RESUMO
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.
Assuntos
Fibrossarcoma/enzimologia , Peptídeo Hidrolases/metabolismo , Ativadores de Plasminogênio/metabolismo , Plasminogênio/metabolismo , Células Tumorais Cultivadas/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Meios de Cultura , Ativação Enzimática , Fibrinolisina/metabolismo , Humanos , Cinética , Peso Molecular , Ligação ProteicaRESUMO
We have identified a soluble form of the human urokinase plasminogen activator (uPA) receptor (uPAR) in the ascitic fluids from patients with ovarian cancer. After purification of uPAR from the ascitic fluids by ligand-affinity chromatography (pro-uPA Sepharose), the uPAR was initially identified by cross-linking to a radiolabeled amino-terminal fragment of human uPA. The uPAR purified from the ascitic fluid has no bound ligand (uPA), as similar amounts can be purified by ligand-affinity chromatography as by immuno-affinity chromatography. uPAR from ascitic fluids partitions in the water phase after a temperature-dependent phase separation of a detergent extract. It therefore lacks at least the lipid moiety of the glycophospholipid anchor present in cellular-bound uPARs. It is highly glycosylated and the deglycosylated form has the same electrophoretic mobility as previously characterized cellular uPAR from other sources. The immunoreactivity of the purified uPAR from the ascitic fluid is indistinguishable from that of characterized uPAR, demonstrated by Western blotting with three different anti-uPAR monoclonal antibodies. The uPAR was found in 11 of 11 ascitic fluids from patients with ovarian cancer and in elevated amounts in the plasma from 2 of 3 patients. The concentration of soluble uPAR in the ascitic fluid was estimated to range between 1 and 10 ng/ml. Human soluble uPAR, derived from the tumor cells, was also found in the ascitic fluid and serum from nude mice xenografted intraperitoneally with three different human ovarian carcinomas.
Assuntos
Líquido Ascítico/química , Carcinoma/química , Neoplasias Ovarianas/química , Receptores de Superfície Celular/isolamento & purificação , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Feminino , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Nus , Transplante de Neoplasias , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Solubilidade , Transplante HeterólogoRESUMO
Histological samples from 60 invasive ductal breast carcinomas were investigated for immunoreactivity for the receptor for urokinase-type plasminogen activator (uPAR) with the use of two monoclonal antibodies recognizing different epitopes. In 51 cases, uPAR immunoreactivity was observed, and in 49 of these specimens, a population of periductal tissue macrophages showed pronounced uPAR immunoreactivity in areas with infiltrating and intraductal carcinoma. In the 2 remaining positive specimens no stromal immunoreactivity was seen. The carcinoma cells were found to contain uPAR immunoreactivity in 8 of the 51 positive cases, including the two specimens that did not show stromal immunostaining. Immunoactivity was not found in the epithelial cells of carcinoma in situ components occasionally seen in the specimens, but stromal macrophage-like cells which had invaded such lesions were positive. In most specimens a subpopulation of tissue neutrophils was also positive. Normally appearing epithelium in all specimens investigated was negative, and no other tissue elements were stained in any of the samples. Ten samples of normal female breast tissue were negative. This is the first report on the immunohistochemical distribution of uPAR in human cancer tissue, and the results provide evidence for a role of the urokinase receptor in providing tissue macrophages a means of directing proteolysis at sites of breast cancer invasion. This macrophage-mediated proteolytic activity is suggested to be involved in the invasion and subsequent distant spreading of this malignancy.
Assuntos
Neoplasias da Mama/química , Carcinoma Intraductal não Infiltrante/química , Macrófagos/química , Receptores de Superfície Celular/análise , Animais , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Receptores de Superfície Celular/imunologia , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
Cell lines derived from human squamous lung carcinoma release large amounts of a soluble glycoprotein into the culture media, having very high molecular weight (greater than 2 X 10(6] and mucin-like properties. A monoclonal antibody called 43-9F has been generated that recognizes a carbohydrate epitope on the glycoconjugate. The epitope is also present on a diverse set of smaller glycoproteins (Mr 50,000-200,000) distributed primarily on the surface of the squamous lung carcinoma cells. A sensitive assay using the 43-9F antibody in a dot blot procedure has been devised that is able to detect an amount of antigen less than that possessed by a single squamous lung carcinoma cell. This assay, and also conventional immunofluorescence and immunohistochemical assay procedures, have been used to screen different normal cells, normal tissues, cancer cells, and tumor biopsy specimens for the antigen. In the normal lung the 43-9F antigen is found only on cells of some of the seromucous glands. In the normal digestive system it is associated in certain organs only with a limited population of mucosal epithelial cells. Other organ systems lack any reactive cells. The cells of most human non-small cell lung carcinomas and their released glycoconjugates have large amounts of the 43-9F epitope, while small cell lung carcinomas and the glycoconjugates released by small cell lung cancer cells lack the epitope. The oligosaccharide recognized by the 43-9F antibody may therefore provide a useful marker to distinguish the different lung carcinomas and for investigating the different cells of origin of these tumors.
Assuntos
Anticorpos Monoclonais , Carcinoma Pulmonar de Células não Pequenas/análise , Glicoproteínas/análise , Neoplasias Pulmonares/análise , Especificidade de Anticorpos , Carcinoma de Células Escamosas/análise , Carcinoma de Células Escamosas/imunologia , Linhagem Celular , Epitopos/análise , Glicoproteínas/imunologia , Humanos , Técnicas de Imunoadsorção , Peso Molecular , Neuraminidase/metabolismo , Ensaio RadioliganteRESUMO
We have studied the prognostic value of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and type 1 plasminogen activator inhibitor (PAI-1) in tumor extracts from 84 patients with squamous cell lung carcinoma and 38 patients with large cell lung carcinoma, measuring each molecule with sandwich enzyme-linked immunosorbent assays. High uPAR levels were significantly associated with short overall survival in patients with squamous cell lung carcinomas when the median value was used as a cutoff point (P = 0.038), while no statistically significant prognostic impact of uPA and PAI-1 levels was found in this group of patients. The combination of high uPAR and high PAI-1 levels did, however, have a particular significant association with short overall survival (P = 0.008). None of the 3 components was found to have a statistically significant prognostic impact in the group of 38 large cell lung cancer patients. There was a positive correlation between uPAR and PAI-1 levels in both groups and between uPA and uPAR levels in the large cell carcinoma patients. In a multivariate analysis, high uPAR was found to be an independent prognostic variable in squamous cell carcinoma patients, with a relative risk of 2.1 (95% confidence interval, 1.1-4.0) and tumor size the only other significant prognostic parameter. These data suggest that uPAR is an important prognostic factor in squamous cell lung carcinoma. In addition to the potential direct clinical usefulness, this information could be helpful in understanding the biology of this disease and developing new therapeutic approaches.
Assuntos
Carcinoma de Células Grandes/química , Carcinoma de Células Grandes/mortalidade , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/mortalidade , Neoplasias Pulmonares/química , Neoplasias Pulmonares/mortalidade , Inibidor 1 de Ativador de Plasminogênio/análise , Receptores de Superfície Celular/análise , Ativador de Plasminogênio Tipo Uroquinase/análise , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Estudos Retrospectivos , Análise de SobrevidaRESUMO
We have recently described the urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) as strong prognostic variables in breast cancer (J. A. Foekens et al., Cancer Res., 52: 6101-6105, 1992; J. Grondahl-Hansen et al., Cancer Res., 53: 2513-2521, 1993; J. A. Foekens et al., J. Clin. Oncol., 11: 899-908, 1994). A specific cell surface receptor (uPAR) binds uPA and strongly enhances plasmin generation, and the amount of uPAR in the tumor tissue might therefore be a rate-limiting factor in the extracellular proteolysis involved in tumor invasion. Here, we report on the prognostic value of uPAR in cytosolic (uPARc) and Triton (uPARt) extracts prepared from 505 primary breast tumors. The median observation time was 54 (range: 12-125) months. uPAR levels were determined by a sandwich ELISA. Univariate analysis showed that high uPAR levels (above the median value) were significantly associated with a shorter overall survival, showing a stronger discriminatory effect for uPARc [relative hazard rate (RHR): 1.47; P = 0.012)] as compared with uPARt (RHR, 1.33; P = 0.059), while no statistically significant differences were found for relapse-free survival. Multivariate analysis including all patients showed that when including other biochemical variables (estrogen receptor, progesterone receptor, PS2, cathepsin D, uPA, and PAI-1), the only retained independent variable via backward elimination was PAI-1 for both relapse-free survival and overall survival. When analyzed separately in clinically relevant subgroups, the prognostic value of uPAR was particularly strong in a subgroup of 201 node-positive postmenopausal women, showing considerably shorter overall (RHR: 2.39; P < 0.0001) and relapse free (RHR: 1.91; P = 0.0006) survival for patients with high uPARc content. High uPARt levels were also significantly associated with shorter overall survival in this subgroup of patients (RHR: 1.5; P = 0.047), but not with relapse-free survival (P = 0.64). Multivariate analysis, including the basic model, estrogen and progesterone receptors, PS2, cathepsin D, uPA, PAI-1, uPARc, and uPARt in the subgroup of postmenopausal node-positive patients, showed that only uPARc and PAI-1 were significant independent prognostic parameters, with respect to overall survival, RHRs being 2.72 (P < 0.0001) and 1.81 (P = 0.005), respectively. In multivariate analysis of relapse-free survival, uPARc, PAI-1, and uPA were independent parameters with respective relative relapse rates of 1.91 (P = 0.002) for uPARc, 1.68 (P = 0.02) for PAI-1, and 1.6 (P = 0.03) for uPA. These data lend support to the hypothesis that uPAR is an important molecule in plasmin-mediated extracellular matrix degradation leading to cancer cell dissemination and death of the patient.
Assuntos
Neoplasias da Mama/química , Proteínas de Neoplasias/análise , Receptores de Superfície Celular/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Citosol/química , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
Radiolabeled pro-urokinase plasminogen activator (pro-uPA) was cross-linked to a specific protein on the surface of human monocyte-like U937 cells in a reaction catalyzed by tissue transglutaminase. The conjugate formed with this unknown component had a much higher molecular weight (apparent M(r) 250,000-300,000) than the complex of pro-uPA and the urokinase plasminogen activator receptor (uPAR). There was a strong preference for the pro-form of uPA. The conjugate was recognized by antibodies against uPA but not by anti-uPAR antibodies. Nevertheless, the blocking of uPAR with a monoclonal antibody abolished the formation of the conjugate, thus showing a role of uPAR in this process.
Assuntos
Reagentes de Ligações Cruzadas , Monócitos/enzimologia , Transglutaminases/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Humanos , Linfoma Difuso de Grandes Células B , Peso Molecular , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/análise , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transglutaminases/isolamento & purificação , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/análise , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificaçãoRESUMO
We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro-u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system.
Assuntos
Ativadores de Plasminogênio/metabolismo , Plasminogênio/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos Monoclonais , Linhagem Celular , Quimotripsina/metabolismo , Epitopos/imunologia , Fibrinolisina/análise , Humanos , Cinética , Testes de Precipitina , Receptores de Superfície Celular/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Relação Estrutura-AtividadeRESUMO
The urokinase plasminogen activator (uPA) is involved in extracellular matrix degradation during cancer invasion. Binding of uPA to a specific cell surface receptor (uPAR) is a key step in this process. We have previously reported that high levels of uPAR in squamous cell lung cancer tissue extracts are associated with poor prognosis (Pedersen et al., Cancer Res 1994, 54, 4671-4675). Recently we found that uPAR is present in blood plasma from healthy donors as determined by enzyme-linked immunosorbent assay (ELISA) and chemical cross-linking. We now report that uPAR in plasma from 17 patients with non-small cell lung cancer (NSCLC) was significantly higher than in 30 healthy controls (P = 0.0004), while no significant increase was found in plasma from 14 patients with small cell lung cancer (SCLC). The increased levels of uPAR in the plasma from NSCLC patients is likely to be due to release of uPAR from the tumour tissue, and may, therefore, be related to prognosis.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Precursores Enzimáticos/sangue , Neoplasias Pulmonares/sangue , Ativadores de Plasminogênio/sangue , Receptores de Superfície Celular/sangue , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Carcinoma de Células Pequenas/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Prognóstico , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.
Assuntos
Anticorpos Monoclonais/farmacologia , Técnicas Biossensoriais , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Células CHO/metabolismo , Células CHO/ultraestrutura , Cricetinae , Humanos , Radioisótopos do Iodo , Ligantes , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Solubilidade , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal antibodies recognizing the non-ligand binding part of this receptor, and it detects both free and occupied uPAR, in contrast to ligand-binding assays used previously. In a variant of the assay, the occupied fraction of uPAR is selectively detected with a uPA antibody. To be used as a standard, a soluble variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer tissue. Recent studies have shown that a high uPA level in tumor extracts is in some cancers associated with poor prognosis. The present assay will now allow similar prognostic studies of uPAR levels.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Receptores de Superfície Celular/análise , Aminoácidos/análise , Animais , Anticorpos Monoclonais , Células CHO , Carcinoma/química , Neoplasias do Colo/química , Cricetinae , Humanos , Camundongos , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Células Tumorais CultivadasRESUMO
Pro-urokinase (pro-uPA) and activated uPA are confined to focal adhesions and cell-cell contacts. We studied the distribution of the uPA receptor (uPAR) on human fibroblasts (HES) and rhabdomyosarcoma (RD) cells by immunofluorescence and immunoelectron microscopy. Two monoclonal antibodies (MAb) utilized were against uPAR: MAb R4, which reacts with occupied and unoccupied uPAR, was concentrated at focal adhesions; MAb R3 reacting with unoccupied receptor stained cell surfaces diffusely. MAb R4 stained cell-cell contacts, tips of microspikes, and co-localized with vinculin. Of the matrix and integrin components tested, alpha v beta 3 integrin was found at focal adhesions but more centrally than uPAR. Since uPAR is anchored to the plasma membrane through a GPI lipid, we studied its mobility by antibody-induced clustering. This revealed that unoccupied uPAR was relatively mobile; MAb R3 redistributed it to clusters. In contrast, uPAR R4 and uPA antibodies at the focal contact sites remained mostly within focal contacts. Addition of exogenous uPA resulted in loss of R3 staining and increase of uPA in focal adhesions. These results suggest that occupancy of the receptor with uPA is associated with localization to cell contact sites and restricted lateral mobility.
Assuntos
Fibroblastos/química , Fibroblastos/citologia , Receptores de Superfície Celular/análise , Rabdomiossarcoma/química , Rabdomiossarcoma/patologia , Anticorpos Monoclonais , Comunicação Celular/fisiologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/ultraestrutura , Epitélio/química , Epitélio/ultraestrutura , Fibroblastos/ultraestrutura , Imunofluorescência , Humanos , Integrinas/análise , Microscopia Imunoeletrônica , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/análise , Rabdomiossarcoma/ultraestrutura , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/análise , Vinculina/análiseRESUMO
The receptor for urokinase plasminogen activator (uPAR) is a key molecule in cell surface-directed plasminogen activation. uPAR binds urokinase plasminogen activator (uPA) and thereby focuses plasminogen activation on the cell surface. Plasmin dissolves fibrin deposits and facilitates cell migration during tissue repair processes by degrading the extracellular matrix. During human implantation and placental development, plasmin is considered important for both trophoblast migration/invasion and for fibrin surveillance. This study examined the expression of uPAR in normal and ectopic human placentae by immunohistochemistry. In first and third trimester normal placentae as well as in tubal ectopic placental tissues, a high uPAR expression was seen in the trophoblast associated with deposits of fibrin-type fibrinoid. Extravillous trophoblast of the basal plate, of the cell islands, and of the cell columns was also positive for uPAR in the first trimester whereas at term the expression of the protein was decreased. Moreover, uPAR immunostaining was observed in decidual cells throughout normal gestation and in endometrial tissues of patients with ectopic pregnancies. These findings suggest that uPAR participates in placental development and in trophoblast invasion particularly in the first trimester of pregnancy and that uPAR is involved in repair mechanisms of the trophoblast and fibrin surveillance.
Assuntos
Fibrina/metabolismo , Placenta/metabolismo , Ativadores de Plasminogênio/metabolismo , Gravidez Ectópica/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Córion/metabolismo , Endométrio/metabolismo , Tubas Uterinas/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/metabolismo , Queratinas/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
The binding of urokinase-type plasminogen activator (u-PA) to a specific cell surface receptor (uPA-R) has been shown to enhance plasminogen activation, a process involved in extracellular matrix degradation and cell migration during angiogenesis and tumor growth. We investigated the expression of u-PA and uPA-R in renal cell carcinomas (n = 11). By immunohistochemistry using monoclonal and polyclonal anti-uPA-R antibodies, we found that tumoral capillary endothelial cells (von Willebrand factor and CD31 positive cells) overexpressed uPA-R, whereas vascular endothelial cells of the normal human kidney do not. In addition, tumor-associated macrophages (CD68-positive cells) strongly expressed uPA-R. In contrast, few tumoral cells and stromal fibroblasts expressed uPA-R. By in situ hybridization using a cDNA S35-labeled probe specific for uPA-R, we confirmed the local expression of uPA-R messenger RNA. We also detected the induction of u-PA in tumoral capillary endothelial cells and in tumor-associated macrophages. In two cases, tumoral cells themselves were also stained by anti-u-PA antibodies in focal areas. Finally tissue-type plasminogen activator (t-PA) was also overexpressed by tumoral capillary endothelial cells as compared with endothelial cells of normal human kidney vessels. These findings indicate an active invasive phenotype of endothelial cells in renal cell carcinoma and suggest a role for the plasminogen activation system in tumoral angiogenesis and invasion.
Assuntos
Carcinoma/patologia , Endotélio Vascular/citologia , Neoplasias Renais/patologia , Macrófagos/metabolismo , Ativadores de Plasminogênio/biossíntese , Receptores de Superfície Celular/biossíntese , Carcinoma/metabolismo , Endotélio Vascular/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Renais/metabolismo , RNA Mensageiro/análise , RNA Neoplásico/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Regulação para CimaRESUMO
Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures; some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.
Assuntos
Neoplasias da Mama/metabolismo , Catepsina D/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Anticorpos Monoclonais/imunologia , Membrana Basal/metabolismo , Neoplasias da Mama/ultraestrutura , Catepsina D/imunologia , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Lisossomos/enzimologia , Microscopia de Fluorescência , Receptores de Superfície Celular/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Coloração e Rotulagem , Fixação de Tecidos , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
Using immunohistochemistry and in-situ hybridization, we studied the expression of the components of the plasminogen activation system during progression to malignant melanoma with fresh melanocytic lesions. Expression of these components is confined to late stages of melanoma. t-PA expression is limited to rare cases of metastatic melanoma. The other components are frequently expressed concomitantly in the same tumour. Urokinase (u-PA) is expressed in stromal cells and only in tumour cells at invasive foci, urokinase receptor (u-PAR) in tumour cells, plasminogen activator inhibitor type I (PAI-1) in the intratumoral extracellular matrix and plasminogen activator inhibitor type II (PAI-2) in tumour cells and stromal cells. In order to investigate the role of u-PAR as a prognostic marker, we have developed an assay for quantitation of the receptor. As a first step towards structural investigations, we have determined the disulfide cross-links of the first domain of uPAR.
Assuntos
Melanoma/metabolismo , Ativadores de Plasminogênio/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Biomarcadores Tumorais , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Hibridização In Situ , Melanoma/patologia , Melanoma/secundário , Receptores de Ativador de Plasminogênio Tipo UroquinaseAssuntos
Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Sítios de Ligação , Western Blotting/métodos , Linhagem Celular , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida/métodos , Glicosilfosfatidilinositóis/análise , Humanos , Indicadores e Reagentes , Cinética , Linfoma , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Radioimunoensaio/métodos , Ensaio Radioligante/métodos , Receptores de Superfície Celular/isolamento & purificação , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Succinimidas , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificaçãoRESUMO
A simple, mild and synthetically useful method for the desulfurization of cyclic thioureas and related compounds, existing as thiol-thione tautomeric mixtures, by hydrogen peroxide in acetic acid is proposed. The effect of substituting different solvents for the acetic acid was investigated.
Assuntos
Acetatos/química , Peróxido de Hidrogênio/química , Tioureia/química , Ácido Acético , Estrutura Molecular , Oxirredução , Solventes/química , Enxofre/química , Tioureia/análogos & derivadosRESUMO
The multipotent drug suramin, which is currently being studied as an anticancer agent, was found to inhibit the interaction between the urokinase-type plasminogen activator (u-PA) and its cellular receptor. 50% inhibition of binding was obtained with a suramin concentration between 30 and 60 micrograms/ml when using U937 cells and a ligand concentration of 0.3 nM. This concentration of the drug is well below the serum levels found in suramin-treated patients. Inhibition of binding was also demonstrated at the molecular level, using chemical cross-linking or an enzyme-linked immunosorbent assay-type technique based on the ligand interaction. The inhibition was not caused by a mere polyanion effect since polysulfates such as heparin, heparan sulfate, and pentosan polysulfate were non-inhibitory or showed only a very weak inhibition. However, polysulfonated compounds with structures resembling suramin (i.e. trypan blue and Evans blue) did prove inhibitory. The inhibition found with suramin showed a concentration dependence consistent with a mixed competitive and noncompetitive mechanism. The off-rate of prebound ligand was accelerated by the drug. It is speculated that the present effect may contribute to the anti-invasive properties of suramin by destroying the cellular potential for localized plasminogen activation and proteolytic matrix degradation.
Assuntos
Receptores de Superfície Celular/metabolismo , Suramina/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ligação Competitiva , Membrana Celular/metabolismo , Células Cultivadas , Reagentes de Ligações Cruzadas , Ensaio de Imunoadsorção Enzimática , Humanos , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
The urokinase plasminogen activator receptor (uPAR) is a membrane protein comprised of three extracellular domains. In order to study the importance of this domain organization in the ligand-binding process of the receptor we subjected a recombinant, soluble uPAR (suPAR) to specific proteolytic cleavages leading to liberation of single domains. Treatment of the receptor with pepsin resulted in cleavage between residues 183 and 184, thus separating the third domain (D3) from the rest of the molecule, which was left as an intact fragment (D(1 + 2)). D(1 + 2) proved capable of ligand binding as shown by chemical cross-linking, but quantitative binding/competition studies showed that the apparent ligand affinity was 100- to 1000-fold lower than that of the intact suPAR. This loss of affinity was comparable with the loss found after cleavage between the first domain (D1) and D(2 + 3), using chymotrypsin. This result shows that in addition to D1, which has an established function in ligand binding (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high affinity in the intact receptor. Real-time biomolecular interaction analysis revealed that the decrease in affinity was caused mostly by an increased dissociation rate of the ligand complex of D(1 + 2). Zero length cross-linking, using carbodiimide-induced, direct condensation, was used to identify regions within suPAR engaged in molecular ligand contact. The purified suPAR was cross-linked to the radiolabeled amino-terminal fragment (ATF) of urokinase, followed by cleavage with chymotrypsin. In accordance with the cleavage pattern found for the uncomplexed receptor, this treatment led to cleavage between D1 and D(2 + 3). Analysis of the radiolabeled fragments revealed the expected ligand labeling of D1 but a clear labeling of D(2 + 3) was also found, indicating that this part of the molecule is also situated in close contact with ATF in the receptor-ligand complex. The latter contact site may contribute to the role of molecular regions outside D1 in high affinity binding.