[CANCER RESEARCH 59, 4440–4445, September 1, 1999]
Regulation of the Urokinase-type Plasminogen Activator System by the vonHippel-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 ofMedical Oncology, Rotterdam Cancer Centre, 3015 GD Rotterdam [J. A. F.], the Netherlands
ABSTRACT
The urokinase-type plasminogen activator (uPA) system plays an im-portant role in tumor cell invasion, metastases, and angiogenesis. uPA,uPA receptor, and plasminogen activator inhibitor 1 (PAI-1) are prog-nostic factors in different solid tumors, e.g., renal cell carcinomas (RCCs).von Hippel-Lindau (VHL) disease is an inherited cancer syndrome that ischaracterized by extensively vascularized tumors, including hemangio-blastomas and RCCs. In 75% of sporadic RCCs, theVHL gene is alsoinactivated. It has been recognized in sporadic RCC that PAI-1 mRNAlevels are up-regulated and uPA mRNA levels are down-regulated. Wedetermined the role of theVHL tumor suppressor gene in the regulationof 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 activitythan in 786-O cells expressing a mutantVHL gene or lacking VHL. uPAmRNA and protein levels were higher in cells with wt VHL compared withcells with mutant VHL or lacking VHL. In addition, PAI-1 mRNA andprotein levels were dramatically increased in 786-O cells with mutantVHL or lacking VHL, compared with cells expressing wt VHL.
Our results provide further evidence that the VHL gene plays animportant role in the process of angiogenesis by regulation of plasmin-mediated proteolysis of the extracellular matrix and may explain whyVHL-induced RCCs grow slowly and metastasize relatively late.
INTRODUCTION
RCC3 is a common manifestation of VHL disease, a rare hereditarycancer syndrome (1). Other manifestations of VHL disease are he-mangioblastomas of the cerebellum, angiomas of the retina, andpheochromocytomas. Cysts are seen in several organs,e.g., liver,kidneys, brain, pancreas, and epididymis (2, 3). In addition to VHLdisease-related tumors, inactivation of theVHL gene is found in themajority (75%) of sporadic RCCs, a minority of sporadic hemangio-blastomas, and colorectal cancer (4–7). When patients are diagnosedwith VHL disease, they are enrolled into a screening program. Renaltumors are, therefore, frequently detected at an early presymptomaticstage and can be followed by ultrasonography, computed tomography,or magnetic resonance imaging. These follow-up studies have re-vealed that most small renal tumors enlarge slowly. Because VHLpatients require repetitive surgery, there is a tendency to wait untiltumors are 3 cm in diameter (8). For years, the treatment of choice forVHL-related RCC was uni- or even bilateral nephrectomy. This is stillthe indicated treatment for diffuse disease or multiple lesions in thekidney. Nephron-sparing surgery has been advocated in small RCC topreserve renal function and delay end-stage renal failure (9). Recently,
arguments for even less aggressive treatment of RCC in VHL patientswere provided. This was based on the observations that metastases ofRCC in VHL patients are significantly rarer than in sporadic RCC andthat they occur only in tumors that are larger than 7 cm (10). Themolecular basis for this clinical observation is still unknown.
TheVHL gene was cloned in 1993 by Latifet al. (11) and appearsto be a tumor suppressor gene located on chromosome 3p25–25. TheVHL gene encodes two proteins: pVHL30 and pVHL19, with molec-ular masses of 30 and 19 kDa, respectively (12–14) The VHL proteinis expressed in nearly all human tissues and has no homology with anyknown protein (15). Proteins that bind pVHL have been studied inattempts to learn more about the function of theVHL gene. pVHLbinds to elongins B and C, and this complex is a negative regulator ofthe transcription elongation factor elongin or SIII (16–18). Veryrecently, it was shown that other proteins, such as CUL2 and Rbx 1,are binding to this complex. This so-called VCB complex is highlysimilar with E3 ubiquitin ligase complexes, which are key regulatorsof protein degradation (19–22).
Because extensive vascularization is characteristic for VHL-relatedtumors, functional analysis of theVHL gene has been directed at itsrole in angiogenesis. Both the VHL-related and sporadic RCCs andhemangioblastomas express high levels of the angiogenic factorVEGF and its receptors (23–25).In vitro studies subsequently showedthat human RCC cells lacking the wt VHL gene product express highlevels of hypoxia-inducible mRNAs such as VEGF, PDGF-B, andglucose transporter 1. Introduction of wt VHL cDNA resulted in adown-regulation of these mRNAs (26–28). In addition, pVHL regu-lates the transforming growth factorb1 gene and interacts withfibronectin, an extracellular glycoprotein that binds to members of theintegrin family of cell surface receptors (29, 30).
In the process of angiogenesis and tumor invasion, the uPAsystem plays an important role. This is illustrated by severalclinical studies that show that uPA, uPAR, and PAI-1 levels ofdifferent cancers are prognostic factors for patient survival andtumor relapse (31, 32). For RCC, it has been shown that PAI-1 isa strong and independent prognostic factor in predicting earlyrelapse of the tumor and overall survival (33, 34). In these studies,the investigators could discriminate between high- and low-riskgroups for disease-free survival by measuring the PAI-1 proteincontent in tumor tissue (34). PAI-1 mRNA expression levels weremeasured in paired samples of RCC and adjacent normal kidneytissue. PAI-1 mRNA levels were significantly higher in tumorsamples compared with normal kidney (35). On the other hand, a.3-fold lower expression of uPA mRNA was observed in RCCcompared with normal kidney tissue (36).
The aim of our study was to determine whether theVHL gene isinvolved in the regulation of the uPA system in RCC. Our resultsindicate that inactivation of theVHL gene results in decreased uPAand increased PAI-1 levels in RCC cells. These findings providefurther support that the VHL gene plays a central role in theregulation of genes involved in angiogenesis and may explain whyRCC in VHL patients grow relatively slowly and metastases occuronly occasionally.
Received 3/30/99; accepted 7/7/99.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby markedadvertisementin accordance with18 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.
2 To whom requests for reprints should be addressed, at Department of InternalMedicine, Laboratory of Medical Oncology, University Hospital Utrecht, P.O. Box85500, 3508 GA Utrecht, The Netherlands. Phone: 31 30 2508568; Fax: 31 30 2523741;E-mail: [email protected].
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 growthfactor 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.
Cell Culture. 786-O cells are RCC cells that lack a functionalVHL 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), weregrown in DMEM-10% FCS supplemented with G418 (1 mg/ml) at 37°C in anatmosphere with 10% CO2 (12).
For performing the different experiments, cell medium was replaced withfresh serum-free DMEM with or without 100 ng/ml TPA (Sigma ChemicalCo., St. Louis, MO) or 5mg/ml dactinomycin (Lyovac Cosmogen, MSD AgverBV, Haarlem, The Netherlands).
Assay of Overall uPA Activity. Overall uPA activity was measured oncells grown in 96-well plates. Cells were washed twice with PBS, and uPAactivity was determined by incubating cells at 37°C with plasminogen (8mg/ml; Sigma) and the plasmin-specific substrate S-2251 (Chromogenix AB,Molndal, Sweden). The release of paranitroaniline from S-2251 was deter-mined in each well by measuring the absorbance at 405 nm using a microplatereader. Controls included incubation of cells without plasminogen. uPA activ-ity was quantitated using a standard curve generated with human uPA (Sigma).
RNA Extraction and Northern Blot Analysis. Total RNA was extractedfrom cultured cells with RNAzol (Tel-Test, Friendswood, TX) according to themanufacturer’s protocol. RNA samples (15mg/lane) were separated in a 1%agarose-formaldehyde gel and transferred to a Hybond nylon membrane (Am-ersham, Buckinghamshire, United Kingdom). Membranes were prehybridizedat 42°C in a prehybridization buffer [50% deionized formamide, 53 SSPE (13SSPE5 0.15 M NaCl, 0.01M NaH2PO4, and 1 mM EDTA), 53 Denhardt’ssolution, 0.5% SDS, and 10% dextran sulfate] and denatured salmon spermDNA for 2–4 h and hybridized for 18 h in the same buffer containing thecDNA probes. cDNA fragments were labeled using a Prime-It RmT RandomPrimer Labeling Kit (Stratagene, La Jolla, CA) and [a-32P]dCTP. The cDNAprobes 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), andb-actin. The membranes were washed and exposed to aphosphorus imager screen (Molecular Dynamics, Sunnyvale, CA). uPA,uPAR, and PAI-1 mRNA expression levels were quantitated with an Image-Quant program (Molecular Dynamics); variations in loading were quantitatedby comparing expression levels withb-actin mRNA expression.
uPA, uPAR, and PAI-1 ELISAs. Conditioned media and cell lysates wereharvested from cells grown for 24 h in serum-free DMEM without phenol red.After the conditioned medium was collected, cells were counted and lysed inNP40 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 in280°C untilfurther use. All samples were measured in duplicate. uPA and PAI-1 antigenswere measured by ELISA described by Grebenschikovet al. (37). uPARantigens were measured by an ELISA described by Ronneet al. (38). Theamount of uPA and uPAR protein present in cell lysates and conditioned mediawas expressed in pg per 105 cells, and the amount of PAI-1 protein wasexpressed as ng per 105 cells.
Immunohistochemistry. Seven RCCs from VHL patients and seven spo-radic RCCs were removed by surgery at the University Hospital Utrecht. Alltumor samples were routinely formalin-fixed and paraffin-embedded. Four-mmsections of these tumors were deparaffinized in xylene and rehydrated inethanol. The endogenous peroxidase activity was blocked with methanolcontaining 1.5% H2O2 for 15 min. Primary and secondary antibodies werediluted in PBS-1% BSA. Primary antibodies used were mouse mAbs againstuPA (mAb 3689; American Diagnostica) and against uPAR (mAb R3; a kindgift of the Finsen Laboratory, Copenhagen, Denmark). Sections were incu-bated with the primary antibodies for 1 h atroom temperature. For detectionof the uPA and uPAR antigen, sections were subsequently incubated for 30min with rabbit antimouse peroxidase (DAKO, Glostrup, Denmark) and swineantirabbit peroxidase (DAKO). Immunoreactivity was visualized with diami-nobenzidine (Sigma). Sections were counterstained with Mayer’s hematoxylin,rehydrated, and mounted.
Statistics. uPA, uPAR, and PAI-1 protein concentrations in conditionedmedia, and cell lysates were presented as the means of three independentexperiments6 SE. Statistical analysis were performed by use of the Student’st test.
RESULTS
Overall uPA Activity in RCC Cells with or without a Func-tional VHL Gene. Because proteases of the plasminogen activatorsystem might play a role in VHL tumor progression, we measured theoverall uPA activity in 786-O cells. The uPA activity was defined asthe net result of positive and negative regulators of uPA system.786-O cells are RCC cells that lack a functionalVHL gene. Reintro-duction of wt VHL in 786-O cells inhibited their ability to formtumors in nude mice (12). The overall uPA activity of 786-O RCCcells expressing wt VHL, mutant VHL, and no VHL were measuredon cells grown in 96-well plates. The overall uPA activity in 786-Ocells with wt VHL (5.676 0.8 microunits per 104 cells) was morethan three times higher than that in 786-O cells lacking VHL(1.78 6 0.39 microunits per 104 cells) or with mutant VHL(0.816 0.17 microunits per 104 cells; P , 0.001; Fig. 1).
uPA, uPAR, and PAI-1 mRNA Levels in 786-O RCC Cells withor without wt VHL. To investigate whether there is a role for theVHL gene in the regulation of the different constituents of the uPAsystem, we measured uPA, uPAR, and PAI-1 mRNA levels in thedifferent cell lines. Northern blot analyses showed that in 786-O cellswith 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 express-ing 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 thedifferent cell lines. The mRNA levels of uPAR did not differ betweenthe different cell lines (Fig. 2). To study whether the differences inuPA and PAI-1 mRNA levels were the result of changes in mRNAstability, we incubated 786-O cells with actinomycin D, and uPA andPAI-1 mRNA expression was followed over time. No difference in therate of transcript decay of uPA and PAI-1 was found (data not shown).
uPA, uPAR, and PAI-1 Protein Levels in 786-O RCC Cells withor without wt VHL. uPA, uPAR, and PAI-1 protein levels weremeasured in cell lysates and conditioned media of 786-O cells with wtVHL, mutant VHL, or lacking VHL by ELISA. In cell lysates, uPAlevels were.3-fold higher in cells with wt VHL than in cells withmutant VHL or lacking VHL (Fig. 3A). In conditioned media of cellswith wt VHL, uPA levels were also significantly increased comparedwith cell lines lacking a functionalVHL gene (Fig. 3B). In cell lysates,PAI-1 protein levels were comparable for all three cell lines (Fig. 3C).However, PAI-1 protein levels were dramatically increased in condi-tioned media of 786-O cells with mutated VHL or no VHL comparedwith wt VHL (Fig. 3D). No differences were observed between uPAR
Fig. 1. Overall uPA activity of 786-O RCC cells expressing wt VHL, mutant VHL, orno VHL. uPA activity was measured on cells grown in 96-well plates.Columns, means ofeight independent experiments, expressed as microunits of uPA activity per 10,000 cells;bars, SE.ppp, P , 0.001.
protein levels in cell lysates of the cell lines tested (Fig. 3E). Inconditioned media of cells expressing mutant VHL or no VHL, verylow levels of the uPAR protein were measured (Fig. 3F).
TPA Stimulation of the uPA System in 786-O Cells.It has beenrecognized that wt VHL but not mutant VHL interacts with PKC (39).TPA, which activates PKC, is known to induce the transcription ofuPA mRNA in many different cells (40). To study whether wt VHL
is required for the transcriptional regulation of uPA, we studied theeffect of TPA on uPA expression of 786-O cells.
First, we examined the effect of TPA on uPA mRNA expression inthe different 786-O cells. All cells responded to 2 and 24 h of TPA(100 ng/ml) treatment by increasing their uPA mRNA expression untilcomparable levels were reached. After 2 and 24 h of TPA treatment,1.5- and 3-fold increases, respectively, of uPA mRNA expression
Fig. 2. Northern blot analysis of uPA (A), uPAR(B), and PAI-1 (C) mRNA in 786-O cells express-ing wt VHL, mutant VHL, or no VHL. To correctfor variations of loading, the blots were rehybrid-ized with b-actin cDNA. Expression levels werequantitated with an ImageQuant program (Molec-ular Dynamics) usingb-actin expression as a con-trol.
Fig. 3. uPA, PAI-1 and uPAR protein levels in 786-O cellsexpressing wt VHL, mutant VHL, or no VHL. uPA, PAI-1, anduPAR protein levels were measured by ELISA in conditioned mediaand cell lysates of 786-O cells. uPA protein levels were significantlyhigher in cell lysates (A) and conditioned media (B) of cells with wtVHL than they were in cells with mutant or no VHL. No differenceswere observed for PAI-1 protein expression in cell lysates (C);however, PAI-1 protein levels were dramatically increased in con-ditioned media of cells with mutant or no VHL compared with cellswith wt VHL (D). No differences were measured for uPAR proteinlevels in cell lysates (E). In conditioned media of cells expressingmutant VHL or no VHL, low levels of the uPAR protein weremeasured (F).Columns, means of three different experiments meas-ured in duplicate, expressed as pg per 105 cells for uPA and uPARand as ng per 105 cells for PAI-1; bars, SE.p, P , 0.05; pp,P , 0.01; ppp, P , 0.001.
were observed in cells with and without wt VHL (data not shown).Subsequently, TPA-induced changes in the uPA system were meas-ured at protein levels. After stimulation of cells for 24 h with TPA,uPA protein levels were significantly increased in cell lysates as wellas in conditioned media of 786-O cells expressing wt VHL, mutantVHL, or no VHL (Fig. 4). These results indicate that stimulation of786-O RCC cells by TPA induces an up-regulation of uPA mRNAand protein, regardless of the presence of the wtVHL gene.
Immunohistochemistry. Seven VHL-related and seven sporadicRCCs of the clear cell type were tested for their immunoreactivitywith mAbs against uPA and uPAR. The mAbs against uPA and uPARdemonstrated a plasma membrane-bound staining in tumor cells. Inthe adjacent normal kidney, a strong cytoplasmic staining was ob-served in epithelial cells of the proximal and distal tubuli. Theexpression of uPA and uPAR was lower in tumor cells compared with
the expression of these antigens in the adjacent normal kidney (Fig.5). Both the VHL-related and the sporadic RCCs showed the sameexpression pattern, and no differential expression was observed withinthe tumors.
DISCUSSION
The uPA system is of great importance in the process of tumor cellinvasion, metastases, and angiogenesis. In this study, we report a rolefor theVHL tumor suppressor gene in the regulation of this system inRCC. For this study, we used a RCC cell line, 786-O, which lacks afunctional VHL gene. In 786-O cells in which a wtVHL gene wastransfected, overall uPA activity was more than three times higherthan in cells lacking a functionalVHL gene. Similar changes in overalluPA activity were observed in KC12 cells, a RCC cell line derived
Fig. 4. The effect of TPA on uPA protein levelsin 786-O cells. Stimulation of cells for 24 h withTPA (100 ng/ml) resulted in a significant increaseof uPA protein levels in cell lysates (A) and inconditioned media (B) in all cell lines.Columns,means of three different experiments measured induplicate, expressed as pg per 105 cells; bars, SE.p, P , 0.05; pp, P , 0.01; ppp, P , 0.001.
Fig. 5. Immunohistochemical staining for uPA (A andB) and uPAR (C andD) in VHL-related RCC and adjacent normal kidney. In the adjacent normal kidney of the tumor, a strongstaining 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 withthe 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.
from a VHL patient (41), when transfected with the wtVHL gene(data not shown).
We subsequently analyzed the different constituents of the uPAsystem: uPA, uPAR, and PAI-1 in 786-O cells. These studies revealedthat uPA mRNA and protein levels were higher in cells with afunctional VHL gene. Conversely, in cells lacking functional VHLPAI-1, mRNA and protein levels were significantly increased. uPARmRNA and protein levels did not show differences between the celllines. Immunohistochemical studies of both VHL-related and sporadicRCC showed a decreased expression of uPA in kidney tumors com-pared with adjacent normal kidney. Our results are in agreement withearlier observations that uPA mRNA levels were lower and PAI-1mRNA levels were higher in sporadic kidney tumors compared withadjacent normal kidney (35, 36). In;75% of sporadic RCC, bothalleles of theVHL tumor suppressor gene are inactivated, either bymutations or methylation (42, 43). Therefore, our results suggest adirect correlation between mutations of VHL on the regulation of theuPA system in RCC and imply that VHL regulates the proteolyticactivity in the kidney.
The activity of plasminogen activators is controlled by the inhibi-tors PAI-1 and PAI-2. The role of PAI-1 in cancers is still poorlyunderstood. Recently, it was demonstrated that, in PAI-1-deficientmice, local invasion and tumor vascularization of transplanted malig-nant keratinocytes were impaired. These results imply that PAI-1 isessential for cancer cell invasion and angiogenesis (44). High PAI-1levels in RCC are correlated with poor patient survival (34). PAI-1mRNA is up-regulated in RCC compared with adjacent normal kidney(35). These data are consistent with our findings in the 786-O cells; incells with mutant or absent VHL PAI-1, mRNA levels were signifi-cantly increased. Reintroduction of wt VHL down-regulated PAI-1mRNA substantially.
Presently, it is unclear how the VHL gene product regulates variousgenes and gene products. Different mechanisms of actions have beenproposed for the VHL protein: transcription elongation, interactionwith transcription factors, regulation of mRNA stability, and involve-ment in the degradation of proteins (17–21, 27, 45). Several groupshave shown that the VHL gene product regulates hypoxia-induciblemRNA such as VEGF, PDGF-B, and glucose transporter 1 at the levelof mRNA stability (27, 28). It has been recognized that PAI-1 isup-regulated under hypoxic conditions (46). However, in this study,we could not detect differences in PAI-1 mRNA stability in thepresence or absence of a functionalVHL gene. Recent studies pro-vided evidence that VHL regulation of VEGF also occurs at the levelof transcription. It was demonstrated that wt VHL can interact withthe transcription factor Sp1 and that this interaction inhibits Sp1activity. The VEGF promoter contains Sp1-binding sites, and thiscomplex inhibits VEGF promoter activity (45, 47). The rat PAI-1promoter contains a Sp1-binding site, which is important for tran-scriptional activation of this gene (48).
No differences were observed in the cell lines we studied for uPARmRNA levels and uPAR protein levels in cell lysates. In conditionedmedia of cells expressing wt VHL, no uPAR protein could be de-tected. In conditioned media of cells expressing mutant VHL orlacking VHL, very low levels of uPAR protein were measured. Thismight be the result of shedding of the receptor.
As a first step toward elucidating how VHL may regulate thisuPA-mediated activation of plasminogen, we have examined whetherPKC is involved. Phorbol esters, which activated PKC, have beenrecognized for their ability to potently increase levels of severalmRNAs, including uPA (40). Furthermore, it has been recognized thatwt VHL but not mutant VHL forms a cytoplasmic complex with thePKC isoformsz andd. Formation of wt VHL/PKC complexes preventPKC translocation to the cell membrane, thereby interrupting a sig-
naling cascade that involves mitogen-activated protein kinase (39).Stimulation of 786-O cells with TPA showed a significant up-regu-lation of uPA mRNA levels in cells with and without a functionalVHL gene. The increased uPA mRNA expression after TPA treatmentwas comparable, regardless of the presence of a functionalVHL gene.These results suggest that a functionalVHL gene is not essential forthe transcriptional regulation of uPA in RCC cells. This is in agree-ment with our observation that TPA induces a significant increase ofuPA protein levels in either cell lysates and conditioned media of cellswith wt VHL, mutant VHL, or no VHL. However, in cells with wtVHL, the absolute uPA protein levels are much higher in cell lysateand in conditioned medium. This difference could not be explained bydifferential mRNA expression and might be regulated at the level ofprotein degradation.
Vascularization creates an access of tumors to the circulation, andintravasation of tumor cells is the next step in the process leading tometastases. Recently, it was demonstrated in a intravasation assay thatsurface uPA/uPAR is indispensable for invasion of the blood vesselwall (49). Cells with low uPA at the cell surface intravasated verypoorly. In our study we showed that RCC cells, lacking a functionalVHL gene, express lower levels of uPA than cells with a functionalVHL gene. This might explain, to a certain extent, the clinical obser-vations that: (a) in patients with VHL-related RCC, metastases arerarer than they are in sporadic RCC; and (b) at initial diagnosis, RCCcan be very large without evidence of metastases. It is tempting tospeculate that, despite the well-vascularized phenotype of RCC, in-travasation of tumor cells lacking a functionalVHL gene is impaired.
In conclusion, our results provide further evidence that theVHLgene plays an important role in the regulation of angiogenesis atvarious levels: regulation of the angiogenic factors such as VEGF,PDGF-B, and transforming growth factor-b; deposition of the extra-cellular matrix protein fibronectin; and proteolysis of extracellularmatrix through regulation of proteases involved in the uPA system.
ACKNOWLEDGMENTS
We are grateful to Othon Iliopoulos and William G. Kaelin (Dana-FarberCancer Institute, Boston, MA) for the kind gift of the 786-O cell lines. Wethank Harry A. Peters and Anieta M. Sieuwerts (Rotterdam Cancer Center,Rotterdam, The Netherlands) for performing the uPA, uPAR, and PAI-1ELISAs and the Finsen Laboratory (Copenhagen, Denmark) for reagents forthe uPAR ELISA and the mAb R3 against human uPAR.
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