Production of plasminogen activator by established cell lines of mouse origin

The correlation between malignant transformation and increased plasminogen activator synthesis has been studied in a variety of established cell lines. In contrast to the behavior of secondary mouse embryo cultures, which always show increased fibrinolytic activity when transformed, no such correlation was found within the BALB/c 3T3 line and its transformed derivatives. Cell lines were established from tumors initiated in BALB/c mice by several transformed cell lines. These lines were generally found to contain no more plasminogen activator than the cells used for inoculation. A correlation was found between transformation and plasminogen activator synthesis within Swiss 3T3 cell lines. However, the correlation was not maintained by serum revertants of transformed Swiss 3T3 cells.     

Plasminogen activator in chick embryo muscle cells: induction of enzyme by RSV, PMA and retinoic acid.

To explore the generality of the effects of sarcoma viruses, tumor-promoting phorbol esters and retinoic acid, we have studied plasminogen activator production in differentiating chick myogenic cultures. Although slightly higher than in chick fibroblast cultures, the level of spontaneously synthesized enzyme is low; it reaches a peak shortly after maximum cell fusion has been completed and then declines. Rous sarcoma virus (RSV) transformation of differentiating myotubes was accomplished by infecting myoblasts with a temperature-sensitive mutant, maintaining cultures at the nonpermissive temperature until completion of fusion and shifting to permissive temperatures at selected times thereafter. RSV transformation, phorbol myristate acetate (PMA) and retinoic acid all induced high levels of plasminogen activator production by differentiating myotubes in the absence of DNA synthesis. In comparison with fibroblasts, virus-induced enzyme synthesis by myogenic cultures proceeded more slowly but ultimately reached comparably high levels. Whereas cAMP strongly repressed RSV- and PMA-induced plasminogen activator production by chick fibroblasts, it weakly stimulated enzyme synthesis by myotubes. This suggests that enzyme induction by RSV and PMA is not mediated primarily through effects on cAMP metabolism.     

Transformation phenotype of polyoma virus-transformed rat fibroblasts: plasminogen activator production is modulated by the growth state of the cells and regulated by the expression of an early viral gene function

The expression of two transformation parameters, namely, ability to grow in agar and plasminogen activator production, was studied in several rat fibroblasts transformed by either wild-type or thermo-sensitive (tsa and ts25) polyoma viruses. The production of plasminogen activator was found to be dependent upon the growth state of the infected cells during a period of several days after infection. The analysis of the transformed phenotype of 25 tsa transformants and of 19 ts25 transformants independently isolated under various growth conditions led to the conclusion that there is no correlation between the regulation processes involved in plasminogen activator production and ability to grow without anchorage. The results obtained also suggested that the production of plasminogen activator is under the control of a functional large T antigen.     

Cyclic AMP phosphodiesterase inhibitors depress production of plasminogen activator by Chinese hamster ovary cells.

Oncogenic transformation in a limited number of cell systems has been shown by others to be associated with an increased production of extracellular proteolytic activators that convert the plasma proenzyme, plasminogen, to the active protease, plasmin. In the present study, two cyclic AMP phosphodiesterase inhibitors (theophylline, papaverine) markedly depressed the production of intracellular and extracellular plasminogen activator by Chinese hamster ovary cells of the CHO-Kl line in serum-free medium. Prostaglandin E₁ had a moderately similar effect on the production of only extracellular plasminogen activator. The ability to control experimentally the level of production of plasminogen activator should be of value in elucidating the possible biological role of the proteolytic action of plasmin on the surface of CHO cells, and the cell surface alterations which accompany oncogenic transformation.     

Acetyl-LDL stimulates macrophage-dependent plasminogen activation and degradation of extracellular matrix

The ability of acetyl-LDL to stimulate macrophage-dependent plasminogen activation and degradation of extracellular matrix was examined. We have found that expression of plasminogen activator activity in response to the scavenger receptor ligand varied among cell populations. Exposure to acetyl-LDL stimulated plasminogen activator expression by cells which constitutively released low levels of activator. These include a virally transformed macrophage-like cell line (RAW246.7), concanavalin A and C. parvum-activated macrophages. The stimulation of plasminogen activator activity was independent of cellular lipid accumulation since nonlipoprotein inhibitors of acetyl-LDL binding to the scavenger receptor stimulated activator expression in great excess to that observed with acetyl-LDL. In contrast, acetyl-LDL was unable to induce soluble plasminogen activator activity in cells which normally do not express it. These include a macrophage-like cell line (J774A.1) and resident peritoneal macrophages. Furthermore, acetyl-LDL was unable to modulate the copious secretion of activator by inflammatory macrophages elicited with thioglycolate. When macrophages were tested for their ability to degrade smooth muscle cell derived matrix, solubilization by resident, elicited, and activated cells was variously increased in the presence of plasminogen. Furthermore, exposure to acetyl-LDL enhanced plasmin-dependent degradation by resident cells and activated cells, whereas matrix degradation by elicited cells was unaffected.     

Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides

Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface.     

Inhibition of a plasminogen activator from oncogenic virus-transformed mouse cells by rabbit antibodies against the enzyme

Antisera were raised in rabbits against an electrophoretically pure 48 000 dalton plasminogen activator from mouse cells transformed by an oncogenic virus. The IgG fraction of the antisera inhibited 48 000 dalton mouse plasminogen activators from a variety of sources (neoplastic and nonneoplastic), a 29 00) dalton plasminogen activator from mouse urine and a 48 000 dalton plasminogen activator from rat urine. No inhibition was observed of a 75 000 dalton plasminogen activator extracted from mouse lung, of mouse plasmin or of plasminogen activators from human urine and from oncogenic-virus transformed chicken cells. The IgG antibodies were stronger and more specific inhibitors of the 48 000 dalton mouse plasminogen activator than any previously tested compounds.     

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.     

Normal and malignant cells, including neurons, deposit plasminogen activator on the growth substrata

The results of four different assay methods showed that both normal and malignant plasminogen activator-secreting cells deposited substantial amounts of this protease on tissue-culture substrata, including collagen coatings. The cells studied were Rous sarcoma virus (RSV)-transformed vole fibroblasts, a malignant neural cell line (NG108-15) capable of neurite formation, and normal mouse-regenerating sensory neurons. Deposited plasminogen activator was detected by a fibrin overlay assay at sites from which cells growing on coverslips had been gently dislodged, showing that active enzyme is left beneath cells and in the immediate pericellular area. For neuronal cells, fibrinolytic zones were detected not only at the previous positions of cell bodies but also along the terrain conditioned by neurite extension, suggesting that a trail of plasminogen activator is left behind during growth cone movement. Substratum-bound enzyme could be solubilized in buffers containing sodium dodecyl sulfate (SDS) or Triton X-100 and demonstrated by zymography following electrophoresis or assayed for amidolytic activity with a chromogenic substrate (Kabi S-2251). The results suggest that plasminogen activator may be considered a component of substrate-adhesion material. Secretory proteases deposited directly on matrix molecules would seem strategically positioned to participate in local degradation of components of the extracellular environment.     

Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells.

Fibrin overlay methods are described which can detect the plasminogen activator produced by single transformed cells or small colonies of transformed cells. These methods were applied to malignant cells derived from humans, mice, hamsters, rats, and chicks. The lysis observed was plasminogen dependent. Transformation of chicken cells by Rous sarcoma virus was detected 4 days after infection. The number of lysis zones produced was proportional to the virus inoculum and was identical to the number of morphologically determined foci. These methods may also have application in model systems for scoring transformation by chemicals. Transformed mouse and chicken cells were detected at the single cell level and the number of lysis zones produced was dependent on the number of cells present, the time of incubation, and the concentration of plasminogen.     

Plasminogen activator from cells transformed by an oncogenic virus: inhibitors of the activation reaction.

This paper describes an assay for direct measurement of plasminogen activation and its application for determining the kinetic constants and for screening potential inhibitors of the reaction. The assay is based on the conversion of the single chain of 125I-labelled plasminogen to the two chains of 125I-labelled plasmin (EC 3.4.21.7), the latter then being separated from each other and from the plasminogen substrate by electrophoresis under reducing conditions in SDS-polyacrylamide gels. The Km of activator from transformed murine cells for human plasminogen was 180 nM. A broad range of compounds was tested as potential inhibitors of plasminogen activation and of plasmin-catalyzed fibrinolysis respectively, and the two reactions differed qualitatively and quantitatively in their response to previous agents. The principal qualitative difference was in the susceptibility of the reactions to a spectrum of naturally-occurring macromolecular inhibitors: all of the macromolecular inhibitors that blocked the action of plasmin were without effect on murine activator or human urokinase (EC 3.4.99.26). A variety of small molecules inhibited both of the reactions tested, and showed significant quantitative differences; some of these were active at micron concentrations. The exacting specificity of plasminogen activators for macromolecules, both substrates and inhibitors, encourages the expectation that effective inhibitors of great specificity may be isolated from as yet undiscovered natural sources.     

Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.     

Malignant transformation in Vitro of chinese hamster embryonic fibroblasts with the Schmidt-Ruppin strain of Rous sarcoma virus and karyological analysis of this process

Using a method of cocultivation of embryonic Chinese hamster cells (CHEF) with Rous sarcoma cells and infection of CHEF by RSV-SR, it was possible to obtain malignant transformation of hamster cells. The morphologically altered cells became apparent within 15–36 days. In the cells transformed by cocultivation, the genome of RSV was determined by the method of contact of the transformed cell and the chicken cell in vivo; the malignant character of the transformed cells was demonstrated by transfer to a homologous newborn host. Repeated attempts to detect virus production in transformed Chinese hamster cells failed. Prior to malignant transformation and in early transformed cultures the diploid stem-line was maintained. A slight decrease in the proportion of diploid cells in transformed cultures was revealed in some experiments and is discussed. Prolonged cultivation of these cells, as also of control fibroblasts, shifts the stem-line to the hyperdiploid or hypotetraploid region. The mechanism of malignant transformation by RSV is discussed with regard to the action of the viral genome and alteration of the genetic make-up of the cell by the virus.     

Decreased rate of S-adenosyl-L-homocysteine metabolism: an early event related to transformation in cells infected with Rous sarcoma virus.

An increase of methylase activity is often related to neoplastic transformation. SAH, the natural inhibitor of transmethylases, does not inhibit cell transformation induced by RSV, in contrast to one of its synthetic analogues, SIBA. This inefficiency was thought to be due to the rapid metabolism of SAH by transformed cells. We now show, that, on the contrary, 70 % of the added amount of SAH disappears in one hour in cell-free extracts of normal cell against only 14 % in extracts of transformed cells. This decreased rate of degradation occurred one day post infection. Cells infected with the non transforming RAV₁ degrade SAH at the same rate as normal cells. A decrease of SAH-hydrolase and adenosine deaminase activity was also observed in infected cells. The decrease of the first enzyme seems to be related to the transformed state, whereas that of the second enzyme seems to depend only on infection, since it is also observed in cells infected with RAV₁.     

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.     

Tissue-type plasminogen activator binding to human endothelial cells. Evidence for two distinct binding sites

The endothelium may contribute to fibrinolysis through the binding of plasminogen activators or plasminogen activator inhibitors to the cell surface. Using a solid-phase radioimmunoassay, we observed that antibodies to recombinant tissue-type plasminogen activator (rt-PA) and plasminogen activator inhibitor type 1 (PAI-1) bound to the surface of cultured human umbilical vein endothelial cells (HUVEC). HUVEC also specifically bound added radiolabeled rt-PA with apparent steady-state binding being reached by 1 h at 4 degrees C. When added at low concentrations (less than 5 nM), rt-PA bound with high affinity mainly via the catalytic site, forming a sodium dodecyl sulfate-stable 105-kDa complex which dissociates from the cell surface over time and which could be immunoprecipitated by a monoclonal antibody to PAI-1. rt-PA bound to this high affinity site retained less than 5% of its expected plasminogen activator activity. At higher concentrations, binding did not require the catalytic site and was rapidly reversible. rt-PA initially bound to this site retained plasminogen activator activity. These studies suggest that tissue-type plasminogen activator and PAI-1 are expressed on the surface of cultured HUVEC. HUVEC also express unoccupied binding sites for exogenous tissue-type plasminogen activator. The balance between the expression of plasminogen activator inhibitors and these unoccupied binding sites for plasminogen activators on the endothelial surface may contribute to the regulation of fibrinolysis.     

Stimulation of plasminogen activator production by dimethyl sulfoxide in Chinese hamster ovary cells

The production of plasminogen activator activity in an auxotrophic mutant of the Chinese hamster ovary cell line was found be greatly stimulated by low concentrations of dimethyl sulfoxide. The production of both cell-associated and excreted plasminogen activator activities was stimulated maximally by dimethyl sulfoxide at a concentration of 2.5%. The stimulation of plasminogen activator activity production was found to be completely inhibited by actinomycin D and cycloheximide but not by mitomycin C, implying that new protein and RNA syntheses were required for this process. Using specific antibodies against plasminogen activator, the presence of a tissue-type plasminogen activator could only be detected in dimethyl sulfoxide treated cells. The dimethyl sulfoxide induced plasminogen activator production was observed only in a mutant auxotrophic for adenosine, glycine, and thymidine but not in wild-type cells. The ability of dimethyl sulfoxide to induce the synthesis of plasminogen activator was lost when the cells were hybridized with another complementary auxotrophic mutant. This implies that the ability of dimethyl sulfoxide to stimulate the production of plasminogen activator may be related to the auxotrophic mutation in this cell.     

Hormonal regulation of membrane phenotype

Incubation of rat hepatoma cells (HTC) in tissue culture with glucocorticoids alters several membrane properties characteristic of transformed cells, without affecting the growth rate of these cells. Variant cell lines resistant to dexamethasone inhibition of plasminogen activator production have been isolated using an agar-fibrin overlay technique to detect plasminogen activator production by individual colonies of HTC cells. The resistance to dexamethasone is not secondary to abnormal or absent glucocorticoid receptors, but due to a lesion in a later step in hormone action specific for plasminogen activator. These variants should prove useful for the study of the mechanism of steroid action as well as for the analysis of the role of proteases in the hormonal regulation of membrane function.     

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.