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Animal Models |
From the Departments of Dermatology*
and
Pathology,
Emory University School of
Medicine, Atlanta, Georgia; the Department of
Surgery,
Children’s Hospital, Harvard
Medical School, Boston, Massachusetts; the Department of
Pathology,§
Beth Israel Deaconness Medical
Center, Harvard Medical School, Boston, Massachusetts; and the
Department of Genetics and Pathology,¶
Rudbeck
Laboratory Uppsala University, Uppsala, Sweden
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, angiosarcoma may arise as a result of exposures to various toxins. The most commonly implicated causative agents are vinyl chloride and thorotrast, both of which are associated with hepatic angiosarcomas.7,8 These angiosarcomas, and experimental angiosarcomas arising in rodents from exposure to vinyl chloride, have been shown to have activated Ras mutations in a number of cases.8-10 Radiation of hemangiomas has been shown to result in the later development of angiosarcoma.11 Finally, lymphedema from a variety of causes, including postsurgical (Stewart-Treves syndrome), congenital, and even post-traumatic lymphedema is associated with the development of angiosarcoma.12-14
Animal models have proven useful in determining molecular events necessary for the development of angiosarcoma. We have previously generated a model of rapidly growing angiosarcoma through the sequential introduction of a temperature-sensitive SV40 large T antigen and activated H-Ras into murine endothelial cells.15 Upon injection into nude mice, endothelial cells containing SV40 large T antigen alone, MS1 cells, develop dormant hemangiomas that do not grow in size. Endothelial cells (SVR) with two oncogenes, SV40 large T antigen and H-ras, form rapidly growing angiosarcomas that cause death of mice.15 To evaluate whether overexpression of VEGF in endothelial cells that expressed the VEGF receptors VEGFR-1 and VEGFR-2 would lead to malignant transformation, we overexpressed primate VEGF using a retrovirus in MS1 cells. The resulting cells, MS1 VEGF cells, form slowly growing angiosarcomas upon injection into mice. These tumors display invasiveness and continued growth in vivo. These MS1 VEGF angiosarcomas, like the angiosarcomas that express large T antigen and Ras, show high level expression of VEGFR-1 and VEGFR-2 in vivo. Our studies suggest that expression of VEGF in cells that express receptors for VEGF is a transforming event in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MS1 murine endothelial cells15 (no. 2279, American Type Culture Collection, Manassas, VA) were infected with a retrovirus encoding primate VEGF and puromycin resistance, or puromycin resistance alone, and selected in 1 µg/ml puromycin. MS1 cells are murine endothelial cells immortalized with a temperature-sensitive SV40 large T antigen, and SVR cells were derived by transforming MS1 cells with oncogenic H-ras.15 Clones were pooled, and supernatants were assayed for the presence for human VEGF using an enzyme-linked immunosorbent assay specific for VEGF (R&D Systems, Minneapolis, MN). The cells containing VEGF or puromycin (Sigma Chemical Co., St. Louis, MO) resistance alone were named MS1 VEGF and MS1puro, respectively.
Receptor Cross-Linking Studies
MS1 and SVR endothelial cells were grown to confluence in 5-cm plates. Medium was aspirated, plates were washed with 10 ml cold phosphate-buffered saline (PBS) and 1.2 ml binding buffer (DMEM 20 mmol/L, HEPES, pH 7.4, 0.1% gelatin), and radio-iodinated VEGF (10 ng/ml) was added according to the method of Soker et al.16 Cells were shaken for 2 hours at 4°C. Plates were washed twice with 8 ml cold PBS, and 20 µl of 20 mmol/L disuccinimidyl suberate in dimethylsulfoxide (DMSO) were added and the plates incubated 15 minutes at room temperature. Two hundred microliters of stop buffer (10 mmol/L Tris-HCl, pH 7.5, 20 mmol/L glycine, 2 mmol/L EDTA) were added, and the plates were incubated for 1 hour, then washed twice with 8 ml PBS.
The cells on the plate were lysed by the addition of 700 µl scrape buffer consisting of 20 ml PBS, 1.5 mmol/L EDTA, and 2 mmol/L phenylmethylsulfonyl fluoride (PMSF) twice, followed by scraping with a rubber policeman. The lysed cells were spun at the maximum speed of a microfuge for 10 minutes. The supernatant was removed, and the pellet was suspended in 40 µl lysis buffer (10 mmol/L Tris-HCl, pH 7.0, 1% Nonidet P-40, 1 mmol/L EDTA, 2 mmol/L PMSF). An equal volume of sample buffer (0.125 mol/L Tris-HCl, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% ß-mercaptoethanol/bromphenol blue) was added, and the samples were boiled for 3 minutes. The samples were loaded on a 6% acrylamide gel and assayed by autoradiography.
In Vivo Tumorigenesis
One million cells were injected subcutaneously into the right flank of nude male mice (Massachusetts General Hospital, Boston, MA) 5 to 6 weeks old. Three mice were injected per cell line. Tumor size was measured by Vernier caliper, and tumor volume was calculated by the formula (w2 x l) x 0.52, where w (width) represents the smallest diameter of the tumor.15 Tumors were excised at 5 months, fixed in formalin, and stained with hematoxylin and eosin.
In Situ Hybridization (ISH)
ISH was performed on 4-mm-thick sections of formalin-fixed, paraffin-embedded tissue. Details of ISH have been reported previously.17-19 Briefly, slides were passaged through xylene and graded alcohols; 0.2 mol/L HCl; Tris/EDTA with 3 µg/ml proteinase K/0.2% glycine, 4% paraformaldehyde in PBS, pH 7.4; 0.1 mol/L triethanolamine containing 1/200 (v/v) acetic anhydride; and 2x SSC. Slides were hybridized overnight at 50°C with 35S-labeled riboprobes in the following mixture: 0.3 mol/L NaCl, 0.01 mol/L Tris, pH 7.6, 5 mmol/L EDTA, 0.02% w/v Ficoll, 0.02% w/v polyvinylpyrollidone, 0.02% w/v bovine serum albumin fraction V/50% formamide, 10% dextran sulfate, 0.1 mg/ml yeast tRNA, and 0.01 mol/L dithiothreitol (DTT). Post-hybridization washes included 2x standard saline citrate (SSC)/50% formamide and 10 mmol/L dithiothreitol at 50°C, 4x SSC, 10 mmol/L Tris, and 1 mmol/L EDTA with 20 µg/ml ribonuclease at 37°C; and 2x SSC, 50% formamide, 10 mmol/L EDTA at 65°C and 2x SSC. Slides were then dehydrated through graded alcohols containing 0.3 mol/L ammonium acetate, dried, coated with Kodak NTB 2 emulsion, and stored in the dark at 4°C for 2 weeks. The emulsion was developed with Kodak D19 developer and the slides were counterstained with hematoxylin. Antisense single-stranded 35S-labeled human VPF/VEGF RNA probe and its sense control have been described previously.18 The antisense probe hybridizes specifically with a region of VPF/VEGF RNA mRNA common to all known VPF/VEGF splice variants. 35S-labeled single-stranded antisense and sense RNA probes for mouse VPF/VEGF mRNA and the mouse VPF/VEGF receptors VEGFR-1 and VEGFR-2 mRNAs have been described previously.19
In Vitro Kinase Assay
In vitro kinase assays were performed according to the method of Waltenberger et al.20 Subconfluent cell cultures were starved overnight in DMEM (Life Technologies, Inc.) containing 0.1% fetal calf serum (Sigma, St. Louis, MO) and then stimulated or not with VEGF 165 (100 ng/ml; Peprotech, Rocky Hill, NJ) for 7 minutes at 37°C. The cells were rinsed with ice-cold PBS containing 100 µmol/L Na3VO4 and lysed for 10 minutes on ice in NP-40 lysis buffer (20 mmol/L Hepes, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 10% glycerol, 300 µmol/L Na3VO4, 1% aprotinin, 1 mmol/L PMSF).
Lysates were clarified at 10,000 x g for 15 minutes at
4°C, and the supernatants were incubated with antibodies against
either VEGFR-2 (RS-2) or phosphotyrosine (4G10, Upstate Biotechnology,
Waltham, MA) for 1 hour at 4°C, followed by a final incubation for 45
minutes with immobilized protein A (Immunosorb; EC Diagnostics,
Uppsala, Sweden). The precipitates were washed three times in NP-40
lysis buffer and twice in PBS containing 100 µmol/L
Na3VO4, then incubated for
15 minutes at room temperature in 40 µl kinase buffer (20 mmol/L
Hepes, pH 7.5, 10 mmol/L MgCl2, 2 mmol/L
MnCl2, 0.05% Triton X-100, 1 mmol/L DTT)
containing 5 µCi [
-32P]ATP (Amersham). The
kinase reactions were terminated by addition of 40 µl 2x sample
buffer (8% SDS, 0.4 mol/L Tris/HCl, pH 8.8, 1 mol/L sucrose, 10 mmol/L
EDTA, 0.02% bromphenol blue, 4% ß-mercaptoethanol) and the samples
were boiled at 95°C for 4 minutes and analyzed by SDS-PAGE in a 10%
SDS-polyacrylamide gel. After fixation in methanol/acetic acid, the gel
was treated with 1 mol/L KOH for 1 hour at 55°C, fixed again, dried,
and analyzed by autoradiography.
Metabolic Labeling
Semiconfluent MS1 and MS1 VEGF cultures were washed with methionine- and cysteine-deficient DMEM (Life Technologies) and subsequently incubated for 2 hours at 37°C in the same medium, complemented with 100 µCi/ml [35S]methionine/cysteine mix (Amersham Pharmacia Biotech, Uppsala, Sweden). The cultures were rinsed once with ice-cold PBS to terminate the labeling and then lysed in 0.5% NP 40/20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 10 mmol/L EDTA, 1% aprotinin, and 0.2 mmol/L PMSF. Cell lysates were immunoprecipitated with a rabbit antiserum raised against a 25-amino acid residue peptide corresponding to a part of the kinase insert in VEGFR-2,20 followed by incubation with immobilized protein A. Control lysates were incubated with protein A-Sepharose alone. Samples were analyzed by SDS-PAGE. The gel was treated with Amplify (Amersham Pharmacia Biotech), dried, and analyzed using a BioImager (Fuji, Tokyo, Japan).
Western Blot Analysis of c-Ets-1 Expression
MS1 VEGF cells were treated with 5 µg/ml SU-1498 (Calbiochem, La Jolla, CA) in DMSO or with DMSO alone for 24 hours. Cells were lysed in lysis buffer containing 50 mmol/L PBS, pH 7.2, 5 mmol/L EDTA, 5% glycerol, and 0.3 mmol/L PMSF. The protein concentration was determined by Protein Assay (Bio-Rad, Hercules, CA) using bovine serum albumin as a standard. Samples were mixed with Laemmli sample buffer and heated at 95°C for 5 minutes before SDS-PAGE (Bio-Rad MiniVertical Electrophoresis standard protocol) and transferred to PVDF membrane (Bio-Rad). The membrane were blocked with blocking buffer (5% dry milk, 10 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.1% Tween-20) and subsequently incubated with the appropriate antibodies for immunoblotting. Anti-Ets-1 polyclonal antibody 21 0.2 µg/ml and peroxidase 0.4 µg/ml conjugated anti-Rabbit lgG were from Santa Cruz Biotechnology (Santa Cruz, CA).21
Assay of Anchorage-Independent Growth
Anchorage-independent growth was determined by adding equal numbers of viable cells to a plate containing 0.35% Noble agar in 5% fetal bovine serum-containing media at a density of 5 x 105 cells per 6-cm2 plate. The number of colonies formed in agar was recorded 2 weeks after plating the cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. VEGFR-2 levels were not altered by
various stimuli in vitro, including Ras activation and
hypoxia.
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. The human probe used in the Northern
blot analysis does not cross-hybridize with endogenous murine VEGF.
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. Tumors
derived from MS1 and MS1puro cells resembled hemangiomas. They
consisted of well-circumscribed tumor masses within the subcutaneous
tissue. Fibrous septa divided the tumor masses into coarse nodules
similar to the lobular pattern seen in many capillary hemangiomas. Each
nodule consisted of well-formed, discrete capillary vessels lined by
differentiated endothelium. The vessels did not intercommunicate nor
dissect through the subcutaneous tissues as one sees in angiosarcoma
(Figure 4A)
. On the other hand, those
tumors derived from MS1 VEGF cells formed large masses occupying both
the dermis and subcutaneous tissue with poorly circumscribed borders.
The proliferating endothelial cells formed a ramifying sinusoidal
network of vessels, which dissected through collagen and around
adnexal structures (Figure 4B)
. Despite this aggressive pattern of
growth the tumors consisted entirely of areas in which vasoformative
features were identified and lacked solid or undifferentiated areas.
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, the VEGFR-2 precursor and mature forms of 200 and 220 kd,
respectively,22
were immunoprecipitated both from
MS1 and MS1 VEGF cells. It is noteworthy that the mature form was less
pronounced in the MS1 VEGF cells than in the MS1 cells, in accordance
with a rapid turnover of the receptor due to binding of the
endogenously produced VEGF121 in the MS1 VEGF cells.
A downstream target of VEGF-VEGFR interactions is the transcription
factor Ets-1.21
To demonstrate the possibility of an
autocrine loop between exogenously produced VEGF and VEGFR-2, we
incubated MS1 VEGF cells with SU1498, a specific inhibitor of VEGFR-2
receptor phosphorylation. This blocks signal transduction from VEGFR-2.
In the presence of SU1498, levels of Ets-1 were decreased, suggesting
that VEGF-VEGFR-2 interactions contributed to baseline levels of Ets-1
expression, and interruption of this autocrine interaction with SU1498
led to decreased expression of Ets-1 (Figure 7)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, VEGF protein and mRNA have been demonstrated in a variety of endothelial tumors, including benign hemangiomas of infancy and angiosarcomas.39-41 Because these tumors have also been demonstrated to express VEGF receptors, both autocrine and paracrine roles for VEGF in endothelial tumorigenesis have been implied. VEGF has been shown to have autocrine activity in retinal pigment epithelial cells in vitro,42 but this is the first report of transforming activity of VEGF in vivo. We demonstrate that overexpression of human VEGF in immortalized endothelial cells is sufficient to convert them from benign hemangiomas to malignant angiosarcomas. These angiosarcomas differ from those derived from sequential introduction of SV40 large T antigen and Ras in terms of their slower rate of in vivo growth. Angiosarcomas derived from endothelial cells containing both large T antigen and H-Ras reach a volume of 1 cm3 in approximately 3 to 4 weeks,15 whereas angiosarcomas containing large T antigen and VEGF 121 require approximately 5 months to reach the same size. MS1VEGF cells do not form colonies in soft agar, thus demonstrating that in this system, growth in vivo does not correlate with soft agar growth.
The VEGF secreted by these tumors is biologically active, as judged from the fact that MS1 VEGF cells fail to respond to exogenous VEGF 165 with an increase in autophosphorylation of VEGFR-2 as compared with the response in the control MS1 cells. The lack of increased phosphorylation of VEGFR-2 is likely to be due to an intracellular binding of endogenously produced VEGF 121. Binding of ligand to the receptor could take place in any intracellular component where VEGF 121 would have access to extracellular domains of VEGFR-2, such as the endoplasmic reticulum or the Golgi apparatus. Activation of the kinase activity of receptor tyrosine kinases is known to lead to rapid degradation, via either via lysosomal degradation or ubiquitination and proteosome-mediated degradation. It is, therefore, expected that this type of chronic stimulation would not lead to a detectable increase in the level of autophosphorylated receptors. Furthermore, if the level of endogenously produced ligand is sufficiently high to saturate receptor binding sites, exogenously added growth factor would not have access to receptors. Similar effects of intracrine stimulation in cells expressing a growth factor and its cognate receptor have been described before.43 In addition, the tumors derived from MS1 VEGF cells demonstrate continued synthesis of primate MS1 VEGF in vivo.
Because VEGFR-2 protein could be identified in immunoprecipitates from metabolically labeled MS1 VEGF cells, the failure to induce kinase activity with exogenous VEGF strongly indicates that VEGF produced by MS1 VEGF cells interacts with endogenous VEGFR-2. To further demonstrate the presence of an autocrine VEGF loop in these cells, MS1 VEGF cells were incubated with SU1498,44 a specific inhibitor of flk-1 tyrosine kinase. Incubation of cells with this inhibitor decreased levels of c-ets-1, a transcription factor that has been shown to be up-regulated by VEGF 121.
A large number of angiosarcomas have alterations in the activity of the p53 tumor suppressor gene.9,10,45 This may be a result of loss of p53 through mutation or deletion, or amplification of the MDM2 gene, which functionally inactivates p53.40 p53-null mice also develop angiosarcoma at a high frequency.46 Our immortalized endothelial cells express SV40 large T antigen, which functionally impairs p53 activity and may serve as a model of an angiosarcoma precursor. Chemically induced angiosarcomas in rodents and humans have been shown to have mutations in activated Ras, leading to constitutive Ras activity, suggesting that p53 inactivation and Ras activation may be synergistic in rapidly growing angiosarcomas. Little is known of the molecular pathogenesis of more slowly growing endothelial malignancies. We show that overexpression of primate VEGF in immortalized endothelial cells that express VEGFR-1 and VEGFR-2 leads to malignant transformation, but to a novel, slowly growing tumor, in contrast to the vast majority of tumors that can be propagated in animals. This model may shed light on the pathogenesis of slow growing malignancies and establishes VEGF as a oncogenic factor in endothelial cells.
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Acknowledgements |
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Footnotes |
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Funded in part by grants from the KAO Corporation, the Dermatology Foundation, the American Skin Association, the Emory Skin Disease Research Core Center P30 AR 42687 (National Institutes of Health), and the National Institute of Arthritis and Musculoskeletal and Skin Diseases KO8 AR02096–01 and RO3 AR44947–03 (all to J. L. A.).
Accepted for publication January 6, 2000.
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