Receptor Gene Expression in Human Renal Cell Carcinoma

American Journal ofPathology, Vol. 147, No. 1, July 1995 Copyright © American Societyfor Investigative Pathology

Modulation of Urokinase and Urokinase Receptor Gene Expression in Human Renal Cell Carcinoma

Stephan N. Wagner,* Michael J. Atkinson,* Sephanie Thanner,* Christine Wagner,* Manfred Schmitt,t Olaf Wilhelm,t Michael Rotter,$ and Heinz H6fler*t From the Institut fur Pathologie, GSF-Forschungszentrum far Umwelt und Gesundheit, Oberschleissheim; and Frauenklinie and Institut far Pathologie,t Technische Universitdt Manchen, Munchen, Germany

In vivo and in vitro experimental models have suggested a major role for the urokinase-type plasminogen activator (uPA) in tumor ceU invasion and metastasis. The uPA proteolytic activity of tumor cells has been shown to be largely determined by the extent ofthe expression and saturation ofthe uPA receptor. We have analyzed the expression and celular localization of both uPA and uPA receptor at the protein and mRNA levels in 33 paired samples of renal ceU carcinoma (RCC) and non-tumorous kidney tissue. In comparison with adjacent normal non-tumorous kidney tissues RCC tumor ceUs modestly overexpressed uPA-receptor mRNA and showed signiffcantly decreased uPA mRNA expression. However, the immunoreactive uPA content of tumor ceUs was comparable to that ofthe surrounding normal non-tumorous kidney tissue. Assuming constancy ofthe uPA-receptor affinityfor uPA this indicates that a proportion of the RCCassociated uPA may be derived from an exogenous source and subsequently concentrated at the tumor ceU surface via uPA receptor expression. The modest increase in uPA receptor expression may lead to a normalization of uPA antigen content in RCC, however, it is not sufficient to substantialy increase tumor tissue-uPA content over the level of normal non-tumorous kidney tissue. (Am JPathol 1995, 14 7:183-192)

Interactions between neoplastic cells and their environment are required for tumor development, spread,

and tumor-associated processes such as angiogenesis. Local proteolytic degradation of the extracellular matrix is believed to be essential for all of these events.1 Renal cell carcinomas (RCCs) are potentially metastatic epithelial tumors whose clinical prognosis is difficult to establish. While metastatic spread can be present in up to 25 to 30% of newly diagnosed patients, others may develop metastatic disease after many years. Metastatic progression involves local invasion, pseudo-capsule penetration, perinephric infiltration, and characteristically, vascular invasion.2'3 These events have been shown in vitro to depend upon tumor-associated proteolysis. Plasmin is a candidate for providing at least some of the proteolytic activity required for RCC invasion, including the activation of collagenase zymogens.4 Significantly, inhibitors of plasmin are able to block the invasiveness of RCC tumor cells in vitro.5 The proteolytic activity of plasmin itself is available for most human tumor cells through the action of the neutral serine protease urokinase-type plasminogen activator (uPA), which generates proteolytically active plasmin from the ubiquitous zymogen plasminogen.6 uPA-specific antibodies7,8 or uPA-specific inhibitors910 have been shown to block invasion by several metastatic human tumor cell lines in vitro. Similar results were obtained with experimental tumors in vivo.11 12 A central role for uPA in tumor progression is further supported by correlative data measuring uPA in vivo. Extracts of several human tumor tissues exhibited higher uPA levels than their normal counterparts (see ref. 6 and references therein). A number of reports have indicated the presence of a high-affinity binding site for uPA (uPA receptor) on the surface of several diverse cell types.8'13'14 The Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 207/F9/F10). This work was performed during the tenure of a research fellowship to S. N. Wagner from the Deutsche Forschungsgemeinschaft (Wa-705/1-1). Accepted for publication March 28, 1995. Address reprint requests to Dr. Stephan N. Wagner at his present address, Hautklinik, Universitatsklinikum Essen, Hufelandstr. 55 D-45122 Essen, Germany.

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uPA receptor binds with high affinity to the aminoterminus of uPA, a domain distinct from the enzymatically active center.13'14 On some cell types the uPA receptor has been reported to direct extracellular proteolysis by localizing uPA to the focal cell-substratum and cell-cell contact sites.15'16 uPA receptor may be involved in uPA turnover by mediating internalization and degradation of uPA/uPA-inhibitor complexes16-18 and may also participate in signal transduction pathways.19 Consequently, uPA receptor expression and regulation of the urokinase activity at the tumor cell surface may be an important step in metastatic spread. Indeed, the invasive and metastatic capacity of tumor cells can be inhibited by saturation of the uPA receptor with inactive uPA or uPA antagonists in vivo and in vitro.20'21 Receptor-bound uPA may be derived either from constitutively expressed endogenous uPA14 or from external sources. Thus, saturation of uPA receptors by exogenously added uPA, or by uPA secreted from co-cultured fibroblasts, promoted tumor cell invasiveness in vitro.8'22'23 There are also some indications that overexpression of the uPA receptor may play a role in the increased accumulation of uPA at the tumor cell surface.24

We have investigated the expression and distribution of uPA and its receptor in 33 cases of RCC and in the corresponding normal non-tumorous kidney tissue. In RCC, tumor cells but not the tumor stromal components expressed uPA and uPA receptor messenger RNAs (mRNA) and antigens. While the immunoreactive uPA content of tumor cells was comparable to that of normal non-tumorous kidney tissue, mRNA expression of uPA was significantly lower. Compared with non-tumorous kidney tissue, expression of uPA receptor was modestly increased in the RCC tumor cells. This suggests that a significant portion of RCC-associated uPA may be derived from an exogenous source and concentrated at RCC tumor cell surfaces by virtue of the increased uPA receptor

tological examination. Histological diagnosis was made independently by two of the authors according to standard criteria2 (Table 1).

Northern Blot Analysis Total cellular RNA was isolated from the frozen tumor and non-tumor samples by guanidinium thiocyanate extraction and cesium chloride centrifugation.25 15 pg total RNA per lane were size-fractionated on 1% agarose, 2 mol/L formaldehyde gels and transferred to nylon membranes by vacuum blotting with 20 x SSC (1 x SSC is 150 mmol/L NaCI, 15 mM sodium citrate at pH 7.6). The membranes were ultraviolet cross-linked and prehybridized in the presence of 4 x SSPE (1 x SSPE is 180 mmol/L NaCI, 8 mmol/L NaH2PO4, 1 mmol/L EDTA), 1% sodium dodecyl sulfate (SDS), 5 x Denhardt's solution, 500 pg/ml denatured salmon DNA, and 50% deionized formamide. The RNA was probed by hybridization at 420C with 32p labeled cDNA under prehybridization conditions overnight. Filters were washed for 30 minutes in 2 x SSC, 0. 1% SDS at room temperature and two times for 60 minutes in 0.1 x SSC, 0.1% SDS at 580C for uPA receptor, 650C for uPA, and 680C for f-actin. Filters were exposed to Kodak X-AR film (Eastman Kodak Co., Rochester, NY) at -700C with intensifying screens. Hybridization signals were quantified by scanning autoradiographs (Ultrascan XL, Pharmacia-LKB, Freiburg, Germany). uPA and uPA receptor expression was corrected for variations in RNA loading by comparing with f3-actin mRNA expression.

The probes used were the 0.32 kb BamHI/Pstl fragment of the pHUK-1 human urokinase cDNA clone,26 the 0.26-kb Psfl fragment of the p-uPA-R-1 cDNA clone encoding the human uPA receptor,27 and the Table 1. Histological Features of 33 RCC Tissue Samples

expression.

Materials and Methods Tissue Preparation Renal tissue samples were obtained from 33 patients by radical nephrectomy. RCC tumor and the corresponding non-tumorous areas were extirpated and split into three aliquots, one part being quick frozen in liquid nitrogen for RNA extraction, another part fixed in freshly prepared 4% paraformaldehyde for in situ hybridization, and the third fixed in 10% buffered formalin (pH 7.0) and embedded in paraffin for his-

Parameters

Number of patients

(%)

Histological cell type Clear cell Granular cell Mixed cell Growth pattern

11 17

Compact Tubulopapillary Tumor grade GI G II G III TNM staging pT 1 pT 2 pT 3a pT 3b

5

(33) (52) (15)

21 12

(63) (36)

7 17 9

(21) (52) (27)

0 16 9 8

(49) (27) (24)

(0)

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3.6-kb Hindlil fragment of human 1-actin sequences (PXHac69).28 Probes were radiolabeled with [a-32P]dATP to a specific activity of 2 x 109 cpm/pg using a T7 DNA polymerase random priming kit (Stratagene, Heidelberg, Germany).

erated by in vitrotranscription of 1 pg of the linearized plasmids in the presence of 100 pCi of [a-35S]UTP using T7 and Sp6 RNA polymerases where appropriate (in vitro transcription system, Promega/Serva).

Immunohistochemistry In Situ Hybridization In situ hybridization was performed using a modification of the method of Hofler et al.29 Briefly, slides were coated with 1% gelatin, 0.1% chrome-alum, dried at room temperature, fixed with 1% paraformaldehyde, dried at 65°C for 12-16 hours, and stored dust-free at room temperature. 7-pm cryostat sections were mounted on these slides, rapidly fixed in 4% paraformaldehyde in PBS for 10 minutes, washed in PBS, and dried at 370C overnight. Sections were rehydrated in PBS and digested in proteinase K (1 pg/ml in 100 mM Tris-HCI pH 8.0, 50 mM EDTA) for 15 minutes at 370C. Proteinase K was inactivated by fixation with 4% paraformaldehyde in PBS for 5 minutes. Sections were acetylated for 10 minutes at room temperature in 0.25% acetic anhydride and 100 mmol/L triethanolamine, pH 8.0. Nucleic acids were denatured in 50% deionized formamide, 2 x SSC for 15 min at 370C. Hybridization was carried out using the sense or antisense labeled riboprobes (40 pg/pl) in 20 p1 of 50% deionized formamide, 2 x SSC, 10% dextrane sulfate, 0. 1% SDS, 250 pg/ml denatured salmon sperm DNA, and 4 mmol/L dithiothreitol. Sections were hybridized at 430C for 12 to 16 hours in a humidified chamber. Residual radioactive probe was removed using 4 x SSC for 3 x 20 minutes at 420C and RNAse A (20 pg/ml) in 500 mmol/L NaCI, 10 mmol/L TRIS-HCI, pH 8.0, and 1 mmol/L EDTA for 30 minutes at 370C. High stringency washing (30 minutes at 420C in 2 x SSC and 0.1 x SSC, respectively) was followed by dehydration in ethanol containing 300 mmol/L ammonium acetate. For signal detection, slides were covered with NTB-2 autoradiographic emulsion (Eastman Kodak Co.) diluted 1:2 in distilled water, airdried, and stored in light-proof boxes with dessicant at 40C. After 14 to 21 days of exposure slides were developed in Kodak Dl 9 developer (Eastman Kodak Co.), fixed in 30% sodium thiosulfate, and counterstained in methylene blue. Ribonucleic acid probes were prepared by subcloning the appropriate cDNA fragments in RNA transcription vectors. The 0.32-kb BamHl/Pstl fragment of pHUK-1 was subcloned into pGEM 3Z (Promega/Serva, Heidelberg, Germany). The 0.26-kb Psf fragment of p-uPA-R-1 was subcloned into pBluescript 11 SK(+) (Stratagene, Heidelberg, Germany). Sense and antisense riboprobes were gen-

uPA antigen expression in tissue sections was localized by immunohistochemistry using the monoclonal antibody #3689 (American Diagnostica, Greenwich, CT). This antibody is reactive with the B-chain of uPA, and recognizes pro-uPA, activated high-molecular weight-uPA, and uPA/plasminogen activator inhibitor complexes.30 The uPA receptor antigen expression was visualized using an anti-uPA receptor monoclonal antibody, #3936 (American Diagnostica). This antibody has been compared with other uPA-receptor reactive monoclonal antibodies, and its specificity has been demonstrated by Western blotting as well as immunohistochemical analysis.31 Immunostaining was performed on 5 pm formalin-fixed, paraffinembedded tissue sections and visualized by APAAP staining. Negative controls included 1) omission of the first, second, and/or third antibody; and 2) substitution of the primary antibody by a rabbit preimmune or nonimmune IgG or a monoclonal mouse IgG of irrelevant specificity. The specificity of each of the primary antibodies was further confirmed by preabsorption of the primary antibodies with appropriate recombinant pro-uPA or uPA-receptor protein.

Enzyme-Linked Immunosorbent Assay (ELISA) uPA antigen levels were measured by ELISA according to the method described by Janicke et al.32 Briefly, snap-frozen specimens of 300 to 500 mg wet weight were pulverized, resuspended in 1% Triton X-100 in TBS (20 mmol/L Tris-HCI, 125 mmol/L NaCI, pH 8.5), and cleared by ultracentrifugation. 100 p1 of appropriately diluted supernatants were added to microtiter plates (Immulon, Denkendorf, Germany) coated with the uPA-specific monoclonal antibody #3689. Bound uPA was detected by an additional biotinylated anti-uPA antibody, monoclonal antibody #377 (American Diagnostica). After addition of streptavidin-peroxidase, peroxidase-mediated conversion of 3,3'5,5'-tetramethylbenzidine was measured at 450 nm with a 46-well absorbance microtiter plate reader (Titertek Multiscan, ICNFlow, Meckenheim, Germany). The lower detection limit is 50 pg uPA/ml. This procedure recognizes pro-uPA and activated HMW-uPA, as well as uPA/

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plasminogen activator inhibitor-I complexes,30 and is not affected significantly by tissue proteases. Protein content was measured with the BCA protein assay kit (Pierce, Rockford, IL).

Statistics Statistical analysis was performed where appropriate with the Wilcoxon signed rank test, the Kruskal-Wallis test, and the Mann-Whitney test using the StatWorks statistical software package (Data Metrics Inc., Philadelphia, PA). Significance was established at the level of P < 0.05.

Results uPA mRNA Expression For direct comparison of uPA mRNA levels Northern blot analysis of both tumor and non-tumorous tissue samples was performed. Previous data indicate that uPA is not evenly expressed in normal kidney tissue. RNA extraction from randomly chosen kidney segments may thus lead to serious variation in uPA mRNA content. We therefore performed in situ hybridization to localize uPA transcripts in normal human kidney tissue and observed strong segmental expression in tubular epithelium of kidneys cortex as well as medulla (S. N. Wagner, M. J. Atkinson, C. Wagner, M. Schmitt, 0. Wilhelm, and H. Hofler, submitted for publication). Consequently, we performed RNA extractions from whole kidney samples containing both cortical and medullary tissue. In each sample studied a single transcript at the anticipated 2.3 kb length of mature uPA mRNA was detected (Figure 1A). Quantitative analysis disclosed that in 30 of the 33 tumor samples steady state uPA mRNA level was below that detected in the corresponding non-tumorous kidney

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tissue. The mean uPA content of RCC tissue samples (mean uPA expression 0.45 ± 0.33 optical density (OD) units) was more than threefold lower than that observed in non-tumorous kidney tissue (mean uPA expression 1.50 ± 1.13 OD units, P< 0.001; Figure 1A). There was no correlation between uPA mRNA content and tumor grading, staging, or growth pattern (data not shown).

uPA Receptor mRNA Expression All tissue samples expressed a single transcript corresponding to the 1.4 kb uPA receptor mRNA (Figure 1 B). While there was some inter-individual variation in uPA receptor signal intensity, in 28 of the 33 paired samples studied tumor tissue uPA-receptor mRNA levels were modestly greater than those of the corresponding non-tumorous kidney tissue. The mean uPA-receptor mRNA content in RCC tissue samples was 1.81 ± 1.13 OD units and in non-tumorous kidney tissues 1.08 ± 0.54 OD units (P< 0.001, Figure 1B). Previously, the presence of alternatively spliced uPAreceptor mRNA has been described in murine gastric mucosa.33 Although the cDNA probe used in these studies would detect such splice variants we did not observe mRNA heterogeneity at the resolution of our system. There was no correlation of uPA-receptor mRNA levels with tumor grading, staging, or growth pattern (data not shown).

uPA Antigen Content To disclose differential expression patterns of uPA protein in normal kidney tissue we performed immunohistochemical analysis. uPA immunoreactivity has been observed in all segments of tubular epithelium (S. N. Wagner, M. J. Atkinson, C. Wagner, M. Schmitt, 0. Wilhelm, and H. Hofler, submitted for publication). We therefore analyzed protein content of whole tissue

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Figure 1. Northern blot analysis of uPA and uPA-receptor mRNAs in paired RCC tumor (t) and non-tumorous kidney (n) tissue samples. Upper panel: 15 ,ug of the respective total RNA were analyzed using uPA (A) and uPA-receptor (B) cDNA probes. The migration positions of 18S rRNA (1.86 kb) and 28S rRNA (4.71 kb) are also indicated. Lower panel: control of RNA loading by rebybridization with 3-actin cDNA probe. The autoradiographs were exposedfor 14 days (membranes hybridized with uPA or (3-actin cDNA probes) or 21 days (membranes hybridized with uPAreceptor cDNA probe).

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extracts representing cortical and medullary kidney tissue. uPA expression at the protein level was quantified in both RCC and non-tumorous kidney tissues by ELISA and values were calculated for total protein content. Despite the decreased expression of uPA mRNA in RCC, we observed that the concentrations of uPA antigen measured in tumor (0.87 ± 0.82 ng uPA/mg protein) and non-tumorous kidney tissues (1.03 ± 0.71 ng uPA/mg protein, P = 0.084) were comparable. As with uPA and uPA receptor transcripts there was no correlation between uPA protein content and tumor grading, staging, or growth pattern (data not shown).

Immunohistochemical Localization of uPA and uPA-Receptor Antigen Immunohistochemical analysis of RCCs revealed the presence of both uPA and uPA receptor antigen in tumor cells. There was a remarkably consistent colocalization of the uPA and uPA-receptor antigens. The anti-uPA and anti-uPA receptor antibodies recognized both a diffuse cytoplasmic- and a plasma membrane-bound immunoreactivity in the granular-

cell subtype of RCC (Figure 2). In contrast, membrane-bound uPA and uPA receptor labeling predominated in clear-cell type tumor cells, where both uPA and uPA receptor were more strongly expressed at the cell surface than in the cytoplasm (Figure 2). This may be due to the removal of the abundant glycogen deposits and lipid droplets in this cell type during fixation.2 A differential expression of uPA and uPA receptor antigen between tumor center and periphery was not observed. Membrane-bound immunoreactivity may represent uPA/uPA-receptor complexes, whereas intracellular immunostaining may be due to endogenously synthesized antigens and/or internalized uPA/uPA-receptor complexes.16'18 Stromal cells within both RCCs and non-tumorous kidney tissues were stained only sporadically by either antibody.

In Situ Hybridization of uPA and uPA-Receptor mRNA As uPA is secreted by a variety of different cells6 and may bind to cell surface receptors on different cell types,8'13 uPA immunolocalization may not coincide

Figure 2. Localization of the uPA and uPA-receptor antigens in RCC tumor samples. (A and B) Paraffin sections of clear cell RCC (A arrow denotes tumor cell membrane staining; arrowheads denote negative reaction in stromal tissue elements) and granular eosinophilic cell RCC (B, arrowhead denotes negative staining of stromal tissue elements) were stained with monoclonal anti-uPA antibody #3689. (C and D) Paraffin sections of clear cell RCC (C, arrow denotes membrane-bound reaction of tumor cells) and granular eosinophilic cell RCC (D, arrowhead denotes negative reaction in stromal tissue elements) were stained with monoclonal anti-uPA-receptor antibody #3936. APAAP method, negative controls included preabsorption ofprimary antibodies with the appropriate recombinant antigen. Bar, 50 pm.

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with its site of synthesis. To identify the sites of uPA and uPA receptor synthesis at the cellular level the appropriate mRNAs were localized by in situ hybridization. Stromal elements within and around RCC tumor cell masses showed no significant hybridization with either probe (Figure 3). In contrast, RCC tumor cells themselves exhibited a strong specific signal with both uPA and uPA-receptor antisense cRNA probes (Figure 3). The subcellular localization of transcripts was confined predominantly to the cytoplasm of tumor cells. There was no detectable differential mRNA expression of uPA and uPA receptor between tumor center and periphery.

been described previously in squamous cell carcinoma of the skin3435 and in astrocytomas,36 whereas in human colon adenocarcinoma uPA was reported to be localized primarily to the tumor surrounding stroma.7 While the site of uPA expression seems to vary between different tumor types, the expression pattern of uPA receptor seems to be similar. As previously reported for human colon adenocarcinoma and breast cancer cells,38,39 we have documented a tumor cell-specific expression of uPA receptor in RCC. Furthermore, we provide evidence for a consistent co-localization of uPA and uPA receptor in RCC tumor cells at both the mRNA and the antigen level. Although increased uPA antigen level may be encountered in RCC,40 neither our study nor that of Hata et al41 have detected significant increases in the uPA content of RCCs compared with normal nontumorous kidney tissue. These results are in apparent contradiction to several human tumor tissues that exhibit higher levels of uPA than their normal counterparts (see ref. 6 and references therein). This may be due to two reasons. First, the high endogenous level

Discussion Using in situ hybridization and immunohistochemical staining we have established that, in primary human RCC tissues, both uPA transcripts and antigen are mostly confined to the RCC tumor cells themselves, and are found only sporadically in stromal elements within or around the tumor. A tumor cell-specific localization of uPA at the mRNA and antigen level has

Ii

Figure 3. Localization of uPA and uPA receptor mRNA in granular cell RCC, dark-field illumination. (A and B) Paraformaldebyde-fixed tissue sections were hybridized with antisense uPA riboprobe (A, arrows denote strong hybridization to tumor cells; arrowheads denote background signal to stromal tissue elements) and antisense uPA-receptor riboprobe (B, arrows denote strong hybridization to tumor cells; arrowhead denotes background signal to stromal tissue elements). (C and D) Negative controls performed by hybridization with sense uPA (C) or uPA receptor (D) riboprobes. Autoradiography was performed for 2 to 3 week. Bars, 50 um.

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in normal kidney tissue may mask changes in RCC uPA content. Normal kidney tissue is considered to be a major source of uPA in various mammalian species, and uPA is present at high concentrations in kidney tissue and the urine as well.43 In the present study, we found uPA level in normal non-tumorous kidney tissue to be significantly higher than those reported for benign breast tissue lesions, normal colon tissue, or normal gastric mucosa.324445 Secondly, uPA is not expressed at high level in RCC. Our results demonstrate significantly lower uPA antigen tissue level in RCC as those reported for several other human tumor tissues.3244,45 Low endogenous expression of uPA in RCC is also indicated by the significantly decreased uPA mRNA level compared with normal non-tumorous kidney tissue. We believe that the low levels of uPA mRNA indicate low endogenous uPA protein production. Thus, uPA mRNA levels are influenced by transcriptional and post-transcriptional mechanisms,46X8 and there is no evidence for translational control. Moreover, a close correlation between uPA mRNA steady state and protein levels has been observed, eg, in human kidney and epidermoid carcinoma cell lines, murine fibroblasts, and metastatic rat mammary adenocarcinoma cells.4648 As uPA is regulated at the transcriptional level by a variety of cytokines and growth factors, one is tempted to speculate that altered expression of such cytokines and growth factors in RCC may be involved in down-regulation of RCC mRNA expression. The effects of these factors on uPA synthesis appear to be highly variable, depending upon the tumor type and the cell culture system studied.6'49 Thus, it remains to be established whether any of these factors are involved in uPA down-regulation in RCC. Alternatively, the more than threefold decreased expression of uPA mRNA in RCC may indicate a de-differentiation process occurring during renal carcinogenesis, similar to the observed downregulation of other differentiation markers such as the mRNAs encoding pro-epidermal growth factor, HER2/neu and glutathione S-transferase-x.5>52 Transcription factors AP-1 and HNF1 -related FPC-binding protein have been implicated in the regulation of the human and the porcine uPA gene, respectively.53'54 Interestingly, both the expression of the AP-1 encoding c-fos oncogene and the expression of HNF1 are decreased in RCC.51'55 Whether the decreased expression of HNF1 may also play a role in the regulation of the human uPA gene in RCC remains to be established. Our results further indicate a dissociation of uPA mRNA and protein level detected in RCC tissue. Despite exhibiting significantly lower levels of uPA

mRNA, RCC contained quantities of uPA antigen comparable to surrounding normal non-tumorous kidney tissue. A similar situation has been described in the murine lung, which contains high levels of uPA antigen, despite minimal levels of uPA mRNA.56 A possible explanation for the dissociation of uPA mRNA and protein level is provided by uPA receptormediated uptake of uPA from tissues surrounding the tumor, as implicated in the concentration of uPA by human colon adenocarcinoma cells.37'39 uPA receptor mRNA expression may be assumed to reflect uPA receptor protein level, as has already been demonstrated in human A549 epithelial lung carcinoma cells57 and human umbilical vein endothelial cells.31 Thus, we conclude that the elevated uPA receptor content in RCC may result in the accumulation of uPA from exogenous sources leading to normalization of uPA antigen level. However, the expression of uPA receptor seems to be only modestly increased over normal non-tumorous kidney tissue, and alone would not be sufficient to substantially increase uPA antigen content to the level of other tumor tissues. At the moment it is hard to see whether expression of the plasminogen activator system may result in a proteolytic advantage of RCC tumor cells over normal renal tissue. Quantitative measurements of uPA transcripts and antigen level do not necessarily reflect the conditions required for plasminogen activation. uPA is released from tumor cells as a virtually inactive proenzyme that requires proteolytic activation.58 The antibody used in the present study recognizes both prouPA and active uPA. Thus, we cannot exclude an increase of enzymatically active uPA versus inactive pro-uPA in RCC. Furthermore, uPA-catalyzed plasminogen activation is inhibited by fast-acting plasminogen activator inhibitors,59 of which at least PAI-1 is increased in RCC (S. N. Wagner, M. J. Atkinson, S. Thanner, M. Schmitt, 0. Wilhelm, M. Rotter, and H. Hofler, submitted for publication). An evaluation of the possible involvement of plasminogen activation in RCC progression therefore requires further studies analyzing the role of these additional components in plasminogen activation.

Acknowledgments We thank Dr. U. Weidle (Boehringer, Mannheim, Germany) for the Psl fragment of the p-uPA-R-1 cDNA clone encoding the human urokinase receptor, Dr. W. GOnzler (GrOnenthal, Stolberg, Germany) for recombinant pro-uPA, Dr. R. Hoffmann (Technical University, Munich, Germany) for surgical tissue specimens, and Dr. R. Busch (Technical University, Munich, Germany) for her help in statistical analysis.

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