Fluorescence imaging of invasive head and neck carcinoma cells with integrin αvß6-targeting RGD-peptides: an approach to a fluorescence-assisted intraoperative cytological assessment of bony resection margins.

We assessed the use of peptides containing arginylglycylaspartic acid (RGD) that target integrin αvß6 as a potential approach for a fluorescence-assisted intraoperative cytological assessment of bony resection margins (F-AICAB) in patients who had bone-infiltrating squamous cell carcinoma (SCC) of the head and neck. This was assessed to demarcate invasive carcinoma cells that stained for αvß6. Specimens from bony resection margins (n=362) were defined as either malignant or benign according to the results of cytological and histological examinations. Integrin αvß6-targeting fluorescence-labelled RGD peptides were added to the cytological samples and the accuracy of the resulting signal assessed by comparing it with the cytological findings. The value of F-AICAB was evaluated to find out if it could help to improve future diagnoses, tests, and treatments. Integrin αvß6 was strongly expressed in invasive SCC cells and qualified as a marker for bone-infiltrating carcinoma cells. It showed a high affinity to bind to invasive SCC cells and enabled swift and specific demarcation of αvß6-stained carcinoma cells. It was also diagnostic, with a sensitivity of 100% (95% CI 81.3% to 99.3%), specificity of 98.3% (95% CI 94.4% to 99.0%), positive predictive value of 92% (95% CI 70.2% to 94.3%), and negative predictive value of 100% (95% CI 96.9% to 99.9%), compared with the cytological findings. The targeting of specific integrin subtypes with selective, synthetic ligands, adapted for multimodal imaging, is a promising new approach to diagnosis. Further studies are necessary to provide more evidence for successful clinical translation and to establish the impact on clinical procedures.

Tissue inhibitor of metalloproteinases-1 induces a pro-tumourigenic increase of miR-210 in lung adenocarcinoma cells and their exosomes.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) recently emerged as a pro-metastatic factor highly associated with poor prognosis in a number of cancers. This correlation seemed paradox as TIMP-1 is best described as an inhibitor of pro-tumourigenic matrix metalloproteinases. Only recently, TIMP-1 has been revealed as a signalling molecule that can regulate cancer progression independent of its inhibitory properties. In the present study, we demonstrate that an increase of both exogenous and endogenous TIMP-1 led to the upregulation of miR-210 in a CD63/PI3K/AKT/HIF-1-dependent pathway in lung adenocarcinoma cells. TIMP-1 induced P110/P85 PI3K-signalling and AKT phosphorylation. It also led to increase of HIF-1α protein levels positively correlating with HIF-1-regulated mRNA expression and upregulation of the microRNA miR-210. Downstream targets of miR-210, namely FGFRL1, E2F3, VMP-1, RAD52 and SDHD, were decreased in the presence of TIMP-1. Upon the overexpression of TIMP-1 in tumour cells, miR-210 was accumulated in exosomes in vitro and in vivo. These exosomes promoted tube formation activity in human umbilical vein endothelial cell (HUVECs), which was reflected in increased angiogenesis in A549L-derived tumour xenografts. Activation and elevation of PI3K, AKT, HIF-1A and miR-210 in tumours additionally confirmed our in vitro data. This new pro-tumourigenic signalling function of TIMP-1 may explain why elevated TIMP-1 levels in lung cancer patients are highly correlated with poor prognosis.

[Tumor-associated prognostic factors of the plasminogen activator family: determination and clinical value of u-PA, t-PA, PAI-1, and PAI-2]. / Tumorassoziierte Prognosefaktoren der Plasminogenaktivator-Familie. Bestimmung und klinische Wertigkeit von u-PA, t-PA, PAI-1 und PAI-2.

Proteolytic factors belonging t the plasminogen activator family (plasmin, u-PA, t-PA, u-PAR, PAI-1, and PAI-2), which usually are involved in blood clotting and degradation of blood clots, are also present in healthy and diseased tissue of the kidney, lung, liver, gastro-intestinal tract, breast, prostate, ovary, and brain. These factors are engaged in brain development, angiogenesis and vascular invasion, wound healing as well as in placenta development and embryogenesis. Plasminogen activators u-PA and t-PA, their inhibitors PAI-1 and PAI-2, and the u-PA-receptor (u-PAR, CD87) are often elevated in solid malignant tumour tissues compared to their normal counterparts. In breast cancer patients, an elevated tumour tissue extract antigen content of u-PA, PAI-1, and u-PAR is associated with increased tumour aggressiveness and poor prognosis; in contrary, an elevated content of t-PA and PAI-2 indicates a favourable prognosis. For clinical relevant determination of these proteolytic factors in tumour tissue extracts, only enzymo-immunometric tests (ELISA) are recommended. Enzymometric and enzymographic tests are actually conducted only in an experimental, preclinical context.

High level synthesis of recombinant soluble urokinase receptor (CD87) by ovarian cancer cells reduces intraperitoneal tumor growth and spread in nude mice.

Focussing of the serine protease urokinase (uPA) to the tumor cell surface via interaction with its receptor (uPAR) is an important step in tumor invasion and metastasis. The human ovarian cancer cell line OV-MZ-6#8 was stably transfected with expression plasmids either encoding cell-associated uPAR (GPI-uPAR) or a soluble form of uPAR (suPAR) lacking its glycan lipid anchor. In vitro, high level synthesis of functionally active recombinant suPAR inhibited cell proliferation and led to reduced cell-associated fibrin matrix degradation, whereas fibrinolytic activity was increased in OV-MZ-6#8 cells overexpressing GPI-uPAR. Both OV-MZ-6#8-derived clones were inoculated into the peritoneum of nude mice and tested for tumor growth and spread. High level synthesis of recombinant suPAR (without altering the physiological expression levels of GPI-uPAR and uPA in these cells) resulted in a significant reduction of tumor burden (up to 86%) in the xenogeneic mouse model. In contrast, overexpression of GPI-uPAR in tumor cells did not affect tumor growth. Our results demonstrate that high levels of suPAR in the ovarian cancer cell vicinity can act as a potent scavenger for uPA, thereby significantly reducing tumor cell growth and cancer progression in vivo.

Integrin alpha(v)beta(3)/vitronectin interaction affects expression of the urokinase system in human ovarian cancer cells.

The urokinase type plasminogen activator (uPA), together with its receptor uPAR and the plasminogen activator inhibitor type-1 (PAI-1) plays a pivotal role during tumor invasion and metastasis. Integrins, via interaction with the extracellular matrix (ECM), control cell adhesion and motility. The two systems are functionally linked because uPAR and PAI-1 bind to the ECM component vitronectin (VN). Because integrin signaling alters gene expression patterns, we investigated whether the expression levels of uPA, uPAR, and PAI-1 are affected by ECM/integrin interactions. Expression of uPA, uPAR, and PAI-1 was significantly enhanced when human ovarian cancer cells (OV-MZ-6) were cultivated on fibronectin or collagen type IV. In contrast, VN induced down-regulation of uPA and uPAR while increasing PAI-1 by up to 4-fold. VN-dependent decrease of uPA protein was paralleled by a significant reduction of uPA promoter activity that was even more pronounced upon alpha(v)beta(3) overexpression and depended on the presence of intact Rel protein-binding sites. The activity of Rel transcription factors was also significantly reduced upon alpha(v)beta(3)-mediated cell adhesion to VN. The activity of the Rel-unresponsive PAI-1 promoter was up to 5-fold induced as a function of alpha(v)beta(3)/VN interaction. Thus, the balance between available concentrations of uPA, uPAR, PAI-1, and integrins in human ovarian cancer cells might provide a switch within the regulation of their invasive phenotype.

beta(3)A-integrin downregulates the urokinase-type plasminogen activator receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the u-PAR promoter.

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that beta(3)-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition beta(3)-integrin regulates u-PAR expression. Overexpression of beta(3)-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (-1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced beta(3)-integrin expression. A region between -398 and -197 bp of the u-PAR promoter was critical for beta(3)-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at -248 bp substantially impaired the ability of beta(3)-integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when beta(3)-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the beta(3)A-integrin isoform but not by other beta(3)-integrin isoforms and required the cytoplasmic membrane NITY(759) motif. Moreover, overexpression of the short but not the long isoform of the beta(3)-integrin adapter protein beta(3)-endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of beta(3)-integrin and u-PAR at the cell surface, beta(3) signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

RFLP Molecular Analysis of the Urokinase-Type Plasminogen Activator Gene.

Several cell biological studies have shown that the invasiveness of a variety of tumors depend on the regulated expression of proteolytic enzymes that degrade the surrounding extracellular matrix and dissociate cell-cell and/or cell-matrix attachments. One such enzyme, the serine protease urokinase-type plasminogen activator (uPA), converts enzymatically inactive plasminogen into the widely acting protease plasmin, which degrades several extracellular matrix components and also activates proenzyme forms of matrix metalloproteases. Thus, uPA is a central molecule in pericellular proteolysis (1-1). uPA (as well as other factors of the plasminogen activator system, the cell surface-associated uPA receptor [uPAR], and the plasminogen activator inhibitor type-1 [PAI-1]) is an important prognostic factor predicting relapse-free and/or survival in patients with a variety of solid malignant tumors including ovarian cancer; in all cases, high levels of uPA are associated with a poor prognosis (4-6).

Radiolabeled alpha(v)beta3 integrin antagonists: a new class of tracers for tumor targeting.

UNLABELLED: The alpha(v)beta3 integrins play an important role during tumor metastasis and tumor-induced angiogenesis. Targeting of this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Cyclo(-Arg-Gly-Asp-D-Phe-Val-) has been shown to be a selective alpha(v)beta3 integrin antagonist with high affinity. In this study we describe the synthesis and biological evaluation of [125I]-3-iodo-D-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) ([125I]P2), [125I]-3-iodo-Tyr5-cyclo(-Arg-Gly-Asp-D-Phe-Tyr-) ([125I]P4) and the negative control peptide [1251]-3-iodo-D-Tyr4-cyclo(-Arg-D-Ala-Asp-Tyr-Val-) ([125I]P6). METHODS: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl amino acid coupling protocols. Radioiodination was performed using the iodogen method. The in vitro binding assays were performed using isolated, immobilized alphaIIbeta3 and alpha(v)beta3 integrins. Expression of the alphaVbeta3 receptor on the different tumors was validated by immunohistochemical methods using alpha(v) and alpha(v)beta3 specific antibodies. For biodistribution studies, nude mice with melanoma M21 or mammary carcinoma MaCaF and BALB/c mice with osteosarcoma were used. RESULTS: The in vitro binding assays demonstrate that the introduction of tyrosine and subsequent iodination have no influence on the high affinity and selectivity for alpha(v)beta3. Immunohistochemical staining clearly indicates the presence of the alpha(v)beta3 integrins on the tumor tissue of the melanoma and the osteosarcoma. Pretreatment and displacement studies show specific binding of [125I]P2 on melanoma M21-bearing nude mice and osteosarcoma-bearing BALB/c mice but less specific binding on mammary carcinomas. [125I]P2 exhibits fast elimination kinetics. The accumulation in the tumor 10 min postinjection is 2.07 +/- 0.32 %ID/g for the melanoma M21 and 3.50 +/- 0.49 %ID/g for the osteosarcoma and decreases to 1.30 +/- 0.13 %ID/g and 2.03 +/- 0.49 %ID/g 60 min postinjection, respectively. [125I]P4 shows even faster elimination kinetics, resulting in a tumor accumulation of 0.40 +/- 0.10 %ID/g 60 min postinjection for the osteosarcoma-bearing BALB/c mice. Both peptides reveal predominately hepatobiliary excretion. For [1251]P2, this also is confirmed by autoradiography. The negative control peptide [125I]P6 shows no specific activity accumulation. CONCLUSION: [125I]P2 exhibits high affinity and selectivity for the alpha(v)beta3 integrin in vitro and in vivo and, thus, represents the first radiolabeled alpha(v)beta3 antagonist for the investigation of angiogenesis and metastasis in vivo.

UVB increases urokinase-type plasminogen activator receptor (uPAR) expression.

Keratinocytes synthesize and secrete urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. When bound to urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator proteolytically converts surface bound plasminogen to plasmin, which in turn cleaves many extracellular components leading to pericellular proteolysis. The activation of the urokinase system has been observed during re-epithelialization of skin wounds and in lesions of the autoimmune blistering skin disease pemphigus. As pemphigus is photoinducible, we investigated the effect of ultraviolet B on urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor expression in the epidermal keratinocyte cell line A431. Ultraviolet B increased cellular and secreted urokinase-type plasminogen activator protein (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) 24 h postirradiation. Northern blot analysis indicated that ultraviolet B increased urokinase-type plasminogen activator receptor mRNA. Compared with a more rapid mRNA induction by epidermal growth factor (maximal after 4 h) the ultraviolet B response was maximal after 24 h and prolonged up to 36 h. The mRNA induction was not dependent on protein synthesis as judged by cycloheximide incubation. Ultraviolet B did not influence urokinase-type plasminogen activator receptor mRNA stability (actinomycin D incubation). A transiently transfected chloramphenicol acetyltransferase-reporter construct containing a -398/+51 urokinase-type plasminogen activator receptor promoter fragment was activated when cells were exposed to ultraviolet B. This induction was almost completely abolished by mutating a -182/-176 AP-1 binding sequence. Ultraviolet B increased the binding capacity at this AP-1 motif in electrophoretic mobility shift assays. These data identify a distinct transcriptional mechanism by which ultraviolet B induces urokinase-type plasminogen activator receptor. The epidermal induction of components of the proteolytic urokinase system by ultraviolet B may help explain the photoinducibility of pemphigus lesions.

Prognostic significance of urokinase (uPA) and its inhibitor PAI-1 for survival in advanced ovarian carcinoma stage FIGO IIIc.

Strong evidence has accumulated on the prognostic value of tumour-associated proteolytic factors in patients afflicted with solid malignant tumours, including advanced ovarian cancer. We evaluated the prognostic impact of the protease urokinase plasminogen activator (uPA) and its inhibitor PAI-1 on overall survival in patients with advanced ovarian cancer stage FIGO IIIc in order to select patients at risk. uPA and PAI-1 antigen were determined by ELISA in primary tumour tissue extracts of 86 ovarian cancer patients FIGO stage IIIc enrolled in a prospective study. Univariate and multivariate analyses were performed using the Cox proportional hazard model. The time-varying coefficient model of Gray was used to assess the time-dependent strength of prognostic factors tumour mass, uPA and PAI-1 on overall survival. In all patients, uPA and PAI-1 (optimized cut-offs of 2.0 and 27.5 ng mg(-1) protein respectively), in addition to the traditional prognostic parameters of residual tumour mass, nodal status, grading and ascites volume, were of prognostic significance in univariate analysis for overall survival. Even in patients with residual tumour mass (n = 43), the statistically independent prognostic impact of PAI-1 persisted, allowing further discrimination between low- and high-risk patients. In multivariate analysis, residual tumour mass (P < 0.001, relative risk (RR) 4.5), PAI-1 (P < 0.001; RR 3.1) and nodal status (P = 0.022, RR 2.6) turned out to be strong, statistically independent prognostic parameters. Evaluation of the time-dependent prognostic impact of residual tumour mass and PAI-1 on overall survival (n = 86, 50 months) revealed that the prognostic power of these factors increased with time. In patients with advanced ovarian cancer, both residual tumour mass and PAI-1 are statistically independent strong prognostic factors. Even within patient subgroups with or without residual tumour mass, PAI-1 allowed selection of patients at risk who might benefit from individualized therapy protocols.

Rel transcription factors contribute to elevated urokinase expression in human ovarian carcinoma cells.

Elevated levels of the urokinase-type plasminogen activator (uPA) in tumor cells are conductive to tumor cell spread and metastasis. In a previous study we observed that suppression of RelA dramatically reduced endogenous uPA synthesis in the human ovarian cancer cell line OV-MZ-6. Because the uPA promoter contains three potential Rel-like protein binding motifs (RRBE, 5'-NF-kappaB, and 3'-NF-kappaB) we conducted the first thorough systematic uPA promoter analysis to examine the direct impact of Rel proteins on uPA gene transcription. Disruption of RRBE resulted in a approximately 40% decrease in uPA promoter activity, mutation of the 5'-NF-kappaB motif led to an additional 20% decrease. The 3'-NF-kappaB motif was not active. Overexpression of RelA significantly enhanced uPA promoter activity, whereas IkappaB-alpha overexpression reduced uPA promoter activity by 40%. These data were supported by the finding that endogenous uPA was also increased sixfold by overexpression of RelA and decreased by 30% upon overexpression of IkappaB-alpha. Transfection of OV-MZ-6 cells with antisense deoxynucleotides directed to RelA expression reduced uPA promoter activity by at least 40%. Our data clearly suggest that by binding to uPA promoter elements, Rel transcripton factors contribute directly to elevated uPA gene expression in human ovarian cancer cells, thereby promoting the multiple functions of uPA during tumor growth and metastasis.

Urokinase induces proliferation of human ovarian cancer cells: characterization of structural elements required for growth factor function.

Ovarian cancer metastasis is associated with an increase in the urokinase-type plasminogen activator (uPA) and its receptor uPAR. We present evidence that binding of uPA to uPAR provokes a mitogenic response in the human ovarian cancer cell line OV-MZ-6 in which endogenous uPA production had been significantly reduced by stable uPA 'antisense' transfection. High molecular weight (HMW) uPA, independent of its enzymatic activity, produced an up to 95% increase in cell number concomitant with 2-fold elevated [3H]thymidine incorporation as did the catalytically inactive but uPAR binding amino-terminal fragment of uPA, ATF. uPA-induced cell proliferation was significantly decreased by blocking uPA/uPAR interaction by the monoclonal antibody IIIF10 and by soluble uPAR. The efficiency of the uPAR binding synthetic peptide cyclo19,31 uPA19-31 to enhance OV-MZ-6 cell growth proved this molecular domain to be the minimal structural determinant for uPA mitogenic activity. Dependence of uPA-provoked cell proliferation on uPAR was further demonstrated in Raji cells which do not express uPAR and were thus not induced by uPA. However, upon transfection with full-length uPAR, Raji cells acquired a significant growth response to HMW uPA and ATF.

Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis (review).

Tumor cell migration and invasion into the surrounding tissue depend on the invasive capacity of cells leading to the loosening of cell-cell and cell-substratum contacts via cell surface associated proteolytic enzyme systems. Plasmin is one of the enzymes involved in these complex events. It is generated by the cleavage of the proenzyme plasminogen upon the action of the urokinase-type plasminogen activator (uPA). uPA is synthesized and secreted by tumor cells and normal cells and interacts with a specific cell surface receptor (uPAR) thereby focalizing enzymatic activity to the cell surface. The activity of uPA is controlled by plasminogen activator inhibitors type-1 and type-2. A strong statistically independent prognostic impact has been attributed to uPA and its inhibitor PAI-1 in a variety of malignancies. Besides its proteolytic activity, uPA in concert with uPAR exert biological effects characteristic for molecules with signal transducing properties including chemotaxis, migration/invasion, adhesion, and mitogenesis.

Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy.

Extravasation and intravasation of solid malignant tumors is controlled by attachment of tumor cells to components of the basement membrane and the extracellular matrix, by local proteolysis and tumor cell migration. Strong clinical and experimental evidence has accumulated that the tumor-associated serine protease plasmin, its activator uPA (urokinase-type plasminogen activator), the receptor uPA-R (CD87), and the inhibitors PAI-1 and PAI-2 are linked to cancer invasion and metastasis. In cancer, increase of uPA, uPA-R, and/or PAI-1 is associated with tumor progression and with shortened disease-free and/or overall survival in patients afflicted with malignant solid tumors. uPA and/or its inhibitor PAI-1 appear to be one of the strongest prognostic markers so far described. Strong prognostic value to predict disease recurrence and overall survival has been documented for patients with cancer of the breast, ovary, cervix, endometrium, stomach, colon, lung, bladder, kidney, brain, and soft-tissue. Due to the strong correlation between elevated uPA and/or PAI-1 values in primary cancer tissues and the tumor invasion/ metastasis capacity of cancer cells, proteolytic factors have been selected as targets for therapy. Various very different approaches to interfere with the expression or reactivity of uPA or CD87 at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, enzyme inhibitors, and recombinant or synthetic uPA and uPA-R analogues.

Thrombin induces thrombomodulin mRNA expression via the proteolytically activated thrombin receptor in cultured bovine smooth muscle cells.

Thrombin, an important mitogen governing smooth muscle cell proliferation, binds to cultured bovine aortic smooth muscle cells (BASMCs) via both the proteolytically activated thrombin receptor (PATR) and thrombomodulin (TM). Although TM mRNA expression and functional activity is regulated by thrombin in human endothelial cells and mouse hemangioma cells, it remains unclear in those models whether the increased TM mRNA expression observed upon thrombin stimulation is mediated through the activation of PATR or via TM occupancy. We observed in cultured BASMCs that TM mRNA is increased threefold to sixfold by either thrombin, basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF). The increase in TM mRNA with thrombin is time dependent (maximal at 3 hours), a consequence of increased mRNA stability, and accompanied by increases in cell surface TM functional activity. Thrombin-induced TM mRNA was reproduced by the hexameric thrombin receptor-activating peptide (TRAP6) and augmented by a TM-specific antibody. Together, these data suggest that up-regulation of TM mRNA by thrombin is mediated via the PATR. We speculate that increases in BASMC TM mRNA and activity after thrombin may contribute to the impaired thrombus formation observed after atherosclerotic vascular injury.

Inhibition of the interaction of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) by synthetic peptides.

Focusing of the serine protease urokinase-type plasminogen activator (uPA) to the cell surface via interaction with its specific receptor (uPAR, CD87) is an important step for tumor cell invasion and metastasis. The ability of a synthetic peptide derived from the uPAR-binding region of uPA (comprising amino acids 16-32 of uPA; uPA(16-32)) to inhibit binding of fluorescently labeled uPA to uPAR on human promyeloid U937 cells was assessed by quantitative flow cytofluorometric analysis (FACS) and compared to the inhibitory capacities of other synthetic peptides known to interfere with uPA/uPAR-interaction. An about 3000-fold molar excess of uPA(16-32) resulted in 50% inhibition of pro-uPA binding to cell surface-associated uPAR. Using a solid-phase uPA-ligand binding assay employing recombinant soluble uPAR coated to microtiter plates, the minimal binding region of wild-type uPA was determined. The linear peptide uPA(19-31) and its more stable disulfide-bridged cyclic form (cyclo(19,31)uPA(19-31)) displayed uPAR-binding activity whereas other peptides such as uPA(18-30), uPA(20-32) or uPA(20-30) did not react with uPAR. Cyclic peptide derivatives of cyclo(19,31)uPA(19-31) in which certain amino acids were deleted and/or replaced by other amino acids as well as uPAR-derived wild-type peptides did also not inhibit uPA/uPAR-interaction. Therefore, the present investigations identified cyclo(19,31)uPA(19-31) as a potential lead structure for the development of uPA-peptide analogues to block uPA/uPAR-interaction.

Mitogen crosstalk accompanying urokinase receptor expression in stimulated vascular smooth muscle cells.

Thrombin and other mitogens regulate the expression of the urokinase-type plasminogen activator receptor (uPAR) protein and mRNA levels in bovine vascular smooth muscle cells (SMC). We investigated interactions between mitogens capable of increasing uPAR mRNA levels in SMC. Up-regulation of uPAR mRNA upon thrombin and basic fibroblast growth factor (bFGF) stimulation was preceded by a 2-3-fold transient increase in bFGF mRNA within 1 h. TGF-beta1 did not result in a significant change in bFGF mRNA levels. Platelet-derived growth factor (PDGF) while substantially enhancing uPAR mRNA levels, diminished bFGF mRNA levels by 3-4-fold. Both thrombin and bFGF induced the message for bFGF-R 2-3-fold. Thrombin also provoked a 3-4-fold rise in TGF-beta1 mRNA levels within 30 min. In summary, on the mRNA level, we demonstrated both positive as well as negative feed-back mechanisms between different mitogens, among them bFGF revealing in addition to autoinduction also up-regulation of the transcript concentration of its own receptor. Thus, cooperation and possible amplification of mitogenic effects might be implicated in the fine-tuned regulation of uPAR mRNA in stimulated bovine aorta SMC.

Inhibition of NF-kappa B-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1.

The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis.

Primary tumor and metastasis in ovarian cancer differ in their content of urokinase-type plasminogen activator, its receptor, and inhibitors types 1 and 2.

The relevance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI) type 1 in predicting the survival probability of patients with advanced ovarian cancer after radical surgery and adjuvant chemotherapy by assessing the patients' primary tumors has recently been shown by us (W. Kuhn et al., Gynecol. Oncol., 55: 401-409, 1994). In the present study, we determined uPA, uPA receptor, PAI-1, and PAI-2 concentrations in primary tumors and tumor-infiltrated omentum and retroperitoneal lymph nodes of ovarian cancer patients. The group consisted of 39 patients with advanced ovarian carcinoma stages Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) IIIc or IV; for comparison 7 patients with early carcinoma stage FIGO I were also included. In metastases of the omentum from ovarian cancer stage FIGO IIIc or IV patients, we noted a 4-fold elevated uPA content, a 2-fold increase in PAI-1, and also a significant increase in uPA receptor and PAI-2 over primary tumors. In metastases of the lymph nodes the levels of the respective antigens were also increased when compared to primary tumors. These data may indicate that elevated levels of components of the fibrinolytic system at sites of metastases may contribute to the aggressive potential of cancer cells by favoring their reimplantation and/or the consolidation of a new tumor stroma.