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Waf1 controls acinar to-duct metaplasia in cerulein-induced pancreatitis
Abstracts / Pancreatology 12 (2012) 502–597
Introduction: The endogenous immune response is influenced by vagotomy. Stimulation or destruction of the vagaus nerve leads to direct release of anti- or pro-inflammatory mediators. These mediators play a key role in the progression of mild edematous to severe necrotizing pancreatitis with systemic complications as pulmonary damage, SIRS and sepsis. The presented study analyzes the influence of pharmacologic stimulation of the vagal nerve system on the severity of experimental necrotizing pancreatitis in rats. Methods: A severe acute necrotizing pancreatitis was induced in rats by using the GDOC-model. Therapy groups received either nicotine, physiostigmin or neostigmin for stimulation of the vagal nerve system. The agents were applied either directly after induction of acute pancreatitis (prophylactic) or 3 hours in a delayed therapy group (therapeutic). The results were compared to animals that had pancreatitis alone or healthy controls. The evaluation was performed 12 hours after induction of acute necrotizing pancreatitis. Results: Histological evaluation of the pancreas in the pancreatitis only group confirmed a severe necrotizing pancreatitis compared to healthy controls regarding edema, inflammation and necrosis. Pharmacologic stimulation of the vagal nerve with nicotine, physiostigmin or neostigmin revealed an attenuated morphologic damage with regard to inflammation (p<0,005, respectively) and necrosis (p<0,05, respectively). The attenuated injury was independent of the application time (prophylactic or therapeutic). Conclusion: Pharmacologic stimulation of the vagal nerve attenuates pancreatic morphological injury in acute necrotizing pancreatitis in rats and should be further evaluated as a treatment approach.
P120. CUX1 stimulates pancreatic cancer progression by modulating the N F k B- de pen d en t c yto k in e ex p re s si o n in t u mo r-a ss oc i a te d macrophages
537
121. Cell cycle inhibitor p21/Waf1 controls acinar to-duct metaplasia in cerulein-induced pancreatitis K. Grabliauskaite, S. Sonda, M. Bain, T. Reding, R. Graf. Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland Introduction: In patients and in animal models of pancreatitis a transdifferentiation converting pancreatic acinar cells to duct-like cells (acinar-to-duct metaplasia (ADM)) has been observed together with acinar cell proliferation. Mouse models suggest that ADM is a precursor lesion of pancreatic adenocarcinoma. p21/Waf1 is a major regulator of cell cycle and activation of this protein leads to the cell cycle arrest. Aims/objectives: In this study we aim to investigate the contribution of p21/Waf1 to ADM and acinar cell proliferation during pancreatitis. Methods: Chronic pancreatitis was induced in WT and p21-/- mice by multiple injections of cerulein over a period of 14 days. The expression of proliferation markers, cell cycle regulators, growth factors and the severity of tissue inflammation wsd analyzed by immunohistochemistry and qRT-PCR. Results: p21/Waf1 expression was upregulated after induction of chronic pancreatitis in WT mice. After cerulein treatment p21-/- mice showed considerable areas of ADM along with downregulation of amylase and p48. A high number of infiltrating leukocytes together with extended fibrosis was observed in metaplastic areas. In p21-/- mice gene expression of the four main cyclins (D,E,A,B) was elevated with the highest increase in cyclin B. However, the increase in the number of proliferating acinar cells was modest in p21-/compared to WT mice. Moreover, the absence of p21/Waf1 was accompanied by upregulation of other cell cycle inhibitors, especially p16. Conclusion: p21/Waf1 is a negative regulator of ADM formation but has limited contribution to the number of proliferating acinar cells. Other cell cycle inhibitors appear to compensate p21/Waf1 loss and prevent completion of mitosis.
B. Kuehnemuth, H. Griesmann, L. Muehlberg, M. Buchholz, T. Gress, P. Michl. Dept. of Gastroenterology, Philipps-University of Marburg, Germany Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive infiltration of inflammatory stroma cells including tumor-associated macrophages (TAM). Previously, we could demonstrate high expression levels of the transcription factor CUX1 in both pancreatic tumor cells and tumor-associated macrophages. In addition, we identified CUX1 as important mediator of tumor progression and invasiveness in pancreatic cancer cells. Objectives: In vivo and in vitro characterization of the effects of CUX1 in tumor-associated macrophages. Methods: CUX1 expression in TAM was analyzed by immunohistochemistry in PDAC tissues. The role of CUX1 in macrophages was evaluated using siRNA and overexpression techniques, and its effect on transcription of secreted cytokines was profiled using a multiplex quantitative RT-PCR approach. CUX1 target genes were validated with RT-PCR, ELISA and reporter assays. The regulation of these genes by CUX1 was examined performing DNA-pulldown experiments. The functional impact of CUX1 and its targets was analyzed using migration and angiogenesis assays. Results: Immunohistochemical co-staining revealed strong expression levels of CUX1 in TAMs of pancreatic cancer tissues. Profiling experiments showed that CUX1 downregulates several cytokines which have been associated with M1 differentiation and tumor suppression. The downregulation of CXCL10 and CCL5 was verified on RNA and protein level, and the transcriptional regulation of CXCL10 by CUX1 could be verified on promoter level. DNA-pulldown experiments showed that CUX1 directly binds to the promotors of CCL5 and CXCL10 and is able to inhibit the binding of NFkB. Functionally, we could show that suppressed CXCL10 led to a enhanced tumor neoangiogenesis. Conclusion: CUX1 promotes the tumor progression of pancreatic cancer by modulating the cytokine profile in tumor-associated macrophages via inhibition of NFkB activity.
P122. Involvement of the RNA-binding proteins Sam68 and SRSF1 in the acquisition of the resistance to gemcitabine in pancreatic adenocarcinoma cells S. Calabretta 1, 2, L. Adesso 1, 2, R. Geremia 2, G. Capurso 1, G. Delle Fave 1, C. Sette 2. 1 Medical Surgical Department of Tecnobiomedical Clinical Sciences and Translational Medicine, University of La Sapienza, Rome, Italy 2 Department of Public Health and Cell Biology, University of Rome Tor Vergata, Via Montpellier, 1, 00133 Rome, Italy
Introduction: The limited effect of conventional chemotherapy in pancreatic adenocarcinoma (PDAC) urges for novel therapies, targeting more directly the molecular aberrations of this disease. The molecular characterization of the drug resistant phenotype of PDAC cells remain unexplored, even though some evidence suggests a correlation with the expression of mesenchymal markers (1). The epithelial-to-mesenchymal transition (EMT) is promoted by finelytuned changes in gene expression, at both transcriptional and splicing levels (2). In addition to the well described role of transcription factors in EMT, recent observations have shown the requirement for select splicing factors in this transtition (3,4). Aim/objectives: Characterization of the molecular events that lead to chemotherapeutic resistance in PDAC cells. Methods: Drug resistant PDAC subpopulation was selected after chronic exposure to gemcitabine. Western blot analyses for the expression of cancer related proteins; RNA-interference of selected genes to investigate their function. Trypan blue staining to analyze cell survival. Results: Chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells displaying a mesenchymal phenotype, which remains less sensitive to drug-induced cell death. These cells express higher levels of Sam68 and SRSF1 splicing factors. Depletion by RNA-
Introduction: The endogenous immune response is influenced by vagotomy. Stimulation or destruction of the vagaus nerve leads to direct release of anti- or pro-inflammatory mediators. These mediators play a key role in the progression of mild edematous to severe necrotizing pancreatitis with systemic complications as pulmonary damage, SIRS and sepsis. The presented study analyzes the influence of pharmacologic stimulation of the vagal nerve system on the severity of experimental necrotizing pancreatitis in rats. Methods: A severe acute necrotizing pancreatitis was induced in rats by using the GDOC-model. Therapy groups received either nicotine, physiostigmin or neostigmin for stimulation of the vagal nerve system. The agents were applied either directly after induction of acute pancreatitis (prophylactic) or 3 hours in a delayed therapy group (therapeutic). The results were compared to animals that had pancreatitis alone or healthy controls. The evaluation was performed 12 hours after induction of acute necrotizing pancreatitis. Results: Histological evaluation of the pancreas in the pancreatitis only group confirmed a severe necrotizing pancreatitis compared to healthy controls regarding edema, inflammation and necrosis. Pharmacologic stimulation of the vagal nerve with nicotine, physiostigmin or neostigmin revealed an attenuated morphologic damage with regard to inflammation (p<0,005, respectively) and necrosis (p<0,05, respectively). The attenuated injury was independent of the application time (prophylactic or therapeutic). Conclusion: Pharmacologic stimulation of the vagal nerve attenuates pancreatic morphological injury in acute necrotizing pancreatitis in rats and should be further evaluated as a treatment approach.
P120. CUX1 stimulates pancreatic cancer progression by modulating the N F k B- de pen d en t c yto k in e ex p re s si o n in t u mo r-a ss oc i a te d macrophages
537
121. Cell cycle inhibitor p21/Waf1 controls acinar to-duct metaplasia in cerulein-induced pancreatitis K. Grabliauskaite, S. Sonda, M. Bain, T. Reding, R. Graf. Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland Introduction: In patients and in animal models of pancreatitis a transdifferentiation converting pancreatic acinar cells to duct-like cells (acinar-to-duct metaplasia (ADM)) has been observed together with acinar cell proliferation. Mouse models suggest that ADM is a precursor lesion of pancreatic adenocarcinoma. p21/Waf1 is a major regulator of cell cycle and activation of this protein leads to the cell cycle arrest. Aims/objectives: In this study we aim to investigate the contribution of p21/Waf1 to ADM and acinar cell proliferation during pancreatitis. Methods: Chronic pancreatitis was induced in WT and p21-/- mice by multiple injections of cerulein over a period of 14 days. The expression of proliferation markers, cell cycle regulators, growth factors and the severity of tissue inflammation wsd analyzed by immunohistochemistry and qRT-PCR. Results: p21/Waf1 expression was upregulated after induction of chronic pancreatitis in WT mice. After cerulein treatment p21-/- mice showed considerable areas of ADM along with downregulation of amylase and p48. A high number of infiltrating leukocytes together with extended fibrosis was observed in metaplastic areas. In p21-/- mice gene expression of the four main cyclins (D,E,A,B) was elevated with the highest increase in cyclin B. However, the increase in the number of proliferating acinar cells was modest in p21-/compared to WT mice. Moreover, the absence of p21/Waf1 was accompanied by upregulation of other cell cycle inhibitors, especially p16. Conclusion: p21/Waf1 is a negative regulator of ADM formation but has limited contribution to the number of proliferating acinar cells. Other cell cycle inhibitors appear to compensate p21/Waf1 loss and prevent completion of mitosis.
B. Kuehnemuth, H. Griesmann, L. Muehlberg, M. Buchholz, T. Gress, P. Michl. Dept. of Gastroenterology, Philipps-University of Marburg, Germany Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive infiltration of inflammatory stroma cells including tumor-associated macrophages (TAM). Previously, we could demonstrate high expression levels of the transcription factor CUX1 in both pancreatic tumor cells and tumor-associated macrophages. In addition, we identified CUX1 as important mediator of tumor progression and invasiveness in pancreatic cancer cells. Objectives: In vivo and in vitro characterization of the effects of CUX1 in tumor-associated macrophages. Methods: CUX1 expression in TAM was analyzed by immunohistochemistry in PDAC tissues. The role of CUX1 in macrophages was evaluated using siRNA and overexpression techniques, and its effect on transcription of secreted cytokines was profiled using a multiplex quantitative RT-PCR approach. CUX1 target genes were validated with RT-PCR, ELISA and reporter assays. The regulation of these genes by CUX1 was examined performing DNA-pulldown experiments. The functional impact of CUX1 and its targets was analyzed using migration and angiogenesis assays. Results: Immunohistochemical co-staining revealed strong expression levels of CUX1 in TAMs of pancreatic cancer tissues. Profiling experiments showed that CUX1 downregulates several cytokines which have been associated with M1 differentiation and tumor suppression. The downregulation of CXCL10 and CCL5 was verified on RNA and protein level, and the transcriptional regulation of CXCL10 by CUX1 could be verified on promoter level. DNA-pulldown experiments showed that CUX1 directly binds to the promotors of CCL5 and CXCL10 and is able to inhibit the binding of NFkB. Functionally, we could show that suppressed CXCL10 led to a enhanced tumor neoangiogenesis. Conclusion: CUX1 promotes the tumor progression of pancreatic cancer by modulating the cytokine profile in tumor-associated macrophages via inhibition of NFkB activity.
P122. Involvement of the RNA-binding proteins Sam68 and SRSF1 in the acquisition of the resistance to gemcitabine in pancreatic adenocarcinoma cells S. Calabretta 1, 2, L. Adesso 1, 2, R. Geremia 2, G. Capurso 1, G. Delle Fave 1, C. Sette 2. 1 Medical Surgical Department of Tecnobiomedical Clinical Sciences and Translational Medicine, University of La Sapienza, Rome, Italy 2 Department of Public Health and Cell Biology, University of Rome Tor Vergata, Via Montpellier, 1, 00133 Rome, Italy
Introduction: The limited effect of conventional chemotherapy in pancreatic adenocarcinoma (PDAC) urges for novel therapies, targeting more directly the molecular aberrations of this disease. The molecular characterization of the drug resistant phenotype of PDAC cells remain unexplored, even though some evidence suggests a correlation with the expression of mesenchymal markers (1). The epithelial-to-mesenchymal transition (EMT) is promoted by finelytuned changes in gene expression, at both transcriptional and splicing levels (2). In addition to the well described role of transcription factors in EMT, recent observations have shown the requirement for select splicing factors in this transtition (3,4). Aim/objectives: Characterization of the molecular events that lead to chemotherapeutic resistance in PDAC cells. Methods: Drug resistant PDAC subpopulation was selected after chronic exposure to gemcitabine. Western blot analyses for the expression of cancer related proteins; RNA-interference of selected genes to investigate their function. Trypan blue staining to analyze cell survival. Results: Chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells displaying a mesenchymal phenotype, which remains less sensitive to drug-induced cell death. These cells express higher levels of Sam68 and SRSF1 splicing factors. Depletion by RNA-