Volume 123, Issue 6, Pages 2052-2063 (December 2002) Stat3 and NF-κB activation prevents apoptosis in pancreatic carcinogenesis  Florian R. Greten, *, Christoph K. Weber, ‡, Tim F. Greten, §, Günter Schneider, *, Martin Wagner, ‡, Guido Adler, ‡, Roland M. Schmid, *  Gastroenterology  Volume 123, Issue 6, Pages 2052-2063 (December 2002) DOI: 10.1053/gast.2002.37075 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Dysregulation of Bcl-xL and Bax in murine ductal pancreatic tumors. Western blot analysis of (A) Bcl-xL and (B) Bax expression comparing pancreatic tumors of transgenic mice (lanes 1–3) with pancreatic tissues of wild-type mice (lanes 4–6). The genotypes of mice analyzed are shown on the left. Molecular weights are indicated by arrows. Ponceau staining of each membrane was performed to assure that equal amounts of proteins had been loaded. Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Expression of Bcl-xL and Bax in tumor-free animals at 180 days. (A) Western blot analysis of Bcl-xL in pancreatic tissues of tumor-free animals killed at the age of 180 days, with each lane representing an individual animal. (B) Immunofluorescence staining for Bcl-xL in 180-day-old mice showing strong expression of Bcl-xL in tubular structures. (C) Nuclear counterstaining of the same tissue section as used in B with Yopro-1. (D) Expression of Bcl-xL in control pancreas and (E) nuclear counterstaining with Yopro-1. (F) Western blot analysis of Bax in pancreatic tissues using the same membrane as in A after stripping. (G) Immunofluorescence staining for Bax in 180-day-old mice showing expression of Bax in acini. (H) Nuclear counterstaining of the same tissue section as used in G with Yopro-1. Note that tubular structures are only visualized in the nuclear staining and are negative for Bax. (I) Expression of Bax in control pancreas and (J) nuclear counterstaining with Yopro-1. For details, see Materials and Methods. (Original magnification: B–E and G–J, 100×.) Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Quantification of Bcl-xL and Bax mRNA in pancreatic tissue from tumor-free animals at 180 days. Relative mRNA levels of (A) Bcl-xL and (B) Bax in tumor-free animals at the age of 180 days as described in Materials and Methods. Quantification is expressed as fold increase of expression compared with wild-type levels. Each data point represents an individual animal. Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Phenotypic characterization of a murine pancreatic tumor cell line, TD-2. (A) Phase-contrast microscopy of TD-2, a cell line derived from a ductal pancreatic carcinoma from a TGF-α/p53+/− mouse. (B) Immunofluorescence analysis of the cells shown in A using an antibody against carbonic anhydrase. (C) Western blot analysis of cytokeratin 8/18 and vimentin in TD-2 cells compared with NIH3T3 cells. (D) EGF-R and (E) Stat3 are constitutively activated in TD-2 cells compared with pancreatic tissue from 180-day-old wild-type mice. (F) Bcl-xL is up-regulated and Bax expression is down-regulated compared with pancreata from wild-type mice. (G) TD-2 cells grow in nude mice. Molecular weights are indicated by arrows. Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Inhibition of Bcl-x promoter activity in TD-2 cells by a dominant negative EGF-R (CD 533), IκBαS32AS36A, and Stat3β. The Bcl-x promoter was cotransfected along with increasing amounts of (A) a dominant negative EGF-R (CD 533) or (B) a nondegradable IκBα (IκBαS32AS36A) or dominant negative Stat3 (Stat3β). Relative luciferase activity is expressed in percent of basal activity. Values are expressed as mean ± SD of at least 3 independent transfections performed as duplicates. To achieve the same DNA concentration in each transfection, cells were also transfected with pcDNA3. Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Reduction of IKK activity and diminished phosphorylation of Stat3 in TD-2 cells after transfection with a dominant negative EGF-R. (A, upper panel) IκB immunocomplex kinase assay of 200,000 TD-2 cells that had been sorted on the basis of GFP expression 48 hours after transfection with either a pIRES-EGFP construct alone or the EGFP construct along with a dominant negative EGF-R (CD 533). A total of 1 μg GST IκBα (1–54) was used as a substrate. (A, lower panel) Western blot analysis of the same membrane incubated with anti-IKKα serving as a loading control. (B) TD-2 cells were transfected with a κB-dependent reporter plasmid (3xIgκLuc) along with increasing amounts of dominant negative EGF-R (CD 533). Relative luciferase activity is expressed in percent of basal activity. Values are expressed as mean ± SD of at least 3 independent transfections performed as duplicates. (C, left panel) Western blot analysis of phospho-Stat3 in 300,000 sorted GFP-positive TD-2 cells 48 hours after transfection with CD 533. Protein lysates were immunoprecipitated using a phosphotyrosine antibody and membrane was incubated with a Stat3 monoclonal antibody. (C, right panel) Control incubation with phosphotyrosine antibody shows equal amounts of precipitated proteins. To achieve the same DNA concentration in each transfection, cells were also transfected with pcDNA3. Molecular weights are indicated by an arrow. Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 IKK activity, phosphorylation of Stat3, and localization of RelA(p65) and phospho-Stat3 in tumor-free animals at 180 days. IκB immunocomplex kinase assay of lysates of tumor-free TGF-α and wild-type animals at the age of 180 days. The lower panel shows a Western blot analysis of the same membrane incubated with anti-IKKα serving as a loading control. (B) Western blot analysis of IκBα expression in tumor-free TGF-α and wild-type animals at the age of 180 days. The lower panel shows Ponceau staining of the membrane. (C) Western blot analysis of phospho-Stat3 in tumor-free TGF-α and wild-type animals at the age of 180 days. Protein lysates were immunoprecipitated using a phosphotyrosine antibody, and membrane was incubated with a Stat3 monoclonal antibody. The lower panel shows equal amounts of precipitated proteins by incubation of the same membrane with a phosphotyrosine antibody. Confocal microscopy analysis of immunofluorescence studies using antibodies against (D) active p65 and (F) tyrosine phosphorylated-Stat3 in tumor-free animals at the age of 180 days. Red fluorescence represents RelA(p65) or phospho-Stat3, and green fluorescence represents nuclear staining with Yopro-1. Yellow areas indicate an overlay of red and green fluorescence showing nuclear staining of either RelA(p65) or phospho-Stat3. No yellow areas are detected in control pancreas for (E) RelA(p65) and (G) phospho-Stat3. (Original magnification 100×.). Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Down-regulation of Bcl-xL expression levels after inhibition of NF-κB and Stat3 activation and induction of apoptosis. (A) Western blot analysis of 100,000 GFP-positive TD-2 cells transfected with either pIRES-EGFP alone, pIRES-EGFP along with Stat3β, pIRES-EGFP along with IκBαS32S36A, or pIRES-EGFP along with Stat3β and IκBαS32S36A. (Lower panel) Incubation of the same membrane using an actin mouse monoclonal antibody. To achieve the same DNA concentration in each transfection, cells were also transfected with pcDNA3. Molecular weights are indicated by an arrow. (B–G) Morphologic evaluation of apoptosis in TD-2 cells. Cells were either transfected with (B and C) pIRES-EGFP alone or with (D–G) pIRES-EGFP along with Stat3β and IκBαS32S36A. After 48 hours, cells were fixed in 4% paraformaldehyde and stained with the DNA specific fluorochrome 4',6-diamidino-2-phenylindole (C, E, and G). Apoptotic cells are characterized by condensed chromatin fragments in the nucleus and by segmentation of the nucleus (E and G), whereas control cells transfected with pIRES-EGFP alone show normal morphology (C). To achieve the same DNA concentration in each transfection, control cells were also transfected with pcDNA3. Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Induction of apoptosis measured by annexin V expression in TD-2 cells. Fluorescence-activated cell sorter analysis of TD-2 cells transfected with a pIRES-EGFP construct alone, the EGFP construct along with Stat3β, a GFP-ΔNIκBα construct alone, or the GFP along with Stat3β. Note that cells transfected with one construct alone were also transfected with pcDNA-3 vector to achieve the same DNA concentration in each transfection. Scatter plots on the left show distribution of all analyzed cells. A live gate was put onto the GFP-positive cells counting 5000 positive cells. Annexin V expression of these 5000 GFP-positive cells is shown in the right curve plots. Amount of annexin V–positive cells is given in percentage of all GFP-positive cells. Plots show a representative result of 3 independent transfections. Gastroenterology 2002 123, 2052-2063DOI: (10.1053/gast.2002.37075) Copyright © 2002 American Gastroenterological Association Terms and Conditions