Search PubMed⌕ Search

Biomedical subjects

K Danø

Publications and source records attributed to K Danø.

At least 73 records · Page 4Linked to original sources
PubMed1994

A cleaved form of the receptor for urokinase-type plasminogen activator in invasive transplanted human and murine tumors.

It was recently found that urokinase-type plasminogen activator (uPA) is involved in the cleavage of its receptor (uPAR) on cultured cells, thereby releasing one of the receptor's 3 domains (the ligand binding domain I) and leaving the 2 others [uPAR(2 + 3)] anchored to the cell surface. With monoclonal antibodies (MAbs) we have now identified human uPAR(2 + 3) in lysates of invasive human MDA-MB-231 mammary carcinomas xenografted into nude mice. The production of peptide antibodies recognizing different domains of murine uPAR made it possible to identify a similar cleaved form of uPAR, murine uPAR(2 + 3), in extracts of primary Lewis lung carcinomas. Cleavage of uPAR also occurs in cultured MDA-MB-231 cells and Lewis lung carcinoma cells. This cleavage is inhibited by anticatalytic antibodies to either human or murine uPA, respectively, indicating that it is catalyzed by either uPA or plasmin generated by uPA. The amount of uPAR(2 + 3) may therefore be directly related to the activity of the uPA system and it is possible that the level of uPAR(2 + 3) in cancer tissue may prove to be a stronger prognostic parameter than the levels of either full-length uPAR or UPA.

Amino Acid Sequence↗
PubMed1994

Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue.

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.

Aged↗
PubMed1994

Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1994

Expression of uPA and its receptor by both neoplastic and stromal cells during xenograft invasion.

Recent studies have shown that molecules involved in generation and regulation of extracellular proteolytic activity are often expressed by non-malignant stromal cells during human cancer invasion. We have studied the expression of the urokinase-type plasminogen activator and the urokinase-type plasminogen activator cell-surface receptor in xenografts of human MDA-MB-231 mammary carcinoma cells growing invasively in nude mice. Northern analysis showed the presence of both human and mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA in tumor extracts. By in situ hybridization, mRNA for human urokinase-type plasminogen activator and its receptor was detected in virtually all the cancer cells, while mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA was expressed by tumor-infiltrating fibroblast-like and macrophage-like cells. In invasive areas the cells expressing the 2 murine mRNAs were either the same or located immediately adjacent to each other. This model system has several advantages for studies of the mechanism by which cancer cells induce or recruit stromal cells to produce molecules involved in proteolysis.

Animals↗
PubMed1994

The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils.

The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface-bound plasminogen to plasmin, a process of presumed importance for a number of biologic processes including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor-alpha results in a rapid increase in the expression of uPAR. This process is accompanied by an increased cell-associated plasminogen activation after preincubation of neutrophils with pro-uPA in vitro. By subcellular fractionation of unstimulated neutrophils, 50% of uPAR is recovered in fractions containing latent alkaline phosphatase, corresponding to an intracellular compartment of easily mobilizable secretory vesicles distinct from both primary and specific granules, whereas the remaining 50% of uPAR is associated with a compartment eluting close to the specific granules. In contrast, the ligand pro-uPA is primarily (approximately 80%) found in the specific granules, but small amounts of pro-uPA/uPA (approximately 20%) coelute with latent alkaline phosphatase. Stimulation of neutrophils with FMLP results in translocation of uPAR as well as of pro-uPA from the secretory vesicles, whereas stimulation with PMA is required to translocate material from specific granules. Flow cytometry of neutrophils saturated with exogenous diisopropyl fluorophosphate-uPA shows a large excess (approximately 90%) of unoccupied uPAR on resting as well as FMLP- and PMA-stimulated neutrophils, suggesting a possible role for exogenous pro-uPA in providing neutrophils with a potential for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi.

Biological Transport↗
PubMed1994

Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay.

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.

Amino Acids↗
PubMed1994

Urokinase and plasminogen activator inhibitor type 1 in pulmonary adenocarcinoma.

The urokinase pathway of plasminogen activation is involved in proteolytic degradation of various tissues, including dissolution of the extracellular matrix and basement membranes during the process of cancer cell invasion. We have studied the prognostic value of urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1) in tumor extracts from 106 patients with adenocarcinoma of the lung. uPA and PAI-1 levels were determined by sandwich enzyme-linked immunosorbent assays. No correlation was found between uPA and PAI-1 (r = 0.23). High PAI-1 levels were significantly associated with short-duration overall survival (P = 0.017), while uPA levels showed no significant association with overall survival. Relating the levels of PAI-1 to other prognostic factors such as stage and age, no significant correlations were found. The prognostic impact of uPA and PAI-1 were investigated together with other prognostic factors in Cox multivariate analysis. PAI-1 was found to be an independent prognostic variable for survival, the relative risks being 1.5 (low versus medium PAI-1 values (95% confidence interval, 1.2-1.8) and 2.2 (low versus high (95% confidence interval, 1.8-2.6)). In patients with stage I disease (69 patients) high levels of PAI-1 were significantly associated with poor prognosis compared to low levels (P = 0.037). These data indicate that PAI-1 is a potentially important prognostic factor in pulmonary adenocarcinoma and may as such be used to select patients with low stage and poor prognosis for adjuvant therapy subsequent to complete surgical resection.

Adenocarcinoma↗
PubMed1994

Expression of the receptor for urokinase-type plasminogen activator in normal and neoplastic blood cells and hematopoietic tissue.

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.

Blood Cells↗
PubMed1994

The receptor for urokinase-type plasminogen activator is expressed by keratinocytes at the leading edge during re-epithelialization of mouse skin wounds.

Expression of urokinase-type plasminogen activator receptor mRNA was examined in vivo in mouse skin wounds by in situ hybridization. Urokinase-type plasminogen activator receptor mRNA was found in keratinocytes at the front of the regenerative epithelial outgrowths at the edge of 12-, 48-, and 96-hour-old wounds. The signal was strongest in the keratinocytes just beginning to move 12 h after wounding. At later time-points the signal was weaker, but still confined to keratinocytes at the wound edges. Using in situ hybridization, no cells expressing urokinase-type plasminogen activator receptor mRNA could be detected in normal epidermis, in normal-looking epidermis adjacent to the wounds, in dermis, in subcutis, or in newly formed granulation tissue. The specificity of the results was supported by the use of antisense RNA from two different non-overlapping cDNA clones and the corresponding sense RNA probes, and by Northern analysis of tissue extracts. Together with previous findings on expression of urokinase-type plasminogen activator and its type 1 inhibitor, the localized and regulated expression of urokinase-type plasminogen activator receptor mRNA during skin wound healing indicates that focal extracellular proteolysis at the cell surface generated by receptor-bound urokinase-type plasminogen activator is implicated in the migration of re-epithelializing keratinocytes.

Animals↗
PubMed1994

Immunohistochemical detection of the receptor for urokinase plasminogen activator in human colon cancer.

Paraffin-wax embedded specimens from 30 cases of colonic adenocarcinoma were investigated for immunoreactivity for the receptor of urokinase-type plasminogen activator (uPAR). In all cases there was a strong signal, predominantly at the invasive foci. The positive cells were mainly tumour-infiltrating macrophages but neutrophils and eosinophils were also strongly stained. The neoplastic cells were positive in 19 of the samples with staining of occasional or a moderate number of cells. In uninvolved, normal-appearing mucosa adjacent to the malignant infiltrates, immunostaining of both macrophages and neutrophils was seen, but the labelling was less intense than that seen in the malignant lesions. Weak to moderate staining of normal intestinal epithelium was also seen at the luminal surface. Comparison between immunoreactivity and in situ hybridization showed a similar distribution of protein and mRNA with two exceptions: first, neutrophils (strongly immunoreactive for uPAR) were negative or only weakly positive for uPAR/mRNA; and second, many cancer cells at invasive foci showed prominent hybridization signals but no detectable uPAR immunoreactivity. Together with previous findings of urokinase plasminogen activator (uPA) protein and mRNA being expressed in tumour-infiltrating fibroblast-like cells at the invasive foci, these results support the view that the uPA pathway of plasminogen activation is involved in tissue degradation in colon cancer. The results also extend and consolidate an emerging picture of non-neoplastic tumour stromal cells producing molecules involved in the generation and regulation of extracellular proteolysis in cancer.

Adenocarcinoma↗
PubMed1994

Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane.

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.

Antibodies, Monoclonal↗
PubMed1994

The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.

All known laminin isoforms are cross-shaped heterotrimeric molecules, consisting of one heavy alpha chain and two light beta and gamma chains. Recently, a cDNA encoding a new gamma chain from laminin 5 (also known as kalinin) was sequenced. This chain, named gamma 2, showed extended homology to the classical gamma 1 chain but differed from this by lacking the terminal globular domain. Recent data, indicating an important role of the gamma 2 chain gene in establishing adhesion contacts between epithelial cells and basement membranes, prompted us to investigate whether the gamma 2 chain gene is aberrantly expressed in cancer tissue, and if so whether its localization could provide clues to its possible role in cancer dissemination. Routinely processed tissue specimens from 36 cases of human cancer were investigated, including 16 cases of colon adenocarcinoma, 7 ductal mammary carcinomas, 4 squamous cell carcinomas, 3 malignant melanomas and 6 sarcomas. In situ hybridization for the detection of mRNAs for the gamma 2 chain and for the classical laminin chains alpha 1, beta 1, and gamma 1 was performed using S-35 labeled antisense RNA probes. As positive control of the specificity of the gamma 2 chain mRNA detection, two different anti-sense probes derived from two nonoverlapping cDNA clones were used. Malignant cells were found to express the gamma 2 chain in 29 of the 30 carcinomas studied and the expression was particularly high in cancer cells located at the invasion front. In contrast, mesenchymally derived cancer cells in three different types of sarcomas did not express the gamma 2 chain. In colon cancer there was a clear histological correlation between the expression of gamma 2 chain by cancer cells and their engagement in tumor budding processes. Laminin chains alpha 1, beta 1, and gamma 1 were weakly expressed throughout cancerous areas with no apparent correlation to sites of invasion. The aberrant expression of the gamma 2 chain gene seen in invasively growing cancer cells point to a role of this molecule in establishing focal adhesions of cancer cells to the extracellular matrix during their migration through surrounding normal tissue.

Adenocarcinoma↗
PubMed1993

A novel, specific pro-urokinase complex on monocyte-like cells, detected by transglutaminase-catalyzed cross-linking.

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.

Cell Line↗
PubMed1993

Specific inhibition of the activity of the urokinase receptor-mediated cell-surface plasminogen activation system by suramin.

Urokinase-type plasminogen activator (uPA) is involved in generating the proteolytic activity necessary for invasive processes, and is dependent on binding to its specific cellular receptor (uPAR) for efficient function. We report here that the polysulphonated napthylurea compound suramin inhibits the activity of this cell-associated proteolytic system, in a manner independent of its antagonism of the uPA-uPAR interaction [Behrendt, Rønne and Danø (1993) J. Biol. Chem. 268, 5985-5989], occurring at a 25-100-fold-lower suramin concentration. This inhibition was found to be due to effects on the activation of both pro-uPA and plasminogen. Suramin inhibited plasmin activation of pro-uPA by a non-competitive mechanism (Ki approx. 2 micrograms/ml), which did not involve a direct effect on plasmin catalytic activity. Similarly, its effect on plasminogen activation was not due to a direct inhibition of uPA. The inhibition of plasminogen activation, which occurred exclusively with receptor-bound uPA, appeared to be due to a reversal of the favourable kinetics which result from the activation of cell-associated plasminogen, although suramin did not inhibit the cellular binding of 125I-labelled plasminogen. This suggests that this effect is due to interference with interactions between components of this system on the cell surface, and that suramin may be useful in gaining further insight into the molecular mechanisms involved in the functional assembly of this proteolytic system. Furthermore the effective inhibition of this system by suramin indicates an anti-invasive potential that may contribute to the anti-tumour effect of suramin in vivo.

Amino Acid Sequence↗
PubMed1993

Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins.

The receptor for human urokinase-type plasminogen activator (uPAR) is synthesized as a 313-residue-long polypeptide containing 28 cysteine residues, the pattern of which defines three homologous repeats within the protein. These entities are believed to represent a novel protein domain structure, of which the NH2-terminal domain of uPAR can be covalently cross-linked to the epidermal growth factor-like module of urokinase after receptor-ligand interaction. The NH2-terminal domain of a recombinant, soluble uPAR derivative, labeled with [35S]cysteine, was isolated after limited proteolysis with chymotrypsin. The four disulfide bonds present within this domain were assigned by a combination of plasma desorption mass spectrometry, amino acid composition, and sequence analyses of peptides generated by trypsin, endoproteinase Asp-N, and thermolysin. The following disulfide bond structure was determined: Cys3-Cys24, Cys6-Cys12, Cys17-Cys45, and Cys71-Cys76. Similar cysteine pairing is likely to be found within other members of this protein superfamily, i.e. the membrane inhibitor of reactive lysis, Ly-6, and the remaining two domains of uPAR. However, an additional pair of cysteines present within these domains probably forms a fifth disulfide bond.

Amino Acid Sequence↗
PubMed1993

An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator.

Using 3' RACE (rapid amplification of cDNA ends), we have isolated a cDNA variant for the receptor for human urokinase plasminogen activator (uPAR). The deduced protein includes the amino-terminal ligand binding domain in uPAR, but lacks the carboxy-terminal membrane attachment by a glycolipid anchor. Genomic DNA analysis showed that the uPAR mRNA variant is generated by alternative splicing. The new variant mRNA is expressed in various human cell lines and tissues and both variants are up-regulated by phorbol ester in A549 cells. We propose that the alternatively spliced uPAR mRNA encodes a soluble uPA binding protein, the possible function of which is discussed.

Amino Acid Sequence↗
PubMed1993

High levels of urokinase-type plasminogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis.

The urokinase pathway of plasminogen activation is supposed to be involved in proteolytic degradation of the extracellular matrix during cancer invasion. The prognostic value of urokinase-type plasminogen activator (uPA) and type 1 plasminogen activator inhibitor (PAI-1) levels in cytosolic extracts of ductal breast carcinomas was studied, retrospectively, in 118 premenopausal and 72 postmenopausal high-risk patients entered into the protocol of Danish Breast Cancer Cooperative Group trials for adjuvant treatment of breast cancer. The median observation time was 8.5 years. uPA and PAI-1 levels were determined by sandwich enzyme-linked immunosorbent assays. There is a strong correlation between these levels (P < 0.001; r = 0.57). Univariate analysis showed that a high uPA level is significantly associated with short overall survival in both premenopausal (P < 0.001) and postmenopausal (P = 0.03) patients, while a high PAI-1 content significantly predicts shorter overall survival in premenopausal (P = 0.005) and postmenopausal (P < 0.001) patients and shorter relapse-free survival in postmenopausal patients (P < 0.001). When the levels of uPA and PAI-1 are related to those of other prognostic parameters, both high uPA and high PAI-1 levels are associated with grade of anaplasia in premenopausal patients and with number of tumor-positive lymph nodes in postmenopausal patients. A high PAI-1 level is associated with low estrogen and progesterone receptor levels in both pre- and postmenopausal patients. The prognostic value of uPA and PAI-1 levels was compared with that of established prognostic parameters by multivariate analysis. In premenopausal patients, high uPA is an independent prognostic parameter for shorter overall survival, the relative risk being 2.0 (95% confidence interval, 1.1-3.7). In postmenopausal patients, a high PAI-1 level is a strong and independent factor in predicting shorter overall survival with a relative risk of 2.9 (95% confidence interval, 1.5-5.8). In this group of patients a high PAI-1 level is also an independent predictor of shorter relapse-free survival (relative risk, 2.1; 95% confidence interval, 1.1-3.9). These data together with previous reports indicate that uPA and PAI-1 are potentially important prognostic factors in breast cancer. This is in good agreement with the supposed function of uPA in cancer invasion. It is proposed that PAI-1 plays a role in protecting the tumor against degrading itself. Alternatively, the PAI-1 level may be a biochemical marker of tumor angiogenesis.

Adult↗