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From the Dipartimento di Medicina Interna, Cattedra di Nefrologia, Università di Torino, and Centro Ricerca Medicina Sperimentale, Torino, Italy
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Abstract |
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It has been recently shown that endothelial cells derived from different tumors are genetically unstable.25 In addition, we showed that endothelial cells derived from human renal carcinomas persistently display abnormal characteristics in terms of survival and angiogenic properties.26 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.
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Materials and Methods |
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TEC lines used in this study were previously described and characterized.26,27 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).26 Moreover, breast TECs were isolated from two ductal and a tubulo-lobular infiltrating breast carcinomas.27 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.24 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.26 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.28 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.29
Cloning of PAX2 Gene
Cloning of the PAX2 gene was performed as previously described.24 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.
Transfection
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.
Reverse Transcriptase-Polymerase Chain Reaction
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
Western Blot analysis was performed for detection of Akt, P-Akt, PTEN, and Pax2, as previously described.24,30 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 x 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-ß-actin polyclonal antibody from Santa Cruz Biotechnology.
Apoptosis Assay
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.
Cell Proliferation Assay
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.
Adhesion Assay
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 x 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, x100) by fluorescence microscopy and expressed as number cells/microscopic field.
Matrigel Invasion Assay
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 x 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, x100) in each well by light microscopy, and expressed as number of cells/microscopic field. Each experiment was done in triplicate.
In Vitro Angiogenesis Assay
The assay was performed as previously described.26 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 x 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.
In Vivo Angiogenesis Assay
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).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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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)
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We previously demonstrated in TECs a decreased expression of PTEN, a tumor suppressor gene that regulates Akt phosphorylation.26
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.
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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.
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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)
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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)
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. These data suggest that Pax2 expression contributes to the angiogenic properties of TECs.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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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.16,20 Indeed, it has been demonstrated that inhibition of Pax2 expression in renal carcinoma cells by anti-sense nucleotides inhibits tumor cell growth.21 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,37 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.26 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.20
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 p53.38 PTEN is a phosphatase that regulates the PI3K-dependent activation of Akt.39 Indeed, Akt phosphorylation was significantly enhanced in TECs and correlated with an increased resistance to apoptotic signals.26 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.15 Several studies have indicated that TECs are resistant to chemotherapeutic and anti-angiogenic drugs.36,40 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.41,42 Our results may provide a rational for the described effects of activin A, which is known to down-regulate Pax2 expression11 and to repress tumor growth through inhibition of angiogenesis.43,44
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.
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Acknowledgements |
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Footnotes |
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Supported by the Associazione Italiana per la Ricerca sul Cancro, the Italian Ministry of University and Research (MIUR) (COFIN04 and FIRB project RBNE01HRS5-001), the Istituto Superiore di Sanità Targeted Project AIDS, the Italian Ministry of Health (Ricerca Finalizzata 02), and by Progetto S. Paolo and MIUR ex60%.
B.B. and G.C. contributed equally to this work.
Accepted for publication October 20, 2005.
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P < 0.005 HMEC-S versus HMEC-c. B: Representative Western blot analysis of P-Akt and Akt expression from cell lysates of TEC-c, TEC-AS, HMEC-c, and HMEC-S. The densitometric analysis of P-Akt/Akt ratio was performed on three individual experiments and data are expressed as mean ± SD. Analysis of variance with Newmann-Keuls multicomparison test was performed: *P < 0.005 TEC-AS versus TEC-c; 
