RESUMO
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.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Invasividade Neoplásica , Regeneração , Neoplasias da Mama/patologia , Neoplasias do Colo/patologia , Neoplasias Cutâneas/patologiaRESUMO
Activation of plasminogen (Plg) has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling events, including wound healing. However, there has been no definitive proof of involvement of Plg in such processes. We now report that healing of skin wounds is severely impaired in mice made deficient in Plg by targeted gene disruption. The results demonstrate that Plg is required for normal repair of skin wounds in mice and support the assumption that it also plays a central role in other disease processes involving extracellular matrix degradation, such as cancer invasion.
Assuntos
Plasminogênio/genética , Plasminogênio/fisiologia , Cicatrização/genética , Cicatrização/fisiologia , Animais , Endopeptidases/metabolismo , Matriz Extracelular/patologia , Regulação da Expressão Gênica , Marcação de Genes , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Mutantes , Pele/lesões , Pele/metabolismo , Pele/patologiaRESUMO
Serine proteases or esterases released from cell cultures into the growth medium were converted to radioactive derivatives by active site labeling with tritiated DFP, both in the presence and absence of other competing active site reagents. The individual labeled enzymes were then identified by SDS-polyacrylamide gel electrophoresis and scintillation autoradiography. Conditioned medium from embryonal mouse fibroblasts transformed by mouse sarcoma virus contained five serine enzymes that were not present in medium from normal cells; two serine enzymes were released by both cell types, and one serine enzyme was found only in medium from normal cells. Two of the enzymes released by transformed cells were identified as plasminogen activators; these accounted for most of the serine enzyme labeling in transformed culture media and for most of the serine enzyme difference between normal and transformed cultures. The culture fluids from two cell strains of human neoplastic origin were examined by the same method. A rhabdomyosarcoma strain released eight serine enzymes (mol wt ranging from 22,500 to 102,000), four of which were plasminogen activators; seven serine enzymes (mol wt 26,000-102,000), including two plasminogen activators, were detected in medium from human melanoma cultures. In terms of electrophoretic mobility two of the plasminogen activators from rhabdomyosarcoma were identical with those from melanoma cultures, while the remaining two rhabdomyosarcoma activators coincided with activators found in commerical urokinase.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias/enzimologia , Peptídeo Hidrolases/metabolismo , Sítios de Ligação , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Esterases/metabolismo , Humanos , Isoflurofato/metabolismo , Cinética , Ativadores de Plasminogênio/metabolismo , Serina , Inibidores da Tripsina/farmacologiaRESUMO
Two mouse urokinase-type plasminogen activator receptor (muPAR) cDNAs were isolated: muPAR1 is homologous to the human urokinase-type plasminogen activator receptor while muPAR2 codes for a 199 residue protein sharing the first 133 residues with muPAR1. Mouse genomic DNA sequencing indicates that the two different mRNAs arise by alternative splicing. In situ hybridization showed differential expression of the two mRNAs in mouse gastric mucosa. muPAR1 mRNA is located in luminal epithelial cells situated close to urokinase-type plasminogen activator-producing connective tissue cells of the lamina propria, pointing to plasmin generation controlled by the cooperation of different cells that may play a role in the release of gastric epithelial cells. muPAR2 mRNA is expressed in the basal epithelial cells, and the deduced protein sequence includes the receptor ligand binding domain, but omits the region involved in glycolipid-mediated membrane anchoring, suggesting that muPAR2 may code for a secreted uPA binding protein.
Assuntos
Mucosa Gástrica/metabolismo , Splicing de RNA , Receptores de Superfície Celular/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/isolamento & purificação , Humanos , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.
Assuntos
Adesão Celular , Membrana Celular/enzimologia , Espaço Extracelular/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular , Ferritinas , Humanos , Técnicas Imunológicas , Microscopia Eletrônica , Proteínas Musculares/metabolismo , VinculinaRESUMO
We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.
Assuntos
Adeno-Hipófise/metabolismo , Ativadores de Plasminogênio/metabolismo , Animais , Feminino , Hormônio do Crescimento/metabolismo , Técnicas Imunoenzimáticas , Técnicas de Imunoadsorção , Masculino , Adeno-Hipófise/citologia , RatosRESUMO
We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10(-6) M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PA-mediated focal proteolysis.
Assuntos
Precursores Enzimáticos/análise , Fibrossarcoma/enzimologia , Glicoproteínas/análise , Ativadores de Plasminogênio/análise , Ativador de Plasminogênio Tipo Uroquinase/análise , Linhagem Celular , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Fibrossarcoma/ultraestrutura , Imunofluorescência , Humanos , Microscopia Eletrônica , Peso Molecular , Inativadores de Plasminogênio , PeleRESUMO
Immunocytochemistry, using rabbit antibodies to a urokinase-type 48-Kdalton Mr mouse plasminogen activator, showed that enzyme immunoreactivity is widely distributed in the normal mouse. Strong staining was obtained in widely disseminated connective tissue cells with a fibroblast-like morphology. Such cells occurred in high numbers in the lamina propria mucosae of the gastrointestinal tract, and in moderate numbers in the connective tissue septa of the pancreas. A few such cells were detected around the larynx, trachea, and bronchi. Immunoreactivity also occurred in epithelial cells of the proximal and distal kidney tubules, the ductus deferens, and in pulmonary pneumocytes. In addition, presumably extracellular staining was seen irregularly along the basement membrane and fibrillar structures in the lamina propria of the small and large intestines. Moreover, decidual cells of the mouse placenta stained strongly, and a moderate staining was observed in epithelial cells of involuting mammary glands, but not in those of noninvoluting glands. No immunoreactivity was observed in endothelial cells. Control experiments included absorption of the antibodies against highly-purified mouse plasminogen activator and the corresponding proenzyme, and the finding of a good correspondence between the number of immunoreactive cells and measurable enzymatic activity determined in adjacent tissue sections. Separation by SDS PAGE followed by immunoblotting revealed only one immunochemically stainable protein band with Mr approximately 48 Kdaltons in extracts from tissues showing immunoreactivity.
Assuntos
Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Sistema Digestório/enzimologia , Feminino , Técnicas Imunológicas , Rim/enzimologia , Pulmão/enzimologia , Masculino , Camundongos , Ativadores de Plasminogênio/metabolismo , Distribuição Tecidual , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
The invasively growing and metastasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.
Assuntos
Neoplasias Pulmonares/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Anticorpos , Feminino , Histocitoquímica , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificaçãoRESUMO
Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.
Assuntos
Matriz Extracelular/metabolismo , Melanoma/patologia , Metástase Neoplásica , Inativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Northern Blotting , Expressão Gênica , Humanos , Técnicas In Vitro , Melanoma/enzimologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Mensageiro/genética , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.
Assuntos
Fibrossarcoma/enzimologia , Peptídeo Hidrolases/metabolismo , Ativadores de Plasminogênio/metabolismo , Plasminogênio/metabolismo , Células Tumorais Cultivadas/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Meios de Cultura , Ativação Enzimática , Fibrinolisina/metabolismo , Humanos , Cinética , Peso Molecular , Ligação ProteicaRESUMO
A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.
Assuntos
Monócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Contagem de Células , Linhagem Celular , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos , Humanos , Técnicas de Imunoadsorção , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Ativadores de Plasminogênio/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.
Assuntos
Inibidores da Angiogênese/farmacologia , Endopeptidases/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Vitronectina/metabolismo , Animais , Endotélio Vascular/efeitos dos fármacos , Fibrinolisina/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Mutantes , Neoplasias Musculares/irrigação sanguínea , Invasividade Neoplásica , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/etiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Vitronectina/genéticaRESUMO
Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.
Assuntos
Clonagem Molecular , DNA/análise , Endotélio/análise , Glicoproteínas/genética , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Códon , Glicoproteínas/análise , Glicoproteínas/imunologia , Humanos , RNA Mensageiro/análiseRESUMO
We have identified a soluble form of the human urokinase plasminogen activator (uPA) receptor (uPAR) in the ascitic fluids from patients with ovarian cancer. After purification of uPAR from the ascitic fluids by ligand-affinity chromatography (pro-uPA Sepharose), the uPAR was initially identified by cross-linking to a radiolabeled amino-terminal fragment of human uPA. The uPAR purified from the ascitic fluid has no bound ligand (uPA), as similar amounts can be purified by ligand-affinity chromatography as by immuno-affinity chromatography. uPAR from ascitic fluids partitions in the water phase after a temperature-dependent phase separation of a detergent extract. It therefore lacks at least the lipid moiety of the glycophospholipid anchor present in cellular-bound uPARs. It is highly glycosylated and the deglycosylated form has the same electrophoretic mobility as previously characterized cellular uPAR from other sources. The immunoreactivity of the purified uPAR from the ascitic fluid is indistinguishable from that of characterized uPAR, demonstrated by Western blotting with three different anti-uPAR monoclonal antibodies. The uPAR was found in 11 of 11 ascitic fluids from patients with ovarian cancer and in elevated amounts in the plasma from 2 of 3 patients. The concentration of soluble uPAR in the ascitic fluid was estimated to range between 1 and 10 ng/ml. Human soluble uPAR, derived from the tumor cells, was also found in the ascitic fluid and serum from nude mice xenografted intraperitoneally with three different human ovarian carcinomas.
Assuntos
Líquido Ascítico/química , Carcinoma/química , Neoplasias Ovarianas/química , Receptores de Superfície Celular/isolamento & purificação , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Feminino , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Nus , Transplante de Neoplasias , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Solubilidade , Transplante HeterólogoRESUMO
BACKGROUND: Degradation of extracellular matrix proteins, such as fibrin, is pivotal to tumor invasion. Inhibition of the interaction between urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and hence pro-u-PA activation, is an attractive approach to anti-invasive cancer therapy. A number of inhibitors exist for the human system, but because of species specificity none of these are efficient in mice. We have recently generated an inhibitory monoclonal antibody (mAb) against mouse u-PAR (mR1) by immunization of u-PAR-deficient mice. OBJECTIVES: To evaluate the effect of mR1 in vivo in a physiological setting sensitive to deregulated fibrinolysis, we have administered mR1 systemically and quantitated the effect on liver fibrin accumulation. METHODS: Wild-type and tissue-type plasminogen activator (t-PA) deficient mice were administered with mR1, or control antibody, during 6 weeks. Thereafter, the livers were retrieved and the amount of liver fibrin measured by unbiased morphometrical analysis of immunofluorescence signal. RESULTS: Systemic administration of mR1 caused significantly increased fibrin signal in anti-u-PAR treated t-PA-deficient mice compared to mock-treated, which mimics the phenotype of u-PAR;t-PA double-deficient mice. Fibrin and fibronectin accumulated within the sinusoidal space and was infiltrated by inflammatory cells. Analysis of small and rare hepatic fibrin plaques observed in t-PA-deficient mice showed infiltrating macrophages that, contrary to surrounding Kuppfer cells, expressed u-PAR. CONCLUSION: We show that u-PAR-expressing macrophages are involved in cell-mediated fibrinolysis of liver fibrin deposits, and that the antimouse-u-PAR mAb is effective in vivo and thus suited for studies of the effect of targeting the u-PA/u-PAR interaction in mouse cancer models.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Fibrina/metabolismo , Fígado/efeitos dos fármacos , Receptores de Superfície Celular/imunologia , Ativador de Plasminogênio Tecidual/genética , Animais , Anticorpos Monoclonais/farmacologia , Imunofluorescência , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
Dexamethasone increases type 1 plasminogen activator inhibitor (PAI-1) activity released from the human fibrosarcoma cell line HT-1080. We demonstrated that dexamethasone caused about 10-fold increases in the intracellular and extracellular levels of PAI-1 protein, as measured by an enzyme-linked immunosorbent assay, in the rate of PAI-1 biosynthesis, and in the PAI-1 mRNA level. The effects on PAI-1 biosynthesis and mRNA level were detectable within 4 h and were maximal 16 to 24 h after the addition of dexamethasone. Cycloheximide did not inhibit the dexamethasone-induced increases in the capacity of the cells to synthesize PAI-1 and in the PAI-1 mRNA level.
Assuntos
Dexametasona/farmacologia , RNA Mensageiro/genética , Ativador de Plasminogênio Tecidual/biossíntese , Linhagem Celular , Cicloeximida/farmacologia , Fibrossarcoma/metabolismo , Humanos , Cinética , Peso Molecular , RNA Mensageiro/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/isolamento & purificaçãoRESUMO
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.