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.
Full text
Available for:
FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has
been postulated to play a central role in the mediation of pericellular ...proteolytic activity. We have studied the kinetics
of plasminogen (Plg) activation catalyzed by uPA specifically bound to its receptor on the human monocytoid cell-line U937
and demonstrate this process to have properties differing widely from those observed for uPA in solution. The solution-phase
reaction was characterized by a Km of 25 microM and for the cell-associated reaction this fell 40-fold to 0.67 microM, below
the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall
catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding
antagonist. In contrast to intact cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937
cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates
was retained. Therefore, the cellular binding of Plg appears to be of critical importance for the efficient activation of
Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to
be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell
surface constitutes the preferential site for Plg activation when uPA is bound to its specific cellular receptor, which therefore
has the necessary characteristics to play an efficient role in the generation of pericellular proteolytic activity.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recombinant human gamma 2 chain of laminin-5 was expressed in Escherichia coli, and used to generate specific polyclonal antibodies which were used to study the distribution of the protein in human ...cancers. A total of 72 biopsies of human cancers were stained, including 23 cases of colon adenocarcinomas, 16 ductal breast carcinomas, 9 malignant melanomas, 14 squamous cell carcinomas of the skin and cervix, and 10 sarcomas. As a control for the specificity of the antibodies, we performed in situ hybridization on adjacent sections of a number of the cases, and in all of these cases the localization of the gamma 2 chain protein and mRNA was identical. We found gamma 2 chain immunoreactivity in cancer cells in all cases of colon adenocarcinomas and squamous cell carcinomas but not in any of the sarcomas, supporting the view that the laminin-5 protein is specific for cells of epithelial origin. Notably, in all of the cases of colon adenocarcinomas, the positive staining was invariably associated with budding cancer cells located at the tip of invading malignant epithelium, whereas the cancer cells deeper in the tumors were most often negative. The staining was cytoplasmic in all cases and only in one case did we see additional extracellular immunoreactivity, indicating that this laminin isoform in cancer tissue is not laid down in the extracellular matrix but probably exerts its function at the cell surface or in its immediate vicinity. Using in situ hybridization to analyze the coexpression of laminin-5 and components of the plasminogen activation system, we found that the histological distribution of laminin-5-positive budding cancer cells at the invasion front in colon adenocarcinomas was identical to that of the receptor for urokinase-type plasminogen activator. These findings suggest that laminin-5 is a marker of invading cancer cells in at least some human malignancies, and that it therefore might represent a valuable marker for the invasive potential of these cancers. The colocalization of laminin-5 and urokinase-type plasminogen activator receptor in a subset of cancer cells in colon cancer also suggests that a controlled up-regulation of a number of gene products is a characteristic of budding colon cancer cells, and that these gene products serve functions crucial for the invasive phenotype of these cancer cells.
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.
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.
The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be
a true member of a family of integral membrane proteins, anchored to the ...plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol
moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-2-hydroxy-1,1-bis(hydroxymethyl)-ethylglycine-sodium
dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound
u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific
phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment
holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or
mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase
separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with 3Hethanolamine and myo-3Hinositol.
5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide
of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been
reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) ...anchor of uPAR, which is attached to the third domain, is an important determinant in governing this reaction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uPAR (suPAR), which lacks the glycolipid anchor. This was not a general difference in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPAR and suPAR are identical except for the C-terminal truncation, the different cleavage patterns suggest that the two uPAR variants differ in the conformation or the flexibility of the linker region between domains 1 and 2. This was supported by the fact that an antibody to the peptide AVTYSRSRYLE, amino acids 84-94 in the linker region, recognizes GPI-uPAR but not suPAR. This difference in the linker region is thus caused by a difference in a remote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, low concentrations of uPA could no longer cleave the modified GPI-uPAR and the reactivity to the peptide antibody was greatly decreased. Naturally occurring suPAR, purified from plasma, was found to have a similar resistance to uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.
In this study in situ hybridization methods were used to examine biopsy samples from 13 adenocarcinomas of the colon for the presence of mRNA for the urokinase-type plasminogen activator (u-PA) and ...its specific cell-surface receptor (u-PAR). In all cases, u-PA mRNA was present in fibroblastlike cells in the stroma adjacent to the invasive tumor nodules. Urokinase-type plasminogen activator mRNA was not detected in the malignant cells. All specimens also contained u-PAR mRNA in cells located at the tumoral-stromal interface of invasive foci, but in contrast at least some of these cells were in all but one case identified as being of malignant origin. Stromal cells, probably tumor-infiltrating macrophages and neutrophils, also were positive in these areas. These results support the view that components of the plasminogen activation system may act to influence proteolytic events occurring at the interface between stroma and malignant cells in adenocarcinomas of the colon in humans.
The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since ...proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, the uPA−uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA−uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K d ≈ 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor−peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met246, His249, His251, and Phe256) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
PDF
