1. Volume 133, Issue 5, Pages 1637-1648 (November 2007)
NADPH Oxidase Promotes Pancreatic Cancer Cell Survival via Inhibiting JAK2 Dephosphorylation by Tyrosine Phosphatases 
Jong Kyun Lee, Mouad Edderkaoui, Patrick Truong, Izumi Ohno, Kee–Taek Jang, Andrea Berti, Stephen J. Pandol, Anna S. Gukovskaya 
Gastroenterology 
Volume 133, Issue 5, Pages (November 2007)
DOI: /j.gastro
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2. Figure 1
Time course of JAK2 phosphorylation and ROS generation induced by GFs in PaCa cells. MIA PaCa-2 (A and B) and PANC-1 (C and D) cells were cultured for the indicated times without and with 100 ng/mL IGF-I or 15% FBS. (A and C) In this and other figures JAK2 phosphorylation was measured by immunoblotting, using antibodies against both phosphorylated and total JAK2. Blots were reprobed for β-actin to confirm equal protein loading. In cells cultured without GFs, JAK2 phosphorylation did not change with time. (B and D) The intensities of phosphorylated and total JAK2 bands were quantified by densitometry, and their ratio was normalized to that at zero time (filled symbols). Intracellular ROS levels were measured by fluorescence-activated cell sorting (FACS) analysis in cells labeled with the ROS-sensitive dye DCF and were normalized to those at zero time (open symbols). Values are means ± SEMs (n = 3). *(JAK2 phosphorylation) and #(ROS levels), P < .05 vs cells at zero time. Data in this and other figures are representative of 3 independent experiments, which all gave similar results.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
NADPH oxidase inhibitor DPI prevents the second, sustained phase of JAK2 phosphorylation induced by GFs in PaCa cells. MIA PaCa-2 and PANC-1 cells were cultured for 72 hours (A, B, D, and E), or for the indicated times (C), without and with 100 ng/mL IGF-I or 15% FBS, in the absence and presence of 15 μmol/L DPI. (A–C) DPI or vehicle was added 15 minutes before the addition of GF. (D and E) DPI was added as indicated, either 15 minutes before or 1 hour after the GF.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Nox4 mediates the sustained phase of JAK2 phosphorylation induced by GFs. MIA PaCa-2 (A and B) and PANC-1 (C–F) cells were transfected (A–D) with scrambled (S) and antisense (AS) Nox4 oligonucleotides or (E and F) with control (C) and Nox4 siRNA plasmids. Cells were cultured for 72 hours with 100 ng/mL IGF-I or 15% FBS. (A, C, and E) The intensities of phosphorylated and total JAK2 bands were quantified by densitometry, and their ratio was normalized to that in the control transfections (ie, S or C, respectively) and presented below the blots. (B, D, and F) The efficiency of transfections was confirmed by immunoblotting, using anti-Nox4 antibody. Blots were reprobed for β-actin to confirm equal protein loading. The intensities of Nox4 and actin bands were quantified by densitometry, and their ratio was normalized to that in control (ie, S or C, respectively) and presented below the blots.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
JAK2 mediates GF-induced inhibition of apoptosis in PaCa cells. Sustained phase of JAK2 phosphorylation is required for the antiapoptotic effect of GFs. Wild-type MIA PaCa-2 and PANC-1 cells (A–C and G) and PANC-1 cells transfected with sense (S) and antisense (AS) JAK2 oligonucleotides (D–F) were cultured for 72 hours without and with 100 ng/mL IGF-I or 15% FBS, in the absence and presence of 15 μmol/L DPI or the JAK2 inhibitor AG490 at 50 μmol/L (A and G) or at indicated concentrations (B and C). The inhibitors were added 15 minutes before the GFs (A–C) or as indicated (G). (A, B, E, and G) Internucleosomal DNA fragmentation was measured using Cell Death Detection enzyme-linked immunoabsorbent assay (ELISA) kit and normalized to that in control cells (ie, wild-type cells cultured without inhibitors (A, B, and G) or cells transfected with sense JAK2 oligonucleotide and cultured without GFs (E). Values are means ± SEMs (n = 3). *P < .05 vs control cells; #P < .05 vs cells without AG490 (A and B), or cells transfected with sense JAK2 oligonucleotide (E) and cultured in the same conditions. (C and F), cells were labeled with AnV/PI and analyzed by FACS. AnV+/PI− cells were considered apoptotic; AnV+/PI+ cells comprise both necrotic cells and apoptotic cells undergoing secondary necrosis.3 (F) The percentage of cells in AnV+/PI− and AnV+/PI+ groups were normalized to that for cells in the same groups transfected with control (sense) JAK2 oligonucleotide. Values are means ± SEMs (n = 3). *P < .05 vs cells of the same group transfected with sense JAK2 oligonucleotide. (D) Phosphorylated and total JAK2 were measured by immunoblotting. Blots were reprobed for β-actin to confirm equal protein loading.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
STAT1 mediates prosurvival effects of JAK2 in PaCa cells. MIA PaCa-2 and PANC-1 cells wild-type (A), transfected with sense (S) and antisense (AS) JAK2 oligonucleotides (B and E), transfected with STAT1 siRNA or control scrambled siRNA (C, D, F, and G) were cultured for 72 hours without and with 100 ng/mL IGF-I or 15% FBS. (C and F) Internucleosomal DNA fragmentation was measured using Cell Death Detection ELISA kit and normalized to that in cells transfected with scrambled siRNA. Values are means ± SEMs (n = 3). *P < .05 vs cells transfected with control scrambled siRNA and cultured in the same conditions. (A, B, D, E, and G) Phosphorylated and total JAK2, phosphorylated and total STAT1, survivin, and XIAP were measured by immunoblotting.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
GFs inhibit PTP activity in PaCa cells through NADPH oxidase-dependent mechanism. Wild-type MIA PaCa-2 cells (A, B, and D) and PANC-1 cells transfected with sense (S) and antisense (AS) Nox4 oligonucleotides (C) were cultured for 72 hours (A–C) or for the indicated times (D) without and with 100 ng/mL IGF-I or 15% FBS, in the absence and presence of the phosphatase inhibitor, bisperoxyvanadate (bpV; 10 μmol/L), or 15 μmol/L DPI. (D) Cells were cultured without GFs for 72 hours (no FBS stimulation) and then stimulated with FBS for 3 minutes, or cultured with FBS for 72 hours. (A) JAK2 phosphorylation was measured by immunoblotting. (B–D) Total PTP activity was measured in cell lysates, using p-nitrophenylphosphate as a substrate, and normalized (B and D) to that in control cells (ie, cells cultured without GFs and DPI) or (C) to that in cells transfected with sense Nox4 oligonucleotide. Values are means ± SEMs (n = 4). *P < .05 vs control cells (ie, cells cultured without GFs and DPI); #P < .05 vs wild-type cells without DPI (B) or cells transfected with sense Nox4 oligonucleotide (C) and cultured in the same conditions.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
LMW-PTP is oxidized and inactivated by GF-induced NADPH oxidase in PaCa cells. (A) LMW-PTP was measured by immunoblotting in PaCa cell lines. (B–G) LMW-PTP was overexpressed in MIA PaCa-2 and PANC-1 cells by transfection with wild-type (WT) HA-tagged LMW-PTP expression plasmid. Cells were cultured after transfection for 72 hours without and with 100 ng/mL IGF-I or 15% FBS, in the absence and presence of 15 μmol/L DPI. (B) The transfection efficiency was confirmed by immunoblotting with anti-HA antibody. (C) PTP activity was measured in LMW-PTP immunoprecipitates obtained with anti-HA antibody, using p-nitrophenylphosphate as a substrate. Values are means ± SEMs (n = 4). *P <.05 vs control cells (ie, cultured without GFs and DPI). (D) Cells were incubated for 10 minutes with H2O2 at the indicated concentrations. Cell lysates were prepared and subjected to SDS-PAGE in the absence of reducing agents (ie, β-mercaptoethanol). Where indicated, 100 mmol/L DTT was added to the sample buffer. The blots were probed with anti-HA antibody and then reprobed for β-actin to confirm equal protein loading. (E–G) Cells were cultured for 72 hours at the indicated conditions. Where shown, the cells were treated with 10 μmol/L H2O2 for 10 minutes before collection. Cell lysates were prepared and subjected to SDS-PAGE in the absence of reducing agents (ie, β -mercaptoethanol). The blots were probed with anti-HA antibody. Western blots are representative of 3 independent experiments, which all gave similar results. The numbers on the left are protein molecular sizes in kilodaltons.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
PTP inhibition with dominant-negative LMW-PTP enhances the sustained JAK2 phosphorylation induced by IGF-I. MIA PaCa-2 and PANC-1 cells were transfected with HA-tagged LMW-PTP expression plasmids, either WT or catalytically inactive C12S mutant (mut), and then cultured for 72 hours without and with 100 ng/mL IGF-I or 15% FBS. (A) The transfection efficiency was confirmed by immunoblotting with anti-HA antibody. (B–E) JAK2 phosphorylation was measured by immunoblotting. The intensities of phosphorylated and total JAK2 bands were quantified by densitometry, and their ratio was normalized to that in cells transfected with wild-type LMW-PTP and cultured in the same conditions. Representative of 3 independent experiments, which all gave similar results.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
LMW-PTP colocalizes with Nox4 in human pancreatic adenocarcinoma tissue and in PaCa cell lines. MIA PaCa-2 and PANC-1 cells (A), human normal pancreatic tissue (A), and human pancreatic adenocarcinoma (B) were stained with an anti-Nox4 antibody and Alexa488 (green)–conjugated secondary antibody or with an antibody against LMW-PTP and Alexa555 (red)–conjugated secondary antibody. There was little staining with the secondary antibody alone, which was subtracted. The slides were viewed under laser scanning confocal microscope (×800). The sections of human pancreatic adenocarcinoma tissue sections were stained with H&E (B, lower right panel). Representative of similar results obtained on tissue specimens from 4 patients.
Gastroenterology  , DOI: ( /j.gastro )
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11. Figure 10
NADPH oxidase–mediated inhibition of PTPs enhances JAK2 phosphorylation and thus the antiapoptotic effect of IGF-I in PaCa cells. Schematic showing the NADPH oxidase–mediated signaling mechanism of the antiapoptotic effect of IGF-I in PaCa cells. IGF-I stimulates NADPH oxidase (Nox4) in PaCa cells, leading to oxidation (ie, inactivation) of PTPs such as LMW-PTP, and resulting in sustained activation of the antiapoptotic JAK2–STAT1/3.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions