- Email: [email protected]
S1815 Tissue Microarray Analysis in Pancreatic Adenocarcinoma
(0.1 μM), carbachol (1 mM) and bombesin (10 μM). Cells were stimulated for one hour, following a two hour pre-incubation with CRT0066101. PKD phosphorylation and activity were measured as well as zymogen activation, amylase secretion and cell injury. CRT0066101 dose-dependently inhibited secretagogue-induced PKD activation and autophosphorylation at Ser916 with IC50 ~ 3.75-5 mM but had no effect on PKC-dependent phosphorylation of the PKD activation loop (Ser744/748). Importantly, we observed that secretagogue-induced trypsinogen and chymotrypsinogen activation were significantly decreased by the inhibitor (~76% and 71% from maximal for CCK; 45% and 68% from maximal for carbachol; 68% and 81% from maximal for bombesin; trypsin and chymotrypsin activities are given respectively). Similarly, the inhibitor reduced secretagogue-induced amylase secretion (43% reduction for CCK; 41% reduction for carbachol; 50% reduction for bombesin). The inhibitor did not affect basal zymogen activation or secretion. In addition, PKD inhibition did not affect secretagogue-induced cell injury. CONCLUSIONS: Our results indicate that CRT0066101 is a potent and specific PKD inhibitor and identify PKD as a potential mediator of zymogen activation and secretion induced by a range of secretagogues in pancreatic acinar cells.
S1815
AGA Abstracts
Tissue Microarray Analysis in Pancreatic Adenocarcinoma Andrada Seicean, Daniel Popa, Radu Seicean, Ovidiu Balacescu The pathogenesis of pancreatic ductal adenocarcinoma involves the multi-stage development of molecular aberrations affecting signaling pathways that regulate cancer growth and progression. Tissue microarray analysis of pancreatic tumors allows simultaneous assessment of genetic disorders, which can lead to identification of biomarkers of poor prognosis. Aim: To characterize the gene expression of pancreatic adenocarcinoma compared to normal tissue from the same patient. Materials and methods. We investigated seven samples of T3 pancreatic adenocarcinoma obtained intraoperatively and compared them to normal pancreatic tissue from the same patients. RNA was extracted and assessed qualitatively and quantitatively, followed by amplification of cDNA using reverse transcriptase, cRNA synthesis, and hybridization of microarray slides. For each sample 100 ng of total RNA was available. The overexpressed and underexpressed genes were classified by their known function in the cell. We selected six genes for analysis, some described previously in pancreatic pathology, and used RT-PCR for validation. Results: Five genes were overexpressed and 45 were underexpressed in the pancreatic adenocarcinoma samples. The overexpressed gene progastricsin PGC, with a role in extracellular matrix degradation, was validated by RT-PCR. Among the underexpressed genes we validated the protein kinase genes BCK1 and FABP4, with roles in desmoplastic reaction CD36, which modulates insulin secretion; UNC13A, involved in formation of presynaptic vesicles and probably nervous hyperexcitability; and RNH1, with a role in the inhibition of angiogenesis. The gene MUC12, with a role in epithelial growth, was underexpressed in microarray analysis, but overexpressed on validation. Conclusion: Microarray tissue analysis of pancreatic adenocarcinoma showed more underexpressed than overexpressed genes. After validation , the overexpressed gene progastricsin PGC was shown to be one factor responsible for tumor progression and metastasis formation. The possible role in pancreatic cancer of loss of MUC12, known to be an early event of carcinogenesis in colorectal cancer, should be further clarified.
S1818 CCK and TPA Stimulate PKCθ Interaction with Proteins Implicated in Determining Growth/Survival/Apoptosis in Pancreatic Acinar Cells Veronica Sancho, Samuel A. Mantey, Nieves Gonzalez, Tapas K. Pradhan, Robert T. Jensen Introduction: The novel PKCθ, described in hematopoietic and muscle cells, has recently been shown to occur in pancreatic acinar cells and to be activated by pancreatic stimulants. In other tissues, PKCθ activation is an important mediator in cell proliferation, apoptosis, differentiation and metabolism, but in pancreatic acinar cells the possible role of of PKCθ in these or other processes is unknown. Aim: To determine the possible signaling role of PKCθ in mediating proliferative and apoptotic cascades by determining its ability to interact with key signaling proteins important in the regulation of these processes. Methods: Rat pancreas acini were obtained by enzymatic digestion. Interaction between PKCθ and other proteins after stimulation with CCK or TPA was determined by co-immunoprecipitation (CoIP) and subsequent Western blotting and immunodetection using specific antibodies. Results: The specificity of the antibodies used to CoIP PKCθ was determined by immunoprecipitation of samples from pancreas acini in parallel with human recombinant PKCθ, and then detecting by Western blotting using a specific PKCθ antibody. Both CCK and the PKC activator, TPA, stimulated T538 phosphorylation of PKCθ. At a concentration of 10nM CCK at 2.5 min the maximal effect was seen, whereas with TPA, this occurred at 1μΜ and 2.5 min. Therefore these conditions were used to assess by CoIP possible interactions with other proteins. In the absence of any stimulus a direct association of PKCθ with AKT, an enzyme that plays a central role in regulation proliferation/apoptosis, was found. Incubation with CCK or TPA stimulated an increased association of PKCθ with AKT and the increase was greater with CCK than TPA. Both CCK and TPA stimulated an association of PKCθ with Bcl-10, an adaptor protein that forms complexes with other proteins and induces apoptosis, and the increase in the association was similar for CCK and TPA. Both CCK and TPA, also stimulated an association of PKCθ with Src, which has been shown being important in mediating in the regulation of secretion, growth and protein synthesis in pancreas acini. The increase association of PKCθ with Src was greater with CCK than TPA. Conclusions: These results demonstrate that both CCK and TPA stimulate PKCθ activation and its association with key regulators of pancreatic signaling molecules involved in growth and/or apoptosis (AKT, Bcl-10 and Src). These results suggest that it is important to assess the role of this recently described pancreatic novel PKC, in addition to other well-established PKCs, in mediating these processes, and in response to pancreatic acinar injury, such as occurs in pancreatitis.
S1816 GDNF Influences Pancreatic β-Cell Development Through Increased Pancreatic Endocrine Progenitor Expression Simon Mwangi, Bindu P. Chandrasekharan, Mallappa Anitha, Yousef Usta, Shanthi V. Sitaraman, Shanthi Srinivasan Background and aims: Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor produced by glial cells that plays a critical role in the development and survival of the enteric nervous system. Our previous studies have shown that GDNF transgenic (GDNFtg) mice that over-express GDNF in pancreatic glia under the control of the glial fibrillary acidic protein have larger β-cell mass and improved glucose control than their wild type (WT) littermates. The role that GDNF plays in early β-cell development is not known. We examined the influence of GDNF on the expression of markers for pancreatic endocrine cell progenitors and β-cell differentiation. Methods: Pancreata were obtained from embryonic day 15.5 (E15.5) to 7 days-old GDNF-tg mice and WT littermates, and gene expression assessed by Real-Time PCR on laser capture microscopy isolated pancreatic islets. E15.5 WT mice pancreas rudiments were also cultured for 24h in the presence or absence of GDNF, stained with dithiozone to identify insulin expressing cells, and gene expression assessed by Real-Time PCR. The hamster HIT-T15 β-cell line was transfected with Pdx1 siRNA and markers of β-cell differentiation (Pax6) assessed by Western blotting and immunostaining to examine the role of PDX1 in GDNF-mediated β-cell differentiation. Results: E15.5 WT mice pancreas rudiments cultured in the presence of GDNF for 24h showed better islet differentiation and increased insulin staining. Analysis of gene expression revealed 2.5 fold higher insulin mRNA expression in islets from rudiments cultured in the presence of GDNF than in vehicle only (P<0.05). In islets isolated by LCM from 7 day old mice, mRNA of markers of endocrine progenitors such as Neurogenin3, NeuroD1, Pdx1 and Sox9, respectively, were 7.2, 6.5, 5.7, and 3.2 fold higher in GDNF-tg mice than in WT littermates (P<0.05). Insulin expression was also higher in these mice (15 fold; P<0.05). GDNF exposure (48h) increased differentiation in HIT-T15 cells as seen by higher levels of Pax6 and insulin both by immunostaining and Western blotting compared to cells cultured in the presence of vehicle alone (P<0.05). This effect of GDNF was abolished when the cells were transfected with Pdx1 siRNA, indicating a critical role of Pdx-1 in β-cell differentiation. Conclusions: We have demonstrated that GDNF influences early β-cell development through increased endocrine progenitor formation. Pdx-1 plays a critical role in GDNF mediated β-cell development and differentiation. Understanding the mechanism of GDNF influence on β-cell development may lead to new therapeutic strategies for the treatment or prevention of diabetes.
S1819 Ethanol Impairs Pancreatic Amylase Secretion and Cell-Cell Adhesion via SRC Pathways, Vav-2 and Catenin P120 in CCK-Stimulated Pancreatic Acini Tomoyuki Iwata, Fumihiko Nozu, Michio Imawari Background: We previously demonstrated that Src-RhoA-Phosphoinositide 3-kinase (PI3K) pathways are involved in Gα13-mediated pathways via Rho guanine nucleotide exchange factor, Vav-2 and recruit cell-cell adhesions via cadherin and catenin in CCK-stimulated pancreatic acini. Although ethanol abuse is one of the common risk factors for acute and chronic pancreatitis, the precise interactions between cell-cell adhesion proteins and ethanol have not been clarified in pancreatic acini. Aim: We attempted to evaluate the association of Src-RhoA-PI3K pathways, Gα13, Vav-2, p120, E-cadherin (EC) and ethanol in CCKstimulated pancreatic acini. Methods: Isolated acini were obtained from male Sprague-Dawley rats. Intact acini were preincubated with ethanol and incubated with CCK. Protein expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC were analyzed by Western immunoblotting (WB) and their interactions were examined by immunoprecipitation (IP). Amylase secretion was also measured. Results: In WB study, CCK (10 pM - 10 nM) enhanced the expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC. Although the pretreatment of 5 mM ethanol did not alter the expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC in CCK-treated pancreatic acini, the pretreatment of 20 and 100 mM ethanol inhibited the expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC in response to CCK stimulation. In IP study, p120 was co-immunoprecipitated with EC, Src, RhoA, PI3K and Gα13 in response to CCK stimulation and Vav-2 was co-immunoprecipitated with Src, RhoA,PI3K and Gα13 in response to CCK stimulation. 5 mM ethanol did not alter the association of p120 with EC, Src, RhoA, PI3K and Gα13 and the association of Vav-2 with Src, RhoA, PI3K and Gα13. On the other hand, 20 and 100 mM ethanol diminished the association of p120 with EC, Src, RhoA, PI3K and Gα13 and the association of Vav-2 with Src, RhoA, PI3K and Gα13. CCK stimulated amylase secretion in biphasic manner and the maximum secretion was seen with the concentration of CCK 100 pM. The pretreatment of 5 mM ethanol did not inhibit amylase secretion in CCK-treated pancreatic acini without altering basal secretion. On the other hands, the pretreatment of 20 and 100 mM ethanol inhibited amylase secretion and shifted the maximum response dose of CCK from 100 pM to 1 nM without altering basal secretion. Conclusion: We conclude that ethanol impairs pancreatic amylase secretion and cell-cell adhesion via Src-RhoA-Phosphoinositide 3-kinase pathways, Gα13-mediated pathways, Vav-2 and p120 in CCK-stimulated pancreatic acini.
S1817 Protein Kinase D Modulates Secretagogue-Induced Zymogen Activation and Amylase Secretion in Rat Pancreatic Acinar Cells Edwin C. Thrower, Jingzhen Yuan, Courtney Jones, Ashar Usmani, Meghan K. Kelly, Christopher Ireson, Stephen J. Pandol, Sushovan Guha BACKGROUND: Previous studies have shown that novel protein kinase C (PKC isoforms) mediate zymogen activation, a key early event in acute pancreatitis (AP). In pancreatic acinar cells novel PKC isoforms have been shown to phosphorylate a distinct serine/threonine kinase, protein kinase D (PKD) at residues S744/748. PKD also autophosphorylates at residue S916. These phosphorylation events are involved in activation of PKD. We hypothesize that PKD is the downstream target of PKC and is involved in premature zymogen activation in rat pancreatic acinar cells. AIM: The purpose of our study was to test our hypothesis that PKD is involved in secretagogue-induced premature zymogen activation in rat pancreatic acinar cells. METHODS AND RESULTS: Using a high-throughput screening strategy, we discovered a small molecule inhibitor CRT0066101 that specifically blocks PKD activation. We used CRT0066101 to determine whether PKD is involved in secretagogue-induced zymogen activation. Isolated rat pancreatic acinar cells were stimulated with high concentrations of the following secretagogues to induce zymogen activation: cholecystokinin (CCK)
AGA Abstracts
A-276
S1815
AGA Abstracts
Tissue Microarray Analysis in Pancreatic Adenocarcinoma Andrada Seicean, Daniel Popa, Radu Seicean, Ovidiu Balacescu The pathogenesis of pancreatic ductal adenocarcinoma involves the multi-stage development of molecular aberrations affecting signaling pathways that regulate cancer growth and progression. Tissue microarray analysis of pancreatic tumors allows simultaneous assessment of genetic disorders, which can lead to identification of biomarkers of poor prognosis. Aim: To characterize the gene expression of pancreatic adenocarcinoma compared to normal tissue from the same patient. Materials and methods. We investigated seven samples of T3 pancreatic adenocarcinoma obtained intraoperatively and compared them to normal pancreatic tissue from the same patients. RNA was extracted and assessed qualitatively and quantitatively, followed by amplification of cDNA using reverse transcriptase, cRNA synthesis, and hybridization of microarray slides. For each sample 100 ng of total RNA was available. The overexpressed and underexpressed genes were classified by their known function in the cell. We selected six genes for analysis, some described previously in pancreatic pathology, and used RT-PCR for validation. Results: Five genes were overexpressed and 45 were underexpressed in the pancreatic adenocarcinoma samples. The overexpressed gene progastricsin PGC, with a role in extracellular matrix degradation, was validated by RT-PCR. Among the underexpressed genes we validated the protein kinase genes BCK1 and FABP4, with roles in desmoplastic reaction CD36, which modulates insulin secretion; UNC13A, involved in formation of presynaptic vesicles and probably nervous hyperexcitability; and RNH1, with a role in the inhibition of angiogenesis. The gene MUC12, with a role in epithelial growth, was underexpressed in microarray analysis, but overexpressed on validation. Conclusion: Microarray tissue analysis of pancreatic adenocarcinoma showed more underexpressed than overexpressed genes. After validation , the overexpressed gene progastricsin PGC was shown to be one factor responsible for tumor progression and metastasis formation. The possible role in pancreatic cancer of loss of MUC12, known to be an early event of carcinogenesis in colorectal cancer, should be further clarified.
S1818 CCK and TPA Stimulate PKCθ Interaction with Proteins Implicated in Determining Growth/Survival/Apoptosis in Pancreatic Acinar Cells Veronica Sancho, Samuel A. Mantey, Nieves Gonzalez, Tapas K. Pradhan, Robert T. Jensen Introduction: The novel PKCθ, described in hematopoietic and muscle cells, has recently been shown to occur in pancreatic acinar cells and to be activated by pancreatic stimulants. In other tissues, PKCθ activation is an important mediator in cell proliferation, apoptosis, differentiation and metabolism, but in pancreatic acinar cells the possible role of of PKCθ in these or other processes is unknown. Aim: To determine the possible signaling role of PKCθ in mediating proliferative and apoptotic cascades by determining its ability to interact with key signaling proteins important in the regulation of these processes. Methods: Rat pancreas acini were obtained by enzymatic digestion. Interaction between PKCθ and other proteins after stimulation with CCK or TPA was determined by co-immunoprecipitation (CoIP) and subsequent Western blotting and immunodetection using specific antibodies. Results: The specificity of the antibodies used to CoIP PKCθ was determined by immunoprecipitation of samples from pancreas acini in parallel with human recombinant PKCθ, and then detecting by Western blotting using a specific PKCθ antibody. Both CCK and the PKC activator, TPA, stimulated T538 phosphorylation of PKCθ. At a concentration of 10nM CCK at 2.5 min the maximal effect was seen, whereas with TPA, this occurred at 1μΜ and 2.5 min. Therefore these conditions were used to assess by CoIP possible interactions with other proteins. In the absence of any stimulus a direct association of PKCθ with AKT, an enzyme that plays a central role in regulation proliferation/apoptosis, was found. Incubation with CCK or TPA stimulated an increased association of PKCθ with AKT and the increase was greater with CCK than TPA. Both CCK and TPA stimulated an association of PKCθ with Bcl-10, an adaptor protein that forms complexes with other proteins and induces apoptosis, and the increase in the association was similar for CCK and TPA. Both CCK and TPA, also stimulated an association of PKCθ with Src, which has been shown being important in mediating in the regulation of secretion, growth and protein synthesis in pancreas acini. The increase association of PKCθ with Src was greater with CCK than TPA. Conclusions: These results demonstrate that both CCK and TPA stimulate PKCθ activation and its association with key regulators of pancreatic signaling molecules involved in growth and/or apoptosis (AKT, Bcl-10 and Src). These results suggest that it is important to assess the role of this recently described pancreatic novel PKC, in addition to other well-established PKCs, in mediating these processes, and in response to pancreatic acinar injury, such as occurs in pancreatitis.
S1816 GDNF Influences Pancreatic β-Cell Development Through Increased Pancreatic Endocrine Progenitor Expression Simon Mwangi, Bindu P. Chandrasekharan, Mallappa Anitha, Yousef Usta, Shanthi V. Sitaraman, Shanthi Srinivasan Background and aims: Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor produced by glial cells that plays a critical role in the development and survival of the enteric nervous system. Our previous studies have shown that GDNF transgenic (GDNFtg) mice that over-express GDNF in pancreatic glia under the control of the glial fibrillary acidic protein have larger β-cell mass and improved glucose control than their wild type (WT) littermates. The role that GDNF plays in early β-cell development is not known. We examined the influence of GDNF on the expression of markers for pancreatic endocrine cell progenitors and β-cell differentiation. Methods: Pancreata were obtained from embryonic day 15.5 (E15.5) to 7 days-old GDNF-tg mice and WT littermates, and gene expression assessed by Real-Time PCR on laser capture microscopy isolated pancreatic islets. E15.5 WT mice pancreas rudiments were also cultured for 24h in the presence or absence of GDNF, stained with dithiozone to identify insulin expressing cells, and gene expression assessed by Real-Time PCR. The hamster HIT-T15 β-cell line was transfected with Pdx1 siRNA and markers of β-cell differentiation (Pax6) assessed by Western blotting and immunostaining to examine the role of PDX1 in GDNF-mediated β-cell differentiation. Results: E15.5 WT mice pancreas rudiments cultured in the presence of GDNF for 24h showed better islet differentiation and increased insulin staining. Analysis of gene expression revealed 2.5 fold higher insulin mRNA expression in islets from rudiments cultured in the presence of GDNF than in vehicle only (P<0.05). In islets isolated by LCM from 7 day old mice, mRNA of markers of endocrine progenitors such as Neurogenin3, NeuroD1, Pdx1 and Sox9, respectively, were 7.2, 6.5, 5.7, and 3.2 fold higher in GDNF-tg mice than in WT littermates (P<0.05). Insulin expression was also higher in these mice (15 fold; P<0.05). GDNF exposure (48h) increased differentiation in HIT-T15 cells as seen by higher levels of Pax6 and insulin both by immunostaining and Western blotting compared to cells cultured in the presence of vehicle alone (P<0.05). This effect of GDNF was abolished when the cells were transfected with Pdx1 siRNA, indicating a critical role of Pdx-1 in β-cell differentiation. Conclusions: We have demonstrated that GDNF influences early β-cell development through increased endocrine progenitor formation. Pdx-1 plays a critical role in GDNF mediated β-cell development and differentiation. Understanding the mechanism of GDNF influence on β-cell development may lead to new therapeutic strategies for the treatment or prevention of diabetes.
S1819 Ethanol Impairs Pancreatic Amylase Secretion and Cell-Cell Adhesion via SRC Pathways, Vav-2 and Catenin P120 in CCK-Stimulated Pancreatic Acini Tomoyuki Iwata, Fumihiko Nozu, Michio Imawari Background: We previously demonstrated that Src-RhoA-Phosphoinositide 3-kinase (PI3K) pathways are involved in Gα13-mediated pathways via Rho guanine nucleotide exchange factor, Vav-2 and recruit cell-cell adhesions via cadherin and catenin in CCK-stimulated pancreatic acini. Although ethanol abuse is one of the common risk factors for acute and chronic pancreatitis, the precise interactions between cell-cell adhesion proteins and ethanol have not been clarified in pancreatic acini. Aim: We attempted to evaluate the association of Src-RhoA-PI3K pathways, Gα13, Vav-2, p120, E-cadherin (EC) and ethanol in CCKstimulated pancreatic acini. Methods: Isolated acini were obtained from male Sprague-Dawley rats. Intact acini were preincubated with ethanol and incubated with CCK. Protein expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC were analyzed by Western immunoblotting (WB) and their interactions were examined by immunoprecipitation (IP). Amylase secretion was also measured. Results: In WB study, CCK (10 pM - 10 nM) enhanced the expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC. Although the pretreatment of 5 mM ethanol did not alter the expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC in CCK-treated pancreatic acini, the pretreatment of 20 and 100 mM ethanol inhibited the expressions of Src, RhoA, PI3K, Gα13, Vav-2, p120 and EC in response to CCK stimulation. In IP study, p120 was co-immunoprecipitated with EC, Src, RhoA, PI3K and Gα13 in response to CCK stimulation and Vav-2 was co-immunoprecipitated with Src, RhoA,PI3K and Gα13 in response to CCK stimulation. 5 mM ethanol did not alter the association of p120 with EC, Src, RhoA, PI3K and Gα13 and the association of Vav-2 with Src, RhoA, PI3K and Gα13. On the other hand, 20 and 100 mM ethanol diminished the association of p120 with EC, Src, RhoA, PI3K and Gα13 and the association of Vav-2 with Src, RhoA, PI3K and Gα13. CCK stimulated amylase secretion in biphasic manner and the maximum secretion was seen with the concentration of CCK 100 pM. The pretreatment of 5 mM ethanol did not inhibit amylase secretion in CCK-treated pancreatic acini without altering basal secretion. On the other hands, the pretreatment of 20 and 100 mM ethanol inhibited amylase secretion and shifted the maximum response dose of CCK from 100 pM to 1 nM without altering basal secretion. Conclusion: We conclude that ethanol impairs pancreatic amylase secretion and cell-cell adhesion via Src-RhoA-Phosphoinositide 3-kinase pathways, Gα13-mediated pathways, Vav-2 and p120 in CCK-stimulated pancreatic acini.
S1817 Protein Kinase D Modulates Secretagogue-Induced Zymogen Activation and Amylase Secretion in Rat Pancreatic Acinar Cells Edwin C. Thrower, Jingzhen Yuan, Courtney Jones, Ashar Usmani, Meghan K. Kelly, Christopher Ireson, Stephen J. Pandol, Sushovan Guha BACKGROUND: Previous studies have shown that novel protein kinase C (PKC isoforms) mediate zymogen activation, a key early event in acute pancreatitis (AP). In pancreatic acinar cells novel PKC isoforms have been shown to phosphorylate a distinct serine/threonine kinase, protein kinase D (PKD) at residues S744/748. PKD also autophosphorylates at residue S916. These phosphorylation events are involved in activation of PKD. We hypothesize that PKD is the downstream target of PKC and is involved in premature zymogen activation in rat pancreatic acinar cells. AIM: The purpose of our study was to test our hypothesis that PKD is involved in secretagogue-induced premature zymogen activation in rat pancreatic acinar cells. METHODS AND RESULTS: Using a high-throughput screening strategy, we discovered a small molecule inhibitor CRT0066101 that specifically blocks PKD activation. We used CRT0066101 to determine whether PKD is involved in secretagogue-induced zymogen activation. Isolated rat pancreatic acinar cells were stimulated with high concentrations of the following secretagogues to induce zymogen activation: cholecystokinin (CCK)
AGA Abstracts
A-276