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[Cancer Research 59, 4440-4445, September 1, 1999]
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[Cancer Research 59, 4440-4445, September 1, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Regulation of the Urokinase-type Plasminogen Activator System by the von Hippel-Lindau Tumor Suppressor Gene1

Maartje Los, Saida Zeamari, John A. Foekens, Martijn F. B. G. Gebbink and Emile E. Voest2

Department of Internal Medicine, Laboratory of Medical Oncology, University Hospital Utrecht, 3508 GA Utrecht [M. L., S. Z., M. F. B. G. G., E. E. V.]; and Department of Medical Oncology, Rotterdam Cancer Centre, 3015 GD Rotterdam [J. A. F.], the Netherlands


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The urokinase-type plasminogen activator (uPA) system plays an important role in tumor cell invasion, metastases, and angiogenesis. uPA, uPA receptor, and plasminogen activator inhibitor 1 (PAI-1) are prognostic factors in different solid tumors, e.g., renal cell carcinomas (RCCs). von Hippel-Lindau (VHL) disease is an inherited cancer syndrome that is characterized by extensively vascularized tumors, including hemangioblastomas and RCCs. In 75% of sporadic RCCs, the VHL gene is also inactivated. It has been recognized in sporadic RCC that PAI-1 mRNA levels are up-regulated and uPA mRNA levels are down-regulated. We determined the role of the VHL tumor suppressor gene in the regulation of the uPA system in RCC. In 786-O RCC cells expressing the wild-type (wt) VHL gene, we measured a 3-fold higher overall urokinase activity than in 786-O cells expressing a mutant VHL gene or lacking VHL. uPA mRNA and protein levels were higher in cells with wt VHL compared with cells with mutant VHL or lacking VHL. In addition, PAI-1 mRNA and protein levels were dramatically increased in 786-O cells with mutant VHL or lacking VHL, compared with cells expressing wt VHL.

Our results provide further evidence that the VHL gene plays an important role in the process of angiogenesis by regulation of plasmin-mediated proteolysis of the extracellular matrix and may explain why VHL-induced RCCs grow slowly and metastasize relatively late.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RCC3 is a common manifestation of VHL disease, a rare hereditary cancer syndrome (1) . Other manifestations of VHL disease are hemangioblastomas of the cerebellum, angiomas of the retina, and pheochromocytomas. Cysts are seen in several organs, e.g., liver, kidneys, brain, pancreas, and epididymis (2 , 3) . In addition to VHL disease-related tumors, inactivation of the VHL gene is found in the majority (75%) of sporadic RCCs, a minority of sporadic hemangioblastomas, and colorectal cancer (4, 5, 6, 7) . When patients are diagnosed with VHL disease, they are enrolled into a screening program. Renal tumors are, therefore, frequently detected at an early presymptomatic stage and can be followed by ultrasonography, computed tomography, or magnetic resonance imaging. These follow-up studies have revealed that most small renal tumors enlarge slowly. Because VHL patients require repetitive surgery, there is a tendency to wait until tumors are 3 cm in diameter (8) . For years, the treatment of choice for VHL-related RCC was uni- or even bilateral nephrectomy. This is still the indicated treatment for diffuse disease or multiple lesions in the kidney. Nephron-sparing surgery has been advocated in small RCC to preserve renal function and delay end-stage renal failure (9) . Recently, arguments for even less aggressive treatment of RCC in VHL patients were provided. This was based on the observations that metastases of RCC in VHL patients are significantly rarer than in sporadic RCC and that they occur only in tumors that are larger than 7 cm (10) . The molecular basis for this clinical observation is still unknown.

The VHL gene was cloned in 1993 by Latif et al. (11) and appears to be a tumor suppressor gene located on chromosome 3p25-25. The VHL gene encodes two proteins: pVHL30 and pVHL19, with molecular masses of 30 and 19 kDa, respectively (12, 13, 14) The VHL protein is expressed in nearly all human tissues and has no homology with any known protein (15) . Proteins that bind pVHL have been studied in attempts to learn more about the function of the VHL gene. pVHL binds to elongins B and C, and this complex is a negative regulator of the transcription elongation factor elongin or SIII (16, 17, 18) . Very recently, it was shown that other proteins, such as CUL2 and Rbx 1, are binding to this complex. This so-called VCB complex is highly similar with E3 ubiquitin ligase complexes, which are key regulators of protein degradation (19, 20, 21, 22) .

Because extensive vascularization is characteristic for VHL-related tumors, functional analysis of the VHL gene has been directed at its role in angiogenesis. Both the VHL-related and sporadic RCCs and hemangioblastomas express high levels of the angiogenic factor VEGF and its receptors (23, 24, 25) . In vitro studies subsequently showed that human RCC cells lacking the wt VHL gene product express high levels of hypoxia-inducible mRNAs such as VEGF, PDGF-B, and glucose transporter 1. Introduction of wt VHL cDNA resulted in a down-regulation of these mRNAs (26, 27, 28) . In addition, pVHL regulates the transforming growth factor ß1 gene and interacts with fibronectin, an extracellular glycoprotein that binds to members of the integrin family of cell surface receptors (29 , 30) .

In the process of angiogenesis and tumor invasion, the uPA system plays an important role. This is illustrated by several clinical studies that show that uPA, uPAR, and PAI-1 levels of different cancers are prognostic factors for patient survival and tumor relapse (31 , 32) . For RCC, it has been shown that PAI-1 is a strong and independent prognostic factor in predicting early relapse of the tumor and overall survival (33 , 34) . In these studies, the investigators could discriminate between high- and low-risk groups for disease-free survival by measuring the PAI-1 protein content in tumor tissue (34) . PAI-1 mRNA expression levels were measured in paired samples of RCC and adjacent normal kidney tissue. PAI-1 mRNA levels were significantly higher in tumor samples compared with normal kidney (35) . On the other hand, a >3-fold lower expression of uPA mRNA was observed in RCC compared with normal kidney tissue (36) .

The aim of our study was to determine whether the VHL gene is involved in the regulation of the uPA system in RCC. Our results indicate that inactivation of the VHL gene results in decreased uPA and increased PAI-1 levels in RCC cells. These findings provide further support that the VHL gene plays a central role in the regulation of genes involved in angiogenesis and may explain why RCC in VHL patients grow relatively slowly and metastases occur only occasionally.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
786-O cells are RCC cells that lack a functional VHL gene. These cells, stably transfected with pRC (no VHL), pRC-HAVHL (wt VHL), or pRC-HAVHL1-115 (mutant VHL containing amino acid residues 1–115; gifts from W. G. Kaelin, Dana-Farber Cancer Institute, Boston, MA), were grown in DMEM-10% FCS supplemented with G418 (1 mg/ml) at 37°C in an atmosphere with 10% CO2 (12) .

For performing the different experiments, cell medium was replaced with fresh serum-free DMEM with or without 100 ng/ml TPA (Sigma Chemical Co., St. Louis, MO) or 5 µg/ml dactinomycin (Lyovac Cosmogen, MSD Agver BV, Haarlem, The Netherlands).

Assay of Overall uPA Activity.
Overall uPA activity was measured on cells grown in 96-well plates. Cells were washed twice with PBS, and uPA activity was determined by incubating cells at 37°C with plasminogen (8 µg/ml; Sigma) and the plasmin-specific substrate S-2251 (Chromogenix AB, Mölndal, Sweden). The release of paranitroaniline from S-2251 was determined in each well by measuring the absorbance at 405 nm using a microplate reader. Controls included incubation of cells without plasminogen. uPA activity was quantitated using a standard curve generated with human uPA (Sigma).

RNA Extraction and Northern Blot Analysis.
Total RNA was extracted from cultured cells with RNAzol (Tel-Test, Friendswood, TX) according to the manufacturer’s protocol. RNA samples (15 µg/lane) were separated in a 1% agarose-formaldehyde gel and transferred to a Hybond nylon membrane (Amersham, Buckinghamshire, United Kingdom). Membranes were prehybridized at 42°C in a prehybridization buffer [50% deionized formamide, 5x SSPE (1x SSPE = 0.15 M NaCl, 0.01 M NaH2PO4, and 1 mM EDTA), 5x Denhardt’s solution, 0.5% SDS, and 10% dextran sulfate] and denatured salmon sperm DNA for 2–4 h and hybridized for 18 h in the same buffer containing the cDNA probes. cDNA fragments were labeled using a Prime-It RmT Random Primer Labeling Kit (Stratagene, La Jolla, CA) and [{alpha}-32P]dCTP. The cDNA probes used were human uPA and human uPAR (kindly provided by Dr. P. H. A. Quax, Gaubius Laboratory, Leiden, the Netherlands), human PAI-1 (kindly provided by Dr. H. Pannekoek, University of Amsterdam, Amsterdam, the Netherlands), and ß-actin. The membranes were washed and exposed to a phosphorus imager screen (Molecular Dynamics, Sunnyvale, CA). uPA, uPAR, and PAI-1 mRNA expression levels were quantitated with an ImageQuant program (Molecular Dynamics); variations in loading were quantitated by comparing expression levels with ß-actin mRNA expression.

uPA, uPAR, and PAI-1 ELISAs.
Conditioned media and cell lysates were harvested from cells grown for 24 h in serum-free DMEM without phenol red. After the conditioned medium was collected, cells were counted and lysed in NP40 lysis buffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.5% NP40]. Lysates and media were cleared by centrifugation and stored in -80°C until further use. All samples were measured in duplicate. uPA and PAI-1 antigens were measured by ELISA described by Grebenschikov et al. (37) . uPAR antigens were measured by an ELISA described by Ronne et al. (38) . The amount of uPA and uPAR protein present in cell lysates and conditioned media was expressed in pg per 105 cells, and the amount of PAI-1 protein was expressed as ng per 105 cells.

Immunohistochemistry.
Seven RCCs from VHL patients and seven sporadic RCCs were removed by surgery at the University Hospital Utrecht. All tumor samples were routinely formalin-fixed and paraffin-embedded. Four-µm sections of these tumors were deparaffinized in xylene and rehydrated in ethanol. The endogenous peroxidase activity was blocked with methanol containing 1.5% H2O2 for 15 min. Primary and secondary antibodies were diluted in PBS-1% BSA. Primary antibodies used were mouse mAbs against uPA (mAb 3689; American Diagnostica) and against uPAR (mAb R3; a kind gift of the Finsen Laboratory, Copenhagen, Denmark). Sections were incubated with the primary antibodies for 1 h at room temperature. For detection of the uPA and uPAR antigen, sections were subsequently incubated for 30 min with rabbit antimouse peroxidase (DAKO, Glostrup, Denmark) and swine antirabbit peroxidase (DAKO). Immunoreactivity was visualized with diaminobenzidine (Sigma). Sections were counterstained with Mayer’s hematoxylin, rehydrated, and mounted.

Statistics.
uPA, uPAR, and PAI-1 protein concentrations in conditioned media, and cell lysates were presented as the means of three independent experiments ± SE. Statistical analysis were performed by use of the Student’s t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overall uPA Activity in RCC Cells with or without a Functional VHL Gene.
Because proteases of the plasminogen activator system might play a role in VHL tumor progression, we measured the overall uPA activity in 786-O cells. The uPA activity was defined as the net result of positive and negative regulators of uPA system. 786-O cells are RCC cells that lack a functional VHL gene. Reintroduction of wt VHL in 786-O cells inhibited their ability to form tumors in nude mice (12) . The overall uPA activity of 786-O RCC cells expressing wt VHL, mutant VHL, and no VHL were measured on cells grown in 96-well plates. The overall uPA activity in 786-O cells with wt VHL (5.67 ± 0.8 microunits per 104 cells) was more than three times higher than that in 786-O cells lacking VHL (1.78 ± 0.39 microunits per 104 cells) or with mutant VHL (0.81 ± 0.17 microunits per 104 cells; P < 0.001; Fig. 1Citation ).



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  		Fig. 1. Overall uPA activity of 786-O RCC cells expressing wt VHL, mutant VHL, or no VHL. uPA activity was measured on cells grown in 96-well plates. Columns, means of eight independent experiments, expressed as microunits of uPA activity per 10,000 cells; bars, SE. ***, P < 0.001.

 
uPA, uPAR, and PAI-1 mRNA Levels in 786-O RCC Cells with or without wt VHL.
To investigate whether there is a role for the VHL gene in the regulation of the different constituents of the uPA system, we measured uPA, uPAR, and PAI-1 mRNA levels in the different cell lines. Northern blot analyses showed that in 786-O cells with mutant VHL and no VHL the mRNA levels of uPA were 30% lower than uPA mRNA levels of cells with wt VHL.

PAI-1 mRNA levels were 5-fold increased in 786-O cells expressing no VHL or mutant VHL compared with cells expressing wt VHL. Both the PAI-1 transcripts (3.2 and 2.4 kb) were expressed in the different cell lines. The mRNA levels of uPAR did not differ between the different cell lines (Fig. 2)Citation . To study whether the differences in uPA and PAI-1 mRNA levels were the result of changes in mRNA stability, we incubated 786-O cells with actinomycin D, and uPA and PAI-1 mRNA expression was followed over time. No difference in the rate of transcript decay of uPA and PAI-1 was found (data not shown).



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  		Fig. 2. Northern blot analysis of uPA (A), uPAR (B), and PAI-1 (C) mRNA in 786-O cells expressing wt VHL, mutant VHL, or no VHL. To correct for variations of loading, the blots were rehybridized with ß-actin cDNA. Expression levels were quantitated with an ImageQuant program (Molecular Dynamics) using ß-actin expression as a control.

 
uPA, uPAR, and PAI-1 Protein Levels in 786-O RCC Cells with or without wt VHL.
uPA, uPAR, and PAI-1 protein levels were measured in cell lysates and conditioned media of 786-O cells with wt VHL, mutant VHL, or lacking VHL by ELISA. In cell lysates, uPA levels were >3-fold higher in cells with wt VHL than in cells with mutant VHL or lacking VHL (Fig. 3A)Citation . In conditioned media of cells with wt VHL, uPA levels were also significantly increased compared with cell lines lacking a functional VHL gene (Fig. 3B)Citation . In cell lysates, PAI-1 protein levels were comparable for all three cell lines (Fig. 3C)Citation . However, PAI-1 protein levels were dramatically increased in conditioned media of 786-O cells with mutated VHL or no VHL compared with wt VHL (Fig. 3D)Citation . No differences were observed between uPAR protein levels in cell lysates of the cell lines tested (Fig. 3E)Citation . In conditioned media of cells expressing mutant VHL or no VHL, very low levels of the uPAR protein were measured (Fig. 3F)Citation .



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  		Fig. 3. uPA, PAI-1 and uPAR protein levels in 786-O cells expressing wt VHL, mutant VHL, or no VHL. uPA, PAI-1, and uPAR protein levels were measured by ELISA in conditioned media and cell lysates of 786-O cells. uPA protein levels were significantly higher in cell lysates (A) and conditioned media (B) of cells with wt VHL than they were in cells with mutant or no VHL. No differences were observed for PAI-1 protein expression in cell lysates (C); however, PAI-1 protein levels were dramatically increased in conditioned media of cells with mutant or no VHL compared with cells with wt VHL (D). No differences were measured for uPAR protein levels in cell lysates (E). In conditioned media of cells expressing mutant VHL or no VHL, low levels of the uPAR protein were measured (F). Columns, means of three different experiments measured in duplicate, expressed as pg per 105 cells for uPA and uPAR and as ng per 105 cells for PAI-1; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
TPA Stimulation of the uPA System in 786-O Cells.
It has been recognized that wt VHL but not mutant VHL interacts with PKC (39) . TPA, which activates PKC, is known to induce the transcription of uPA mRNA in many different cells (40) . To study whether wt VHL is required for the transcriptional regulation of uPA, we studied the effect of TPA on uPA expression of 786-O cells.

First, we examined the effect of TPA on uPA mRNA expression in the different 786-O cells. All cells responded to 2 and 24 h of TPA (100 ng/ml) treatment by increasing their uPA mRNA expression until comparable levels were reached. After 2 and 24 h of TPA treatment, 1.5- and 3-fold increases, respectively, of uPA mRNA expression were observed in cells with and without wt VHL (data not shown). Subsequently, TPA-induced changes in the uPA system were measured at protein levels. After stimulation of cells for 24 h with TPA, uPA protein levels were significantly increased in cell lysates as well as in conditioned media of 786-O cells expressing wt VHL, mutant VHL, or no VHL (Fig. 4)Citation . These results indicate that stimulation of 786-O RCC cells by TPA induces an up-regulation of uPA mRNA and protein, regardless of the presence of the wt VHL gene.



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  		Fig. 4. The effect of TPA on uPA protein levels in 786-O cells. Stimulation of cells for 24 h with TPA (100 ng/ml) resulted in a significant increase of uPA protein levels in cell lysates (A) and in conditioned media (B) in all cell lines. Columns, means of three different experiments measured in duplicate, expressed as pg per 105 cells; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Immunohistochemistry.
Seven VHL-related and seven sporadic RCCs of the clear cell type were tested for their immunoreactivity with mAbs against uPA and uPAR. The mAbs against uPA and uPAR demonstrated a plasma membrane-bound staining in tumor cells. In the adjacent normal kidney, a strong cytoplasmic staining was observed in epithelial cells of the proximal and distal tubuli. The expression of uPA and uPAR was lower in tumor cells compared with the expression of these antigens in the adjacent normal kidney (Fig. 5)Citation . Both the VHL-related and the sporadic RCCs showed the same expression pattern, and no differential expression was observed within the tumors.



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  		Fig. 5. Immunohistochemical staining for uPA (A and B) and uPAR (C and D) in VHL-related RCC and adjacent normal kidney. In the adjacent normal kidney of the tumor, a strong staining for uPA (A) and uPAR (C) was observed in the epithelial cells of the proximal and distale tubuli. The expression of uPA and uPAR was lower in tumor cells compared with the expression of these antigens in the adjacent normal kidney. In tumor cells, immunoreactivity for uPA (B) and uPAR (D) was predominantly observed at the cell membrane. T, tumor; N, nontumorous kidney.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The uPA system is of great importance in the process of tumor cell invasion, metastases, and angiogenesis. In this study, we report a role for the VHL tumor suppressor gene in the regulation of this system in RCC. For this study, we used a RCC cell line, 786-O, which lacks a functional VHL gene. In 786-O cells in which a wt VHL gene was transfected, overall uPA activity was more than three times higher than in cells lacking a functional VHL gene. Similar changes in overall uPA activity were observed in KC12 cells, a RCC cell line derived from a VHL patient (41) , when transfected with the wt VHL gene (data not shown).

We subsequently analyzed the different constituents of the uPA system: uPA, uPAR, and PAI-1 in 786-O cells. These studies revealed that uPA mRNA and protein levels were higher in cells with a functional VHL gene. Conversely, in cells lacking functional VHL PAI-1, mRNA and protein levels were significantly increased. uPAR mRNA and protein levels did not show differences between the cell lines. Immunohistochemical studies of both VHL-related and sporadic RCC showed a decreased expression of uPA in kidney tumors compared with adjacent normal kidney. Our results are in agreement with earlier observations that uPA mRNA levels were lower and PAI-1 mRNA levels were higher in sporadic kidney tumors compared with adjacent normal kidney (35 , 36) . In ~75% of sporadic RCC, both alleles of the VHL tumor suppressor gene are inactivated, either by mutations or methylation (42 , 43) . Therefore, our results suggest a direct correlation between mutations of VHL on the regulation of the uPA system in RCC and imply that VHL regulates the proteolytic activity in the kidney.

The activity of plasminogen activators is controlled by the inhibitors PAI-1 and PAI-2. The role of PAI-1 in cancers is still poorly understood. Recently, it was demonstrated that, in PAI-1-deficient mice, local invasion and tumor vascularization of transplanted malignant keratinocytes were impaired. These results imply that PAI-1 is essential for cancer cell invasion and angiogenesis (44) . High PAI-1 levels in RCC are correlated with poor patient survival (34) . PAI-1 mRNA is up-regulated in RCC compared with adjacent normal kidney (35) . These data are consistent with our findings in the 786-O cells; in cells with mutant or absent VHL PAI-1, mRNA levels were significantly increased. Reintroduction of wt VHL down-regulated PAI-1 mRNA substantially.

Presently, it is unclear how the VHL gene product regulates various genes and gene products. Different mechanisms of actions have been proposed for the VHL protein: transcription elongation, interaction with transcription factors, regulation of mRNA stability, and involvement in the degradation of proteins (17, 18, 19, 20, 21 , 27 , 45) . Several groups have shown that the VHL gene product regulates hypoxia-inducible mRNA such as VEGF, PDGF-B, and glucose transporter 1 at the level of mRNA stability (27 , 28) . It has been recognized that PAI-1 is up-regulated under hypoxic conditions (46) . However, in this study, we could not detect differences in PAI-1 mRNA stability in the presence or absence of a functional VHL gene. Recent studies provided evidence that VHL regulation of VEGF also occurs at the level of transcription. It was demonstrated that wt VHL can interact with the transcription factor Sp1 and that this interaction inhibits Sp1 activity. The VEGF promoter contains Sp1-binding sites, and this complex inhibits VEGF promoter activity (45 , 47) . The rat PAI-1 promoter contains a Sp1-binding site, which is important for transcriptional activation of this gene (48) .

No differences were observed in the cell lines we studied for uPAR mRNA levels and uPAR protein levels in cell lysates. In conditioned media of cells expressing wt VHL, no uPAR protein could be detected. In conditioned media of cells expressing mutant VHL or lacking VHL, very low levels of uPAR protein were measured. This might be the result of shedding of the receptor.

As a first step toward elucidating how VHL may regulate this uPA-mediated activation of plasminogen, we have examined whether PKC is involved. Phorbol esters, which activated PKC, have been recognized for their ability to potently increase levels of several mRNAs, including uPA (40) . Furthermore, it has been recognized that wt VHL but not mutant VHL forms a cytoplasmic complex with the PKC isoforms {zeta} and {delta}. Formation of wt VHL/PKC complexes prevent PKC translocation to the cell membrane, thereby interrupting a signaling cascade that involves mitogen-activated protein kinase (39) . Stimulation of 786-O cells with TPA showed a significant up-regulation of uPA mRNA levels in cells with and without a functional VHL gene. The increased uPA mRNA expression after TPA treatment was comparable, regardless of the presence of a functional VHL gene. These results suggest that a functional VHL gene is not essential for the transcriptional regulation of uPA in RCC cells. This is in agreement with our observation that TPA induces a significant increase of uPA protein levels in either cell lysates and conditioned media of cells with wt VHL, mutant VHL, or no VHL. However, in cells with wt VHL, the absolute uPA protein levels are much higher in cell lysate and in conditioned medium. This difference could not be explained by differential mRNA expression and might be regulated at the level of protein degradation.

Vascularization creates an access of tumors to the circulation, and intravasation of tumor cells is the next step in the process leading to metastases. Recently, it was demonstrated in a intravasation assay that surface uPA/uPAR is indispensable for invasion of the blood vessel wall (49) . Cells with low uPA at the cell surface intravasated very poorly. In our study we showed that RCC cells, lacking a functional VHL gene, express lower levels of uPA than cells with a functional VHL gene. This might explain, to a certain extent, the clinical observations that: (a) in patients with VHL-related RCC, metastases are rarer than they are in sporadic RCC; and (b) at initial diagnosis, RCC can be very large without evidence of metastases. It is tempting to speculate that, despite the well-vascularized phenotype of RCC, intravasation of tumor cells lacking a functional VHL gene is impaired.

In conclusion, our results provide further evidence that the VHL gene plays an important role in the regulation of angiogenesis at various levels: regulation of the angiogenic factors such as VEGF, PDGF-B, and transforming growth factor-ß; deposition of the extracellular matrix protein fibronectin; and proteolysis of extracellular matrix through regulation of proteases involved in the uPA system.


   ACKNOWLEDGMENTS
 
We are grateful to Othon Iliopoulos and William G. Kaelin (Dana-Farber Cancer Institute, Boston, MA) for the kind gift of the 786-O cell lines. We thank Harry A. Peters and Anieta M. Sieuwerts (Rotterdam Cancer Center, Rotterdam, The Netherlands) for performing the uPA, uPAR, and PAI-1 ELISAs and the Finsen Laboratory (Copenhagen, Denmark) for reagents for the uPAR ELISA and the mAb R3 against human uPAR.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Netherlands Organization for Scientific Research, University Hospital Utrecht and University of Utrecht, Grant NWO 920-03-024. Back

2 To whom requests for reprints should be addressed, at Department of Internal Medicine, Laboratory of Medical Oncology, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Phone: 31 30 2508568; Fax: 31 30 2523741; E-mail: e.e.voest{at}digd.azu.nl Back

3 The abbreviations used are: RCC, renal cell carcinoma; VHL, von Hippel-Lindau; wt, wild-type; VEGF, vascular endothelial growth factor; PDGF-B, platelet-derived growth factor B chain; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; PAI-1, plasminogen activator inhibitor 1; TPA, 12-O-tetradecanoylphorbol-13-acetate; mAb, monoclonal antibody; PKC, protein kinase C. Back

Received 3/30/99. Accepted 7/ 7/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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