Volume 119, Issue 5, Pages 1358-1372 (November 2000) De novo expression of vascular endothelial growth factor in human pancreatic cancer: Evidence for an autocrine mitogenic loop  Zofia Von Marschall, Thorsten Cramer, Michael Höcker, Rahel Burde, Thomas Plath, Michael Schirner, Regina Heidenreich, Georg Breier, Ernst–Otto Riecken, Bertram Wiedenmann, Stefan Rosewicz  Gastroenterology  Volume 119, Issue 5, Pages 1358-1372 (November 2000) DOI: 10.1053/gast.2000.19578 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of VEGF in normal pancreas and pancreatic carcinoma. Paraffin-embedded tissue sections were (A–D) stained with polyclonal VEGF antibody or hybridized with 35S-labeled (E) antisense or (F) sense cRNA for VEGF. No specific immunostaining was observed in ductal cells of (A) nontransformed pancreas or (C and D) chronic pancreatitis tissues. VEGF was overexpressed by ductal tumor cells detected by means of (B) immunohistochemistry and (E) in situ hybridization. (F) Hybridization with sense cRNA did not show any significant hybridization signal. The exposure time for in situ hybridization was 14 days. Original magnifications: A, 60×; B and C, 20×; D–F, 40×. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Expression of KDR/flk-1 and flt-1 in pancreatic carcinoma. (A) Immunostaining with anti-CD31 antibody showed a considerable amount of endothelial cells and blood vessels surrounding ductal tumor structures. No specific staining for KDR/flk-1 was observed in endothelia or in ductal cells of (B) nontransformed pancreas or (I) chronic pancreatitis tissue. In pancreatic carcinoma, KDR/flk-1 staining was observed in (C) endothelial and (D and E) ductal tumor cells. Flt-1 expression was observed in (F and G) endothelia of tumors and (H) ductal tumor cells, but no flt-1 expression was detected in either ductal cells or endothelial cells of chronic pancreatic tissue (J). Arrows indicate blood vessels as evidenced by intraluminal erythrocytes. Original magnifications: E and G, 20×; A, B, C, J, and F, 40×; D, H, and I, 60×. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression of VEGF in human pancreatic carcinoma cell lines. (A) Analysis of VEGF mRNA splice forms. VEGF isoforms were analyzed by RT-PCR using oligonucleotide primers designed to detect all known isoforms. As a negative control, PCR was performed without prior RT (RT−). Aliquots from each PCR product were electrophoresed on a 1% agarose gel. Size of amplification products was determined by a 100-bp ladder. The indicated bands of 403, 535, and 607 bp correspond to VEGF121, VEGF165, and VEGF189, respectively. Data are representative of 3 experiments. (B) Expression of VEGF protein. A VEGF-specific ELISA was used to determine VEGF concentrations in the supernatants of the indicated cell lines and normalized to milligrams of protein. Data are the means ± SEM of 3 experiments. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Expression of flt-1 in human pancreatic carcinoma cell lines. (A) Flt-1 mRNA expression was analyzed by RT-PCR using flt-1–specific primers. β-Actin was amplified as a positive control, and parallel RT-PCR without prior RT (RT−) was performed as a negative control. A representative ethidium bromide stain of 3 experiments is shown. The band of 511 bp corresponds to the expected size for flt-1. (B) VEGF-dependent phosphorylation of flt-1. The indicated pancreatic carcinoma cell lines and HUVECs, which were used as a positive control, were stimulated with VEGF (+) or vehicle (−) for 10 minutes, lysed, immunoprecipitated with an flt-1–specific polyclonal antibody, separated by SDS-PAGE, immunoblotted with antiphosphotyrosine antibody (PY20), and visualized using the ECL detection system. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Expression of KDR/flk-1 in human pancreatic carcinoma cell lines. (A) KDR/flk-1 mRNA expression was analyzed by RT-PCR using KDR/flk-1–specific primers. RT-PCR without prior RT (RT−) was performed as a negative control. The band of 403 bp corresponds to the expected size for KDR/flk-1. (B) VEGF-dependent phosphorylation of KDR/flk-1 in AsPc-1 and Dan-G cells. KDR/flk-1 was immunoprecipitated from control cells and cells stimulated with VEGF165, using a receptor-specific antibody, separated by SDS-PAGE, immunoblotted with antiphosphotyrosine antibody (PY20), and visualized using the ECL detection system. Molecular size was deduced from a molecular-weight ladder, electrophoresed in parallel. (C) Reprobing of the blot with a KDR/flk-1 antibody confirmed the equivalent loading of lanes. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) VEGF increases Thr202/Tyr204 phosphorylation of p44 and p42 MAPK in Dan-G cells. Dan-G cells were maintained in serum-free medium for 24 hours and then treated with 1 ng/mL VEGF165 or 20% FCS for the indicated times. Equal amounts of protein (30 μg/lane) were then subjected to Western blotting using the phospho-specific p44/42 MAPK antibody. Reprobing of the blot with anti–Erk-1 and anti–Erk-2 antibodies confirmed the equivalent loading of lanes. A representative of 2 independent experiments, yielding similar results, is shown. (B) VEGF stimulates c-fos–promotor activity in Dan-G cells. Dan-G cells were transfected with 0.75 μg of the c-fos–luc construct plasmid and then incubated with or without VEGF165 (1 ng/mL) or PMA (10−6 mol/L) for 36 hours. Cell extracts were prepared, and luciferase activity was determined. Luciferase activity is expressed as increase in luciferase activity compared with unstimulated control and represents the mean ± SEM from 3 separate transfections. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of VEGF on pancreatic carcinoma cell proliferation. (A) Dan-G cells, (B) AsPc-1 cells, and (C) Capan-1 cells were seeded at a density of 104/well in 96-well plates and treated in the absence of serum for 48 hours with the indicated concentrations of VEGF165 or vehicle. [3H]Thymidine (1 μCi/well) was added for the last 24 hours, and incorporation of [3H]thymidine compared with unstimulated control cells was determined. Each experiment was performed in triplicate. The data represent the mean ± SEM of 6–12 experiments. *Significantly different from control (P < 0.05). Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Inhibition of VEGF-mediated DNA synthesis of Dan-G cells by VEGF antagonists. Serum-deprived Dan-G cells were treated with vehicle or stimulated with 1 ng/mL VEGF165, alone or in combination with the indicated concentration of the following agents: anti-VEGF–neutralizing antibody (mAb), isotype-matched control antibody (IgG), soluble receptor fragments of KDR (sKDR), and flt-1 (sflt-1) for 48 hours. Incorporation of [3H]thymidine was then compared with results in unstimulated controls. Each experiment was performed in triplicate. Shown are the means ± SEM of at least 3 experiments. *Significantly different from controls (P < 0.05). Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 Expression of dominant-negative flk-1 receptors inhibits growth in AsPc-1 cells. (A) Representative ethidium bromide–stained agarose gel showing RT-PCR products amplified from cDNA derived from wild-type AsPc-1 cells (wt), mock-transfected cells (m), and cells transfected with pRc-dn-flk-1 (f1, f2). The band of 473 bp corresponds with the expected size of the truncated flk-1 receptor cDNA. (B) Inhibition of VEGF-induced phosphorylation of p44 and p42 MAPKs in dn-flk-1–expressing AsPc-1 cells. Subconfluent wild-type AsPc-1 cells, mock-transfected cells, and clone dn-flk-1-1 and -2 were maintained in serum-free medium for 24 hours and then treated with 1 ng/mL of VEGF165 for 10 minutes. Thirty micrograms of protein per lane was then subjected to Western blotting using the phospho-specific p44/42 MAPK antibody. Top panel, quantitative analysis of 4 experiments as determined by laser densitometry. Data are given as means ± SEM. Bottom panel, representative Western blot and the corresponding loading control. The weaker signal of MAPK phosphorylation in the VEGF-treated dn-flk-1-1 clone is attributable to the smaller amount of loaded protein compared with control. (C) Effects of dominant-negative flk-1 on anchorage-dependent growth. Wild-type cells, mock-transfected cells, and clones dn-flk-1-1 and -2 were plated at 104 cells/well in 96-well plates with growth medium. Cells were then allowed to adhere for 24 hours. Growth medium was replaced by serum-free medium, and cell numbers were determined in a hemocytometer. Data represent the means ± SEM of 3 experiments, each performed in triplicate. *P < 0.05 compared with wild-type and mock-transfected cells. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 10 (A) Expression of dominant-negative flk-1 receptors in Dan-G cells. A representative ethidium bromide–stained agarose gel shows RT-PCR products amplified from cDNA derived from wild-type Dan-G cells (wt), mock-transfected cells (m), and cells transfected with pRc-dn-flk-1 (f1, f2). The band of 473 bp corresponds to the expected size of the truncated flk-1 receptor cDNA. (B) Effects of dominant-negative flk-1 expression on anchorage-dependent growth. Wild-type cells, mock-transfected cells, and clones dn-flk-1-1 and -2 were plated at 5 × 103 cells/well. After 24 hours, growth medium was replaced by serum-free medium, and cell numbers were determined in a hemocytometer. Data represent means ± SEM of 3 experiments, each performed in triplicate. *P < 0.05, **P < 0.01 compared with wild-type and mock-transfected cells. ***P < 0.001. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 11 IGF-I– and EGF-mediated MAPK phosphorylation in AsPc-1 cell clones. Dn-flk-1 cell clones and mock-transfected AsPc-1 cell were maintained in serum-free medium for 24 hours and then treated with or without IGF-I (50 ng/mL) or EGF (20 ng/mL) for 10 minutes. Thirty micrograms of protein per lane was then subjected to Western blotting using a phospho-specific p44/p42 MAPK antibody. (A) A Western blot and the corresponding loading control. (B) Quantitative analysis as determined by laser densitometry. Gastroenterology 2000 119, 1358-1372DOI: (10.1053/gast.2000.19578) Copyright © 2000 American Gastroenterological Association Terms and Conditions