Volume 127, Issue 1, Pages 250-260 (July 2004) Successful growth and characterization of mouse pancreatic ductal cells: functional properties of the Ki-RASG12V oncogene  Franz S. Schreiber, Therese B. Deramaudt, Thomas B. Brunner, Michael I. Boretti, Keith J. Gooch, Doris A. Stoffers, Eric J. Bernhard, Anil K. Rustgi  Gastroenterology  Volume 127, Issue 1, Pages 250-260 (July 2004) DOI: 10.1053/j.gastro.2004.03.058

Figure 1 Morphologic features of PDCs. Light microscopy (100×) showed a cobblestone appearance, a typical feature of PDCs. No apparent morphologic differences were observed between early passage WT and Ki-RASG12V PDCs cultured on (A) collagen or on (B) plastic, and morphology did not change with serial passages (data not shown). Electron microscopy of Ki-RASG12V PDCs revealed features typical for PDCs: apical microvilli (AM), tight junctions (TJ), a relatively small volume of unspecialized cytoplasm, nucleus (N). (C) Magnification, 500× and (D) 2500×. No secretory vesicles, a feature of pancreatic acinar cells, were detectable, thereby indicating the purity of the duct cell population. Gastroenterology 2004 127, 250-260DOI: (10.1053/j.gastro.2004.03.058)

Figure 2 Cyst formation on a collagen type I matrix Ki-RASG12V PDCs grown on collagen type I-formed 3-dimensional structures (magnification, 200×). Gastroenterology 2004 127, 250-260DOI: (10.1053/j.gastro.2004.03.058)

Figure 3 Expression of pancreatic ductal markers in WT PDCs and Ki-RASG12V PDCs. (A) Quantitative reverse-transcription polymerase chain reaction analysis for CA II and CFTR. Expression levels of CA II and CFTR from normal whole pancreas and cultured PDCs were normalized to expression of β-actin. (B) Western blot analysis for CA II. Pancreas and kidney tissues were used as positive controls. National Institutes of Health 3T3 fibroblasts served as negative control. β-actin served as a protein loading control. (C) Immunocytochemical staining for K19 was positive in WT PDCs and Ki-RASG12V PDCs (magnification, 400×). Gastroenterology 2004 127, 250-260DOI: (10.1053/j.gastro.2004.03.058)

Figure 4 Growth curves for WT PDCs, Ki-RASG12V PDCs, and WT PDCs, or Ki-RASG12V PDCs infected with a retroviral vector expressing the puromycin selection marker (WT/puro PDCs or Ki-RASG12V/puro PDCs) or p53V143A mutation (p53V143A PDCs or Ki-RASG12V/p53V143A PDCs). Cells were counted at indicated time points. Experiments were performed independently 3 times, each in triplicate, with generation of SD. Gastroenterology 2004 127, 250-260DOI: (10.1053/j.gastro.2004.03.058)

Figure 5 Western blot analysis of (A) signaling molecules and (B) cell-cycle regulators in WT PDCs and Ki-RASG12V PDCs. Hyperphosphorylation (ppRb) and hypophosphorylation (pRb) forms of pRb are indicated. Gastroenterology 2004 127, 250-260DOI: (10.1053/j.gastro.2004.03.058)

Figure 6 Clonogenic survival of WT PDCs and Ki-RASG12V PDCs. (A) Comparison of WT PDC and Ki-RASG12V PDC clonogenic survival. Points labeled 4/19 were obtained at passage 19 (WT PDCs) to 21 (Ki-RASG12V PDCs), the undated curves were performed at passage 24 (WT PDCs) and 27 (Ki-RASG12V PDCs). Survival changed slightly in both lines with passage number, but the relative radiosensitivity remained constant at different passage numbers. (B) Radiosensitization of Ki-RASG12V PDCs by FTI treatment. Plating efficiency without irradiation was decreased 4-fold from 0.016 to 0.04 by 1.2 μmol/L R115777 treatment. All curves using R115777 are corrected for the plating efficiency changes owing to the drug and the survival differences shown, and thus reflects radiosensitivity differences, not drug effects on clonogenicity. (C) Comparison of Ki-RASG12V/p53V143A PDCs with p53V143A PDCs. (D) Radiosensitization of Ki-RASG12V/p53V143A PDCs by FTI R115777. (E) p53V143A PDCs are not radiosensitized by FTI treatment. Gastroenterology 2004 127, 250-260DOI: (10.1053/j.gastro.2004.03.058)

Figure 7 Inhibition of Ras processing after R115777 treatment of p53V143A PDCs and Ki-RASG12V PDCs/p53V143A PDCs and its effects on cellular signaling. (A) Cells we re-treated for 24 hours with 1.25 μmol/L R115777 (FTI lanes) or dimethyl sulfoxide carrier (control lanes) before harvest for protein isolation. Unprenylated Ras is indicated with arrows. (B) Cells we re-treated for 24 hours with 1.25 μmol/L R115777 (FTI lanes) or dimethyl sulfoxide carrier (control lanes) before irradiation at a dose rate of 1.7 Gy/min. Conditions for irradiation and Western blot analysis were performed as described in the Materials and Methods section. Gastroenterology 2004 127, 250-260DOI: (10.1053/j.gastro.2004.03.058)