Title: Expression of Pax2 in Human Renal Tumor-Derived Endothelial Cells Sustains Apoptosis Resistance and Angiogenesis
Abstract: The transcription factor Pax2 is known to play a key role during renal development and to act as an oncogene favoring renal tumor growth. We recently showed that endothelial cells derived from human renal carcinomas display abnormal characteristics of survival and angiogenic properties. In the present study we found that renal tumor-derived endothelial cells, but not normal endothelial cells, expressed Pax2 protein and mRNA. To down-regulate Pax2 expression, we transfected tumor-derived endothelial cells with an anti-sense PAX2 vector whereas we transfected normal human microvascular endothelial cells with a sense PAX2 vector to induce Pax2 expression. The inhibition of Pax2 expression in tumor-derived endothelial cells induced an increase in tumor suppressor PTEN expression and a decrease in Akt phosphorylation. In addition, decreased apoptosis resistance, adhesion, invasion, and in vitro and in vivo angiogenesis were observed. Con-versely, Pax2 induction in normal endothelial cells conferred to these cells a proinvasive, proangiogenic phenotype similar to that of tumor-derived endothelial cells. These results indicate that Pax2 is involved in renal tumor angiogenesis and its expression may antagonize that of the PTEN tumor suppressor gene, affecting the Akt-survival pathway and promoting angiogenesis. The transcription factor Pax2 is known to play a key role during renal development and to act as an oncogene favoring renal tumor growth. We recently showed that endothelial cells derived from human renal carcinomas display abnormal characteristics of survival and angiogenic properties. In the present study we found that renal tumor-derived endothelial cells, but not normal endothelial cells, expressed Pax2 protein and mRNA. To down-regulate Pax2 expression, we transfected tumor-derived endothelial cells with an anti-sense PAX2 vector whereas we transfected normal human microvascular endothelial cells with a sense PAX2 vector to induce Pax2 expression. The inhibition of Pax2 expression in tumor-derived endothelial cells induced an increase in tumor suppressor PTEN expression and a decrease in Akt phosphorylation. In addition, decreased apoptosis resistance, adhesion, invasion, and in vitro and in vivo angiogenesis were observed. Con-versely, Pax2 induction in normal endothelial cells conferred to these cells a proinvasive, proangiogenic phenotype similar to that of tumor-derived endothelial cells. These results indicate that Pax2 is involved in renal tumor angiogenesis and its expression may antagonize that of the PTEN tumor suppressor gene, affecting the Akt-survival pathway and promoting angiogenesis. The paired-box 2 (PAX2) gene belongs to a family of nine homeobox genes1Dahal E Koseki H Balling R Pax genes and organogenesis.BioEssays. 1997; 19: 755-765Crossref PubMed Scopus (311) Google Scholar, 2Chi N Epstein JA Getting your Pax straight: Pax proteins in development and disease.Trends Genet. 2002; 18: 41-47Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar that bind to DNA to initiate transcription of specific genes. In the embryo, Pax2 expression is restricted to the developing kidney, central nervous system, eyes, and ears.3Dressler GR Deutsch U Chowdhury K Nornes HO Gruss P Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system.Development. 1990; 109: 787-795PubMed Google Scholar, 4Nornes HO Dressler GR Knapik EW Deutsch U Gruss P Spatially and temporally restricted expression of Pax2 during murine neurogenesis.Development. 1990; 109: 797-809PubMed Google Scholar In developing kidney, Pax2 is detectable in the caudal mesonephric duct, ureteric bud, and in the mesenchymal condensates induced in the ureteric bud.2Chi N Epstein JA Getting your Pax straight: Pax proteins in development and disease.Trends Genet. 2002; 18: 41-47Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar Pax2 is down-regulated when the mesenchymal cells differentiate into tubular epithelial cells, and it is no longer expressed in the mature kidney.5Eccles MR Wallis LJ Fidler AE Spurr NK Goodfellow PJ Reeve AE Expression of PAX2 gene in human foetal kidney and Wilms' tumor.Cell Growth Differ. 1992; 3: 279-289PubMed Google Scholar, 6Torres M Gomez-Pardo E Dressler GR Gruss P Pax-2 controls multiple steps of urogenital development.Development. 1995; 121: 4057-4065Crossref PubMed Google Scholar, 7Rohenpieler UW Dressler GR Pax2 is required for mesenchyme-to-epithelium conversion during kidney development.Development. 1993; 119: 711-720PubMed Google Scholar The relevance of Pax2 in the development of kidney is suggested by the severe kidney abnormalities, similar to congenital nephritic syndrome, observed in transgenic mice in which Pax2 expression was deregulated.8Dressler GR Wilkinson JE Rothenpieler UW Petterson LT William-Simons L Westphal H Deregulation of Pax2 expression in transgenic mice generates severe kidney abnormalities.Nature. 1993; 362: 65-67Crossref PubMed Scopus (280) Google Scholar Failure to repress Pax2 was found in hyperproliferative cystic tissue in a mouse model of polycystic kidney disease.9Ostrom L Tang MJ Gruss P Dressler GR Reduced Pax2 gene dosage increases apoptosis and slows the progression of renal cystic disease.Dev Biol. 2000; 219: 250-258Crossref PubMed Scopus (98) Google Scholar In humans, Pax2 overexpression was associated with fetal and infantile multicystic dysplastic kidneys and autosomal dominant polycystic kidney disease.10Winyard PJD Risdon RA Sams VR Dressler GR Woolf AS The PAX2 transcription factor is expressed in cystic and hyperproliferative dysplastic epithelia in human kidney malformations.J Clin Invest. 1996; 98: 451-459Crossref PubMed Scopus (167) Google Scholar Moreover, Pax2 is up-regulated in adults during tubular epithelial cell regeneration after renal injury11Maeshima A Maeshima K Nojima Y Kojima I Involvement of Pax2 in the action of activin A on tubular cell regeneration.J Am Soc Nephrol. 2002; 13: 2850-2859Crossref PubMed Scopus (75) Google Scholar, 12Imgrund M Grone E Grone HJ Kretzler M Holzman L Schlondorff D Rothenpieler UW Re-expression of the developmental gene Pax2 during experimental acute tubular necrosis in mice.Kidney Int. 1999; 56: 1423-1431Crossref PubMed Scopus (171) Google Scholar when the tubular epithelial cells dedifferentiate to an embryonic phenotype.13Woolf AS Winyard PJD Advances in the cell biology and genetics of human kidney malformations.J Am Soc Nephrol. 1998; 9: 1114-1125PubMed Google Scholar Functional studies suggest that Pax2 is involved in cell survival and proliferation.14Zhang SL Guo J Moini B Ingelfinger JR Angiotensin II stimulates Pax2 in rat kidney proximal tubular cells: impact on proliferation and apoptosis.Kidney Int. 2004; 66: 2181-2192Crossref PubMed Scopus (36) Google Scholar In renal collecting duct cells, Pax2 was found to suppress caspase-2-induced apoptosis and to stimulate cell proliferation.15Torban E Eccles MR Favor J Goodyer PR PAX2 suppresses apoptosis in renal collecting duct cells.Am J Pathol. 2000; 157: 833-842Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Moreover, a pro-oncogenic potential has been ascribed to the PAX gene family16Dressler GR Pax2, kidney development and oncogenesis.Med Pediatr Oncol. 1996; 27: 440-444Crossref PubMed Scopus (47) Google Scholar, 17Sanyanusin P Schimmenti LA McNoe LA Ward TA Pierpont ME Sullivan MJ Dobyns WB Eccles MR Mutations of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vescicoureteral reflux.Nat Genet. 1995; 9: 358-364Crossref PubMed Scopus (581) Google Scholar; in fact, several chromosomal translocations involving members of the PAX gene family have been described in various human cancers, suggesting that altered regulation of PAX gene products can promote cellular transformation.18Barr FG Chromosomal translocations involving paired box transcription factors in human cancer.Int J Biochem Cell Biol. 1997; 29: 1449-1461Crossref PubMed Scopus (35) Google Scholar, 19Dressler GR Douglass EC Pax2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor.Proc Natl Acad Sci USA. 1992; 89: 1179-1183Crossref PubMed Scopus (368) Google Scholar, 20Muratovska A Zhou C He S Goodyer P Eccles MR Paired-box genes are frequently expressed in cancer and often required for cancer cell survival.Oncogene. 2003; 22: 7989-7997Crossref PubMed Scopus (208) Google Scholar In particular the inappropriate expression of Pax2 has been identified in Wilms tumor,19Dressler GR Douglass EC Pax2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor.Proc Natl Acad Sci USA. 1992; 89: 1179-1183Crossref PubMed Scopus (368) Google Scholar renal cell carcinoma,21Gnarra JR Dressler GR Expression of Pax2 in human renal cell carcinomas and growth inhibition by antisense oligonucleotides.Cancer Res. 1995; 55: 4092-4098PubMed Google Scholar breast cancer,22Silberstein GB Dressler GR Van Horn K Expression of the PAX2 oncogene in human breast cancer and its role in progesterone-dependent mammary growth.Oncogene. 2002; 21: 1009-1016Crossref PubMed Scopus (62) Google Scholar and prostate cancer.23Khoubehi B Kessling AM Adshead JM Smith GL Smith RD Ogden CW Expression of the developmental and oncogenic PAX2 gene in human prostate cancer.J Urol. 2001; 165: 2115-2120Abstract Full Text Full Text PDF PubMed Google Scholar We recently detected Pax2 expression in an angiogenic tumor, Kaposi's sarcoma. In addition we demonstrated that the expression of Pax2 protein by Kaposi's sarcoma cells correlated with an enhanced resistance against apoptotic signals and with a proinvasive phenotype.24Buttiglieri S Deregibus MC Bravo S Cassoni P Chiarle R Bussolati B Camussi G Role of Pax2 in apoptosis resistance and proinvasive phenotype of Kaposi's sarcoma cells.J Biol Chem. 2004; 279: 4136-4143Crossref PubMed Scopus (41) Google Scholar It has been recently shown that endothelial cells derived from different tumors are genetically unstable.25Hida K Hida Y Amin DN Flint AF Panigrahy D Morton CC Klagsbrun M Tumor-associated endothelial cells with cytogenetic abnormalities.Cancer Res. 2004; 64: 8249-8255Crossref PubMed Scopus (404) Google Scholar In addition, we showed that endothelial cells derived from human renal carcinomas persistently display abnormal characteristics in terms of survival and angiogenic properties.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar In the present study we evaluated whether renal tumor-derived endothelial cells (TECs) expressed the PAX2 gene product and whether Pax2 was involved in their proinvasive and proangiogenic phenotype. For this purpose, TECs, which were found to express Pax2, were transfected with an anti-sense PAX2 vector to inhibit the synthesis of Pax2 protein. In addition, normal human microvascular endothelial cells (HMECs), which were found not to express Pax2, were transfected with a sense PAX2 vector to induce the synthesis of Pax2. Using these cell lines, we studied the effect of Pax2 expression on Akt-dependent pathway, cell survival, proliferation, adhesion, invasion, and in vitro and in vivo angiogenic properties. TEC lines used in this study were previously described and characterized.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar, 27Grange C, Bussolati B, Bruno S, Fonsato V, Sapino A, Camussi G: Isolation and characterization of human breast tumor-derived endothelial cells. Oncol Rep (in press)Google Scholar Briefly, renal TECs were isolated from six different renal clear-cell carcinomas using anti-CD105 antibody coupled to magnetic beads, by magnetic cell sorting (MACS System; Miltenyi Biotec, Auburn, CA).26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar Moreover, breast TECs were isolated from two ductal and a tubulo-lobular infiltrating breast carcinomas.27Grange C, Bussolati B, Bruno S, Fonsato V, Sapino A, Camussi G: Isolation and characterization of human breast tumor-derived endothelial cells. Oncol Rep (in press)Google Scholar The expression of Pax2 in the tumors from which TECs were isolated was evaluated by immunohistochemistry using the anti-Pax2 antibody from Covance (Princeton, NJ) as previously described.24Buttiglieri S Deregibus MC Bravo S Cassoni P Chiarle R Bussolati B Camussi G Role of Pax2 in apoptosis resistance and proinvasive phenotype of Kaposi's sarcoma cells.J Biol Chem. 2004; 279: 4136-4143Crossref PubMed Scopus (41) Google Scholar The cell lines were characterized as endothelial cells by morphology, positive staining for von Willebrand factor antigen, CD105, CD146, and vascular endothelial-cadherin; and negative staining for cytokeratin and desmin.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar TEC lines were maintained in culture in endothelial basal medium (EBM) complete medium supplemented with epidermal growth factor (10 ng/ml), hydrocortisone (1 mg/ml), bovine brain extract (all from Cambrex Bioscience, Walkersville, MD), and 10% fetal calf serum (FCS). HMECs were obtained from derma or from normal renal tissue using anti-CD31 antibody coupled to magnetic beads, by magnetic cell sorting (MACS System, Miltenyi Biotec). HMECs from derma were immortalized by infection of primary cultures with a replication-defective adeno-5/SV40 virus as previously described.28Conaldi PG Serra C Mossa A Falcone V Basolo F Camussi G Dolei A Toniolo A Persistent infection of human vascular endothelial cells by group B coxsackieviruses.J Infect Dis. 1997; 175: 693-696Crossref PubMed Scopus (57) Google Scholar The previously characterized renal carcinoma cell line K1, obtained from a renal clear-cell carcinoma, was maintained in Dulbecco's modified Eagle's medium with 10% FCS.29Bussolati B Russo S Deambrosis I Cantaluppi V Volpe A Ferrando U Camussi G Expression of CD154 on renal cell carcinomas and effect on cell proliferation, motility and platelet-activating factor synthesis.Int J Cancer. 2002; 100: 654-661Crossref PubMed Scopus (45) Google Scholar Cloning of the PAX2 gene was performed as previously described.24Buttiglieri S Deregibus MC Bravo S Cassoni P Chiarle R Bussolati B Camussi G Role of Pax2 in apoptosis resistance and proinvasive phenotype of Kaposi's sarcoma cells.J Biol Chem. 2004; 279: 4136-4143Crossref PubMed Scopus (41) Google Scholar Briefly, 2 μg of RNA were reverse-transcribed using oligo(dT) primers and 15 U of reverse transcriptase enzyme (Eppendorf, Hamburg, Germany). Five μl of cDNA were amplified with forward and reverse primers (forward, 5′-ATGGATATGCACTGCAAAGCAGA-3; reverse, 5′-CTAGTGGCGGTCATAGGCAG-3′) covering the initial and terminal part of the coding sequence, respectively (NCBI GenBank no. GI409138), with TaqDNA polymerase (Invitrogen, San Diego, CA). Amplified DNA was ligated with the T/A cloning system into pTARGET mammalian expression vector (Promega, Madison, WI) for expression under the control of the cytomegalovirus promoter. We identified a clone with PAX2 in correct orientation (S) and a clone with PAX2 in inverse orientation (AS). DNA sequences were compared with the NCBI database using the BLAST program. HMECs or TECs were seeded in a T25 flask to reach confluence and transfected with 8 μg of DNA and 20 μl of LipofectAMINE 2000 reagent (Invitrogen) in Dulbecco's modified Eagle's medium plus 10% FCS without antibiotics, according to the protocol suggested by the manufacturer. As a control, cells were transfected with empty plasmid. Transfected cells were stably selected by culturing in the presence of 1 mg/ml geneticin (Sigma Chemical Co., St. Louis, MO). Successful transfection was evaluated by immunofluorescence and Western blotting. TECs and HMECs were stably transfected up to passage 10. Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions; 2 μg of RNA were reverse-transcribed using oligo(dT) primers and 15 U of reverse transcriptase enzyme (Eppendorf). Two μl of cDNA were amplified with forward (5′-ATGGATATGCACTGCAAAGCAGA-3′) and reverse (5′-CTAGTGGCGGTCATAGGCAG-3′) primers and TaqDNA polymerase (Invitrogen). Reactions were performed for 30 cycles at a melting temperature of 52°C and analyzed with an ethidium bromide 1.5% agarose gel. Western Blot analysis was performed for detection of Akt, P-Akt, PTEN, and Pax2, as previously described.24Buttiglieri S Deregibus MC Bravo S Cassoni P Chiarle R Bussolati B Camussi G Role of Pax2 in apoptosis resistance and proinvasive phenotype of Kaposi's sarcoma cells.J Biol Chem. 2004; 279: 4136-4143Crossref PubMed Scopus (41) Google Scholar, 30Deregibus MC Cantaluppi V Doublier S Brizzi MF Deambrosis I Albini A Camussi G HIV-1-Tat protein activates phosphatidylinositol 3-kinase/AKT-dependent survival pathways in Kaposi's sarcoma cells.J Biol Chem. 2002; 77: 25195-25202Crossref Scopus (91) Google Scholar Cells were lysed at 4°C for 1 hour in a lysis buffer (50 mmol/L Tris-HCl, pH 8.3, 1% Triton X-100, 10 μmol/L phenylmethyl sulfonyl fluoride, 10 μmol/L leupeptin, and 100 U/ml aprotinin) and centrifuged at 15,000 × g. The protein contents of the supernatants were measured by the Bradford method. Aliquots containing 10 μg of protein of the cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and electroblotted onto nitrocellulose membrane filters. The blots were blocked with 5% nonfat milk in 20 mmol/L Tris-HCl, pH 7.5, 500 mmol/L NaCl plus 0.1% Tween (TBS-T). The membranes were subsequently immunoblotted overnight at 4°C with the relevant primary antibodies at the appropriate concentration. After extensive washings with TBS-T, the blots were incubated for 1 hour at room temperature with peroxidase-conjugated isotype-specific secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), washed with TBS-T, developed with ECL detection reagents (Amersham, Buckinghamshire, UK) for 1 minute, and exposed to X-Omat film (Eastman Kodak Co., Rochester, NY). The following antibodies were used: anti-Pax2 polyclonal antibody (Covance), anti-human Akt1 and phospho-Akt1 (P-Akt) from Upstate Biotechnology (Lake Placid, NY), anti-PTEN antibody from Cell Signaling Technology (Beverly, MA), and anti-β-actin polyclonal antibody from Santa Cruz Biotechnology. Apoptosis was evaluated using the terminal dUTP nick-end labeling assay (ApoTag; Oncor, Gaithersburg, MD). After serum withdrawal and/or treatment with 1 to 100 ng/ml of vincristine for 24 hours, cells were suspended in phosphate-buffered saline (PBS) and fixed in 1% paraformaldehyde in PBS, pH 7.4, for 15 minutes at 4°C followed by precooled ethanol/acetic acid (2:1) for 5 minutes at −20°C. Cells were treated with terminal deoxynucleotide transferase enzyme and incubated in a humidified chamber for 1 hour at 37°C and then treated with warmed fluorescein isothiocyanate-conjugated anti-digoxigenin for 30 minutes at room temperature. After washing, samples were mounted in medium containing 1 μg/ml of propidium iodide and the cells were analyzed by immunofluorescence. Cells were seeded into 96-well plates at a density of 8000 cells/well in EBM plus 10% FCS, and the proliferation was evaluated after 72 hours. DNA synthesis was detected as incorporation of 5-bromo-2′-deoxyuridine (BrdU) into the cellular DNA using an enzyme-linked immunosorbent assay kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. Briefly, after washing, cells were incubated with 10 μmol/L BrdU for 6 to 12 hours at 37°C, 5% CO2, in a humidified atmosphere. Cells were then fixed with 0.5 mol/L ethanol/HCl and incubated with nuclease to digest DNA. BrdU incorporated into the DNA was detected using an anti-BrdU peroxidase-conjugated monoclonal antibody and visualized with a soluble chromogenic substrate. Optical density was measured with an enzyme-linked immunosorbent assay reader at 405 nm. Cells were cultured to confluence in gelatin-coated 24-well tissue culture plates. Before the assay, the monolayers were starved in EBM plus 0.1% bovine serum albumin. Renal carcinoma cells (K1) were detached with nonenzymatic solution (Sigma), labeled with the intravital red fluorescent cell linker PKH26 (Sigma) and added to the endothelial monolayers at the density of 5 × 105 cells. The adhesion assay was allowed to proceed for 30 minutes at 37°C, 5% CO2, in a humidified atmosphere in static conditions. The plates were then washed three times with PBS to remove the unattached cells. Each experiment was done in triplicate. Labeled cells attached to the endothelial monolayer were counted in five fields (magnification, ×100) by fluorescence microscopy and expressed as number cells/microscopic field. The invasion ability was evaluated using Transwell chambers (Costar, Cambridge, MA), in which the upper and the lower chambers were separated by 8-μm pore-size polyvinylpyrrolidone-free polycarbonate filters. Briefly, before the invasion assay, filters were coated with 100 μg/well of Matrigel (Becton Dickinson, Bedford, MA) diluted in culture medium. The lower compartment was loaded with medium plus 10% FCS. HMEC-c, HMEC-S, TEC-c, and TEC-AS cells (5 × 104 cells/well) were seeded onto the upper compartment and were incubated for 48 hours at 37°C, 5% CO2, in a humidified atmosphere. Cells migrated to the underside of the filters were fixed with methanol, stained with Giemsa solution (Diff-Quik kit; Harleco, Gibbstown, NJ), counted in five fields (magnification, ×100) in each well by light microscopy, and expressed as number of cells/microscopic field. Each experiment was done in triplicate. The assay was performed as previously described.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar Briefly, 24-well plates were coated with growth factor-reduced Matrigel (Becton Dickinson) at 4°C and incubated for 30 minutes at 37°C, 5% CO2, in a humidified atmosphere. Cells (HMEC-c, HMEC-S, TEC-c, and TEC-AS) were seeded on the Matrigel-coated wells in RPMI plus 5% FCS at the density of 5 × 104 cells/well. After a 6-hour or 24-hour incubation, cells were observed with a Nikon inverted microscope (Nikon Corporation, Tokyo, Japan), and the experimental results were recorded. The extent of capillary-like structures was measured with the MicroImage analysis system (Cast Imaging srl, Venice, Italy) and expressed in arbitrary units. For the in vivo studies, an in vivo Matrigel angiogenesis assay was performed, in which cells were subcutaneously injected into SCID mice (Charles River, Jackson Laboratories, Bar Harbor, ME; n = 5 for each experimental condition). Briefly, cells were harvested using trypsin solution (Sigma), washed with PBS, resuspended in 250 μl of Dulbecco's modified Eagle's medium, and added to 250 μl of growth factor-reduced Matrigel at 4°C. Cells were injected subcutaneously into the mid-abdominal region of SCID mice via a 26-gauge needle and a 1-ml syringe. At day 6, mice were killed, and the plugs were recovered and processed for histology. Typically, gels were cut out by retaining peritoneal lining for support, fixed in 10% buffered formalin, and embedded in paraffin. Sections (3 μm) were cut, stained with hematoxylin and eosin or with a Masson trichrome reaction, and examined under a light microscopy system. Morphometric analysis was performed to count vessels that were expressed as percent area per field. Vessel structures were counted only if showing a patented lumen with red globuli and/or leukocytes. The vessels area was planimetrically assessed using the MicroImage analysis system (Casti Imaging srl). The human nature of endothelial cells was assessed by immunofluorescence with an anti-HLA class I polyclonal antibody (Santa Cruz Biotechnology). The expression of Pax2 was evaluated in different lines of TECs derived from six renal and three breast carcinomas. All TEC lines derived from renal carcinomas expressed Pax2 as demonstrated by both Western blot (Figure 1A) and reverse transcriptase-polymerase chain reaction (data not shown). In contrast, TECs derived from breast carcinomas did not express Pax2 (Figure 1A). This correlated with Pax2 expression in the renal carcinomas but not in the breast carcinomas used for the extraction of TECs, as detected by immunohistochemistry (Figure 1, B and C). Normal HMECs obtained from derma or kidney did not express Pax2 (Figure 1A). To evaluate the relevance of Pax2 expression by renal TECs, a renal TEC line was stably transfected with a PAX2 anti-sense vector (TEC-AS), to obtain the inhibition of Pax2 expression, or with a control vector (TEC-c). Moreover, HMECs were stably transfected with a PAX2 sense vector (HMEC-S), to obtain the expression of Pax2, or with a control vector (HMEC-c). TEC-AS no longer expressed Pax2 protein or mRNA (Figure 1, E, J, and K) in respect to control TEC-c transfected with the empty vector (Figure 1, D, J, and K). HMEC-S transfected with PAX2 sense vector expressed both Pax2 protein and Pax2 mRNA (Figure 1, G, J, and K) at variance with HMEC-c transfected with empty vector (Figure 1, F, J, and K). We previously demonstrated in TECs a decreased expression of PTEN, a tumor suppressor gene that regulates Akt phosphorylation.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar To test whether Pax2 expression influences PTEN and Akt-dependent pathway, we compared the PTEN levels and Akt phosphorylation, after serum starvation, in TECs and HMECs expressing or not Pax2. The results indicate that the expression of Pax2, as it occurred in TEC-c, correlated with a decreased expression of PTEN and an increased phosphorylation of Akt (Figure 2). When the expression of Pax2 was down-regulated by anti-sense transfection, the levels of PTEN were increased and the ratio P-Akt/Akt markedly reduced. Conversely, transfection of HMECs with PAX2 sense markedly depressed PTEN expression and enhanced P-Akt/Akt ratio in respect to HMEC-c (Figure 2). These results suggest a regulatory role of Pax2 on a tumor suppressor gene such as PTEN and on the Akt-dependent survival pathway. One of the most peculiar characteristics of normal endothelial cells is the occurrence of apoptosis in the absence of serum. To verify the effect of Pax2 expression on endothelial cell apoptosis, TEC-c, TEC-AS, HMEC-c, and HMEC-S were incubated for 24 hours in the absence of serum or with increasing doses of vincristine (1 to 100 ng/ml). As shown in Figure 3A, cells that expressed high levels of Pax2 protein (TEC-c and HMEC-S) were protected against apoptosis induced by serum deprivation or by vincristine. In contrast, TEC-AS and HMEC-c exhibited a higher sensitivity to apoptosis. In addition, the transfection with PAX2 anti-sense vector reduced the growth of TEC-AS in respect to TEC-c, whereas the transfection with PAX2 sense vector enhanced the growth of HMEC-S in respect to HMEC-c (Figure 3B). These results suggest that Pax2 promotes the resistance of tumor endothelial cells to apoptosis and stimulates cell proliferation. TECs exhibited enhanced adhesive properties to carcinoma cells in respect to normal endothelial cells and increased ability to invade Matrigel (Figure 4). The transfection with PAX2 anti-sense vector markedly reduced the adhesion of carcinoma cell line K1 on TEC-AS in respect to TEC-c (Figure 4A). Conversely, the expression of Pax2 induced a significant increase of K1 adhesion to HMEC-S in comparison with HMEC-c. Similar results were obtained in the invasion assay, in which loss of Pax2 protein expression in TEC-AS significantly inhibited the ability of TECs to invade Matrigel. In contrast, the acquirement of Pax2 expression by HMEC-S induced the ability to invade Matrigel in normal endothelial cells (Figure 4B). We compared TEC-AS and HMEC-S with TEC-c and HMEC-c for the ability to organize when plated on Matrigel in capillary-like structures. Within a few hours TECs formed an extensive network of ring-like structures (Figure 5, A and B) whereas TEC-AS markedly lost this ability (Figure 5, A and C). Moreover, HMEC-S significantly increased the ability of normal endothelial cells to organize in capillary-like structures (Figure 5, A, D, and E). To evaluate the involvement of Pax2 in in vivo angiogenesis, TEC-c and TEC-AS were injected subcutaneously within diluted Matrigel in SCID mice. After 6 days, plugs were recovered and processed for histological analysis. We previously demonstrated that TECs, at variance with normal endothelial cells, spontaneously organized in human microvessels containing blood cells.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar The loss of Pax2 in TEC-AS was associated with a significant reduction in this ability (Figure 6). These data suggest that Pax2 expression contributes to the angiogenic properties of TECs. In the present study, we demonstrated that human endothelial cells derived from renal carcinomas expressed Pax2 and that its expression contributed to the apoptosis resistance and the proangiogenic properties characteristic of these cells. Renal cell carcinoma is characterized by resistance to radiation and chemotherapy31Motzer RJ Bander N Nanus DM Renal-cell carcinoma.N Engl J Med. 1996; 335: 865-875Crossref PubMed Scopus (1578) Google Scholar and by an abundant angiogenesis with development of abnormal blood vessels.32Mancilla-Jimenez R Papillary renal cell carcinoma: a clinical, radiologic and pathologic study of 34 cases.Cancer. 1976; 38: 246-270Google Scholar, 33Dekel Y Koren R Kugel V Livne PM Gal R Significance of angiogenesis and microvascular invasion in renal cell carcinoma.Pathol Oncol Res. 2002; 8: 129-132Crossref PubMed Scopus (49) Google Scholar, 34Yoshino S Kato M Okada K Clinical significance of angiogenesis, proliferation and apoptosis in renal cell carcinoma.Anticancer Res. 2000; 20: 591-594PubMed Google Scholar We recently demonstrated that tumor endothelial cells derived from renal carcinomas differ from normal microvascular endothelial cells because they do not undergo senescence and maintain a constant activated phenotype with the ability to grow and organize in the absence of serum in capillary-like structures.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar In vivo, when implanted subcutaneously in SCID mice, these cells are able to form vascular structures connected with the mouse vasculature.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar These observations are consistent with the finding that TECs derived from different tumors are phenotypically and genetically different from normal endothelium25Hida K Hida Y Amin DN Flint AF Panigrahy D Morton CC Klagsbrun M Tumor-associated endothelial cells with cytogenetic abnormalities.Cancer Res. 2004; 64: 8249-8255Crossref PubMed Scopus (404) Google Scholar, 35St. Croix B Rago C Velculescu V Traverso G Romans KE Montgomary E Lal A Riggins GJ Lengauer C Vogelstein B Kinzler KW Genes expressed in human tumor endothelium.Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1646) Google Scholar and may be resistant to chemotherapeutic drugs.36Iwahana M Utoguchi N Mayumi T Goryo M Okada K Drug resistance and P-glycoprotein expression in endothelial cells of newly formed capillaries induced by tumors.Anticancer Res. 1998; 18: 2977-2980PubMed Google Scholar In the present study, we found that renal endothelial cells derived from renal carcinomas expressed Pax2, a transcription factor that has been involved in tumor growth.16Dressler GR Pax2, kidney development and oncogenesis.Med Pediatr Oncol. 1996; 27: 440-444Crossref PubMed Scopus (47) Google Scholar, 20Muratovska A Zhou C He S Goodyer P Eccles MR Paired-box genes are frequently expressed in cancer and often required for cancer cell survival.Oncogene. 2003; 22: 7989-7997Crossref PubMed Scopus (208) Google Scholar Indeed, it has been demonstrated that inhibition of Pax2 expression in renal carcinoma cells by anti-sense nucleotides inhibits tumor cell growth.21Gnarra JR Dressler GR Expression of Pax2 in human renal cell carcinomas and growth inhibition by antisense oligonucleotides.Cancer Res. 1995; 55: 4092-4098PubMed Google Scholar Our observation that the endothelial compartment in Pax2-positive renal carcinomas also expressed Pax2 may suggest that tumor endothelial cells in renal carcinoma derive from dedifferentiated tumor cells, as suggested for leukemia,37Gunsilius E Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells.Adv Exp Med Biol. 2003; 522: 17-24Crossref PubMed Scopus (33) Google Scholar and therefore express Pax2. Alternatively, TECs may represent a dedifferentiated endothelial population displaying an embryonic renal phenotype that results in Pax2 expression. Indeed we previously showed that TECs express the embryonic marker HLA-G, a nonclassical HLA antigen expressed on fetal and maternal placental endothelial cells.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar Our finding that TECs derived from the three Pax2-negative breast carcinomas did not express Pax2 suggests that the endothelial expression of this transcription factor is limited to tumors that overexpress Pax2. However, previous studies have suggested that PAX2 gene function is required only at certain stages of tumor cell development because it may be transiently expressed by several nonrenal tumors.20Muratovska A Zhou C He S Goodyer P Eccles MR Paired-box genes are frequently expressed in cancer and often required for cancer cell survival.Oncogene. 2003; 22: 7989-7997Crossref PubMed Scopus (208) Google Scholar Herein we investigated the functional implication of Pax2 expression by endothelial cells. We found that down-regulation of Pax2 expression by TECs transfected with PAX2 anti-sense resulted in an enhanced expression of the tumor suppressor gene PTEN, which is down-regulated in TECs in respect to normal endothelial cells. These results suggest that the expression of Pax2 may control the transcription of PTEN. In addition, it has been previously demonstrated that Pax2 possesses an oncogenic potential due to down-regulation of other tumor suppressor genes, such as p53.38Maulbeker CC Gruss P The oncogenic potential of Pax genes.EMBO J. 1993; 12: 2361-2367PubMed Google Scholar PTEN is a phosphatase that regulates the PI3K-dependent activation of Akt.39Stambolic V Suzuki A De La Pompa JL Brothers GM Mirtsos C Sasaki T Ruland J Penninger JM Siderovski DP Mak TW Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2107) Google Scholar Indeed, Akt phosphorylation was significantly enhanced in TECs and correlated with an increased resistance to apoptotic signals.26Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (237) Google Scholar In the present study we demonstrated that down-regulation of Pax2 expression correlated with a decreased Akt phosphorylation and an enhanced sensitivity to apoptosis induced by serum deprivation or by chemotherapeutic drugs such as vincristine. These results are consistent with a previously reported anti-apoptotic activity of PAX2 gene expression in tubular epithelial cells.15Torban E Eccles MR Favor J Goodyer PR PAX2 suppresses apoptosis in renal collecting duct cells.Am J Pathol. 2000; 157: 833-842Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Several studies have indicated that TECs are resistant to chemotherapeutic and anti-angiogenic drugs.36Iwahana M Utoguchi N Mayumi T Goryo M Okada K Drug resistance and P-glycoprotein expression in endothelial cells of newly formed capillaries induced by tumors.Anticancer Res. 1998; 18: 2977-2980PubMed Google Scholar, 40Jung YD Ahmad SA Akagi Y Takahashi Y Liu W Reinmuth N Shaheen RM Fan F Ellis LM Role of the tumor microenvironment in mediating response to anti-angiogenic therapy.Cancer Metastasis Rev. 2000; 19: 147-157Crossref PubMed Scopus (59) Google Scholar Because of the involvement of PI3K/Akt survival pathway in such resistance, the combined use of anti-angiogenic and anti-PI3K drugs has been suggested.41Chang F Lee JT Navolanic PM Steelman LS Shelton JG Blalock WL Franklin RA McCubrey JA Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy.Leukemia. 2003; 17: 590-603Crossref PubMed Scopus (978) Google Scholar, 42Vivanco I Sawyers CL The phosphatidylinositol 3-kinase-Akt pathway in human cancer.Nature Rev. 2002; 2: 489-501Google Scholar Our results may provide a rational for the described effects of activin A, which is known to down-regulate Pax2 expression11Maeshima A Maeshima K Nojima Y Kojima I Involvement of Pax2 in the action of activin A on tubular cell regeneration.J Am Soc Nephrol. 2002; 13: 2850-2859Crossref PubMed Scopus (75) Google Scholar and to repress tumor growth through inhibition of angiogenesis.43Breit S Ashman K Wilting J Rossler J Hatzi E Fotsis T Schweigerer: The N-myc oncogene in human neuroblastoma cells: down-regulation of an angiogenesis inhibitor identified as activin A.Cancer Res. 2000; 60: 4596-4601PubMed Google Scholar, 44Panopoulou E Murphy C Rasmussen H Bagli E Rofstad EK Fotsis T Activin A suppresses neuroblastoma xenograft tumor growth via antimitotic and antiangiogenic mechanisms.Cancer Res. 2005; 65: 1877-1886Crossref PubMed Scopus (61) Google Scholar In the present study we found that the expression of Pax2 not only correlated with an enhanced survival of endothelial cells but also with an enhanced in vitro and in vivo angiogenic activity. In fact, suppression of Pax2 production induced the loss of the proinvasive and proangiogenic phenotype in TECs. Conversely, the acquisition of Pax2 in normal endothelial cells induced a phenotype similar to that of TECs. In conclusion, these results indicate that Pax2 is involved in renal tumor angiogenesis because its expression may down-regulate PTEN, a tumor suppressor gene that modulates the Akt-survival pathway, and may control steps involved in angiogenesis, such as survival, proliferation, invasion, and cell organization. We thank Prof. Anna Sapino and Prof. Ugo Ferrando (University of Turin, Turin, Italy) for kindly providing tumor specimens.