Rous-sarcoma-virus-transformed fibroblasts having low levels of plasminogen activator.

Several clones of transformed chick embryo fibroblasts infected with wild-type B77-C or Prague-C strain of Rous sarcoma virus have been isolated from soft agar suspension. These clones were screened for plasminogen activator activity by overlaying monolayer cultures with medium containing agar, casein, and chicken plasminogen. Twenty-three percent of all of the isolated clones showed little caseinolytic activity, 42% had intermediate activity, and 35% had high activity. Although the clones with low plasminogen activator activity had no more than twice the activity shown by uninfected fibroblasts, they did not differ significantly from clones possessing high levels of plasminogen activator in their morphology, 2-deoxyglucose transport, or efficiency of colony formation in soft-agar.     

Plasminogen Activator Production Accompanies Loss of Anchorage Regulation in Transformation of Primary Rat Embryo Cells by Simian Virus 40

We have isolated several lines of rat embryo cells transformed by simian virus 40. All these lines are fully transformed with regard to saturation density and serum sensitivity, but they differ greatly in their anchorage dependence, as assayed by efficiency of plating in methyl cellulose suspension. This set of lines reveals a consistent relation of plasminogen activator production to plating efficiency in methyl cellulose. T-antigen-positive transformed lines that synthesize activator grow in methyl cellulose suspension, while T-antigen-positive transformed lines that do not synthesize activator fail to form colonies in suspension. Normal rat embryo cells produce very little plasminogen activator and do not grow in methyl cellulose. Sera that permit high levels of plasmin formation and activity support growth in semi-solid medium better than sera whose plasminogen is activated poorly and/or sera that contain inhibitors to plasmin.     

src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

Sera from certain rabbits bearing Schmidt-Ruppin strain Rous sarcoma virus (RSV)-induced tumors precipitated p60(src) from chicken cells transformed by the homologous virus as well as by other strains [Prague strain RSV, Bryan high-titer strain RSV, and Bratislava 77 strain of avain sarcoma virus (ASV)], the molecular weights (M(r)s) ranging from 60,000 to 64,000. The p60(src) immunoprecipitated from cells transformed by each of these strains incorporated [gamma-(32)P]ATP into the M(r) 53,000 subunit of IgG, though with differing activities. No such protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was observed when the following immunoprecipitates were used: from uninfected cells, from untransformed cells infected by Rous-associated virus, or from cells transformed by acute leukosis viruses, avian erythroblastosis virus, or myelocytoma virus 29. The kinase reaction had a pH optimum at pH 5.9 and an apparent K(m) for ATP of 4.9 +/- 2 muM, and was dependent on Mg(2+) (K(b) = 46 +/- 12 mM), for which Ca(2+) was no substitute. The kinase was cyclic AMP independent.In order to test whether the protein kinase reaction is directly catalyzed by p60(src), we compared the in vitro temperature sensitivities of the kinase activities from cells infected by transformation-temperature-sensitive mutant and parental wild-type virus. The first-order rate constant for the inactivation of the kinase from extracts of cells infected by the mutant virus was 2-fold greater than that from cells infected by wild-type virus. This result implicates the protein kinase as an enzymatic activity of the src gene product, the p60(src). Concomitant with the loss of the kinase activity by heat inactivation, p60(src) loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p60(src) kinase is itself dependent on phosphorylation for its activity.     

Metastasizing fibrosarcomas in hamsters (BMH) after injection of B77 sarcoma virus transformed mouse cells

Injection of virogenic mouse cells B77-1026 into newborn Syrian hamsters resulted in arising of progressively growing autochthonous fibrosarcomas. From hamster tumors five stable tumor cell lines (BMH/1--BMH/5) were established in vitro. All cells of the newly established tumor cell lines had hamster karyotype, they were able to grow in soft agar and did not contain rescuable B77 viral genome. BMH tumor cells injected into syngeneic newborn as well as young adult hamsters produced tumors at the site of application and metastasized frequently into viscera. From metastases in different organs further tumor cell lines and single cell clones were established in vitro. All these tumor cell lines and clones exhibited higher metastatic capacity than the parent cell lines.     

Infection of human embryo cells with avian sarcoma virus B77 in vitro.

Human embryo cells were infected with avian sarcoma virus B77--Hu(B77). The virus genome was detected in the Hu(B77) cells during subsequent cell passages in the uncloned cells as well as in single-cell clones. Despite the presence of the virus genome in cells, the growth properties did not differ from uninfected cells. The Hu(B77) cells did not reveal the transformed phenotype in vitro.     

Detection and partial characterization of a chymostatin-sensitive endopeptidase in transformed fibroblasts.

A chymostatin-sensitive step in the release of plasminogen activator from transformed fibroblasts has been described recently. By using synthetic peptidyl substrates, we have detected and characterized a chymostatin-sensitive peptidase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus. The activity represents a neutral endopeptidase that exhibits phenylalanine specificity and is inhibited by diisopropyl fluorophosphate. A detailed inhibitor profile of the enzyme activity shows that it is distinct from other chymotrypsin-like phenylalanine-preferring peptidases. The endopeptidase activity in transformed fibroblasts is increased over that of parallel cultures of normal fibroblasts. The mechanism of enzyme inhibition by chymostatin is indicated by these studies, and the possible role of the enzyme in modulating plasminogen activator secretion is discussed.     

Microinjection analysis of envelope-glycoprotein messenger activities of avian leukosis viral RNAs.

Virion RNA from the avian leukosis virus Rous-associated virus 2 (RAV-2) and poly(A)-containing RNAs from RAV-2-infected chick embryo fibroblasts were microinjected into fibroblasts transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV), which is deficient in viral envelope glycoprotein. Production of infectious RSV following these injections depended upon the viral envelope-messenger activity of the injected RNA. This system constituted a sensitive and rigorous assay system for viral envelope-messenger RNA. It was found that 21S mRNA from RAV-2-infected cells expressed the highest activity, while 35S mRNA expressed comparatively little. In addition, RAV-2-virion RNA expressed little messenger activity. The rate of formation of infectious RSV following 21S mRNA injections reached a peak near 9 hr, which was followed by a rapid decline. Evidence has been obtained that a small fraction of both 35S virion RNA and 35S mRNA from virus-infected cells was encapsulated into virus particles following their injection into virus-producing cells.     

Isolation of defective mutant of avian sarcoma virus

A colony of transformed cells was isolated from chick-embryo cells infected with a stock of nondefective Schmidt-Ruppin strain of Rous sarcoma virus. The virus recovered from this colony was a stable defective mutant very similar to the Bryan strain of Rous sarcoma virus in the following characteristics: (i) noninfectiousness of virus particles released from transformed cells that lack helper factor; (ii) formation of infectious pseudotypes by coinfection with avian leukosis virus or by interaction with endogenous-helper factor in chicken cells; (iii) ability of the noninfectious form of virus to transform chick-embryo cells in the presence of ultraviolet light-inactivated Sendai virus; (iv) absence of glycoprotein in the noninfectious form; (v) failure to produce nondefective virus by recombination with avian leukosis virus; and (vi) segregation of polymerase-negative virus.The morphology of transformed cells is characteristic of those infected by the Schmidt-Ruppin strain. The demonstration of segregation of such a defective virus from nondefective sarcoma virus and failure to detect revertants of this mutant suggest that the deletion of some genes may be involved in this mutation.     

Plasminogen-independent fibrinolysis by proteases produced by transformed chick embryo fibroblasts.

The fibrinolytic activity of proteases secreted by chick embryo fibroblasts infected with Rous sarcoma virus was studied by use of a procedure in which a fibrin clot was formed with highly purified fibrinogen and thrombin above the cell layer. This procedure results in the formation of fibrin that is apparently a more suitable substrate for studies on fibrinolysis than is fibrin prepared by other methods. Since neither plasminogen nor serum were included in the assay system in the present studies, the fibrinolytic activity observed cannot be ascribed to the conversion of the plasminogen in serum to plasmin by a plasminogen activator produced by transformed cells. Our procedure, therefore, measures proteolytic activities other than those reported by previous investigators. Maintenance of some of the transformed phenotypes of Rous sarcoma virus transformed chick embryo fibroblasts such as morpholigical change and increased rate of glucose uptake apparently does not depend on the presence of plasminogen in the culture medium.     

Product of the src gene is not phosphorylated in avian sarcoma virus B77 infected but untransformed human cells

Human embryo cells infected with the avian sarcoma virus B77 [Hu(B77)] but untransformed, contain the whole rescuable virus genome integrated in the host cell DNA. The cellular DNA induced the transformation of the infectious B77 virus after transfection of chicken cells. In the Hu(B77) cells the src gene product was expressed as a 58 kD protein which possessed phosphokinase activity but was not phosphorylated in comparison with the src production of B77 transformed rat cells RBI in which p60src is expressed. The addition of vanadate to tissue culture fluid reversibly elicited cell transformation in both control and virus infected human cells, but did not influence phosphorylation of the src gene product. The secretion of phosphoproteins in the investigated cells was different. Whereas transformed rat cells released a 62 kD transformation related phosphoprotein, the human cells did not. In tissue culture fluid from Hu(B77) cells an elevated amount of a 22 kD phosphoprotein was found in comparison with control cells. The implications of these findings are discussed with respect to the role of the v-src gene product in malignant cell transformation.     

Protease-activated “prodrugs” for cancer chemotherapy

Many types of malignant cells and human tumors display increased concentrations of the protease plasminogen activator that converts plasminogen to the highly active protease, plasmin. Because plasmin rapidly cleaves various low molecular weight compounds coupled to appropriate peptide specifiers, we hypothesized that coupling of such peptide specifiers to anticancer drugs might create “prodrugs” which would be locally activated by tumor-associated plasmin and consequently would be less toxic to normal cells. To provide an initial test of this concept we have synthesized peptidyl prodrugs of the structure D-Val-Leu-Lys-X in which the peptidyl portion has been designed to allow the prodrug to serve as an excellent plasmin substrate and X is an anticancer drug—either the glutamine analog (αS,5S) α-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid (AT-125) or the alkylating agent N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard). Treatment of these prodrugs with plasmin generated the free peptide and the free drug, demonstrating that these prodrugs are plasmin substrates. The prodrugs and free drugs were tested in an in vitro system against either normal chicken embryo fibroblasts, which display a low level of plasminogen activator, or their virally transformed counterparts, which produce high levels of plasminogen activator. In each case the peptidyl prodrugs displayed at least a 5-fold increase in selectivity for the transformed cells compared to the free drug. The greater selectivity of action of the peptidyl prodrugs against transformed cell cultures suggests that these or similar prodrugs that are substrates for tumor-associated proteases may show increased therapeutic effectiveness in the treatment of tumors that produce sufficiently increased amounts of plasminogen activator.     

A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus.

The src genes of four natural isolates of avian sarcoma viruses differ from cellular proto-src in two genetic substitutions: the promoter of the cellular gene is replaced by a retroviral counterpart, and at least six codons from the 3' terminus are replaced by retroviral or heterologous cell-derived elements. Since virus constructs with a complete proto-src coding region failed to transform avian cells but acquired transforming function by point mutations of various codons, it has been proposed that point mutation is sufficient to convert proto-src to a transforming gene. However, promoter substitution is sufficient to convert two other proto-onc genes, proto-ras and proto-myc, to retroviral transforming genes. In view of this, we have reexamined whether promoter substitution, point mutation, or both are necessary to convert proto-src into a retroviral transforming gene. It was found that a recombinant virus (RpSV), in which the src gene of Rous sarcoma virus (RSV) was replaced by the complete coding region of proto-src, transformed quail and chicken embryo cells. The oncogene of RpSV differs from the src gene of RSV in three genetic properties: (i) it is weaker--e.g., transformed cells are flatter; (ii) it is slower--e.g., focus formation takes 9 to 12 days compared to 4 days for RSV; and (iii) its host range is narrower than that of RSV--e.g., only subsets of heterogeneous embryo cells are transformed by RpSV even after weeks or months. Replacement of the proto-src 3' terminus of RpSV by that of src from RSV generates a recombinant virus (RpvSV) that equals RSV in transforming function. It is concluded that a retroviral promoter, naturally substituted via illegitimate recombination with retroviruses, is sufficient to convert at least three proto-onc genes, src, myc, and ras, to retroviral transforming genes.     

v-src induces clonal sarcomas and rapid metastasis following transduction with a replication-defective retrovirus.

v-src is an effective carcinogen when expressed from Rous sarcoma virus (RSV) in vivo. Whereas RSV tumors require sustained oncogene expression, their growth is largely a balance between viral recruitment of tissues and host immune destruction of infected cells. We have therefore examined the tumorigenic potential of v-src in the absence of viral recruitment and viral antigen expression. v-src was introduced with high efficiency into chicken wing web tissues using replication-defective (rd) retroviral vectors. Clonal sarcomas were induced rapidly, and, furthermore, v-src potentiated metastatic progression in approximately 0.1%-1% of tumor clones with unexpectedly short latency. rd vectors proved effective not only in transducing v-src into tissues but also as insertional markers of tumor clonality. The rd vector present in most primary and metastatic tumors was a highly truncated form of RSV derived by viral transmission of spliced v-src mRNA; this vector should thus avoid viral recruitment and host anti-viral immune reaction through its complete lack of viral structural genes. Under such conditions v-src maintains strong carcinogenicity in vivo when restricted to clonal tumor growth and can confer rapid metastatic potential on a discrete subset of tumor clones.     

Transformation-dependent activation of urokinase-type plasminogen activator by a plasmin-independent mechanism: involvement of cell surface membranes.

Transformed cells produce elevated levels of the urokinase-type plasminogen activator (u-PA), which has been linked with the invasive or migratory phenotype of these cells. The u-PA is secreted and normally maintained in the inactive, single-chain form (scu-PA) and it has been assumed that natural activation occurs via a plasmin-mediated cleavage converting scu-PA to the active, two-chain form (tcu-PA). We now demonstrate that secreted scu-PA in Rous sarcoma virus-transformed chicken embryo fibroblast (RSVCEF) cultures is activated by an endogenous, plasmin-independent mechanism. Normal CEFs and CEFs infected with a temperature-sensitive RSV mutant and incubated at the nonpermissive temperature do not activate scu-PA. Conditioned medium harvested from plasmin-free cultures of RSVCEFs contains active tcu-PA as determined by two independent methods. The scu-PA is progressively converted with time in culture and requires the presence of intact cells or a plasma membrane-enriched fraction. When added to RSVCEF cultures, a synthetic peptide corresponding to residues 20-41 of the growth factor domain of chicken u-PA blocks the conversion to tcu-PA, and scu-PA accumulates in the cultures. These results suggest that scu-PA is secreted by cells, becomes bound to a u-PA receptor, and is proteolytically converted to active tcu-PA by a catalytic mechanism on the surface of RSV-transformed fibroblasts.     

Neoplastic transformation of mouse embryo cells by virus released from tumorigenic mouse cells transformed by avian sarcoma virus B77.

Mouse C3H embryo cells were transformed in vitro by avian sarcoma virus Bratislava 77 (B77) released scantily from a mouse cell line transformed earlier by the same virus. B77 virus transformed C3H embryo cells contained B77 viral genome and were transplantable into syngeneic as well as allogeneic DBA/2J young mice in which autochthonous sarcomas were induced. Tumors in both strains of mice were virogenic. The probable reasons for an increased transformation capacity of B77 virus in mammals are discussed.     

Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma.

The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic potential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic potential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks thereafter and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.     

Differential effects of transforming avian RNA tumor viruses on avian macrophages

Functionally differentiated chicken macrophages were derived by in vitro differentiation of embryonic yolk sac cells and were characterized by several macrophage-specific cell markers. Uniform, infected, virus-producing cultures were obtained after exposure of these macrophages to avian myoblastosis virus (AMV), avian myelocytomatosis virus (MC29), myeloblastosis-associated virus (MAV-2), and Prague strain of Rous sarcoma virus (PR-B RSV). Both AMV and MC29 induced morphological transformation typical of the in vivo leukemias induced by these virus strains. Analysis of the expression of macrophage-specific markers in these two transformed cell types demonstrated that different markers of the mature macrophage were suppressed by each virus, even though the parental cell immediately preceding the transformation event was a mature macrophage in both cases. Cells infected with PR-B RSV and MAV-2 showed no observable difference from uninfected macrophages in terms of morphological characteristics, growth rate, or expression of the differentiated functions of macrophages. Ths system provides demonstrations of a cell type that produces infectious, transforming RSV but fails to respond by functional alterations induced by the transforming gene, src.     

Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells

The late assembly (L) domain of retrovirus Gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate its function through interaction with unknown cellular factors. Here, we report the identification of the Nedd4-like family of E3 ubiquitin protein ligases as proteins that specifically interact with the Rous sarcoma virus (RSV) L domain in vitro and in vivo. We screened a chicken embryo cDNA expression library by using a peptide derived from the RSV p2b sequence, isolating two unique partial cDNA clones. Neither clone interacted with a peptide containing mutations known to disrupt in vivo RSV L domain function or with human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) L domain-derived peptides. The WW domain region of one of the clones, late domain-interacting protein 1 (LDI-1), but not the C2 domain, bound RSV Gag and inhibited RSV Gag budding from human 293 cells in a dominant-negative manner, functionally implicating LDI-1 in RSV particle budding from cells. RSV Gag can be coimmune precipitated from cell extracts with an antisera directed at an exogenously expressed hemagglutinin (HA)-tagged LDI-1 or endogenous Nedd4 proteins. These findings mechanistically link the cellular ubiquitination pathway to retrovirus budding.