RESUMO
BACKGROUND: The urokinase plasminogen activator (uPA) system is involved in tissue remodelling processes and is up-regulated in many types of malignancies. We investigated whether serum levels of different forms of soluble uPA receptor (suPAR) are associated with survival and in particular with prostate cancer and cardiovascular disease mortality. METHODS: Using time-resolved fluorescence immunoassays, we measured intact suPAR [suPAR(I-III)] and intact plus cleaved suPAR [suPAR(I-III) + suPAR(II-III)] and thus calculated the amount of suPAR(II-III) in serum samples from 375 men participating in a prostate cancer screening trial. A total of 312 men were free of prostate cancer and 63 men had prostate cancer diagnosed at the time of screening. The cohort was followed for 15 years. We assessed survival using Kaplan-Meier estimation and Cox proportional hazards regression. RESULTS: The mean age at blood sampling was 64 years. In total, 152 men died during follow-up. SuPAR(I-III) and suPAR(II-III) were significantly positively associated with mortality (P = 0.001 and P < 0.0001, respectively). In a Cox regression model adjusting for age and prostate cancer status, an increase in suPAR(I-III) and suPAR(II-III) by 1-unit (ln-scale) was associated with a hazard ratio (HR) of 2.26 [95% confidence interval (CI) 1.17-4.35] and 2.53 (95% CI 1.56-4.10), respectively. There was a trend towards an increased risk of death from prostate cancer in screening-detected prostate cancer patients with increased values of either suPAR form. However, this difference was not significant and the association disappeared after adjusting for age, tumour stage, tumour grade and prostate-specific antigen. Being in the highest quartile of any of the suPAR forms was associated with a highly significant increased risk of cardiovascular death, with HR adjusted for age of 3.27 (95% CI 1.38-7.73) for suPAR(I-III) quartile 4 versus quartile 1. Conclusions. High concentrations of serum suPAR(I-III) and suPAR(II-III) were associated with poor overall survival. The association was particularly strong for death from cardiovascular disease. No similar association was found for prostate cancer after adjustment for other prognostic factors.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Próstata/diagnóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Idoso , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/mortalidade , Detecção Precoce de Câncer/métodos , Métodos Epidemiológicos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/sangue , Neoplasias da Próstata/mortalidade , Suécia/epidemiologiaRESUMO
Previous studies have found that soluble urokinase plasminogen activation receptor (suPAR) increases during inflammatory and malignant illness and elevated suPAR levels may be associated with poor clinical outcome. The purpose of this study was to investigate plasma levels of suPAR during the course of allogeneic stem cell transplantation (SCT). Twenty SCT patients were included in the study. suPAR was measured by ELISA in daily taken plasma samples during the pretransplant conditioning with chemotherapy and weekly for 1 month after infusion of the graft. suPAR levels before the start of the conditioning were significantly elevated when compared to those of healthy controls. During the conditioning in particular treatment with antithymocyte globulin was associated with significantly increased suPAR levels (P = 0.012). At day +7 after infusion of the graft, suPAR levels had decreased to pretreatment levels. High suPAR levels at day 0 were associated with increased mortality (P = 0.011). The present study found increased suPAR levels during the conditioning in SCT patients. Further, the data indicated that increased suPAR levels may be associated with increased mortality, suggesting suPAR as a candidate for further studies as an outcome predictor in SCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Adulto , Anemia/sangue , Anemia/diagnóstico , Anemia/terapia , Soro Antilinfocitário/farmacologia , Soro Antilinfocitário/uso terapêutico , Sangue/efeitos dos fármacos , Transplante de Medula Óssea , Criança , Pré-Escolar , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Feminino , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/terapia , Humanos , Imunossupressores/farmacologia , Estimativa de Kaplan-Meier , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco de Sangue Periférico , Prognóstico , Condicionamento Pré-Transplante/métodos , Transplante Homólogo , Resultado do Tratamento , Adulto Jovem , Talassemia beta/sangue , Talassemia beta/diagnóstico , Talassemia beta/terapiaRESUMO
BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality in developed countries. It is known that early detection results in improved survival, and consequently there is a need for improved diagnostic tools in CRC. The plasma level of soluble urokinase plasminogen activator receptor (suPAR) was proposed as a marker in CRC patients. This study was undertaken to evaluate the individual molecular forms of suPAR as discriminators in a group of patients undergoing endoscopical examination following symptoms related to colorectal cancer. METHODS: In a case-control study comprising 308 patients undergoing endoscopical examination following CRC-related symptoms, 77 CRC patients with adenocarcinoma were age and gender matched to: 77 patients with adenomas; 77 with other non-malignant findings, and 77 with no findings. The different uPAR forms were measured in citrate plasma collected before endoscopical examination, using three different Time Resolved - Fluorescence Immuno Assays (TR-FIA's). RESULTS: All soluble uPAR forms were found to be significantly higher in cancer patients than in patients presenting with other non-malignant findings; uPAR(I) P=0.0006, suPAR(I-III) P<0.0001 and suPAR(I-III)+(II-III) P<0.0001, whereas no significant difference was found when performing similar comparisons for patients presenting with adenomas. The odds ratio (OR) for the comparison of uPAR(I) in patients with CRC to subjects with other non-malignant findings was 3.44 (95% CI:1.86-6.37). CRC patients had a mean elevated level of 20.9% (95% CI:10.2-32.6) for suPAR(I-III) and 18.5% (95% CI:9.0-28.8) for suPAR(I-III)+(II-III) compared with subjects with non-malignant findings. CONCLUSIONS: The findings confirm reports on increased uPAR expression in cancer patients and in particular elevated levels of suPAR in blood from CRC patients and indicate that suPAR levels in blood are increasing during carcinogenesis. Although none of the measured uPAR forms were cancer specific, our findings suggest that uPAR expression could be useful in the early detection of CRC when combined with other markers and clinical variables.
Assuntos
Neoplasias Colorretais/diagnóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Adenocarcinoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Neoplasias Colorretais/sangue , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
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
BACKGROUND: Degradation of extracellular matrix proteins, such as fibrin, is pivotal to tumor invasion. Inhibition of the interaction between urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and hence pro-u-PA activation, is an attractive approach to anti-invasive cancer therapy. A number of inhibitors exist for the human system, but because of species specificity none of these are efficient in mice. We have recently generated an inhibitory monoclonal antibody (mAb) against mouse u-PAR (mR1) by immunization of u-PAR-deficient mice. OBJECTIVES: To evaluate the effect of mR1 in vivo in a physiological setting sensitive to deregulated fibrinolysis, we have administered mR1 systemically and quantitated the effect on liver fibrin accumulation. METHODS: Wild-type and tissue-type plasminogen activator (t-PA) deficient mice were administered with mR1, or control antibody, during 6 weeks. Thereafter, the livers were retrieved and the amount of liver fibrin measured by unbiased morphometrical analysis of immunofluorescence signal. RESULTS: Systemic administration of mR1 caused significantly increased fibrin signal in anti-u-PAR treated t-PA-deficient mice compared to mock-treated, which mimics the phenotype of u-PAR;t-PA double-deficient mice. Fibrin and fibronectin accumulated within the sinusoidal space and was infiltrated by inflammatory cells. Analysis of small and rare hepatic fibrin plaques observed in t-PA-deficient mice showed infiltrating macrophages that, contrary to surrounding Kuppfer cells, expressed u-PAR. CONCLUSION: We show that u-PAR-expressing macrophages are involved in cell-mediated fibrinolysis of liver fibrin deposits, and that the antimouse-u-PAR mAb is effective in vivo and thus suited for studies of the effect of targeting the u-PA/u-PAR interaction in mouse cancer models.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Fibrina/metabolismo , Fígado/efeitos dos fármacos , Receptores de Superfície Celular/imunologia , Ativador de Plasminogênio Tecidual/genética , Animais , Anticorpos Monoclonais/farmacologia , Imunofluorescência , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
We assayed the levels of soluble urokinase plasminogen activator receptor (su-PAR) with an ELISA in various body fluids from 77 patients with benign or malignant ovarian tumors, histologically classified as follows: benign; possibly malignant (borderline); and well, intermediately, and poorly differentiated malignant. The concentration of su-PAR in fluid from malignant cysts was extremely high, approximately 10-fold higher than the concentration in ascitic fluid and approximately 100-fold higher than that in blood. Also, the concentration in malignant cysts was approximately 10-fold higher than the concentration in benign cysts. Such high concentrations of su-PAR were found not only in truly malignant but also in possibly malignant cysts. Thus, we suggest that the concentration of su-PAR in cystic fluid can serve as a marker, allowing early diagnosis of malignant ovarian cysts. The concentration of su-PAR in fluid aspirated transvaginally with ultrasonographic guidance can be used to discriminate possibly and truly malignant cysts from benign cysts without surgery. The high concentrations of su-PAR in ovarian cystic fluids allowed us to characterize the molecular forms. Cross-linking of a radiolabeled ligand to the receptor demonstrated that at least a fraction of su-PAR was able to bind the ligand. A cleaved form of the receptor constituting domains 2 and 3 was detected with Western blotting. The cleaved receptor, which is devoid of the ligand-binding domain 1, has not previously been demonstrated in body fluids; it has only been demonstrated on cell surfaces. su-PAR domains 2 and 3 were found in cystic fluids from both malignant and benign ovarian tumors.
Assuntos
Líquido Ascítico/metabolismo , Cistos Ovarianos/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , Cistos Ovarianos/sangue , Neoplasias Ovarianas/sangue , Lesões Pré-Cancerosas/sangue , Lesões Pré-Cancerosas/metabolismo , Receptores de Superfície Celular/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase , SolubilidadeRESUMO
We studied the urokinase-type plasminogen activator (uPA) receptor (uPA-R) in normal and neoplastic human thyroid cells. It has recently been shown that cleaved forms of uPA-R display an extremely strong chemotactic activity. Normal human thyroid TAD-2 cells express the intact form of the uPA-R and a truncated form lacking the uPA-binding domain on their surface, in a similar manner to tumor thyroid cell lines. However, in tumor thyroid cell lines, the amount of the truncated form is variable: high in papillary carcinoma cells, very low in follicular carcinoma cells, and not detectable in anaplastic carcinoma cells. Similar studies on primary cell cultures confirm the presence of the truncated form of uPA-R in normal and in papillary carcinoma cells and its partial or total loss in follicular carcinoma cells. The presence of truncated uPA-R correlates to uPA secretion, except in papillary carcinoma cells, which express the truncated form of uPA-R but do not release uPA. uPA-R is also able to act as an adhesion receptor by binding vitronectin (VTN) and interacting with integrins. We observe that removal of uPA-R from the surface of normal thyroid and anaplastic carcinoma cells by phosphatidylinositol-specific phospholipase C or treatment with anti-uPA-R antibodies decreases the adhesion of both cell types to VTN and, less efficiently, to fibronectin or collagen. On the other hand, uPA treatment strongly increases the adhesion of anaplastic carcinoma cells specifically to VTN.
Assuntos
Receptores de Superfície Celular/metabolismo , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adesão Celular , Matriz Extracelular/metabolismo , Humanos , Peso Molecular , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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
We have used immuno-gold labeling and electron microscopy to study the topography of thylakoid membrane polypeptides. Thylakoid vesicles formed by passage through a French press were adsorbed onto a plastic film supported by an electron microscope grid and processed for single or double immuno-gold labeling. After shadowing with platinum, the inside-out and right-side-out vesicles were identified by their distinctive morphologies. Right-side-out vesicles were labeled by a monoclonal antibody recognizing an epitope located in the trypsin-cleaved, N-terminal portion of the LHC II apoprotein, and by an antibody to CF1. A monoclonal antibody to the alpha-subunit of cytochrome b-559 reacted with a synthetic tridecapeptide corresponding to the C-terminal portion of the polypeptide. Both this antibody and a polyclonal antibody to the synthetic peptide labeled inside-out vesicles exclusively, indicating that the polypeptide C-terminus was exposed on the lumenal (exoplasmic) surface of the membrane.
Assuntos
Complexo Antígeno-Anticorpo/análise , Clorofila/metabolismo , Cianobactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Complexo de Proteína do Fotossistema II , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Cianobactérias/genética , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/imunologia , Membranas Intracelulares/ultraestrutura , Complexos de Proteínas Captadores de Luz , Microscopia Eletrônica , Dados de Sequência Molecular , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética , Plantas/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Despite improved therapy of advanced colorectal cancer, the median overall survival (OS) is still low. A surgical removal has significantly improved survival, if lesions are entirely removed. The purpose of this retrospective explorative study was to evaluate the prognostic value of histological growth patterns (GP) in chemonaive and patients receiving neo-adjuvant therapy. Two-hundred-fifty-four patients who underwent liver resection of colorectal liver metastases between 2007 and 2011 were included in the study. Clinicopathological data and information on neo-adjuvant treatment were retrieved from patient and pathology records. Histological GP were evaluated and related to recurrence free and OS. Kaplan-Meier curves, log-rank test and Cox regression analysis were used. The 5-year OS was 41.8% (95% CI 33.8-49.8%). Growth pattern evaluation of the largest liver metastasis was possible in 224 cases, with the following distribution: desmoplastic 63 patients (28.1%); pushing 77 patients (34.4%); replacement 28 patients (12.5%); mixed 56 patients (25.0%). The Kaplan-Meier analyses demonstrated that patients resected for liver metastases with desmoplastic growth pattern had a longer recurrence free survival (RFS) than patients resected for non-desmoplastic liver metastases (p=0.05). When patients were stratified according to neo-adjuvant treatment in the multivariate Cox regression model, hazard ratios for RFS compared to desmoplastic were: pushing (HR=1.37, 95% CI 0.93-2.02, p=0.116), replacement (HR=2.16, 95% CI 1.29-3.62, p=0.003) and mixed (HR=1.70, 95% CI 1.12-2.59, p=0.013). This was true for chemonaive patients as well as for patients who received neo-adjuvant treatment.
Assuntos
Neoplasias Colorretais/patologia , Tumor Desmoplásico de Pequenas Células Redondas/patologia , Neoplasias Hepáticas/secundário , Recidiva Local de Neoplasia/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Terapia Combinada , Tumor Desmoplásico de Pequenas Células Redondas/mortalidade , Tumor Desmoplásico de Pequenas Células Redondas/cirurgia , Intervalo Livre de Doença , Feminino , Hepatectomia , Humanos , Fígado/patologia , Fígado/cirurgia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
Proteolytic activity and inflammation in the tumour microenvironment affects cancer progression. In colorectal cancer (CRC) liver metastases it has been observed that three different immune profiles are present, as well as proteolytic activity, determined by the expression of urokinase-type plasminogen activator (uPAR).The main objectives of this study were to investigate uPAR expression and the density of macrophages (CD68) and T cells (CD3) as markers of inflammation in resected CRC liver metastases, where patients were neo-adjuvantly treated with chemotherapy with or without the angiogenesis inhibitor bevacizumab. Chemonaive patients served as a control group. The markers were correlated to growth patterns (GP) of liver metastases, i.e. desmoplastic, pushing and replacement GP. It was hypothesised that differences in proteolysis and inflammation could reflect tumour specific growth and therapy related changes in the tumour microenvironment. In chemonaive patients, a significantly higher level of uPAR was observed in desmoplastic liver metastases in comparison to pushing GP (p = 0.01) or replacement GP (p = 0.03). A significantly higher density of CD68 was observed in liver metastases with replacement GP in comparison to those with pushing GP (p = 0.01). In liver metastases from chemo treated patients, CD68 density was significantly higher in desmoplastic GP in comparison to pushing GP (p = 0.03). In chemo and bevacizumab treated patients only a significant lower CD3 expression was observed in liver metastases with a mixed GP than in those with desmoplastic (p = 0.01) or pushing GP (p = 0.05). Expression of uPAR and the density of macrophages at the tumour margin of liver metastasis differ between GP in the untreated patients. A higher density of T cells was observed in the bevacizumab treated patients, when desmoplastic and pushing metastases were compared to liver metastases with a mix of the GP respectively, however no specific correlations between the immune markers of macrophages and T cells or GP of liver metastases could be demonstrated.
RESUMO
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 receptor (uPAR) is a receptor for both urokinase plasminogen activator (uPA) and the adhesion protein vitronectin. There are two forms of cell surface-bound uPAR; intact uPAR and a cleaved form, uPAR(2+3), which is formed by uPA-catalyzed cleavage of uPAR. In ligand-blotting experiments we found that vitronectin binds uPAR but not uPAR(2+3). In real-time biomolecular interaction analysis using recombinant, soluble uPAR (suPAR) both plasma and multimeric forms of vitronectin bound to intact, antibody-immobilized suPAR. Monoclonal antibodies against domain 1 of uPAR blocked suPAR binding to vitronectin and vitronectin did not interact with suPAR(2+3). Both suPAR(2+3) and the isolated domain 1 failed to compete with the intact suPAR in binding to vitronectin. We therefore conclude that the intact receptor is required for efficient vitronectin binding.
Assuntos
Receptores de Superfície Celular/metabolismo , Vitronectina/metabolismo , Anticorpos Monoclonais , Ligação Competitiva , Técnicas Biossensoriais , Humanos , Ligantes , Neuraminidase , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Receptores de Superfície Celular/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Solubilidade , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
A cDNA clone encoding the barley photosystem I polypeptide which migrates with an apparent molecular mass of 16 kDa on SDS-polyacrylamide gels has been isolated. The 634 bp sequence of this clone has been determined and contains one large open reading frame coding for a 15,457 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10,821 Da. The amino acid sequence of the transit peptide indicates that the polypeptide is routed towards the stroma side of the thylakoid membrane. The hydropathy plot of the polypeptide shows no membrane-spanning regions.
Assuntos
Clorofila/genética , Clonagem Molecular , DNA/genética , Grão Comestível/genética , Hordeum/genética , Proteínas de Plantas/genética , Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Genes , Hordeum/metabolismo , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteína do Fotossistema I , Plantas/metabolismoRESUMO
In order to invade and spread cancer cells must degrade extracellular matrix proteins. This degradation is catalysed by the concerted action of several enzymes, including the serine protease plasmin. Several experimental studies have shown that inhibition of plasmin formation reduces cancer cell invasion and metastasis, indicating a critical role of this proteolytic pathway in these processes. In order to further study the role of plasmin in cancer progression, we have characterized urokinase-type plasminogen activator (uPA) mediated plasmin formation in three human breast cancer cell lines. Using monoclonal antibodies against uPA and its receptor uPAR, we have investigated the contribution of uPA and uPAR to invasive capacity in an in vitro invasion assay. MDA-MB-231 BAG cells were found to express high protein levels of uPA, uPAR and PAI-1. MDA-MB 435 BAG cells produced low amounts of uPA, PAI-1 and moderate amounts of uPAR, whereas MCF-7 BAG cells showed low levels of uPA, uPAR and PAI-1 protein. In a plasmin generation assay MDA-MB-231 BAG cells were highly active in mediating plasmin formation, which could be abolished by adding either an anticatalytic monoclonal antibody to uPA (clone 5) or an anti-uPAR monoclonal antibody (clone R3), which blocks binding of uPA to uPAR. The two other cell lines lacked the capacity to mediate plasmin formation. In the Matrigel invasion assay the cells showed activity in this order: MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG. Testing MDA-MB-231 BAG cells in the Matrigel invasion assay revealed that invasion could be inhibited in a dose-dependent manner either by the clone 5 uPA antibody or by the clone R3 uPAR antibody, suggesting that the cell surface uPA system is actively involved in this invasive process. It is concluded that these three cell lines constitute a valuable model system for in vitro studies of the role of cell surface uPA in cancer cell invasion and has application in the search for novel compounds which inhibit mechanisms involved in uPA-mediated plasmin generation on cancer cells.
Assuntos
Neoplasias da Mama/patologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Anticorpos Monoclonais , Sítios de Ligação , Neoplasias da Mama/metabolismo , Reagentes de Ligações Cruzadas , Meios de Cultivo Condicionados , Feminino , Fibrinolisina/biossíntese , Humanos , Invasividade Neoplásica , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/análise , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/análise , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
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
The urokinase receptor (uPAR) is a glycolipid anchored cell surface glycoprotein that plays a central role in extracellular proteolysis during tissue remodeling processes and cancer invasion. By intramuscular (i.m.) injection of rabbits with plasmid DNA coding for a carboxy-terminally truncated secreted form of the murine uPAR (muPAR), specific anti-sera with a titer of 64,000, as measured by ELISA, have been obtained. Rabbits received a total of 10 monthly injections of 1 mg DNA in phosphate-buffered saline. The antibody titer peaked between the 5th and 7th injection and slowly declined after the 8th injection. After the final immunization the immune response persisted for at least 6 months without further injections. The antibodies generated by DNA immunization were useful for immunohistochemistry and immunoblotting, recognizing the antigen both in its native and in its reduced and alkylated form. Using the antibodies in immunoblotting muPAR was identified in lysates of peritoneal macrophages, spleen and lung tissue. Both the intact and cleaved form of muPAR were identified in lysates of a murine monocyte cell line P388D.1. No cross-reaction with human uPAR was observed. In immunohistochemical analysis of normal mouse lung tissue uPAR immunoreactivity was located in the alveoli and pulmonary vessels, whereas the bronchial epithelium was negative. These results demonstrate that DNA immunization of rabbits using i.m. injection is a very effective and easy method to raise polyclonal antibodies which can be used for characterization and localization of muPAR in mouse tissue.
Assuntos
Formação de Anticorpos , Receptores de Superfície Celular/genética , Vacinas de DNA/imunologia , Animais , Anticorpos/imunologia , Afinidade de Anticorpos , Linhagem Celular , Expressão Gênica , Vetores Genéticos , Humanos , Immunoblotting , Imuno-Histoquímica/métodos , Camundongos , Coelhos , Receptores de Superfície Celular/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Coloração e Rotulagem/métodos , Fatores de TempoRESUMO
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
ELISAs for urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have shown that tumor levels of these molecules are prognostic parameters in breast cancer as well as other types of cancer. These ELISAs measure the total amount of the given component, including preforms, active, inactive, and complex-bound forms. However, the amount of the active forms of a component may more closely reflect the ongoing level of proteolytic activity and thereby be particularly related to prognosis. Since the inactive complex between uPA and PAI-1 can only be formed the active forms of the individual components, we have developed a sensitive and specific uPA:PAI-1 complex ELISA consisting of a sandwich format with two monoclonal antibodies (MAbs) against PAI-1 as capture antibodies and three biotinylated MAbs against uPA as detector antibodies. The data were collected as kinetic measurements of bound alkaline phosphatase activity. A standard of uPA:PAI-1 complex could be specifically measured in the assay with a detection limit of 8 pg/ml and a linear relationship between signal and complex concentration up to 4 ng/ml. Neither free uPA nor free PAI-1 were detected by this assay and the addition to the internal standard of free PAI-1 in amounts up to 20 ng/ml or uPA did not reduce the detection of complex by the assay. This ELISA was applied to extracts from 20 individual breast cancers. Each tumor was extracted in two different buffers and the median concentration of uPA:PAI-1 complex in the optimal extraction buffer was 0.8 ng/mg protein, range 0.4-2.8 ng/mg protein. Extraction of the tumor tissue at a low pH prevented de novo formation of complex from free uPA and PAI-1 in the tissue without destabilizing preformed uPA:PAI-1 complex. During incubation of the assay plate at neutral pH further uPA:PAI-1 complex formation from free components in the extracts was blocked by p-nitrophenyl guanidinobenzoate (NPGB). Thus, the present assay selectively quantifies preformed complex in tumor extracts and will enable us, for the first time, to evaluate the potential prognostic value of the uPA:PAI-1 complex in cancer.