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Growth factor-dependent activation of the Ras-Raf-MEK-MAPK pathway in the human pancreatic carcinoma cell line PANC-1 carrying activated K-ras: implications for cell proliferation and cell migration

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Abstract

Human ductal adenocarcinoma of the pancreas frequently carry activating point mutations in the K-ras protooncogene. We have analysed the activity of the Ras-Raf-MEK-MAPK cascade in the human pancreatic carcinoma cell line PANC-1 carrying an activating K-ras mutation. Serum-starved cells and cells grown in medium with serum did not show constitutively activated c-Raf, MEK-1, or p42 MAPK. Stimulation of cells with epidermal growth factor (EGF) or fetal calf serum (FCS) resulted in activation of N-Ras, but not K-Ras, as well as activation of c-Raf, MEK-1, and p42 MAPK. Preincubation of serum-starved cells with MEK-1 inhibitor PD98059 abolished EGF- and FCS-induced MAPK activation, identifying MEK as the upstream activator of MAPK. PANC-1 cells exhibited marked serum-dependence of anchorage-dependent and -independent cell growth as well as cell migration. EGF, alone or in combination with insulin and transferrin, did not induce cell proliferation of serum-starved PANC-1 cells, indicating that activation of MAPK alone was not sufficient to induce cell proliferation. FCS-induced DNA synthesis was inhibited by 40% by the MEK-1 inhibitor. On the other hand, treatment with either FCS or EGF alone resulted in marked, MEK-dependent increase of directed cell migration. Collectively, our results show that the activating K-ras mutation in PANC-1 cells does not result in constitutively increased Raf-MEK-MAPK signaling. Signal transduction via the Ras-Raf-MEK-MAPK cascade is maintained in these cells and is required for growth factor-induced cell proliferation and directed cell migration.

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Acknowledgements

We thank UR Rapp for providing the c-Raf antiserum, W Kölch for cooperation in establishing the Raf-assay and T Seufferlein for introduction into the MAPK-assay. We wish to thank Y Kloog for providing the N-Ras cDNA, C Block for pcDNA/H-Ras (S17N) and JS Gutkind for pcDNA3/HA-MAPK expression plasmid, respectively. Furthermore, we greatly appreciate the help of B Moepps, S Gierschik and U Bachfischer. This work was supported by a grant of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (project 01-KS-9605/2).

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  1. Department of Pharmacology and Toxicology, University of Ulm, Ulm, 89069, Germany

    Klaudia Giehl, Bettina Skripczynski, Angela Mansard & Peter Gierschik

  2. Department of Internal Medicine I, University of Ulm, Ulm, 89069, Germany

    Andre Menke

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  1. Klaudia Giehl
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Giehl, K., Skripczynski, B., Mansard, A. et al. Growth factor-dependent activation of the Ras-Raf-MEK-MAPK pathway in the human pancreatic carcinoma cell line PANC-1 carrying activated K-ras: implications for cell proliferation and cell migration. Oncogene 19, 2930–2942 (2000). https://doi.org/10.1038/sj.onc.1203612

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