Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells

First-trimester normal human trophoblast cells show some phenotypic similarities to malignant cells, e.g., rapid proliferation and ability to invade neighboring tissue, including basement membrane in situ, but do not have the ability for unlimited growth or metastasis. The present study examined whether the invasive ability of normal trophoblast cells is an intrinsic property of these cells, independent of the microenvironment provided by the pregnant uterus, and if so, whether they share some of the molecular mechanisms of invasion exercized by metastatic malignant cells. The ability of in vitro grown human trophoblast lines to invade an epithelium-free human amniotic membrane was measured from the temporal kinetics of retention of radioactivity within this membrane resulting from a penetration by 125I-iododeoxyuridine-labeled trophoblast cells. The magnitude of this invasion was compared to that of the highly metastatic human JAR-choriocarcinoma cell line and murine B16F10 melanoma line. Trophoblasts were found to share some of the same molecular mechanisms of invasion with the metastatic cell lines. Inhibitors of collagenase, plasmin, plasminogen, and plasminogen activators completely prevented invasion of the amnion by the trophoblast lines as well as by the metastatic JAR and B16F10 lines. Mersalyl, a compound known to activate collagenase, stimulated invasion by all cell lines tested, including under conditions in which plasmin activity was inhibited. In addition, trophoblasts produced significant levels of type IV collagenase and laminin, both of which appear to be important products of metastatic tumor cells required for basement membrane invasion. It may be concluded from these findings that the invasive property of first trimester human trophoblasts is genetically determined; that the magnitude of amnion invasion cannot differentiate between metastatic cell lines and invasive but nonmetastatic cell lines; and that invasiveness is not a sufficient prerequisite for metastatic ability.     

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.     

Tumor-associated urokinase-type plasminogen activator: biological and clinical significance.

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.     

Activation of human breast carcinoma collagenase through plasminogen activator

Latent collagenase activity was detected in the media of a well-characterized line of human breast carcinoma cells maintained for over two years in culture. The media also contained sufficient plasminogen activator to convert extrinsically added plasminogen to plasmin which in turn activated the collagenase. During culture of the breast carcinoma in serum-free medium, collagenase activity was maximum on day 12 whereas plasminogen activator activity changed little with time. Using type I collagen as a substrate, the activated breast tumor collagenase produced $\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments consistent with a mammalian collagenase. These findings suggest a pathologic role of plasminogen activator in the activation of latent collagenase during tumor invasion.A number of investigators have postulated that proteases may play a role in tumor invasion (1–5). Collagenase is one such protease which is active at neutral pH and specifically cleaves triple helical collagen into two ($\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments (6). Secretion of collagenase by tumor cells migrating from the primary mass provides an attractive hypothesis for the mechanism of tumor invasion of surrounding host connective tissue—since the local environment would likely be at neutral pH. Consequently, a number of investigators have reported significant levels of collagenase activity in a wide variety of tumors (7–14). Abramson (13) has correlated aggressive $\text{in vivo}$ growth in carcinomas of the head and neck with collagenase activity, and Kuettner et al. (14) have postulated that inhibitors of collagenase may prevent tumors from invading cartilage.Collagenase is produced in both latent and active forms (6). The latent form can be activated with brief protease treatment (15). Since one of the proteases capable of activating collagenase is plasmin (15), the possibility arose that tumor cells could activate collagenase through plasminogen activator. Plasminogen activator secreted by tumor cells (4, 5) could convert plasminogen zymogen to plasmin which would in turn activate latent tumor collagenase. Testing this hypothesis $\text{in vitro}$ was the subject of the present study.Previous studies on collagenase from human carcinoma (7, 13, 14) have suffered from the drawback that contaminating inflammatory cells and fibroblasts may have been the source of the collagenase. Therefore, we have studied collagenase production from cultured human breast carcinoma cells which have been well characterized to be mammary epithelial in origin, malignant in karyotype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasminogen. Finally, to verify that the tumor-secreted collagenase cleaved type I collagen at a single locus, enzyme degradation products were studied by gel electrophoresis.     

Sulfated Glycosaminoglycans Enhance Tumor Cell Invasionin Vitroby Stimulating Plasminogen Activation

Metastasizing tumor cells invade host tissues by degrading extracellular matrix constituents. We report here that the highly sulfated glycosaminoglycans, heparin and heparan sulfate, as well as the sulfated polysaccharide, fucoidan, significantly enhanced tumor cell invasionin vitrointo fibrin, the basement membrane extract, Matrigel, or through a basement membrane-like extracellular matrix. The enhancement of tumor cell invasion was due to a stimulation of the proteolytic cascade of plasminogen activation since the effect required plasminogen activation and was abolished by inhibitors of urokinase-type plasminogen activator (uPA) or plasmin. Sulfated polysaccharides enhanced five reactions of tumor-cell initiated plasminogen activation in a dose-dependent manner. They amplified plasminogen activation in culture supernatants up to 70-fold by stimulating (i) pro-uPA activation by plasmin and (ii) plasminogen activation by uPA. (iii) In addition, sulfated polysaccharides partially protected plasmin from inactivation by α₂-antiplasmin. Sulfated polysaccharides also stimulated tumor-cell associated plasminogen activation, e.g., (iv) cell surface pro-uPA activation by plasmin and (v) plasminogen activation by cell surface uPA. These results suggest that sulfated glycosaminoglycans liberated by tumor-cell mediated extracellular matrix degradationin vivomight amplify pericellular plasminogen activation and locally enhance tumor cell invasion in a positive feedback manner.     

Matrix-bound plasminogen activator inhibitor type 1 inhibits the invasion of human monocytes into interstitial tissue.

Invasion of tissue by monocytes in the course of cellular immune reactions is a multistep process that is thought to be based on the action of urokinase type plasminogen activator (u-PA), an ubiquitous serine protease able to convert the zymogen plasminogen into the active protease plasmin. Expression and occupation of urokinase-type plasminogen activator receptors (u-PA-R) are known to be up-regulated by IFN-gamma and TNF-alpha, and endogenously occupied u-PA-R were found to be instrumental in monocyte invasiveness. We used the amnion invasion assay to investigate whether monocyte invasiveness is affected by matrix-bound plasminogen activator inhibitors (PAI) and by fluid phase u-PA. We show in this study that preincubation of amnion membranes with 1.5 U/cm2 PAI-1 decreases invasion of IFN-gamma activated monocytes by 70% compared with controls. Anti-vitronectin antibodies, which block PAI-1 binding to the matrix, abrogate the inhibitory effect of PAI-1 on monocyte invasiveness, indicating that active PAI-1 is bound via matrix-associated vitronectin. In contrast, preincubation of the amnion membrane with PAI-2 which does not bind to the extracellular matrix has no effect on monocyte invasiveness. Finally, the inhibitory action of matrix-bound PAI-1 can be abrogated by addition of 5 IU/ml u-PA to the monocytes in the invasion chamber. These findings indicate that monocyte invasiveness might be regulated not only by expression and occupation of u-PA-R but also by matrix-bound PAI-1.     

Bacterial plasminogen activators and receptors

Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broad-spectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers.     

Glycosylation of the laminin receptor (α3β1) regulates its association with tetraspanin CD151: Impact on cell spreading,motility, degradation and invasion of basement membrane by tumor cells

Invasion is the key requirement for cancer metastasis. Expression of β1,6 branched N-oligosaccharides associated with invasiveness, has been shown to promote adhesion to most Extra Cellular Matrix (ECM) and basement membrane (BM) components and haptotactic motility on ECM (fibronectin) but attenuate it on BM (laminin/matrigel) components. To explore the mechanism and to evaluate the significance of these observations in terms of invasion, highly invasive B16BL6 cells were compared with the parent (B16F10) cells or B16BL6 cells in which glycosylation was inhibited. We demonstrate that increased adhesion to matrix components induced secretion of MMP-9, important for invasion. Further, both the subunits of integrin receptors for fibronectin (α5β1) and laminin (α3β1) on B16BL6 cells were shown to carry these oligosaccharides. Although, glycosylation of receptors had no effect on their surface expression, it had same differential effect on cell spreading as haptotactic motility. Absence of correlation between invasiveness and expression of most tetraspanins (major regulators of integrin function) hints at an alternate mechanism. Here we show that glycosylation on α3β1 impedes its association with CD151 and modulates spreading and motility of cells apparently to reach an optimum required for invasion of BM. These studies demonstrate the complex mechanisms used by cancer cells to be invasive.     

Pro-urokinase-type plasminogen activator is a substrate for hepsin

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1') peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 x 10(5) m(-1) s(-1)) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 +/- 1.9 nm uPA h(-1), which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.     

Plasmin inhibitors prevent leukocyte accumulation and remodeling events in the postischemic microvasculature

Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ε-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ε-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall.     

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.     

92-kD type IV collagenase mediates invasion of human cytotrophoblasts

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.     

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 Me Wo 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.     

α2,6 Sialylation associated with increased β1,6-branched N-oligosaccharides influences cellular adhesion and invasion

Expression of β1,6-branched N-linked oligosaccharides have a definite association with invasion and metastasis of cancer cells. However, the mechanism by which these oligosaccharides regulate these processes is not well understood. Invasive variants of B16 murine melanoma, B16F10 (parent) and B16BL6 (highly invasive variant) cell lines have been used for these studies. We demonstrate that substitution of α2,6-linked sialic acids on multiantennary structures formed as a result of β1,6-branching modulate cellular adhesion on both extracellular matrix (ECM) and basement membrane (BM) components. Removal of α2,6 sialic acids either by enzymatic desialylation or by stably down-regulating the ST6Gal-I (enzyme that catalyses the addition of α2,6-linked sialic acids on N-linked oligosaccharides) by lentiviral driven shRNA decreased the adhesion on both ECM and BM components and invasion through reconstituted BM matrigel.     

Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen.

Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.     

Laminin increases the release of type IV collagenase from malignant cells

We have studied the effect of laminin on type IV collagenolytic activity elaborated by malignant cells in culture. Laminin (at concentrations of 4-8 micrograms/ml) added to serum-free culture supernatants of subconfluent A2058 human melanoma cells significantly increased the release of the type IV collagenolytic activity (200-300%). The induction of type IV collagenase was more pronounced (580%) using a fragment of laminin which binds to the cell surface laminin receptor. A monoclonal antibody against the human laminin receptor blocked the effect of laminin on type IV collagenase, suggesting that occupation of the laminin receptor may be necessary for the effect. Increase in the type IV collagenolytic activity mediated by laminin was also demonstrated in two other malignant cell lines, HT fibrosarcoma (168%) and mouse melanoma (B16-F10) (271%). The increase in type IV collagenase was found to be specific for laminin because another cell-binding matrix protein, fibronectin, did not have any effect, and epidermal growth factor and transferrin actually decreased the type IV collagenase in human melanoma culture medium (epidermal growth factor, 50% at 20 ng/ml; and transferrin, 20% at 10 micrograms/ml). These studies suggest that tumor cell binding to laminin, which comprises the first step of basement membrane invasion, will induce the second step, namely the collagenolytic dissolution of the basement membrane.     

Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radioimmunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human an rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply.     

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.     

Binding and activation of plasminogen at the surface of Staphylococcus aureus. Increase in affinity after conversion to the Lys form of the ligand.

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I-Glu-plasminogen. The binding was inhibited by both unlabeled Glu-plasminogen and Glu-plasmin. The Lys form of plasminogen, which lacks the 8-kDa amino-terminal activation peptide, was approximately 100-fold more effective than the Glu form in competing with the binding of 125I-labeled Glu-plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. L-Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine-binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high- and low-affinity binding sites. Kd and the number of high-affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively. 125I-Glu-plasminogen bound to staphylococcal surface was converted to plasmin by tissue-type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low-molecular-mass plasmin inhibitors such as aprotinin, the bound Glu-plasmin was further converted to Lys-plasmin. The surface-bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S-2251. The plasminogen conversion shown by the present experiments not only leads to the surface-bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface-bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface-bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.