Localization and modulation of galactosyltransferase during in vitro proliferation of a human ovarian adenocarcinoma cell line

A cell line, IGROV1, originating from a human ovarian cancer, releases a galactosyltransferase activity in its culture medium during proliferation. The proliferating IGROV1 cells appear as two populations: some cells grow in floating clusters whereas the greater part of them adhere to the culture substrate. The study of galactose transfer by intact cells onto an exogenous glycoprotein acceptor (ovomucoid) shows the presence of surface-associated galactosyltransferase onto the two cellular sub-populations. Opposite to intracellular activity, surface-associated and released galactosyltransferase activities depend on cellular adhesion and proliferation.     

Estimation of cell surface associated protease activity and its application to lymphocytes

A new method has been developed to estimate proteolytic activity available at the cell surface. Radioiodinated protein substrates are covalently linked to modified polystyrene-divinylbenzene beads with various diameters. These beads are presented to viable cells. Secreted enzyme activity is estimated when no contact occurs between beads and cells. Surface associated proteolytic activity is estimated by the increased rate of iodinated peptide release due to a contact between beads and cells. This method was applied to various lymphocyte preparations. In the absence of serum, mouse spleen lymphocytes produce three- to fourfold higher proteolytic activity than lymph node cells. This activity is completely inhibited by serum diluted 1:10. Since the proteolysis is so marked in the case of spleen cells, one must conclude that lymphocytes removed from the serum and treated in buffered mediums at 37° C have enzymatically altered surface properties. Cell surface associated enzyme activity was measured using rat lymph node lymphocytes with less than 0.1% contamination by granulocytes. This predominantly thymus derived, T cell population had 30% increase in proteolysis due to contact between cells and solid-phase localized substrate of casein. The released enzymatic activity was inhibited by diisopropylfluorophosphate, but its effect on the surface associated enzyme activity remains questionable since it perturbs several membrane functions.     

In vivo invasion of modified chorioallantoic membrane by tumor cells: the role of cell surface-bound urokinase

The ability of the chick embryo chorioallantoic membrane (CAM) to withstand invasion by tumor cells can be intentionally compromised by altering its morphological integrity. Using a newly developed quantitative assay of invasion we showed that intact CAMs were completely resistant to invasion by tumor cells, wounded CAMs did not pose a barrier to penetration, and CAMs that were wounded and then allowed to reseal displayed partial susceptibility to invasion. The invasion of resealed CAMs required catalytically active plasminogen activator (PA) of the urokinase type (uPA); the invasive efficiency of tumor cells was reduced by 75% when tumor uPA activity or tumor uPA production was inhibited. The invasive ability of human tumor cells, which have surface uPA receptors but which do not produce the enzyme, could be augmented by saturating their receptors with exogenous uPA. The mere stimulation of either uPA or tissue plasminogen activator production, in absence of binding to cell receptors, did not result in an enhancement of invasiveness. These findings suggest that the increased invasive potential of tumor cells is correlated with cell surface-associated proteolytic activity stemming from the interaction between uPA and its surface receptor.     

Lipoprotein lipase release from BFC-1 beta adipocytes. Effects of triglyceride-rich lipoproteins and lipolysis products.

Lipoprotein lipase (LPL), synthesized by adipocytes and myocytes, must be transported to the luminal endothelial cell surface where it then interacts with circulating lipoproteins. The first step in this extracellular LPL transport pathway is LPL release from the surface of LPL-synthesizing cells. Because hydrolysis of triglyceride (TG)-rich lipoproteins releases LPL from the apical surface of endothelial cells, we hypothesized that the same substances dissociate LPL from adipocytes. 125I-LPL was bound to the surface of brown adipocytes (BFC-1 beta). LPL binding to the adipocyte surface was greater than to endothelial cell surfaces. Using low concentrations of heparin, more LPL was released from endothelial cells than BFC-1 beta, suggesting that the affinity of LPL binding to the adipocytes was greater than LPL affinity for endothelial cells. Greater than 3-fold more LPL was released from the cell surface when very low density lipoproteins (VLDL) were added to culture medium containing 3% bovine serum albumin. LPL remaining on the cell surface decreased with VLDL addition. Endogenously produced LPL activity was also released from the cells by VLDL. Low and high density lipoproteins did not release 125I-LPL or LPL activity from the adipocytes. To assess whether lipolysis was necessary for LPL release, BFC-1 beta were incubated with TG-rich lipoproteins from a patient with apoCII deficiency. The apoCII-deficient lipoproteins did not release LPL unless an exogenous source of apoCII was added. Apolipoproteins E and Cs and high molar ratios of oleic acid:bovine serum albumin did not release surface-associated LPL. Lysolecithin (25 and 100 microM), but not lecithin, monoglycerides, or diglycerides, released adipocyte surface LPL. Because lysolecithin also released LPL during a 4 degrees C incubation, cellular metabolic functions are not required for LPL dissociation from the cells. Lysolecithin also inhibited LPL binding to endothelial cells; however, this effect was abrogated by addition of bovine serum albumin. We hypothesize that lipolysis products from TG-rich lipoproteins release adipocyte surface LPL, which can then be transported to the luminal endothelial cell surface.     

The mechanism of human NK cell-mediated cytotoxicity. Mode of action of surface-associated proteases in the early stages of the lytic reaction

The possible involvement of cell surface-associated proteolytic enzymes in human NK cell-mediated cytotoxicity and the mechanism by which such enzymes exert their activity have been studied. The treatment of intact cells with 3H-DFP under restricted conditions that predominantly bind surface-associated enzymes resulted in the labeling of five to six enzyme bands. Among these were a 35,000-dalton enzyme, which may be a previously identified trypsin-like proteinase engaged in cytotoxicity, and a 58,000-dalton elastase. The latter seems not to be involved in the reaction, as potent inhibitors of this enzyme have negligible effect on cytotoxicity. Of the membrane-associated enzymes, those engaged in cytotoxicity seem to be concealed from the external environment, as pretreatment of the effector cells with protease inhibitors such as trasylol and PMSF have no effect on the reaction. Immediately upon addition of the target cells and the initiation of cytotoxicity, the reaction becomes highly sensitive to inhibitors for a limited time interval of 2 to 5 min when trasylol is employed and 5 to 10 min when TPCK is the inhibitor, suggesting that target cell binding triggers the exposure of the enzymes to the external environment, rendering them accessible to the inhibitors. This short sensitivity period parallels the interval in which the reaction is sensitive to the microfilament inhibitor cytochalasin B. As the reaction proceeds, it becomes increasingly resistant to inhibitors of both proteolysis and cytoskeleton, at the same time suggesting that microfilament action and the unraveling of proteases may be processes that bear a close linkage with one another. The surface-associated elastase on the other hand maintains a constitutive mode of activity distinctive and unrelated to that of enzymes engaged in cytotoxicity. These findings suggest the existence on the surface of the NK lymphocyte of a mechanism that associates the receptor for target cells with an array of enclaved proteolytic enzymes via microfilaments. The resting cytotoxic structures become activated as the receptor attaches to the target cell, triggers the exposure of the proteolytic moiety, and initiates the lytic phase of the reaction.     

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity

Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity.     

Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII

Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.     

Selective inhibition of neutrophil activation by the subendothelial extracellular matrix: Possible role in protection of the vessel wall during diapedesis

Mobilization of circulating neutrophils toward an inflamed area involves adherence of the cells to the vascular endothelium and subsequent penetration through the endothelial cell layer without causing significant damage. To investigate the nature of a possible protective mechanism, granulocytes were incubated with the extracellular matrix (ECM) produced by cultured endothelial cells and tested for release of enzymes, chemoattractants, and free oxygen radicals. In the absence of exogenously added stimuli, the neutrophils adhered to the ECM but there was no detectable release of lysozyme, chemotactic activity, or production of O2-. In contrast, the cells readily released a heparan sulfate-degrading endoglycosidase (heparanase) to an extent comparable with that released in contact with polystyrene surfaces. Neutrophils treated with the calcium ionophore A23187 or with the peptide FMLP produced O2- to a much lesser degree when incubated in contact with ECM-coated surfaces than did those incubated in contact with uncoated polystyrene culture dishes. The ECM itself was devoid of superoxide dismutase activity. Stimulation with opsonized zymosan was not inhibited by the ECM. Experiments with isolated constituents of the ECM revealed that fibronectin but not collagen type IV or laminin could partially inhibit O2- production by Ca2+ ionophore-stimulated neutrophils. Treatment of the ECM with proteolytic enzymes, but not with heparanase, abolished its inhibitory effect on neutrophil activation. These results indicate that the subendothelial basement membrane has the capacity to inhibit release of potentially noxious agents excluding heparanase, suggesting a preferential involvement of this enzyme in neutrophil diapedesis.     

Release of cell surface-associated basic fibroblast growth factor by glycosylphosphatidylinositol-specific phospholipase C.

Heparan sulfate proteoglycans (HSPG) are ubiquitous constituents of mammalian cell surfaces and most extracellular matrices. A portion of the cell surface HSPG is anchored via a covalently linked glycosyl-phosphatidylinositol (Pl) residue, which can be released by treatment with a glycosyl-Pl specific phospholipase C (Pl-PLC). We report that exposure of bovine aortic endothelial and smooth muscle cells to Pl-PLC resulted in release of cell surface-associated, growth-promoting activity that was neutralized by antibasic fibroblast growth factor (bFGF) antibodies. Active bFGF was also released by treating the cells with bacterial heparitinase. Under the same conditions there was no release of mitogenic activity from cells (BHK-21, NIH/3T3, PF-HR9) that expressed little or no bFGF, as opposed to Pl-PLC-mediated release of active bFGF from the same cells transfected with the bFGF gene. The released bFGF competed with recombinant bFGF in a radioreceptor assay. Addition of Pl-PLC to sparsely seeded vascular endothelial cells resulted in a marked stimulation of cell proliferation, but there was no mitogenic effect of Pl-PLC on 3T3 fibroblasts. Studies with exogenously added 125I-bFGF revealed that about 6.5% and 20% of the cell surface-bound bFGF were released by treatment with Pl-PLC and heparitinase, respectively. Both enzymes also released sulfate-labeled heparan sulfate from metabolically labeled 3T3 fibroblasts. Pl-PLC failed to release 125I-bFGF from the subendothelial extracellular matrix (ECM), as compared to release of 60% of the ECM-bound bFGF by heparitinase. Our results indicate that 3-8% of the total cellular content of bFGF is associated with glycosyl-Pl anchored cell surface HSPG. This FGF may exert both autocrine and paracrine effects, provided that it is released by Pl-PLC and adequately presented to high affinity bFGF cell surface receptor sites.     

Angiostatin and plasminogen share binding to endothelial cell surface actin.

Previous studies from this laboratory have demonstrated that plasminogen binds to endothelial cell surface-associated actin via its kringles in a dose-dependent and specific manner. The purpose of this study was to determine whether angiostatin, a proteolytic fragment of plasminogen, shares binding properties with plasminogen. Our results indicated that like plasminogen, angiostatin bound to actin in a time-, concentration-, and kringle-dependent manner. Furthermore, this binding was significantly inhibited by excess plasminogen, suggesting that both proteins shared binding motifs on the actin molecule. Fluorescence studies demonstrated that angiostatin bound to intact endothelial cells through its kringles, and this binding was also inhibited by plasminogen but not by unrelated proteins. Ligand blot analyses on endothelial cell lysates indicated that angiostatin interacted with a 42 kDa protein, which was identified as actin. Furthermore, an anti-actin antibody inhibited binding of angiostatin to endothelial cells by approximately 25%. These results suggest that angiostatin and plasminogen share binding to endothelial cell surface actin and, therefore, that angiostatin has the potential to inhibit plasmin-dependent processes such as cell migration-movement.     

Glycosaminoglycans enhance phorbol ester-induced proteolytic activity and angiogenesis in vitro

Summary It has been reported that endothelial cells suspended in three-dimensional type I collagen gels can be induced to undergo tube formation by 12-o-tetradecanoyl phorbol 13-acetate (TPA). In this report, we show that TPA-induced endothelial cell tube formation can be further enhanced by the addition of other matrix components in the collagen gels. In the presence of TPA, both high molecular weight hyaluronate and chondroitin sulfate elicit a dose-dependent stimulation of tube formation. The enhanced tube formation appears to be due to an increase in the number of cells undergoing morphogenesis as the average length per tube is not obviously increased. Concomitant with the increased cell morphogenesis, there is an increase in proteolytic activity secreted by the cells. Treatment of cells with cycloheximide suppresses hyaluronate- and chondroitin sulfate-enhanced cell morphogenesis and proteolytic activity suggesting that new protein synthesis, perhaps proteases, is necessary for endothelial cell morphogenesis. The possible role of the production of proteolytic activity in endothelial cell tube formation is discussed.     

A low-Mr chemoattractant for vascular endothelial cells

The formation of new blood vessels occurs by sprouting from previously existing microvasculature. The process involved directed migration of the vascular endothelial cells towards chemical signals released from the target tissue. We have used the Boyden chemotaxis chamber method to identify chemotactic signals for fetal bovine vascular endothelial cells. Human placenta organ cultures produce a high-Mr chemoattractant for the endothelial cells from which a low-Mr factor can be liberated with trichloroacetic acid treatment and ethanol extraction. This activity was isolated from extracts of human placenta using Sephadex LH-20, Amberlite XAD-2, and silica gel thin-layer chromatography. The Mr of the factor is less than 400, it is lipophilic and resistant to proteolytic enzymes. The factor induces chemotactic migration of both aortic endothelial cells and capillary endothelial cells from the retina, but has no effect on fibroblasts or leukocytes suggesting a specific function of the compound for the vascular endothelial cells.     

Thrombin stimulates c-sis gene expression in microvascular endothelial cells

We have determined whether expression of the c-sis gene product, platelet-derived growth factor (PDGF), is regulated in cultured renal microvascular endothelial cells by factors to which vascular endothelial cells may be exposed at sites of perivascular cellular proliferation. Thrombin exposure increased endothelial cell levels of c-sis message by 3-5-fold over a time course that peaked at 4 h after exposure. Similarly, thrombin-exposed microvascular endothelial cells released increased amounts of PDGF activity into their media. The thrombin effect was not mediated through the proteolytic activity of thrombin, as proteolytically inactive thrombin stimulated the c-sis expression as well as native thrombin. This stimulation was mimicked by exposure of cells to biologically active phorbol esters, suggesting that thrombin action may be mediated through activation of kinase C (Ca2+/phospholipid-dependent enzyme). Thus, thrombin regulates the expression and release of PDGF activity from endothelial cells in culture and may act in vivo to stimulate mitogen release from endothelial cells, thereby inducing proliferation of perivascular cells.     

Regulation of endothelial cell differentiation and transformation by H-Ras

Angiogenesis is regulated by growth factors which activate tyrosine kinase receptors leading to the activation of a number of intracellular signaling pathways. The specific function of H-Ras during FGF-2 stimulated endothelial cell differentiation, defined as invasive growth and formation of branching networks in fibrin gels, was investigated by using conditionally immortalized endothelial cell lines induced to express H-Ras mutants. Expression of inhibitory N17Ras did not impair differentiation in response to FGF-2 and TNF-alpha. The farnesyltransferase inhibitor FTI-277 inhibited farnesylation of Ras but did not inhibit differentiation of human microvascular endothelial cells or mouse brain endothelial cells. In contrast, activated V12Ras inhibited endothelial cell differentiation and cells displayed a transformed phenotype with an increased rate of proliferation and loss of contact inhibited growth. Furthermore, V12Ras expressing endothelial cells grew as solid tumors when injected subcutaneously into mice. Our data suggest that, in endothelial cells, H-Ras activity is not required for differentiation. However, this activity must be tightly regulated as aberrant activity can disturb the ability of endothelial cells to undergo differentiation.     

Anchorage independent growth and plasminogen activator production by bovine endothelial cells

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.

Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.     

Cell surface-associated proteinases in NK cell-mediated cytotoxicity: enhancement of enzyme expression is unique to activation with interferon-alpha

The human NK cell-mediated cytotoxicity reaction is sensitive to proteinase inhibitors with specificity for chymotrypsin-like enzymes inhibitable by 1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK). Evidence is presented in support of previous data suggesting that this type of cytotoxicity is attributable to enzymes associated with the surface membrane of the NK cell. Activation of the cells with IFN-alpha results in increased cytolytic activity, the suppression of which requires an almost two- to threefold increase in the concentration of proteinase inhibitors. Treatment of NK cells with IFN-alpha results in increased surface binding of [3H]diisopropyl fluorophosphate ([ 3H]DFP). This effect is not inhibited by cycloheximide (50 micrograms/ml), suggesting translocation of preexisting enzymes to the surface membrane. TPCK can compete with [3H]-DFP for binding to the cell surface and can abrogate the increase in [3H]DFP binding observed after IFN-alpha stimulation of the cells. Treatment with IFN-gamma does not increase cell surface-associated proteolytic activity and stimulation with IL-2 results in much smaller increments. The sensitivity of cytotoxicity to proteinase inhibitors is confined to the initial 2-5 min of the reaction. This suggests that cell surface-associated proteinases play a role in the programming of NK cells for lysis, whereas subsequent events may be dependent on secreted enzyme moieties.     

Polyoma Middle T-induced Vascular Tumor Formation: The Role of the Plasminogen Activator/Plasmin System

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.     

STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca²⁺ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca²⁺-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca²⁺ entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca²⁺ channel-mediated Ca²⁺ influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.