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K Danø

Publications and source records attributed to K Danø.

At least 37 records · Page 2Linked to original sources
PubMed1999

Plasma urokinase receptor levels in patients with colorectal cancer: relationship to prognosis.

BACKGROUND: The proteolytic enzyme plasmin, which is generated from the precursor plasminogen by the action of urokinase plasminogen activator, is thought to play a role in tumor cell invasion and metastasis. Urokinase plasminogen activator receptor (uPAR) is functionally involved in the cell surface activation (i.e., cleavage) of plasminogen. Increased tumor tissue levels of uPAR are associated with poor prognosis in several types of cancer. This retrospective study was undertaken to test the relationship between preoperative plasma levels of soluble uPAR (suPAR) and survival in patients with colorectal cancer. METHODS: suPAR levels in preoperative plasma from 591 patients with colorectal cancer were determined by use of a kinetic enzyme-linked immunosorbent assay and analyzed with respect to associations with postoperative survival, Dukes' stage, age, and serum carcinoembryonic antigen level. Plasma suPAR measurements were log transformed for survival analysis, which employed the Kaplan-Meier method and the Cox proportional hazards model. All P values reported are two-sided. RESULTS: Univariate analysis, using the log-transformed suPAR concentrations, demonstrated that there was an increasing risk of mortality with increasing plasma suPAR level (P<.0001). An arbitrary cut point, the median for all patients (1.37 ng/mL), divided patients with Dukes' stage B, C, or D disease into statistically different prognostic groups. In multivariate Cox analysis including Dukes' stage, age, and carcinoembryonic antigen level, the suPAR concentration independently predicted survival (P<.0001). CONCLUSIONS: The preoperative plasma suPAR level independently predicted survival of patients with colorectal cancer. Further studies of plasma suPAR in patients with cancer are needed to evaluate the utility of plasma suPAR measurements and cut points in identifying high-risk patients among those with early stage disease.

Adult↗
PubMed1999

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

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.

Animals↗
PubMed1999

Cancer invasion and tissue remodeling--cooperation of protease systems and cell types.

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non-neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI-1 and MMPs) can be expressed by either the non-neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non-neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Animals↗
PubMed1998

Selection of peptides that bind to plasminogen activator inhibitor 1 (PAI-1) using random peptide phage-display libraries.

Large random hexa- and decapenta-peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa-peptide library on immobilized plasminogen activator inhibitor 1 (PAI-1) specifically selected a minor fraction of concatemers, indicating that binding to PAI-1 requires an extended amino acid sequence. Accordingly, the decapenta-peptide library exclusively yielded PAI-1 binding peptides of 15 amino acid residues. None of these phage-bound peptides prevented the interaction between PAI-1 and its target serine protease urokinase (u-PA). To isolate peptides that block the interaction between PAI-1 and u-PA, phages bound to immobilized PAI-1 were eluted by incubation with u-PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI-1 or u-PA.

Amino Acid Sequence↗
PubMed1998

Reduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice.

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogen-deficient mice were crossed with transgenic mice expressing Polyoma middle T antigen under the control of the mouse mammary tumor virus long terminal repeat. Virgin females carrying the Polyoma middle T antigen uniformly developed multiple, bilateral mammary tumors, regardless of the presence or absence of circulating plasminogen. Both the age at which these tumors became palpable and subsequent tumor growth were indistinguishable between plasminogen-deficient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogen-deficient mice was significantly reduced as compared to littermate controls with respect to frequency of occurrence, total number of metastases, and total metastatic tumor burden. Plasminogen activators, as well as other key factors that govern the conversion of plasminogen to plasmin, were expressed within the mammary tumors, suggesting that the plasminogen/plasmin system may promote metastasis by contributing to tumor-associated extracellular proteolysis. The data provide direct evidence that plasmin(ogen) is a tumor progression factor in PymT-induced mammary cancer, and support the hypothesis that hemostatic factors play an important role in tumor biology.

Animals↗
PubMed1998

The level of urokinase-type plasminogen activator receptor is increased in serum of ovarian cancer patients.

Ascites and serum of patients with ovarian carcinoma contain a soluble form of urokinase-type plasminogen activator receptor (uPAR). We now report that pro-uPA-Sepharose-purified uPAR from ascites of patients with ovarian carcinoma is the full-length molecule missing the glycosyl-phosphatidylinositol anchor, as determined by its amino acid composition. We next examined the significance of determining serum soluble uPAR (suPAR) levels in ovarian cancer patients using a specific ELISA and compared the results with serum concentrations of CA-125, an established diagnostic marker. Serum from pre- and postoperative ovarian cancer patients was assayed for suPAR and CA-125. The majority of the patients with ovarian cancer had enhanced preoperative serum levels of suPAR compared with healthy controls, but suPAR concentrations decreased after operation. Although uPAR was associated with most ovarian carcinomas, it appeared to be a less specific indicator for ovarian cancer than CA-125. On the other hand, suPAR was more specific for other types of solid tumors. Moreover, we have observed some cases of ovarian cancer that showed increase of suPAR but not of CA-125. The prognostic significance of serum suPAR assay for survival of ovarian carcinoma patients was evaluated using Cox's proportional hazards analysis. Our preliminary data show that high preoperative levels of suPAR were associated with worse survival of the patients, whereas CA-125 had no prognostic implications. This is the first report evaluating the assay of serum suPAR levels in ovarian cancer and analyzing its value as a tumor or prognostic marker.

Amino Acids↗
PubMed1998

Photoaffinity labeling of the human receptor for urokinase-type plasminogen activator using a decapeptide antagonist. Evidence for a composite ligand-binding site and a short interdomain separation.

Binding of urokinase-type plasminogen activator (uPA) to its cellular receptor (uPAR) renders the cell surface a favored site for plasminogen activation. Recently, a 15-mer peptide antagonist of the uPA-uPAR interaction, with an IC50 value of 10 nM, was identified using phage display technology [Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. 91, 7129-7133]. In the present study, the molecular aspects of the interaction between this peptide and uPAR have been investigated. We have characterized the real-time receptor binding kinetics for the antagonist using surface plasmon resonance and identified critical residues by alanine replacements. The minimal peptide antagonist thus derived (SLNFSQYLWS) was rendered photoactivatable by replacing residues important for uPAR binding with photochemically active derivatives of phenylalanine containing either (trifluoromethyl)diazirine or benzophenone. These peptides incorporated covalently into purified soluble uPAR upon photoactivation, and this was inhibited by preincubation with receptor binding derivatives of uPA. The intact three-domain structure of uPAR was essential for efficient photoaffinity labeling. Proteolytic domain mapping using chymotrypsin revealed a specific labeling of both uPAR domain I and domains II + III dependent on the position of the photoprobe in the antagonist. On the basis of these studies, we propose the existence of a composite ligand binding site in uPAR combined of residues located in distinct structural domains. According to this model, a close spatial proximity between uPAR domain I and either domains II or III in intact uPAR is required for the assembly of this composite binding site. Since the receptor binding properties of the peptide antagonist closely mimic those of uPA itself, these two ligands presumably share coincident binding site in uPAR.

Amino Acid Sequence↗
PubMed1998

Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation.

Analysis of extracellular matrix degradation systems has led to the insight that in cancer invasion there is often crucial interplay between cancer cells and several types of surrounding non-neoplastic stromal cells. Likewise, in normal tissue remodeling processes, the synthesis of proteolytic components is often distributed between several cell types, and there are strong similarities between neoplastic and non-neoplastic processes in the same tissue. Thus, tissue remodeling events are excellent models for studies of extracellular proteolysis in cancer. This has become even clearer by recent analyses of genetically modified mice.

Breast Neoplasms↗
PubMed1997

The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction.

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.

Antibodies, Monoclonal↗
PubMed1997

ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts.

A sandwich-type ELISA has been developed for the assessment of complexes between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas. The assay is based on a combination of rabbit polyclonal anti-uPA antibodies and a biotinylated mouse anti-uPAR monoclonal antibody (MAb). The detection limit of the assay is approximately 0.5 fmol/ml. A linear dose-response is obtained with up to 40 fmol/ml of uPA:uPAR complexes, while uPA and uPAR separately do not cause any response in the ELISA. A buffer which has been used previously for optimal extraction of uPAR yields the highest amounts of uPA:uPAR complexes. Absorption of tumor extracts with anti-uPA or anti-uPAR MAbs results in a complete disappearance of the ELISA signal, demonstrating the specificity of the ELISA. The recovery of chemically cross-linked uPA:uPAR complexes added to tumor extracts varies between 80% and 105%. The intra- and inter-assay variation coefficients are 5.3% and 9.8%, respectively. Furthermore, a peptide antagonist for uPAR was employed to evaluate de novo uPA:uPAR complex formation during tumor tissue extraction and the immunoassay procedure. Our results strongly indicate that de novo complex formation is a major factor to consider and that complexes analyzed in the presence of this antagonist represent original uPA:uPAR complexes present prior to tumor tissue processing. The present ELISA appears suitable for studying the potential prognostic impact of uPA:uPAR complexes in lung tumor tissue as well as other types of cancer.

Animals↗
PubMed1997

The complex between urokinase plasminogen activator and its type-1 inhibitor in breast cancer extracts quantitated by ELISA.

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.

Animals↗
PubMed1997

Cell-surface acceleration of urokinase-catalyzed receptor cleavage.

The urokinase-type plasminogen activator (uPA) binds to a specific cell-surface receptor, uPAR. On several cell types uPAR is present both in the full-length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either directly or indirectly mediated by uPA itself. In a soluble system, uPA can cleave purified uPAR, but the low efficiency of this reaction has raised doubts as to whether uPA is directly responsible for uPAR cleavage on the cells. We now report that uPA-catalyzed cleavage of uPAR on the cell surface is strongly favored relative to the reaction in solution. The time course of uPA-catalyzed cleavage of cell-bound uPAR was studied using U937 cells stimulated with phorbol 12-myristate 13-acetate. Only 30 min was required for 10 nM uPA to cleave 50% of the cell-bound uPAR. This uPA-catalyzed cleavage reaction was inhibited by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occurred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ala84 and Arg89-Ser90, respectively. The uPA-catalyzed cleavage of uPAR is a new negative-feedback regulation mechanism for cell-surface plasminogen activation. We propose that this mechanism plays a physiological role at specific sites with high local concentrations of uPA, thus adding another step to the complex regulation of this cascade reaction.

Amino Acid Sequence↗
PubMed1997

Elevated plasma levels of urokinase plasminogen activator receptor in non-small cell lung cancer patients.

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.

Biomarkers, Tumor↗
PubMed1997

Co-ordinated expression of MMP-2 and its putative activator, MT1-MMP, in human placentation.

The spatial expression of mRNA for matrix metalloproteinase 2 (MMP-2), its putative activator, the membrane-type 1 matrix metalloproteinase (MT1-MMP), and the MMP-2 substrate type IV collagen was investigated in human placentas of both normal and tubal ectopic pregnancies and in cyclic endometrium using in-situ hybridization. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. In both normal and tubal pregnancies MT1-MMP, MMP-2 and type IV collagen mRNA were highly expressed and co-localized in the extravillous cytotrophoblasts of anchoring villi, in cytotrophoblasts that had penatrated into the placental bed and in cytotrophoblastic cell islands. In addition, the decidual cells of normal pregnancies in some areas co-expressed MT1-MMP and MMP-2 mRNA, with moderate signals for both components. Fibroblast-like stromal cells in tubal pregnancies were positive for MMP-2 mRNA but generally negative for MT1-MMP mRNA. The consistent co-localization of MT1-MMP with MMP-2 and type IV collagen in the same subset of cytotrophoblasts strongly suggests that all three components co-operate in the tightly regulated fetal invasion process. The co-expression of MT1-MMP and MMP-2 mRNA in some of the decidual cells indicates that these cells are also actively involved in the placentation process.

Chorionic Villi↗
PubMed1997

ELISA determination of soluble urokinase receptor in blood from healthy donors and cancer patients.

Measurement of urokinase receptor (uPAR) in tumor extracts has prognostic value, but assay of the soluble uPAR (suPAR) in peripheral blood may offer wider applications in cancer patient management. A tumor extract uPAR ELISA was modified to eliminate nonspecific plasma protein interference, enabling specific detection of suPAR in plasma and sera with >90% recovery of added calibrator. suPAR concentrations in citrate plasma correlated with sera in 93 healthy blood donors (r = 0.84, P <0.0001), with a median value for both of 1.2 microg/L. The plasma median for 19 advanced breast cancer patients was 2.9 microg/L suPAR, and a similar increase was found for 10 advanced colon cancer patients, consistent with release of suPAR from tumors into blood. Repetitive monitoring of suPAR in cancer patients' blood may have value in assessment of prognosis and tumor recurrence.

Adult↗