Volume 124, Issue 7, Pages 1830-1845 (June 2003) Down-regulation of the dual-specificity phosphatase MKP-1 suppresses tumorigenicity of pancreatic cancer cells  Quan Liao, Junchao Guo, Jörg Kleeff, Arthur Zimmermann, Markus W Büchler, Murray Korc, Helmut Friess  Gastroenterology  Volume 124, Issue 7, Pages 1830-1845 (June 2003) DOI: 10.1016/S0016-5085(03)00398-6

Figure 1 MKP-1 mRNA expression analysis in pancreatic tissues. (A) Total RNA (20 μg) isolated from the indicated pancreatic tissues was hybridized with the 32P-labeled MKP-1 cDNA probe and rehybridized with the 7S cDNA probe to verify equivalent RNA loading. (B) Relative MKP-1 mRNA levels were calculated as ODMKP-1/OD7S for each sample. (C) Protein lysates (30 μg) of the indicated pancreatic tissues were subjected to Western blot analysis using an anti-MKP-1 antibody as described in the Materials and Methods section. The molecular size marker (in kilodaltons) is indicated on the left. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 2 MKP-1 immunohistochemistry in the normal pancreas and pancreatic cancer. (A, B) In the normal pancreas, MKP-1 immunoreactivity was weak to absent in ductular and acinar cells. (A) Faint MKP-1 staining was observed in islet cells. (B) Strong MKP-1 immunoreactivity was present in smooth muscle cells of small vessels in the pancreatic parenchyma. (C and D) Moderate to strong MKP-1 immunoreactivity was present in the cancer cells within the pancreatic tumor mass. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 3 MKP-1 immunohistochemistry in metaplastic ducts and PanINs. Moderate to strong MKP-1 immunoreactivity was present in the metaplastic tubular complexes in CP-like areas adjacent to the (A) cancer cells as well as in (B) CP. (C) Moderate and occasionally focal MKP-1 expression was observed in PanIN-1 lesions. Moderate to strong MKP-1 immunoreactivity was observed in (D and E) PanIN-2 and (F) PanIN-3 lesions. (D) A PanIN-1A/B lesion evolving into a PanIN-2 lesion. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 4 MKP-1 protein levels in pancreatic cancer cell lines. Protein lysates (30 μg) of the indicated cell lines were subjected to Western blot analysis by using an anti-MKP-1 antibody as described in the Materials and Methods section. The molecular size marker (in kilodaltons) is indicated on the left. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 5 MKP-1 expression in MKP-1 antisense-transfected cells. (A, B) PANC-1 and (C, D) T3M4 pancreatic cancer cells were stably transfected with the MKP-1 antisense expression plasmid as described in the Materials and Methods section. (A, C) MKP-1 antisense mRNA expression was verified by Northern blot analysis using α32P-labeled MKP-1 cRNA sense probe; exemplary Northern blots are shown. (B, D) Western blot analysis was performed for the indicated cell lines using an anti-MKP-1 antibody as described in the Materials and Methods section (B, D, upper panel). Representative Western blots of 2 independent experiments are shown in B and D. (B, D, lower panel) Relative MKP-1 protein levels in the indicated cell lines were compared with wild-type cells, which were set to 100. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 6 Growth characteristics of MKP-1 antisense-transfected cells. (A, D) Anchorage-dependent growth was determined in wild-type, sham-transfected, and 4 MKP-1 antisense-transfected (A) PANC-1 and (D) T3M4 cells by the MTT assay as described in the Materials and Methods section. (B, C, E) To determine anchorage-independent growth, indicated (C) PANC-1 or (E) T3M4 cells (1,000/well) were seeded in 6-well plates in complete medium containing 0.35% agar. After 14 days, colonies were counted. Typical wells of wild-type PANC-1 and an MKP-1 antisense-transfected PANC-1 clone are shown in B. (C, E) Data are expressed as means ± SEM of triplicate determinations in 3 independent experiments for each cell line. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 7 Tumorigenicity of MKP-1 antisense-transfected cells. (A–C) Exponentially growing cells (4 × 106) were injected at 2 sites subcutaneously in athymic nude mice and tumor growth was measured weekly until week 8. Typical mice at week 8 of the indicated cells are shown in A. Tumor volume was determined by the equation: vol = (l × h × w) × π/4, where l is length, h is height, and w is width of the tumor. (B) Values are means ± SD of 10 tumors/clone. (C) Mean tumor volume (white bar) + SD and individual tumor volumes (black bar) at week 8 for the indicated cells. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 8 Histologic analysis of nude mice tumors. Tumors of (A, C, E) sham-transfected and (B, D, F) MKP-1 antisense-transfected PANC-1 cells were investigated by immunohistochemistry. (A, B) MKP-1 immunohistochemistry showing decreased MKP-1 expression in the (B) MKP-1 antisense-transfected PANC-1 cells in comparison with (A) sham-transfected PANC-1 cells. (C, D) Analysis of microvessel density using the von Willebrand factor antibody as described in the Materials and Methods section. (E, F) Ki67 staining shows a higher percentage of proliferating cells in (E) sham-transfected cells in comparison with the (F) MKP-1 antisense-transfected PANC-1 cells. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 9 Effects of decreased MKP-1 levels on responsiveness toward growth factors and actD. Wild-type, sham transfected, and 4 MKP-1 antisense mRNA-expressing (A–C) PANC-1 and (D–F) T3M4 clones, respectively, were incubated for 48 hours with the indicated concentrations of (A, D) FCS, (B, E) EGF, and (C, F) actD. Cell growth was determined by the MTT assay as described in the Materials and Methods section. Data are expressed as percent change from unstimulated controls and are means ± SEM of 8 determinations per experiment from 3 separate experiments. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 10 Effects of decreased MKP-1 levels on responsiveness toward death receptor mediated apoptosis. Wild-type, sham transfected, and 4 MKP-1 antisense mRNA-expressing (A) PANC-1 and (B) T3M4 clones as indicated, were incubated for 48 hours with 10 ng/mL TNF-α (black), 100 ng/mL Fas-AB (white), and 100 ng/mL TRAIL (grey). Cell growth was determined by the MTT assay as described in the Materials and Methods section. Data are expressed as percent change from unstimulated controls and are means ± SEM of 8 determinations per experiment from 3 separate experiments. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 11 Effects of decreased MKP-1 levels on phosphorylated MAPK levels. (A, C) Western blot analysis using phospho-specific anti-MAPK (p42/44mapk) antibodies was performed as described in the Materials and Methods section. (B, D) Relative phosphorylated MAPK levels in the indicated cell lines were calculated as ODphospho-MAPK/ODp42-MAPK. (A, B) PANC-1 cells; (C, D) T3M4 cells. Statistical analysis of all clones vs. the 2 control cell lines revealed a significant increase in basal phosphorylation in the MKP-1 antisense-transfected PANC-1 cells (P < 0.05), but not in the MKP-1 antisense-transfected T3M4 cells (P = 0.15). Results shown are representative from 2 independent experiments. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 12 Effects of decreased MKP-1 levels on EGF-induced MAPK phosphorylation. Cells were incubated in the absence or presence of EGF (100 ng/mL) for the indicated times. Western blotting was performed with a phospho-specific anti-MAPK (p42/44mapk) antibody, as described in the Materials and Methods section. To confirm equal loading of lanes, the membranes were stripped and reprobed with an anti-p42-MAPK antibody. The positions of MAPKs p44 and p42 are indicated on the right. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)

Figure 13 Effects of decreased MKP-1 levels on EGF-induced MAPK phosphorylation. (A) PANC-1 and (B) T3M4 cells were incubated in the absence or presence of EGF (100 ng/mL) for the indicated times. Phosphorylation of MAPK was determined by Western blotting. Relative phosphorylated MAPK levels in the indicated cell lines were calculated as ODphospho-MAPK/ODp42-MAPK. Statistical analysis of the 2 clones vs. the 2 controls revealed a significant difference (P < 0.05) at 10 and 60 minutes for PANC-1 cells and a significant difference (P < 0.05) at 60 minutes for T3M4 cells. Results are calculated from 2 independent experiments and presented as means ± SEM. Gastroenterology 2003 124, 1830-1845DOI: (10.1016/S0016-5085(03)00398-6)