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Carcinogenesis Advance Access originally published online on August 4, 2005
Carcinogenesis 2006 27(1):73-83; doi:10.1093/carcin/bgi203
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Carcinogenesis vol.27 no.1 © Oxford University Press 2005; all rights reserved.

Antitumor effect of parathyroid hormone-related protein neutralizing antibody in human renal cell carcinoma in vitro and in vivo

Isabelle Talon 1, 2, Véronique Lindner 1, 3, Carole Sourbier 1, Eric Schordan 1, Sylvie Rothhut 1, Mariette Barthelmebs 1, Hervé Lang 1, 2, Jean-Jacques Helwig 1 and Thierry Massfelder 1, *

1 INSERM U727, Section of Renal Pharmacology and Physiopathology, University Louis Pasteur, School of Medicine, Strasbourg, 67085 France, 2 Department of Urology and 3 Department of Pathology, Hôpitaux Universitaires de Strasbourg, Strasbourg, 67091 France

* To whom correspondence and reprint requests should be addressed. Tel: +333 90 24 34 56; Fax: +333 90 24 34 59; Email: thierry.massfelder{at}medecine.u-strasbg.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in 40–80% of human conventional renal cell carcinomas (RCCs). We showed recently that VHL-deficient RCCs expressed large amounts of parathyroid hormone-related protein (PTHrP), and that PTHrP, acting through the PTH1 receptor (PTH1R), plays an essential role in tumor growth. We also showed that PTHrP expression is negatively regulated by the VHL gene products (pVHL). Our goal was to determine whether blocking the PTHrP/PTH1R system might be of therapeutic value against RCC, independent of VHL status and PTHrP expression levels. The antitumor activity of PTHrP neutralizing antibody and of PTH1R antagonist were evaluated in vitro and in vivo in a panel of human RCC lines expressing or not pVHL. PTHrP is upregulated compared with normal tubular cells. In vitro, tumor cell growth and viability was decreased by up to 80% by the antibody in all cell lines. These effects resulted from apoptosis. Exogenously added PTHrP had no effect on cell growth and viability, but reversed the inhibitory effects of the antibody. The growth inhibition was reproduced by a specific PTH1R antagonist in all cell lines. In vivo, the treatment of nude mice bearing the Caki-1 RCC tumor with the PTHrP antibody inhibited tumor growth by 80%, by inducing apoptosis. Proliferation and neovascularization were not affected by the antiserum. Anti-PTHrP treatment induced no side effects as assessed by animal weight and blood chemistries. Current therapeutic strategies are only marginally effective against metastatic RCC, and adverse effects are common. This study provides a rationale for evaluating the blockade of PTHrP signaling as therapy for human RCC in a clinical setting.

Abbreviations: HIF, hypoxia-induced factor; PTHrP, parathyroid hormone-related protein; PTH1R, parathyroid hormone-1 receptor; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; RCC, conventional renal cell carcinoma; VHL, von Hippel–Lindau; pVHL, VHL gene products


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Conventional renal cell carcinoma (RCC) represents 75% of all renal cell carcinomas. In the last 20 years, the incidence rate of RCC has increased by >43% and the death rate by 16% (1). Each year, 200 000 patients are diagnosed worldwide with this neoplasm, resulting in an estimated 100 000 deaths (1,2). Approximately 20–30% of patients have metastases at initial presentation. Of the patients who present with apparent localized disease and are treated with radical or conservative nephrectomy, 50–60% will develop metastatic disease within 5–10 years. Metastatic RCC is resistant to radiotherapy and to systemic chemotherapy or biologic therapy (3,4). At present, interleukin-2 is the systemic therapy of choice for metastatic disease, but the overall response rate is only 15%. Interleukin-2 does show the most favorable immunotherapeutic response, with complete response achieved in 5% of patients, and long-term, relapse-free survival in responding patients greater than for any other therapeutic agent. The 5-year survival rate is 70% for localized RCC, but is only 5% in the metastatic group. Since therapy for advanced renal cancer clearly remains inadequate, new therapeutic options for RCC are urgently needed.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for hereditary human VHL syndrome. Affected individuals are predisposed to develop tumors in a variety of organs, including the kidney (5,6). The VHL gene is also inactivated in up to 80% of sporadic RCC (5–7). Linkage analysis of VHL kindreds mapped the VHL locus to chromosome 3p25–26 in humans (5). Inactivation of the VHL gene is a consequence of somatic mutations or 3p loss of heterozygosity in 30–60% of RCC patients. Hypermethylation and inactivation of the VHL promoter occurs in an additional 10–20% of patients (7–9). The VHL gene is believed to have gatekeeper properties in proximal tubular cells from which RCC originates, not only because VHL patients may have hundreds of individual discrete tumors in the kidney (10), but also because the reintroduction of the VHL gene in the VHL-deficient RCC line 786-0 suppresses their tumorigenicity in nude mice (11).

The protein products of the human VHL gene (pVHL) are VHL (1–213) and VHL (54–213), the latter being generated by internal translation initiation from an in-frame methionine at codon 54 (12). They have identical biological properties. pVHL is a component of an E3 ubiquitin ligase complex that targets the {alpha} subunits of HIF-1 and HIF-2 (hypoxia-induced factors) transcription factors for destruction in the presence of oxygen. As a consequence, tumor cells lacking pVHL overexpress HIF target genes of the metabolic, angiogenic and growth factor families, including VEGF, PDGF or TGF-ß (12). pVHL is thus involved in cell cycle control, cell differentiation and death, regulation of extracellular pH, extracellular matrix formation and angiogenesis, all contributing to carcinogenesis. HIF-1 and -2 {alpha} proteins are frequently present in hypoxic regions of solid tumors. As a consequence of VHL deficiency, high levels of these factors are found in RCC, regardless of the oxygen tension. It follows that many of the hypoxia-responsive genes are upregulated in the majority of RCC (13). For example, RCCs are highly vascularized due to the overexpression of VEGF and TGF-ß. Alterations of the VHL gene are considered to be an early event in RCC tumorigenesis. This is evidenced by the presence of VHL gene mutations or silencing in early pT1 stage of RCC (7,9). For example, Brauch et al. (9), using >150 pT1 to pT3 RCC, have identified VHL mutations or silencing in 45% of RCC. Interestingly, in their study, VHL alterations, although present at all stages, were significantly associated with the pT3 stage. They hypothesized that the large range of somatic VHL alterations rates reported in the literature (40–80%, see above) might be related not only to the ethnic origin of the population studied, but also to the number of pT3 tumors included in the analyzed patient samples. Thus, depending on the population studied, 20–60% of RCC have normal VHL expression, and low levels of HIFs. This indicates that the VHL gene is not sufficient to suppress tumorigenesis and that damage to other genes is probably involved in RCC initiation and development, at least in RCC containing normal VHL.

Parathyroid hormone-related protein (PTHrP), identified as the tumor-derived agent responsible for humoral hypercalcemia of malignancy (HHM) (14), is a polyprotein derived from normal and malignant cells. It regulates cell growth, differentiation and death (15). An early study by Burton et al. (16) showed that PTHrP regulates the proliferation of the RCC line, SKRC-1. We have recently shown (17) in three VHL-deficient human RCC lines (786-0, UOK-126 and UOK-128 cells) expressing high levels of PTHrP that blocking PTHrP with antibodies, or antagonizing its receptor—the parathyroid hormone-1 receptor (PTH1R), inhibits tumor growth in vitro by promoting cell apoptosis. In nude mice subcutaneously implanted with 786-0 cells, anti-PTHrP treatment induced complete regression of the implanted tumors in 70% of mice, inducing cell death. In the same study, we also demonstrated that the VHL suppressor gene negatively regulates PTHrP expression at the post-transcriptional level. These studies identified PTHrP as a critical growth factor for VHL-deficient RCC expressing high levels of PTHrP and suggested that PTHrP might be a new therapeutic target in patients with VHL-deficient tumors.

In the current study, our objective was to define whether blocking the PTHrP/PTH1R system might also have broader therapeutic application against RCC regardless of the VHL status and regardless of PTHrP expression levels. Using a panel of RCC lines either expressing or not expressing pVHL, we provide evidence that anti-PTHrP therapy may have broad therapeutic application against human RCC.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
In vitro studies
Cells and cell culture
Normal human (h) proximal tubular cell line HK-2 (a gift from Dr C.Silve, Paris, France) and human RCC lines 786-0, UOK-126, UOK-128, A498, ACHN, Caki-1 and Caki-2 were maintained in DMEM medium (Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal bovine serum. All cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA) except UOK-126 and UOK-128 that were generously given to us by Dr P.Anglard (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France) (18). The cell lines are derived from human sporadic RCC, and are either VHL-deficient (786-0, UOK-126, UOK-128 and A498 cell lines) or encode active pVHL (ACHN, Caki-1 and Caki-2 cell lines). Unless specified otherwise, cells were used at 70–80% confluence.

RNA extraction and real-time quantitative RT–PCR analysis of PTHrP and PTH1R
Total RNA was extracted from cultured cells using TRIzol (Invitrogen), according to the manufacturer's protocol. The hPTHrP and hPTH1R cDNAs were amplified using the LightCycler-FastStart DNA Master SYBR Green kit (Roche Diagnostics, Meylan, France). In addition, a standard curve was obtained for hGAPDH by serial dilutions of mixed cDNA samples. PCRs were performed in 20 µl mix containing 4 mM MgCl2, 0.5 µM each primer set, 0.2 µM SYBR Green probe, 0.4 µM hybridization probe, 1x FastStart reaction buffer (DNA polymerase, dNTPs, buffer) and 2 µl cDNA (50 ng). PCRs for hPTHrP, hPTH1R and hGAPDH were as follows: 95°C for 10 min followed by 40 cycles at 95°C for 10 s, 60°C for 5 s and 72°C for 7 s (hPTHrP), 18 s (hPTH1R) or 16 s (hGAPDH). cDNA was replaced by PCR-grade water as a negative control. Each sample was run twice and quantified with the LightCycler analysis software according to the manufacturer's protocol (Roche Diagnostics). Relative expression of hPTHrP and hPTH1R in samples were calculated by quantifying hPTHrP and hPTH1R levels normalized to hGAPDH, and results in RCC lines were expressed as fold increase above HK-2 levels set 1. Primers for hPTHrP were: sense, 5'-CGGTGTTCCTGCTGAGCTA-3'; antisense, 5'-TGCGATCAGATGGTGAAGGA-3' (PCR product of 161 bp). Primers for the hPTH1R were: sense, 5'-CTCTTTGGCGTCCACTACATTG-3'; antisense, 5'-TTGAGGAACCCATCGTCCTTG-3' (PCR product of 450 bp). Primers for hGAPDH were: sense, 5'-GGAAGGTGAAGGTCGGAGTC-3'; anti-sense, 5'-GCAGTGATGGCATGGACTG-3' (PCR product of 415 bp).

Analysis of PTHrP expression
PTHrP concentration in the conditioned medium of cultured cells was determined by immunoradiometric assay (IRMA) (Laboratoire de Biochimie Générale et Spécialisée, Hospices Civils, Strasbourg, France) and expressed as pM in confluent cells or pM/24 h/million cells in subconfluent cells (80% confluence).

Western blot analysis of the PTH1R
Protein expression was analyzed as described (17) using 10–30 µg of protein and 5 µg/ml of an affinity-purified polyclonal rabbit antibody directed against hPTH1R (Eurogentec, Angers, France). A polyclonal mouse anti-ß actin antibody (Sigma-Aldrich, St Quentin Fallavier, France) was used for visualization of protein gel loading. Visualization was performed using the ECL method (17).

Cell number measurements
RCC proliferation was assessed by counting adherent cells, as described (17). RCCs were seeded in 24-well plates (20 000 cells/well), grown for 48 h and then treated for 48 h with test substances at the concentrations indicated in the figures or figure legends. Test substances were affinity-purified polyclonal rabbit antibody directed against the N-terminal region of PTHrP (Bachem, Voisins-le-Bretonneux, France), non-immune rabbit IgG (Sigma) which served as control for PTHrP antibody experiments, (Tyr36)-PTHrP(1–36) (Bachem) or the PTH1R antagonist (Asn10, Leu11, D-Trp12)PTHrP(7–34)amide (Bachem). All peptides were dissolved in 10–3 M HCl containing 0.1% bovine serum albumin (BSA) at a final concentration of 250 µM and stored at –70°C in 25 µl aliquots. Before use, peptides were further diluted to the desired concentration in 0.9% NaCl containing 0.1% BSA (diluent).

MTT cell viability measurements
This assay is based on the conversion of the yellow tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to purple formazan crystals by metabolically active cells and provides a quantitative estimate of viable cells (19). RCCs were seeded and treated as above for cell proliferation measurements with test substances at the concentrations indicated in the figures or figure legends. After 48 h of treatment (17), MTT (Sigma) at 0.5 mg/ml (final concentration) was added to each well for 4 h at 37°C. After this period, 1 ml of a solubilization solution (10% SDS in 0.01 M HCl) was added to each well, and plates were incubated for 24 h at 37°C. Spectrophotometric absorbance was measured at 570 nm (reference 690 nm) on 200 µl aliquots using a microplate reader.

Cell death analysis
RCCs were seeded in 96-well plates (2000 cells/well) and were exposed 48 h later to the N-terminal PTHrP-neutralizing antibody (Bachem) or non-immune rabbit IgG (Sigma) at the concentrations indicated in the figures or figure legends or to the PTH1R antagonist (Asn10, Leu11, D-Trp12)PTHrP(7–34)amide (Bachem) at 10–6 M or its diluent for 48 h. Cell death was then analyzed using a photometric enzyme immunoassay allowing the qualitative and quantitative determination of cytoplasmic histone-associated DNA fragments specific for apoptosis, according to the manufacturer's protocol (Roche Diagnostics).

In vivo studies
Implantation
All animal studies were in compliance with French animal use regulations. Male Swiss Nu/Nu nude mice (7-week-old) (Iffa-Credo, St Germain sur l'Arbresle, France) were given injections of 10 million Caki-1 cells subcutaneously into the skin of the back (n = 20). The tumor take rate was 85%. Four weeks after injection, tumors had grown to a volume of 161.3 ± 27.4 mm3. The tumor-bearing mice were then separated into two groups of eight and nine animals of comparable mean tumor volume. One group (n = 8) received daily intraperitoneal injection of 40 µg of non-immune rabbit IgG (Sigma). The other group (n = 9) received daily intraperitoneal injection of 40 µg of the N-terminal PTHrP neutralizing antibody (Bachem). We have shown in our previous study in nude mice bearing VHL-defective RCC tumors (17) that such treatment induced tumor regression. Tumor size was measured using calipers every 2–3 days (17). At the end of the treatment period (19 days), mice were euthanized with ether and blood was harvested for the measurement of plasma electrolytes, creatinine and PTHrP concentrations (Laboratoire de Biochimie Générale et Spécialisée). Tumors were then removed, fixed in formalin and paraffin-embedded. Sections of 4 µm thickness were used. Some sections were stained with hematoxylin–eosin and others with the N-terminal PTHrP neutralizing antibody or an affinity purified polyclonal rabbit antibody directed against the PTH1R (Eurogentec) as we have described previously (17).

Proliferative/apoptotic index
The proliferative index was determined by staining tumor sections with a mouse monoclonal anti-hKi67 antibody (Dako, Trappes, France), as previously described (17). An apoptosis detection kit, based on the TUNEL method (Roche Diagnostics), was used to measure the apoptotic index on sections. To determine both indices the number of total and stained cells in 15 fields (0.25 mm2 each) were quantified in a blinded fashion by an experienced urologic histopathologist (Dr Véronique Lindner, Department of Pathology, University Hospital, Strasbourg, France). Both indices were expressed as a percentage of stained cells to total cells.

Factor VIII and microvascular density
Tumor sections were stained for endothelial cells using a rabbit polyclonal anti-human factor VIII antibody (Dako), as previously described (17). Microvessel density was determined in a blinded manner as described above by counting, for each tumor, both vessel intersecting points and the total number of vessels in 4–5 fields (0.25 mm2 each) showing the highest vascular density.

Statistics
All values are expressed as mean ± SEM. Values were compared using multifactorial analysis of variance followed by the Student–Newman–Keul's test for multiple comparisons. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
PTHrP and PTH1R expression in RCC lines
Using real-time quantitative PCR analysis, we detected PTHrP (Figure 1A, upper panel) and the PTH1R transcripts (Figure 1A, lower panel) in all cell lines. RCC lines showed higher PTHrP expression than HK-2 cells. Interestingly, in VHL-deficient A498 cells, PTHrP levels were similar to those observed in wild-type VHL-expressing RCC lines. PTH1R expression was not higher in all RCC lines compared with HK-2 cells and did not show any difference in relation with the VHL status of the cells. Similar results were obtained with cells grown in the absence of serum (data not shown). This pattern of expression was confirmed at the protein level by IRMA for PTHrP (Figure 1B, upper panel) and western blot for the PTH1R (Figure 1B, lower panel).



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  		Fig. 1. PTHrP and PTH1R expression in RCC lines and in HK-2 cells in vitro. (A) Real-time quantitative PCR analysis of hPTHrP (top) and PTH1R (bottom) mRNA in the various cell lines. GAPDH was used as the housekeeping gene for quantification of both transcripts as detailed in Materials and methods. PTHrP and PTH1R expression in RCC lines were expressed as fold increase above HK-2 cells set to 1. Results are shown as mean ± SEM, n = 3–12 and n = 3–7 for PTHrP and PTH1R quantifications, respectively; *P < 0.05; **P < 0.01 from HK-2 cells. (B) IRMA measurements of immunoreactive PTHrP in conditioned medium of each cell line (top). Results are shown as mean ± SEM, n = 4; **P < 0.01 from HK-2 cells. Western blots of RCC lysates incubated with antibodies against the hPTH1R and ß-actin (bottom). Shown are representative radiographs of at least three independent experiments.

 
Thus, all RCC lines employed constitutively express both PTHrP and the PTH1R. In addition, PTHrP expression was significantly higher in RCCs compared with normal cells, regardless of the VHL status of the cells.

Effect of PTHrP/PTH1R blockade on RCC growth and survival in vitro
Since PTHrP is overproduced by all RCC lines studied here (and most described in the literature), and because our previous studies suggested that either a large N-terminal-containing fragment or full-length PTHrP is required to induce PTH1R-dependent stimulation of RCC growth (17), we choose to expose cells to either various concentrations of a PTHrP antibody recognizing all N-terminal-containing PTHrP peptides, including full-length PTHrP or to a potent and specific PTH1R antagonist, i.e (Asn10, Leu11, D-Trp12)PTHrP(7–34)amide (20). Cells were then analyzed for growth, viability and apoptosis. The antibody decreased cell number (Figure 2A) and viability (Figure 2B) in a concentration-dependent manner and increased cytoplasmic histone-associated DNA fragment specific for apoptosis (Figure 2C) in all cell lines tested, including HK-2 cells. These results suggest that endogenous PTHrP acts on RCC growth and survival by inhibiting cell apoptosis. In all cell lines, except Caki-2 cells, growth was decreased by 40–80% at the highest concentration use of the antibody. Increasing PTHrP antibody concentrations to 50 µg/ml also decreased cell growth in Caki-2 cells by 80% (data not shown). PTHrP(1–36) at 10–6 M alone did not affect cell number (Figure 3A, left panel) and viability (Figure 3A, right panel), but reversed or significantly decreased the inhibitory effect of the PTHrP antibody on cell number (Figure 3B, left panel) and viability (Figure 3B, right panel) in all cell lines tested. These results demonstrate that the effect of the antibody relates specifically to PTHrP blockade. The absence of effect of PTHrP on cell growth is reminiscent of what we had observed previously in VHL-deficient RCC lines (17). Growth inhibition was reproduced by blocking the PTH1R in all cell lines tested, including HK-2 cells. Indeed, the PTH1R antagonist at 10–6 M decreased both cell number (by 20–47%) (Figure 4A) and cell viability (by 15–60 %) (Figure 4B) by inducing cell apoptosis (Figure 4C). This concentration of the PTH1R antagonist was chosen since it had maximally inhibited cell growth in our previous study on VHL-deficient RCCs (17). These results further argue the auto/paracrine nature of the PTHrP-induced cell survival.



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  		Fig. 2. Effect of PTHrP blockade on RCC growth in vitro. Effects of the PTHrP antibody at various concentrations on cell number (A), cell viability (B) and cytoplasmic histone-associated DNA fragments (C) in all RCC lines and in HK-2 cells. IgG, non-specific IgG, 5 µg/ml. The effects in each cell line have been compared with cells treated in control (Ctl) using the vehicle (pure water) that has been used to dissolve and dilute PTHrP antibody and IgG. For the analysis of the effects of the various treatments on cell number (A) and cell viability (B) the control has been set to 100%. For the analysis of cell apoptosis (C), the control (representing basal apoptosis) has been set to 1 according to the manufacturer's protocol. Results were expressed either as percentages from control (A and B) or as fold increase above control level (C) Results are shown as mean ± SEM, n = 4–16 for cell number and MTT and n = 3–7 for histone-associated DNA fragments. *P < 0.05; **P < 0.01 from the corresponding control.

 


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  		Fig. 3. Effect of exogenous PTHrP(1–36) alone and on cell growth inhibition induced by PTHrP neutralizing antibody. (A) Effects of PTHrP(1–36) alone at 10–6 M on cell number (left panel) and cell viability (right panel) in all RCC lines and HK-2 cells. The control (Ctl) represents the effect obtained in cells treated with the vehicle used to dissolve the peptide (0.9% NaCl containing 0.1% BSA) that has been set to 100% in each cell line. Results were expressed as percentage from control. The Ctl bar is the same for each cell line. Thus, in order to avoid unnecessary repeat of the bar for each cell line, we decided to show the Ctl bar only once in the graph shown. Results are shown as mean ± SEM, n = 4. (B) Effects of the PTHrP antibody at 5 µg/ml on cell number (left panel) and cell viability (right panel) in the presence of 10–6 M PTHrP(1–36) in all RCC lines and in HK-2 cells. The control (Ctl) represents the effect obtained in cells treated with the vehicle constituted by a mix of the diluent used to dissolve the peptide (0.9% NaCl containing 0.1% BSA) and of the diluent used to dissolve the PTHrP antibody (pure water) that has been set to 100% in each cell line. Again, since this bar is the same for each cell line and in order to avoid unnecessary repeat of the bar for each cell line, the Ctl bar is shown only once in the graph. Results are shown as mean ± SEM, n = 4. *P < 0.05; **P < 0.01 from the corresponding control.

 


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  		Fig. 4. Effect of PTH1R blockade on RCC growth in vitro. Effects of the PTH1R antagonist at 10–6 M on cell number (A), cell viability (B) and cytoplasmic histone-associated DNA fragments (C) in all RCC lines and HK-2 cells. The effects in each cell line have been compared with cells treated in control (Ctl) using the vehicle (0.9% NaCl containing 0.1% BSA) that has been used to dissolve and dilute the peptidic antagonist. As for the results obtained with the PTHrP antibody (Figure 2), for the analysis of the effects of the antagonist on cell number and cell viability the control has been set to 100%. For the analysis of cell death in response to the antagonist, the control (representing basal apoptosis) has been set to 1 according to the manufacturer's protocol and the effects of the various treatments expressed as fold increase above Ctl. As for Figure 3, in which only one concentration of the test compound has been used, the Ctl bar is represented only once in the graph. Results are shown as mean ± SEM, n = 4 for cell number, MTT and histone-associated DNA fragments analysis. **P < 0.01 from the corresponding control.

 
Taken together, these results show that blockade of the PTHrP/PTH1R signaling pathway induces tumor cell death through apoptosis, regardless of the VHL status of the cells and PTHrP expression level.

Effect of PTHrP blockade on growth of wild-type VHL-expressing RCC tumors in nude mice
To evaluate the biological significance of PTHrP blockade in wild-type VHL-expressing RCC, we used the xenograft athymic mouse model. Based on our previous studies in nude mice bearing VHL-deficient RCC tumors (17), and in order to compare the current studies with our prior studies, mice were treated with daily intraperitoneal injections of 40 µg/mouse of the PTHrP antibody or the non-immune IgG, as detailed in Materials and methods, for a period of 19 days. Treatment with the PTHrP antibody significantly inhibited RCC tumor growth by 80% in all animals compared with IgG-treated animals (Figure 5A and B). No histopathological differences were observed between either group on hematoxylin–eosin staining (data not shown). Areas of necrosis were present in the center of all tumors. Tumors obtained from IgG-treated mice strongly expressed both the PTHrP (Figure 5C, upper left panel) and the PTH1R (Figure 5C, upper right panel). Obvious staining for PTHrP and PTH1R was observed in the cytoplasm of all cells and in the nuclei of many cells. No apparent differences were noted from mice treated with the PTHrP antibody in the staining pattern of tumors for PTHrP (Figure 5C, lower left panel) and PTH1R (Figure 5C, lower right panel). The proliferative index was not different between tumors obtained from PTHrP antibody-treated mice (Figure 6A, upper and lower right panels) and IgG-treated mice (Figure 6A, upper and lower left panels). Quantification is given on the histogram (Figure 6A). In marked contrast, there was a significant increase in the apoptotic index in tumors obtained from PTHrP antibody-treated mice (Figure 6B, upper and lower right panels) compared with IgG-treated mice (Figure 6B, upper and lower left panels). Quantification is given on the histogram (Figure 6B) showing that PTHrP antibody treatment increased the apoptotic index by 35% (P < 0.05). Tumor neovascularization shows no difference between antibody-treated mice (Figure 6C, upper and lower right panels) and IgG-treated mice (Figure 6C, upper and lower left panels). Quantification of tumor neovascularization is given on the histograms representing the number of vessel intersecting points (Figure 6C, upper histogram) and the total number of vessels as a function of surface area (Figure 6C, lower histogram).



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  		Fig. 5. Effect of PTHrP blockade on the growth of wild-type VHL expressing RCC in vivo. (A) Tumor growth (volume increase in percentage from day 0) in mice bearing RCC tumors and treated intraperitoneally for 19 days with non-specific IgG (Ctl IgG, black circles) or the antibody against the N-terminus of PTHrP (PTHrP antibody, open circles). Results are shown as mean ± SEM, n = 8 and 9 tumors in the IgG group and PTHrP antibody group, respectively; **P < 0.01 from Ctl IgG. In this study, tumor growth was inhibited by 80% in mice treated with the PTHrP antibody. (B) Photographs show the implanted tumors in a representative mouse at the first day of drug injection (left, day 0), in a representative IgG-treated mouse (middle, day 19) and in a representative mice treated with the PTHrP antibody (right, day 19). (C) Sections of tumors from IgG- (upper panels) or PTHrP antibody (lower panels)-treated mice shown in (A) immunostained with an antibody against either PTHrP (left panels) or the PTH1R (right panels). A strong staining is seen in the cytoplasm of tumor cells. Staining of PTHrP and of the PTH1R is also obvious in nuclei in many cells throughout the tumors (magnification 400x).

 


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  		Fig. 6. Histopathological analysis of tumors from IgG- and PTHrP antibody-treated mice. (A) Left, tumor sections of IgG- (upper and lower left panels) or PTHrP antibody (upper and lower right panels)-treated mice immunostained with antibody against Ki67 (black nuclei; upper panels, magnification 200x; lower panels, magnification 400x). Right, quantification of Ki67 staining (proliferative index). Results are shown as mean ± SEM, n = 8–9. (B) Left, tumor sections of IgG- (upper and lower left panels) or PTHrP antibody (upper and lower right panels)-treated mice immunostained for DNA fragmentation (TUNEL) (black nuclei; upper panels, magnification 200x; lower panels, magnification 400x). Right, quantification of TUNEL staining (apoptotic index). Results are shown as mean ± SEM, n = 8–9; *P < 0.05 from IgG-treated mice. Anti-PTHrP treatment induced cell apoptosis. (C) Left, tumor sections of IgG- (upper and lower left panels) or PTHrP antibody (upper and lower right panels)-treated mice immunostained for factor VIII (upper panels, magnification 200x; lower panels, magnification 400x). Right, upper histogram, quantification of crossing vessels intersecting points per surface area (0.25 mm2). Results are shown as mean ± SEM, n = 8–9. Right, lower histogram, quantification of vessels per surface area (0.25 mm2). Results are shown as mean ± SEM, n = 8–9.

 
PTHrP concentrations in plasma were 0.6 ± 0.1 pM (n = 6) in nude mice without tumor. PTHrP concentrations were significantly increased to 2.7 ± 0.3 pM (P < 0.001) in Caki-1 tumor-bearing mice. Plasma concentrations of electrolytes, creatinine and albumin, as well as the body weight showed no difference between animals treated in control or with the PTHrP antibody over the period of treatment (Table I). Although circulating PTHrP was elevated in tumor-bearing mice, plasma calcium levels were not different from normal nude mice (data not shown). This result is similar to the ones observed in our previous study (17), and suggests that the increase in PTHrP circulating level obtained in these mice was not high enough to affect calcium homeostasis.


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  		Table I. Treatments of nude mice with the PTHrP antibody do not affect plasma parameters

 
These results demonstrate that blocking the PTHrP signaling pathway is effective against progression of wild-type VHL expressing RCC tumors through an increase in cell apoptosis. In addition, anti-PTHrP treatment has no apparent toxicity.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Studies in human breast, prostate and lung cancers strongly suggest that PTHrP behaves as a factor that regulates tumor growth and invasion (21–23). Our previous studies on human RCC revealed a critical role for this factor in the growth of these tumors as well (17). Indeed, using three different human RCC lines deficient in VHL tumor suppressor gene and expressing high levels of PTHrP, we showed that PTHrP participates in tumor growth and that PTHrP expression is negatively regulated at the level of mRNA stability by pVHL. These results suggest the potential for anti-PTHrP treatment therapy in human directed against VHL-deficient RCC expressing high levels of PTHrP. Since normal VHL expression is documented in 20–60% of patients (see Introduction), the goals of the current study were to explore the PTHrP/PTH1R status in a panel of RCCs expressing or not expressing pVHL, and to determine whether blocking the PTHrP/PTH1R system might be of therapeutic value against RCC independent of the VHL status and of PTHrP expression levels.

In the current study, we report that in addition to VHL-deficient RCCs, PTHrP expression is also significantly increased in RCCs that express pVHL. Thus, PTHrP appears to be upregulated in human RCCs whether they express pVHL or not. In two independent previous studies, PTHrP was shown to be overexpressed in 95% of human RCC (24,25). In these two studies, a total of 40 RCC tumors were investigated by immunohistochemistry either for N-terminal PTHrP (24) or C-terminal PTHrP (25). PTHrP was elevated, although at variable levels from one tumor to another, similar to the observations in the present study. Although the VHL status was not investigated in either of these two studies, taken together with our results, these data strongly suggest that up-regulation of PTHrP is a common feature of human RCC. This result begs the question with respect to the regulation of PTHrP expression in RCC. Although pVHL plays an important role in regulating PTHrP expression level (17), other factors are obviously involved. In accord with this hypothesis, in our previous studies, the reintroduction of the VHL gene in VHL-deficient RCCs decreased PTHrP levels by only 50%, leaving them far higher than levels in normal cells (17). A variety of growth and angiogenic factors have been shown to regulate PTHrP expression, including EGF, PDGF, TGF-{alpha} and -ß and VEGF in different cell types (14,26–31). Since these factors and/or their receptor are also up-regulated in human RCC and participate in RCC tumorigenesis (1,2,32,33), it is tempting to hypothesize that one or more of these factors participate (s) in the control of PTHrP expression in RCC. However, these factors and/or their receptors are also negatively regulated either at the transcriptional or post-transcriptional level, or both, by pVHL, making their potential involvement in the regulation of PTHrP expression in RCC complex (12,33–36).

The fact that PTHrP is constitutively upregulated in RCC implies that factors, such as oncogenes, that regulate its expression are present in RCC. Alternatively, tumor suppressor factors that negatively regulate PTHrP expression might be repressed in RCC. The identity of these additional factors has not been investigated. In addition to a variety of genetic changes, oncogenes or proto-oncogenes such as c-met (hepatocyte growth factor/scatter factor receptor), C-erB1 (EGF receptor), c-jun and members of the ras-family have been implicated in RCC tumorigenesis (37), but their possible relation with PTHrP expression remains uncertain. Rare kindreds with familial RCC not linked to the VHL tumor suppressor gene have been described, suggesting that additional familial RCC susceptibility genes exist (37). One possible candidate is p53 tumor suppressor. RCC tumors rarely acquire p53 mutation, although p53 functions are repressed in a subset of RCC (38). Since PTHrP expression has been shown to be repressed by p53 in the human squamous carcinoma cell line A253 (39,40), we speculate that the loss of p53 function in RCC could lead to an increase in PTHrP expression. Again, however, a role for p53 in RCC tumorigenesis has not been consistently demonstrated. To understand what controls PTHrP expression in a common HHM-associated tumor, Holt et al. (41) studied 12 RCC lines (Caki-1 cells and 11 SKRC cell lines). They showed that the difference in PTHrP expression between cells was caused by differences in the transcription rate of the PTHrP gene and that this was correlated with the methylation status of the PTHrP gene promoter region. However, the VHL status of these cell lines, except Caki-1, was unknown. Taken together, all these findings show that PTHrP is controlled by a variety of mechanisms in RCC. Further studies are needed to understand how each of these pathways contributes to PTHrP expression in RCC not only in vitro but also in vivo at the various tumoral stages. The important question of whether PTHrP is an independent prognostic factor in RCC is currently under investigation in our laboratory.

Immunoreactive PTHrP is localized in proximal and distal tubules in various species, including humans (14,42–45). Consistent with immunocytochemical analysis, PTHrP has been shown to be secreted in the conditioned medium of rabbit proximal tubule cells, proximal tubule-like LLC-PK1 cells, rat renal epithelial cells NRK-52E of proximal origin and human proximal tubule cells HK-2 in culture (45–47). Using either exogenous PTHrP fragments or specific PTHrP neutralizing antibodies, it has been demonstrated that PTHrP is a potent mitogenic factor for proximal tubule cells both in vitro and in vivo (45–47). Our own results described here in HK-2 cells confirm the results obtained by Garcia-Ocana et al. (47) in the same cell line. Our data further suggest that PTHrP participates in the growth of this cell type in vitro through the inhibition of cell apoptosis. PTHrP was shown to increase DNA synthesis in proximal tubule cells through cAMP and PKC-dependent pathways (48) and has been suggested to participate in an auto/paracrine way in the regenerative (protective) and/or damage processes following renal injuries, including renal ischemia, tubulointerstitial nephropathy, chronic and acute renal failure and glomerulonephritis (49).

Our previous results (17) suggested that the secreted PTHrP species which acts as a survival factor for RCC is most likely an N-terminal-PTHrP fragment, and that it acts through the PTH1R. For this reason we performed experiments with a PTHrP antibody recognizing all N-terminal-containing PTHrP species. Using three different methods, we show that PTHrP acts on RCC growth by preventing cells from entering the apoptotic pathway. The inhibitory effect of the PTHrP antibody on cell growth was blocked or significantly inhibited in all cell lines by exogenous PTHrP(1–36), demonstrating that the effect of the antibody relates specifically to PTHrP blockade. Interestingly, high concentrations of exogenous PTHrP(1–36) did not affect cell growth in all cell lines. These data are reminiscent of what we observed in our previous study on VHL-deficient RCC lines expressing high levels of PTHrP (17), and further suggest that all RCC lines secrete PTHrP above a maximally effective level. Finally, using a potent, specific competitive PTH1R antagonist, we show that PTHrP induces tumor cell survival in all cell lines tested through its interaction with the PTH1R. The fact that PTHrP acts as a survival factor for RCC irrespective of the VHL status of the tumor suggest that PTHrP acts so through a common pathway in cells with active or inactive VHL. Interestingly, Caki-2 cells were more resistant to the inhibitory effect of the antibody, despite the fact that these cells produce a similar amount of immunoreactive PTHrP as some of the other cell lines that were more sensitive to the antibody. Since PTH1R expression is also low in this cell line, one possibility is that the PTHrP/PTH1R system is less active in these cells. Another possibility is that PTHrP acts through a signaling pathway defective in this cell line. The molecular mechanism by which PTHrP induces cell survival in RCC tumors is currently under investigation in our laboratory.

In the present study, we used the same dose and protocol for injection of the PTHrP antibody in mice bearing Caki-1 tumor as the one used previously in mice bearing the VHL deficient 786-0 tumor (17), i.e. 40 µg/mouse daily. Although the treatment was very efficient in inhibiting Caki-1 tumor progression, we did not observe the tumor regression in Caki-1 tumors, previously observed in 786-0 tumors. Since Caki-1 cells express lower levels of PTHrP than 786-0 cells, the lower efficiency of the PTHrP antibody on Caki-1 tumors was surprising. However, this result is corroborated with the data observed in vitro using the PTHrP neutralizing antibody. Thus, it is tempting to suggest that the lower efficiency of the PTHrP antibody on Caki-1 tumor growth results from a PTHrP/PTH1R system being less central to cell survival in this cell line. However, we cannot rule out the possibility that increasing the dose of the PTHrP antibody would have led to regression of these tumors as well. Unfortunately, it was not possible to increase the dose for cost reasons. Similarly, although it could have been of value to study a larger panel of RCC tumors in vivo to evaluate the efficacy of antibody blockade for a number of tumors, this was not possible. Another finding that contrasts somehow with our previous results is the lack of effect on tumor neovascularization. Indeed in our previous study, anti-PTHrP/PTH1R treatment slightly but significantly increased neovascularization of 786-0 tumors, suggesting that PTHrP has anti-angiogenic activity. The lack of effect af the PTHrP antibody on tumor vascularization in the current study might be a result of the lower PTHrP expression by Caki-1 tumors, or the presence of pVHL in Caki-1 cells. Knowing the pathogenesis of human RCC, it will be important in the future to examine the presence of PTHrP in RCC metastases, independent of VHL status, and also whether anti-PTHrP treatments might be effective against metastatic spread in RCC.

Together with our recent studies obtained in VHL-deficient RCC tumors expressing high levels of PTHrP (17), the data presented herein constitute a starting point for clinical evaluation of anti-PTHrP strategies against human RCC.


   Acknowledgments
 
This work was supported by grants from INSERM, University Louis Pasteur, the French Ligue Contre le Cancer (Comités du Bas-Rhin et du Haut-Rhin et Comité National, recipient T.M.) and ARC (Association pour la Recherche sur le Cancer, recipient T.M.). We thank Dr P.Anglard (Institut of Genetic and Molecular and Cellular Biology, Illkirch-Graffenstaden, France) for giving us the UOK-126 and UOK-128 cell lines. We thank INSERM U381 (Dr M.Kedinger, Strasbourg) for housing nude mice. We thank Ms F.Thirion and F.Reymann for technical assistance in immunohistochemistry studies. We thank Prof. A.F.Stewart (Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, PA) for the critical reading of the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Vogelzang,N.J. and Stadler,W.M. (1998) Kidney cancer. Lancet, 352, 1691–1696.[CrossRef][Web of Science][Medline]
  2. Pantuck,A.J., Zisman,A. and Belldegrun,A.S. (2001) The changing natural history of renal cell carcinoma. J. Urol., 166, 1611–1623.[CrossRef][Web of Science][Medline]
  3. Motzer,R.J. and Russo,P. (2000) Systemic therapy for renal cell carcinoma. J. Urol., 163, 408–417.[CrossRef][Web of Science][Medline]
  4. Curti,B.D. (2004) Renal cell carcinoma. JAMA, 292, 97–100.[Free Full Text]
  5. Latif,F., Tory,K., Gnarra,J. et al. (1993) Identification of the von Hippel–Lindau disease tumor suppressor gene. Science, 260, 1317–1320.[Abstract/Free Full Text]
  6. Friedrich,C.A. (1999) Von Hippel-Lindau syndrome. A pleomorphic condition. Cancer, 86, 2478–2482.[CrossRef][Web of Science][Medline]
  7. Gnarra,J.R., Tory,K., Weng,Y. et al. (1994) Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat. Genet., 7, 85–90.[CrossRef][Web of Science][Medline]
  8. Herman,J.G., Latif,F., Weng,Y. et al. (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl Acad. Sci. USA, 91, 9700–9704.[Abstract/Free Full Text]
  9. Brauch,H., Weirich,G., Brieger,J. et al. (2000) VHL alterations in human clear cell renal cell carcinoma: association with advanced tumor stage and a novel hot spot mutation. Cancer Res., 60, 1942–1948.[Abstract/Free Full Text]
  10. Lubensky,I.A., Gnarra,J.R., Bertheau,P., Walther,M.M., Linehan,W.M. and Zhuang,Z. (1996) Allelic deletions of the VHL gene detected in multiple microscopic clear cell renal lesions in von Hippel–Lindau disease patients. Am. J. Pathol., 149, 2089–2094.[Abstract]
  11. Iliopoulos,O., Kibel,A., Gray,S. and Kaelin,W.G. (1995) Tumor suppression by the human Von Hippel-Lindau gene product. Nat. Med., 1, 822–826.[CrossRef][Web of Science][Medline]
  12. Kim,W. and Kaelin,W.G. Jr. (2003) The von Hippel–Lindau tumor suppressor protein: new insights into oxygen sensing and cancer. Curr. Opin. Genet. Dev., 13, 55–60.[CrossRef][Web of Science][Medline]
  13. Wiesener,M.S., Münchenhagen,P.M., Berger,I. et al. (2001) Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1 in clear cell renal carcinomas. Cancer Res., 61, 5215–5222.[Abstract/Free Full Text]
  14. Philbrick,W.M., Wysolmerski,J.J., Galbraith,S., Holt,E., Orloff,J.J., Yang,K.H., Vasavada,R.C., Weir,E.C., Broadus,A.E. and Stewart,A.F. (1996) Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol. Rev., 76, 127–173.[Abstract/Free Full Text]
  15. Martin,T.J., Moseley,J.M. and Williams,E.D. (1997) Parathyroid hormone-related protein: hormone and cytokine. J. Endocrinol., 154, 23–37.
  16. Burton,P.B., Moniz,C. and Knight,D.E. (1990) Parathyroid hormone related peptide can function as an autocrine growth factor in human renal cell carcinoma. Biochem. Biophys. Res. Commun., 167, 1134–1138.[CrossRef][Web of Science][Medline]
  17. Massfelder,T., Lang,H., Schordan,E., Lindner,V., Rothhut,S., Welsch,S., Simon-Assmann,P., Barthelmebs,M., Jacqmin,D. and Helwig,J.J. (2004) Parathyroid hormone-related protein is an essential growth factor for human clear cell renal carcinoma and a target for the von Hippel–Lindau tumor suppressor gene. Cancer Res., 64, 180–188.[Abstract/Free Full Text]
  18. Anglard,P., Trahan,E., Liu,S., Latif,F., Merino,M.J., Lerman,M.I., Zbar,B. and Linehan,W.M. (1992) Molecular and cellular characterization of human renal cell carcinoma cell lines. Cancer Res., 52, 348–356.[Abstract/Free Full Text]
  19. Vistica,D.T., Skehan,P., Scudiero,D., Monks,A., Pittman,A. and Boyd,M.R. (1991) Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res., 51, 2515–2520.[Abstract/Free Full Text]
  20. Nutt,R.F., Caulfield,M.P., Levy,J.J., Gibbons,S.W., Rosenblatt,M. and McKee,R.L. (1990) Removal of partial agonism from parathyroid hormone (PTH)-related protein- (7-34)NH2 by substitution of PTH amino acids at positions 10 and 11. Endocrinology, 127, 491–493.[Abstract/Free Full Text]
  21. Luparello,C., Romanotto,R., Tipa,A., Sirchia,R., Olmo,N., Lopez de Silanes,I., Turnay,J., Lizarbe,A. and Stewart,A.F. (2001) Midregion parthyroid hormone-related protein inhibits growth and invasion in vitro and tumorigenesis in vivo of human breast cancer cells. J. Bone Miner. Res., 16, 2173–2181.[CrossRef][Web of Science][Medline]
  22. Dougherty,K.M., Blomme,E.A., Koh,A.J., Henderson,J.E., Pienta,K.J., Rosol,T.J. and McCauley,L.K. (1999) Parathyroid hormone-related protein as a growth regulator of prostate carcinoma. Cancer Res., 59, 6015–6022.[Abstract/Free Full Text]
  23. Hastings,R.H., Burton,D.W., Quintana,R.A., Biederman,E., Gujral,A. and Deftos,L.J. (2001) Parathyroid hormone-related protein regulates the growth of orthotopic human lung tumors in athymic mice. Cancer, 92, 1402–1410.[CrossRef][Web of Science][Medline]
  24. Gotoh,A., Kitazawa,S., Mizuno,Y., Takenaka,A., Arakawa,S., Matsumoto,O., Kitazawa,R., Fujimori,T., Maeda,S. and Kamidono,S. (1993) Common expression of parathyroid hormone-related protein and no correlation of calcium level in renal cell carcinomas. Cancer, 71, 2803–2806.[CrossRef][Web of Science][Medline]
  25. Iwamura,M., Wu,W., Muramoto,M., Ohori,M., Egawa,S., Uchida,T. and Baba,S. (1999) Parathyroid hormone-related protein is an independent prognostic factor for renal cell carcinoma. Cancer, 86, 1028–1034.[CrossRef][Web of Science][Medline]
  26. Cho,Y.M., Lewis,D.A., Koltz,P.F., Richard,V., Gocken,T.A., Rosol,T.J., Konger,R.L., Spandau,D.F. and Foley,J. (2004) Regulation of parathyroid hormone-related protein gene expression by epidermal growth factor-family ligands in primary human keratinocytes. J. Endocrinol., 181, 179–190.[Abstract]
  27. Burton,P.B. and Knight,D.E. (1992) Parathyroid hormone-related peptide can regulate the growth of human lung cancer cells, and may form part of an autocrine TGF-alpha loop. FEBS Lett., 305, 228–232.[CrossRef][Web of Science][Medline]
  28. Sellers,R.S., LeRoy,B.E., Blomme,E.A., Tannehill-Gregg,S., Corn,S. and Rosol,T.J. (2004) Effects of transforming growth factor-beta1 on parathyroid hormone-related protein mRNA expression and protein secretion in canine prostate epithelial, stromal, and carcinoma cells. Prostate, 58, 366–373.[CrossRef][Web of Science][Medline]
  29. Akino,K., Ohtsuru,A., Kanda,K., Yasuda,A., Yamamoto,T., Akino,Y., Naito,S., Kurokawa,M., Iwahori,N. and Yamashita,S. (2000) Parathyroid hormone-related peptide is a potent angiogenic factor. Endocrinology, 141, 4313–4316.[Abstract/Free Full Text]
  30. Esbrit,P., Alvarez-Arroyo,M.V., De Miguel,F., Martin,O., Martinez,M.E. and Caramelo,C. (2000) C-terminal parathyroid hormone-related protein increases vascular endothelial growth factor in human osteoblastic cells. J. Am. Soc. Nephrol., 11, 1085–1092.[Abstract/Free Full Text]
  31. Bakre,M.M., Zhu,Y., Yin,H., Burton,D.W., Terkeltaub,R., Deftos,L.J. and Varner,J.A. (2002) Parathyroid hormone-related peptide is a naturally occurring, protein kinase A-dependent angiogenesis inhibitor. Nat. Med., 8, 995–1003.[CrossRef][Web of Science][Medline]
  32. Gunaratnam,L., Morley,M., Franovic,A., de Paulsen,N., Mekhail,K., Parolin,D.A., Nakamura,E., Lorimer,I.A. and Lee,S. (2003) Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL (–/–) renal cell carcinoma cells. J. Biol. Chem., 278, 44966–44974.[Abstract/Free Full Text]
  33. Sosman,J.A. (2003) Targeting of the VHL-hypoxia-inducible factor-hypoxia-induced gene pathway for renal cell carcinoma therapy. J. Am. Soc. Nephrol., 14, 2695–2702.[Abstract/Free Full Text]
  34. Levy,A.P., Levy,N.S. and Goldberg,M.A. (1996) Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the Von Hippel–Lindau protein. J. Biol. Chem., 271, 25492–25497.[Abstract/Free Full Text]
  35. Knebelmann,B., Ananth,S., Cohen,H.T. and Sukhatme,V.P. (1998) Transforming growth factor alpha is a target for the Von Hippel–Lindau tumor suppressor. Cancer Res., 58, 226–230.[Abstract/Free Full Text]
  36. Ananth,S., Knebelmann,B., Grüning,W., Dhanabal,M., Walz,G., Stillman,I.E. and Sukhatme,V.P. (1999) Transforming growth factor beta1 is a target for the Von Hippel–Lindau tumor suppressor and a critical growth factor for clear cell renal carcinoma. Cancer Res., 59, 2210–2216.[Abstract/Free Full Text]
  37. Zambrano,N.R., Lubensky,I.A., Merino,M.J., Linehan,W.M. and Walther,M.M. (1999) Histopathology and molecular genetics of renal tumors toward unification of a classification system. J. Urol., 162, 1246–1258.[CrossRef][Web of Science][Medline]
  38. Gurova,K.V., Hill,J.E., Razorenova,O.V., Chumakov,P.M. and Gudkov,A.V. (2004) p53 pathway in renal cell carcinoma is repressed by a dominant mechanism. Cancer Res., 64, 1951–1958.[Abstract/Free Full Text]
  39. Galbraith,S.C., Foley,J., Kats,Y., Moy,A., Dann,P., Burtis,W.J., Philbrick,W.M. and Orloff,J.J. (1996) Clonal variation of p53 expression and proliferative phenotype in A253 squamous carcinoma cells. Oncol. Res., 8, 353–361.[Web of Science][Medline]
  40. Foley,J., Wysolmerski,J.J., Broadus,A.E. and Philbrick,W.M. (1996) Parathyroid hormone-related protein gene expression in human squamous carcinoma cells is repressed by mutant isoforms of p53. Cancer Res., 56, 4056–4062.[Abstract/Free Full Text]
  41. Holt,E.H., Vasavada,R.C., Bander,N.H., Broadus,A.E. and Philbrick,W.M. (1993) Region-specific methylation of the parathyroid hormone-related peptide gene determines its expression in human renal carcinoma cell lines. J. Biol. Chem., 268, 20639–20645.[Abstract/Free Full Text]
  42. Soifer,N.E., Van Why,S.K., Ganz,M.B., Kashgarian,M., Siegel,N.J. and Stewart,A.F. (1993) Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia. J. Clin. Invest., 92, 2850–2857.[Web of Science][Medline]
  43. Massfelder,T., Stewart,A.F., Endlich,K., Soifer,N., Judes,C. and Helwig,J.J. (1996) Parathyroid hormone-related protein detection and interaction with NO and cyclic AMP in the renovascular system. Kidney Int., 50, 1591–1603.[Web of Science][Medline]
  44. Largo,R., Gomez-Garre,D., Santos,S., Penaranda,C., Blanco,J., Esbrit,P. and Egido,J. (1999) Renal expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in a rat model of tubulointerstitial damage. Kidney Int., 55, 82–90.[CrossRef][Web of Science][Medline]
  45. Santos,S., Bosch,R.J., Ortega,A., Largo,R., Fernandez-Agullo,T., Gazapo,R., Egido,J. and Esbrit,P. (2001) Up-regulation of parathyroid hormone-related protein in folic acid-induced acute renal failure. Kidney Int., 60, 982–995.[CrossRef][Web of Science][Medline]
  46. Garcia-Ocana,A., De Miguel,F., Penaranda,C., Albar,J.P., Sarasa,J.L. and Esbrit,P. (1995) Parathyroid hormone-related protein is an autocrine modulator of rabbit proximal tubule cell growth. J. Bone Miner. Res., 10, 1875–1884.[Web of Science][Medline]
  47. Garcia-Ocana,A., Galbraith,S.C., Van Why,S.K., Yang,K., Golovyan,L., Dann,P., Zager,R.A., Stewart,A.F., Siegel,N.J. and Orloff,J.J. (1999) Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion. J. Am. Soc. Nephrol., 10, 238–244.[Abstract/Free Full Text]
  48. Garcia-Ocana,A., Gomez-Casero,E., Penaranda,C. and Esbrit,P. (1998) Parathyroid hormone-related protein increases DNA synthesis in proximal tubule cells by cyclic AMP- and protein kinase C-dependent pathways. Life Sci., 62, 2267–2273.[CrossRef][Web of Science][Medline]
  49. Esbrit,P., Santos,S., Ortega,A., Fernandez-Agullo,T., Velez,E., Troya,S., Garrido,P., Pena,A., Bover,J. and Bosch,R.J. (2001) Parathyroid hormone-related protein as a renal regulating factor. From vessels to glomeruli and tubular epithelium. Am. J. Nephrol., 21, 179–184.[CrossRef][Web of Science][Medline]
Received March 30, 2005; revised July 6, 2005; accepted July 28, 2005.


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