insertion (fes gene) flanked by feline leukemia virus sequences at its 5' end (1.6 kbp) and 3' end (1.2 kbp) (Sherr et al., J. Virol. 34:200-212, 1980). DNA transfection techniques have been utilized to determine the regions of the ST-FeSV genome involved in malignant transformation. I have found that the 3.7-kbp 5'-end fragment of the ST-FeSV provirus (which corresponds to the 3.4-kbp 5'-end fragment of the viral genome) is sufficient to transform NIH/3T3 fibroblasts. Enzymes that cleave the ST-FeSV provirus DNA within the feline leukemia virus gag gene sequences or within the fes gene abolished the transforming activity. Preservation of the proviral large terminal repeats was also required for transformation. Transformed NIH/3T3 cells obtained by transfection of total or subgenomic ST-FeSV DNA expressed normal levels of the ST-FeSV gene product ST P85 and of its associated protein kinase activity. Furthermore, these cells contained high levels of phosphotyrosine residues, a biochemical marker associated with cellular transformation induced by certain retroviruses including ST-FeSV. These results, taken together, strongly support the concept that only those ST-FeSV proviral sequences necessary for ST P85 expression are involved in malignant transformation.     

Conversion of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus (ST:FeSV) is associated with the de-novo expression of IL-1 alpha, IL-1 beta, IFN-alpha, TNF-alpha, GM-CSF, and CD4.

In an earlier study, we have demonstrated the conversion of human fibroblasts (HF) to tissue macrophages (TM) by the Snyder-Theilen feline sarcoma virus (ST:(FeSV)) [1]. The present study shows that conversion of cultured HF by the ST:FeSV to TM resulted in the de-novo expression of interleukin-1 alpha, IL-1 beta, interferon-alpha, tumor necrosis factor-alpha, granulocyte-macrophage colony stimulating factor, and CD4. The conversion of HF to TM was also associated with increased expression of non-specific esterases as well as increased amount of ingested lipid material by the TM. Clonotypic and organotypic analyses of cells infected with the ST:FeSV(FeLV) showed a similar degree of conversion to TM among eleven individual clones of skin fibroblasts, and among fibroblasts obtained from eight different organs. These findings bear on the origin (heterogeneity) of TM, the nature of TM-induced cytokines, and the potential role of ST:FeSV-recruited TM during immune reactions in vivo.     

Involvement of a high-molecular-weight polyprotein translational product of Snyder-Theilen Feline sarcoma virus in malignant transformation

The previously described high-molecular-weight polyprotein major translational product of the Snyder-Theilen strain of feline sarcoma virus (FeSV) was shown to possess protein kinase activity with specificity for tyrosine acceptor sites. Cells transformed by Snyder-Theilen FeSV exhibited constitutively elevated levels of phosphotyrosine and a concomitant reduction in epidermal growth factor (EGF) binding sites. By endpoint cloning in microtiter plates, a number of transformation-defective (tf) mutants of the Snyder-Theilen strain of FeSV were isolated. Mink cells nonproductively infected by such mutants were morphologically nontransformed, failed to grow in soft agar, bound EGF as efficiently as control mink cells, and lacked rescuable transforming virus. Although the level of expression of the major viral polyprotein translational product in td mutant-infected clones was comparable to that of wild-type (wt) transformants, the polyprotein in mutant clones lacked detectable protein kinase activity and total cellular phosphotyrosine levels were not elevated significantly above control values. Of a large number of wt Snyder-Theilen FeSV-transformed mink cell clones isolated, the majority were found to revert to a nontransformed morphology upon continuous passage in cell culture. Such nontransformed variants, as well as a Gardner FeSV-transformed mink cell revertant, lacked detectable polyprotein expression and exhibited levels of phosphotyrosine and EGF binding similar to those of control mink cells. These findings provide strong evidence favoring the involvement of the Snyder-Theilen FeSV-encoded high-molecular-weight polyprotein and its associated tyrosine-specific protein kinase activity in transformation.     

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.     

Differences in mechanisms of transformation by independent feline sarcoma virus isolates.

The Gardner and Snyder-Theilen isolates of feline sarcoma virus (FeSV) have previously been shown to encode high-molecular-weight polyproteins with a transforming function and an associated tyrosine-specific protein kinase activity. Cells transformed by these viruses exhibited morphological alterations, elevated levels of phosphotyrosine, and a reduced capacity for binding epidermal growth factor. In addition, polyproteins encoded by both of these FeSV isolates bound to, and phosphorylated tyrosine acceptor sites within, a 150,000-molecular-weight cellular substrate (P150). McDonough FeSV-transformed cells resembled Gardner and Snyder-Theilen FeSV transformants with respect to morphological changes and a reduced capacity for epidermal growth factor binding. in contrast to the other two FeSV isolates, however, McDonough FeSV encoded as its major translational product a high-molecular-weight polyprotein with probable transforming function but without protein kinase activity detectable under similar assay conditions. Moreover, total cellular levels of phosphotyrosine remained unaltered in McDonough FeSV-transformed cells, and the major McDonough FeSV polyprotein translational product lacked binding affinity for P150. These findings argue for differences in the mechanisms of transformation by these independently derived FeSV isolates.     

Hydrocortisone promotes the neodifferentiation of Kirsten murine sarcoma virus transformed human skin fibroblasts to adipose cells: relevance to oncogenic mechanisms

Here we have demonstrated that transformation of human skin fibroblasts (SF) by the Kirsten murine sarcoma virus (KiMSV) is associated with their neodifferentiation into preadipose cells. Hydrocortisone (HC) promotes the transformation/neodifferentiation of such preadipocytes into mature fat cells. The effects of HC on the expression of adipocyte-containing foci and on the total number of transformed foci present in KiMSV-treated cultures appeared to be dose-dependent and was optimal at a concentration of about 500 ng/ml, or 1.25 X 10(-6) M. Although increasing serum concentrations (2-15%) increased the total number of transformed foci, it had no effect on the expression of adipocyte-containing foci in the presence of HC. The virus-induced preadipocytes undergoing partial conversion in the presence of HC were capable of clonal expansion and extensive proliferative activity. In contrast, mature adipocytes were terminally differentiated and as such have lost their ability to proliferate. The results suggest a role for a ras oncogene and HC in the transformation/neodifferentiation of human cells that might ultimately lead to cancer in some fraction of such cells.     

Collagenase production by human skin fibroblasts.

Normal human skin fibroblasts, when cultured in serum free medium, produce collagenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme.