PubMed Health⌕ Search

Biomedical subjects

K Danø

Publications and source records attributed to K Danø.

At least 91 records · Page 5Linked to original sources
PubMed1993

Receptor for urokinase is present in tumor-associated macrophages in ductal breast carcinoma.

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.

Animals↗
PubMed1993

Binding of the urokinase-type plasminogen activator to its cell surface receptor is inhibited by low doses of suramin.

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.

Binding, Competitive↗
PubMed1993

Potentiation of plasminogen activation by an anti-urokinase monoclonal antibody due to ternary complex formation. A mechanistic model for receptor-mediated plasminogen activation.

We have observed that a murine IgG1 monoclonal antibody directed against human urokinase-type plasminogen activator (uPA) greatly potentiates pro-uPA-mediated plasminogen activation. This effect was dependent on the interaction between the immunoglobulin and the kringle domain of pro-uPA and could be competed efficiently by kringle-containing proteolytic fragments of uPA. In addition, the potentiation could also be competed by the lysine analog 6-aminohexanoic acid, an antagonist of plasminogen binding. This unexpected plasminogen binding dependence was found to be due to a carboxyl-terminal lysine residue on the immunoglobulin gamma chain, which by analogy with other proteins represents a potential binding site for plasminogen. Removal of this residue with carboxypeptidase B resulted in a complete abolition of the potentiation. It appears therefore that the potentiatory effect involves a novel mechanism with the antibody acting to provide a specific template for the assembly of a ternary complex involving pro-uPA/uPA and plasminogen, enabling them to interact in a catalytically favorable manner. This interpretation was confirmed by studying the kinetics of plasminogen activation by the complex between active, two-chain uPA and the antibody, which resulted in an overall 50-fold increase in reaction efficiency (kcat/Km), primarily due to a reduction in Km from 20 to 0.1 microM. Pro-uPA activation by plasmin was also accelerated, although to a lesser extent. The potentiation due to complex formation also provides a mechanism for the initiation of this system, dependent only on the low intrinsic proteolytic activity of the zymogen forms. The effects observed here, mediated by ternary complex formation, simulate the effects we have previously observed on assembly of the uPA receptor-mediated cellular plasminogen activation system and may therefore represent a mechanistic model for both its activity and initiation.

Amino Acid Sequence↗
PubMed1993

Enzyme-linked immunosorbent assay of urokinase-type plasminogen activator (uPA) in cytosolic extracts of human breast cancer tissue.

The enzyme urokinase-type plasminogen activator (uPA) plays a role in cancer invasion, and high levels of uPA in detergent extracts of mammary cancer tissue have been reported to be associated with a poor prognosis. We have explored the possibility of using mammary cancer cytosol extracts routinely prepared for steroid receptor analysis for retrospective prognostic studies of uPA. A sandwich enzyme-linked immunosorbent assay (ELISA) for uPA was developed, using polyclonal catching antibodies and a mixture of three biotinylated monoclonal detecting antibodies, that were selected to recognize free uPA, inhibitor-bound uPA, and uPA bound to its cell surface receptor. The assay detects active uPA and its inactive proenzyme form, pro-uPA, equally well. The limit of detection is approximately 1 pg of pro-uPA in a volume of 100 microliters, and there is a linear dose-response up to 100 pg pro-uPA. The efficiency in extracting uPA of a neutral non-detergent buffer used to prepare cytosol extracts was compared with that of 4 other buffers. There was a pronounced difference in the efficiency, the most efficient being a pH 4.2 buffer containing the non-ionic detergent Triton X-100, while the least efficient was the buffer used to prepare cytosols. Nevertheless, uPA immunoreactivity was readily measurable in the cytosols, and there was a close correlation between the amounts of uPA extracted under optimal conditions and those routinely used for steroid hormone receptor analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1993

Distribution and lateral mobility of the urokinase-receptor complex at the cell surface.

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.

Antibodies, Monoclonal↗
PubMed1993

Messenger RNA for two type IV collagenases is located in stromal cells in human colon cancer.

The gene expression of two type IV collagen-degrading enzymes (72-kd and 92-kd type IV collagenases) was investigated in human colon adenocarcinomas by in situ hybridization. In all cases (18 out of 18), messenger RNA for the 72-kd type IV collagenase was present and located in numerous fibroblasts in the stroma surrounding the invasive cancer tissue. In normal-appearing colonic mucosa distant from the cancer tissue, either no expression or only very weak expression of this enzyme was detected. Also the 92-kd type IV collagenase was found in all samples investigated (10 out of 10), exclusively expressed by tissue macrophages. A very strong hybridization signal for messenger RNA for the 92-kd enzyme was found in a subpopulation of tissue macrophages surrounding invading malignant epithelium. In normal-appearing colon tissue, a markedly weaker hybridization signal was observed in macrophages contained in Peyer's patches. No hybridization signals for either of the two type IV collagenases were detected in cancer cells. Together with previous findings on expression of components of the plasminogen activation system, these results indicate that several nonepithelial cell types in the tumor stroma are involved in production of factors involved in extracellular proteolysis during colon cancer invasion.

Adenocarcinoma↗
PubMed1992

Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associated uPAR variant without ligand-binding properties. In extracts of U937 cells there are two uPAR variants which after complete deglycosylation have apparent molecular masses of 35,000 and 27,000. Analysis with monoclonal antibodies showed that these variants represented the intact uPAR and a two-domain form, uPAR(2+3), lacking ligand-binding domain 1. Trypsin treatment showed that both variants are present on the outside of the cells. Addition to the culture medium of an anticatalytic monoclonal antibody to uPA inhibited the formation of the uPAR(2+3), indicating that uPA is involved in its generation. Purified uPAR can be cleaved directly by uPA as well as by plasmin. The uPA-catalyzed cleavage does not require binding of the protease to the receptor through its epidermal growth factor-like receptor-binding domain, since low molecular weight uPA that lacks this domain also cleaves uPAR. This unusual reaction in which a specific binding protein is proteolytically inactivated by its own ligand may represent a regulatory step in the plasminogen activation cascade.

Antibodies, Monoclonal↗
PubMed1992

A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria.

The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.

Amino Acid Sequence↗
PubMed1992

Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor.

The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4 degrees C uPA fluorescence was visible at the cell surface, shift of the temperature to 37 degrees C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator.

Cell-binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase-type plasminogen activator (u-PA). In contrast to the human system, chemical cross-linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand-blotting analysis, two mouse u-PA-binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u-PA receptor (u-PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u-PAR due to cross-hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand-blotting analysis. Binding of mouse u-PA to its receptor showed species specificity in ligand-blotting analysis, since mouse u-PA did not bind to human u-PAR and human u-PA did not bind to mouse u-PAR. The apparent M(r) of mouse u-PAR varied between different mouse cell lines and ranged over M(r) 45,000-60,000. In four of the cell lines, mouse u-PA bound to two mouse u-PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u-PA-binding protein. In the monocyte macrophage cell line P388D.1, trypsin-treatment of intact cells could remove only the large mouse u-PAR variant (M(r) 60,000) indicating that only this type was a cell-surface-exposed molecule. The smaller mouse u-PAR variant (M(r) 45,000), was deglycosylated by the enzyme endo-beta-N-acetylglucosaminidase H and is probably an intracellular precursor form carrying only high-mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u-PAR variant proteins by diisopropylfluorophosphate-inactivated mouse u-PA-Sepharose affinity chromatography yielded two silver-stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.

Animals↗
PubMed1992

Localization of messenger RNA for Mr 72,000 and 92,000 type IV collagenases in human skin cancers by in situ hybridization.

We have examined the expression of 2 type IV collagen degrading enzymes (Mr 72,000 and 92,000 type IV collagenases) in human skin cancer by in situ hybridization. In all cases of infiltrating carcinomas of squamous cell (9 of 9) and basal cell (5 of 5) types, messenger RNA for the Mr 72,000 type IV collagenase was present in numerous fibroblasts. These were especially abundant in the stroma adjacent to the invasive tumor nodules. Malignant cells were negative for mRNA for the Mr 72,000 enzyme in all cases as were all other epithelial as well as endothelial cells. mRNA for the Mr 92,000 type IV collagenase was present in all 9 squamous cell and in 3 of the 5 basal cell carcinomas. In all these cases, a subpopulation of tissue macrophages was found to be positive, while malignant cells showed a signal for Mr 92,000 type IV collagenase in 6 of the squamous cell carcinomas but in none of the basal cell carcinomas. In all cases, the signal for this mRNA was confined to cells located at the tumoral/stromal interface or in the close vicinity of tumor nodules. No mRNA for any of the 2 collagenases was detected in 3 biopsies of normal skin. In vitro studies have indicated that collagenases are involved in the degradation of the extracellular matrix during cancer invasion. The present findings are consistent with such a role of the Mr 72,000 and 92,000 type IV collagenases in squamous and basal cell carcinomas in situ. The findings also demonstrate that degradative enzymes are not necessarily produced by the malignant cells themselves but may be generated by induction or recruitment of nonmalignant stromal cells.

Basal Cell Carcinoma↗
PubMed1992

Okadaic acid strongly increases gene transcription, mRNA and protein level for the urokinase receptor in human A549 cells.

The specific phosphatase inhibitor, okadaic acid, increases the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in 8 out of 13 human cell lines. The strongest increase (90-fold) was observed in A549 lung carcinoma cells, in which it was partly traced back to an increased transcription of the u-PAR gene. There was a parallel but less pronounced increase in the u-PAR protein level. These findings indicate that u-PAR gene transcription is regulated by one or more factors that are constitutively phosphorylated and are dephosphorylated by okadaic acid-sensitive phosphatases. A lack of additivity of u-PAR induction by okadaic acid and by the protein kinase C activator, PMA, in the A549 cells suggests that the regulatory factors affected by okadaic acid are phosphorylated by protein kinase C.

Blotting, Northern↗
PubMed1992

lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis.

A number of human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal. To overcome this problem, we transduced human breast cancer cells with a replication-defective Moloney murine leukaemia retroviral vector (M-MuLV) containing both neoR (neomycin resistance) and lacZ genes. The resulting cell lines were selected for antibiotic (G418) resistance, and cell-sorted for lacZ expression. lacZ continued to be expressed in cultured cells for at least 20 passages without further G418 selection. The lacZ gene codes for beta-D-galactosidase, and cells expressing this gene stain blue with the chromogenic substrate X-gal. The lacZ-expressing cells retained the pre-transduction ability to traverse Matrigel in vitro, to form subcutaneous tumours in nude mice, and to grow invasively with the formation of metastases. X-gal staining showed high specificity, staining the tumour cells but not the surrounding mouse tissue on either whole tissue blocks or histological sections. The staining procedure was highly sensitive, allowing detection of microfoci of human cancer cells, and quantitative estimation of the metastatic capacity of the cells. These results indicate that lacZ transduction of human tumour cells is a powerful means of studying human cancer cell invasion and metastases in vivo.

Animals↗
PubMed1992

Assignment of the urokinase-type plasminogen activator receptor gene (PLAUR) to chromosome 19q13.1-q13.2.

The urokinase-type plasminogen activator receptor (uPAR) is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathological processes. We applied a cDNA probe from the corresponding gene (PLAUR) in a location analysis using a panel of human/rodent cell hybrids and in a multipoint linkage analysis of 40 CEPH families. These two independent studies both found PLAUR to be located on chromosome 19. The cell hybrid study suggested that PLAUR is located at chromosome 19q13-qter, and the multipoint analysis indicated that PLAUR is located at chromosome 19q13.1-q13.2 and surrounded by DNA markers in the following way (with distances given in recombination fractions): D19S27-.11-CYP2A-.06-PLAUR-.03-D19S8-.04-APOC 2-.24-PRKCG. Further, a ligand-binding study performed on cell hybrids verified the species specificity of the uPAR and confirmed the chromosome assignment.

Animals↗