1. Volume 116, Issue 6, Pages 1441-1452 (June 1999)
Transforming growth factor α activates Ha-Ras in human pancreatic cancer cells with Ki- ras mutations 
Thomas Seufferlein*, Johan Van Lint‡, Susanne Liptay§, Guido Adler*, Roland M. Schmid* 
Gastroenterology 
Volume 116, Issue 6, Pages (June 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Effect of TGF-α on EGFR tyrosine phosphorylation and ERK2 activation in MiaPaCa-2 and Panc-1 cells. Cultures of serum-starved (A) MiaPaCa-2 or (B) Panc-1 cells were treated with 50 ng/mL TGF-α for 5 minutes. Control cells received an equivalent amount of solvent. Cells were lysed and lysates immunoprecipitated (IP) with anti-EGFR antibody followed by antiphosphotyrosine Western blotting (upper panels), or ERK2 immune complex kinase assays were performed as described in Materials and Methods (lower panels). In each case, results are representative of 3 independent experiments. (C) Cultures of MiaPaCa-2 (left) and Panc-1 cells (right) were incubated with various concentrations of TGF-α for 5 minutes (upper panels) or 50 ng/mL TGF-α for various times (lower panels) as indicated, and ERK2 immune complex kinase assays were performed as described in Materials and Methods. Results of the kinase assays are the means of duplicates and are expressed as percentages of the maximum TGF-α–stimulated ERK2 activation ( cpm/3 × 106 cells at 5 minutes).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Effect of PD on basal and TGF-α–stimulated activation of ERK1 and ERK2, tyrosine phosphorylation of the EGFR, and activation of p70s6K in MiaPaCa-2 and Panc-1 cells. (A and B) Cultures of (A) MiaPaCa-2 and (B) Panc-1 cells were treated with 20 μmol/L PD (+; ■) for 1 hour. Control cells received an equivalent amount of solvent (−; ■). Cells were subsequently stimulated with 50 ng/mL TGF-α for 5 minutes, and ERK1 (left panels) and ERK2 (right panels) immune complex kinase assays were performed as described in Materials and Methods. Results are representative of 3 independent experiments, each performed in duplicate, and are expressed as percentage of maximum ERK1 or ERK2 stimulation in response to 50 ng/mL TGF-α ( cpm/3 × 106 cells at 5 minutes). (C) MiaPaCa-2 cells (left) or Panc-1 cells (right) were incubated with various concentrations of PD for 1 hour as indicated and subsequently stimulated with 50 ng/mL TGF-α for 5 minutes. Results are representative of 3 independent experiments, each performed in duplicate, and are expressed as percentage of maximum ERK2 stimulation in response to 50 ng/mL TGF-α ( cpm/3 × 106 cells at 5 minutes). (D) MiaPaCa-2 (upper left) or Panc-1 (upper right) cells were incubated with 20 μmol/L PD for 1 hour or received an equivalent amount of solvent (−) and were subsequently stimulated with 50 ng/mL TGF-α for 5 minutes. Cells were lysed and lysates immunoprecipitated with a polyclonal anti-EGFR antibody followed by Western blotting with antiphosphotyrosine monoclonal antibody as described in Materials and Methods. MiaPaCa-2 (lower left) or Panc-1 (lower right) cells were incubated with 20 μmol/L PD (PD; +) or 20 ng/mL rapamycin (Rapa; +) for 1 hour and subsequently stimulated with 50 ng/mL TGF-α for 15 minutes. Cells were subsequently lysed in 2× SDS-PAGE sample buffer, and p70s6k mobility shift was further analyzed by Western blotting using a polyclonal anti-p70s6k antibody as described in Materials and Methods. Arrows indicate hyperphosphorylated (pp70s6k) and hypophosphorylated (p70s6k) forms of p70s6k.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
TGF-α–induced activation of p90rsk and an AP-1 reporter construct requires MEK-1 activity in MiaPaCa-2 and Panc-1 cells. (A) Serum-starved cultures of MiaPaCa-2 and Panc-1 cells were incubated with 20 μmol/L PD for 1 hour (▧) and subsequently stimulated with 50 ng/mL TGF-α. Control cells received an equivalent amount of solvent (■). Cells were then lysed and lysates subjected to p90rsk immune complex kinase assays as described in Materials and Methods. Data are expressed as percentage of maximum stimulation in response to 50 ng/mL TGF-α at 5 minutes (16,000-20,000 cpm/3 × 106 cells) and are the mean of 3 independent experiments ± SE, each performed in duplicate. (B) Cultures of MiaPaCa-2 cells (left) and Panc-1 cells (right) were transfected with an AP-1–luciferase reporter construct as described in Materials and Methods. Cells were subsequently incubated with 20 μmol/L PD for 1 hour (+) before stimulation with 50 ng/mL TGF-α for another 18 hours. Control cells received an equivalent amount of solvent (−). Cells were then lysed, and luciferase activity was determined by luminometry. Data shown are representative of 3 independent experiments, each performed in triplicate.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
(A) TGF-α–induced activation of the ERK cascade is independent of PKC activity. Serum-starved cultures of MiaPaCa-2 cells (left) and Panc-1 cells (right) were incubated with 3.5 μmol/L GF X (▧) for 1 hour or received an equivalent amount of solvent (■). Cells were subsequently stimulated with 50 ng/mL TGF-α or 400 nmol/L phorbol 12,13-dibutyrate (insets) for 5 minutes and lysed; lysates were further analyzed by ERK2 immune complex kinase assays as described in Materials and Methods. Data are expressed as percentage of maximum stimulation of ERK2 by 50 ng/mL TGF-α at 5 minutes ( cpm/3 × 106 cells) or 400 nmol/L phorbol 12,13-dibutyrate at 5 minutes (12,000-15,000 cpm/3 × 106 cells) and are the means of 3 independent experiments ± SE, each performed in duplicate. (B) TGF-α–induced activation of AP-1 requires Ras activity. Cultures of MiaPaCa-2 (left) and Panc-1 (right) cells were transfected with an AP-1–luciferase reporter plasmid together with an empty vector plasmid (■) or a dominant-negative N17 Ras plasmid (▧) as described in Materials and Methods. Cells were subsequently incubated with 50 ng/mL TGF-α for another 18 hours, then lysed, and luciferase activity was determined by luminometry. Data shown are representative of 3 independent experiments, each performed in triplicate.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
TGF-α stimulates transactivation on a Ras-responsive E74 reporter plasmid. Cultures of MiaPaCa-2 cells (left) and Panc-1 cells (right) were transfected with a Drosophila E74-luciferase reporter plasmid together with an empty vector plasmid (■) or a dominant-negative N17 Ras plasmid (▧) as described in Materials and Methods. Cells were subsequently incubated with 50 ng/mL TGF-α for another 18 hours, then lysed, and luciferase activity was determined by luminometry. Data shown are representative of 3 independent experiments, each performed in triplicate.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
(A) Ha-Ras is expressed in MiaPaCa-2 and Panc-1 human pancreatic cancer cell lines. Serum-starved cultures of MiaPaCa-2 cells (left) or Panc-1 cells (right) were incubated with 50 ng/mL TGF-α for 5 minutes or received an equivalent amount of solvent (−). Cells were subsequently lysed in 2× SDS-PAGE sample buffer and further analyzed by Western blotting with a monoclonal anti–Ha-Ras antibody. (B) TGF-α activates Ha-Ras in MiaPaCa-2 and Panc-1 cells. Cells treated with 50 ng/mL TGF-α for 5 minutes (upper panels) or various times (lower panels) were lysed in RIPA buffer as described in Materials and Methods and incubated with a GST-Raf-RBD fusion protein coupled to GST-Sepharose. Active Ha-Ras bound to the Raf-RBD was subsequently analyzed by Western blotting with a monoclonal anti–Ha-Ras antibody. (C) Ha-Ras translocates to the free laminar edge of Panc-1 cells on stimulation with TGF-α. Serum-starved Panc-1 cells were incubated with 50 ng/mL TGF-α for 10 minutes or received an equivalent amount of solvent and were further analyzed by indirect immunofluorescence using a specific anti–Ha-Ras polyclonal antibody as described in Materials and Methods. White arrows point to the free laminar edge of the cells either in untreated (−) or TGF-α–treated cells (TGFα).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 7
(A and B) TGF-α–induced DNA synthesis in MiaPaCa-2 and Panc-1 cells is dependent on MEK-1 activity. Serum-starved cultures of MiaPaCa-2 cells (A, left) and Panc-1 cells (A, right) were incubated with various concentrations of TGF-α for 24 hours in DMEM with [3H]thymidine added for the last 6 hours. [3H]Thymidine incorporation was determined as described in Materials and Methods. Serum-starved cultures of MiaPaCa-2 cells (B, left) and Panc-1 cells (B, right) were incubated with various concentrations of PD as indicated in the presence of 50 ng/mL TGF-α and incubated for 24 hours in DMEM with [3H]thymidine added for the last 6 hours. [3H]Thymidine incorporation was determined as described in Materials and Methods. (C) TGF-α–induced colony formation in MiaPaCa-2 and Panc-1 cells requires MEK-1 and Ras activity. MiaPaCa-2 cells (left) and Panc-1 cells (right) were plated in agarose medium that contained DMEM/0.5% FBS at a density of 2 × 104 cells/dish in the absence or presence of 50 ng/mL TGF-α and/or 20 μmol/L PD (+; ▧) or 50 μmol/L of a selective FTI (+; 2) and incubated for 2 weeks as described in Materials and Methods. Values are the means of colonies formed on 5 separate dishes; bars are ± SE. In all cases, a representative of 3 independent experiments is shown. Where no error bar is shown, it lies within the dimensions of the symbol.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions