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Immunohistochemical characterization of pancreatic tumors induced by DMBA in rats
A472 AGA ABSTRACTS • G1919 TELOMERASE EXPRESSION IN PANCREATIC CARCINOMA. R. Jesenofskv, J-M. L~Shr, P. MiJller, S. Liebe; University of Rostock, Department of Internal Medicine, Germany. Introduction: Telomeres, specialized structures at the end of enkaryotic chromosomes, consist of up to thousands of repeats of the sequence TI'AGGG and associated proteins. In all normal somatic ceils telomere length decreases with every cell division consistent with the inability of DNA polymerases to replicate the ends of linear DNA. This decrease is correlated with aging and cellular senescence in vivo and in vitro. In contrast all tumor cell lines investigated show no net loss of telomeres. Therefore maintenance of telomere length seems to be a prerequisite to overcome the proliferative limitations of normal cells and to become immortal. In the vast majority of tumors and immortal cell lines this goal is reached by the expression of the ribonucleoprotein enzyme telomerase which adds telomeric repeats to the 3 end of chromosomes and thus prevents the decrease of telomere length. The aim of this study was (1) to verify a report of telomerase expression in pancreatic carcinoma and (2) to determine the time of first telomerase expression in pancreatic carcinogenesis to be able to assess telomerase expression as diagnostic marker. Material and Methods: Tissue specimens from pancreatic carcinoma, from chronic pancreatitis and from normal pancreata were investigated. Furthermore telomerase activity of ten human pancreatic tumor cell lines and two bovine pancreatic duct cell lines, one SV40 T-antigen immortalized, the other additionally transfected with an activated ki-ras gene, was examined. Telomerase activity was determined by Telomerase PCR ELISA (Boehringer, Mannheim, Germany) a modification of the TRAP assay. In brief, Telomerase adds telomeric repeats to a biotinylated primer. The products are amplified by PCR with telomere specific primers. The resulting PCR products are immobilized and after denaturation are hybridized with a telomere specific DIG-labeled probe. These hybrids are detected with an antibody against digoxigenin that is conjugated to peroxidase. Finally, the probe is visualized by virtue of peroxidase metabolizing TMB to form a colored reaction product. Additionally the telomerase specific ladder pattern was visualized with streptavidin-alkaline phosphatase and CSPD after electrophoresis and blotting of the PCR products. Results: All human pancreatic tumor cell lines exhibited a high level of telomerase expression. In contrast neither the immortalized nor the additionally ki-ras transfected tumorigenic bovine pancreatic duct ceils showed any telomerase activity. Telomerase activity was detectable in two of five pancreatic carcinomas, none of three chronic pancreatitis samples and in one of six normal pancreatic tissues. Blotting of the PCR products showed the corresponding results. Conclusion: In our bovine model of pancreatic carcinogenesis immortalization and tumorigenicity seem to be independent from telomerase expression. Also the low percentage of telomerase positive pancreatic carcinomas indicates that there might be alternative ways (e.g. ALT, alternative lengthening of telomeres) involved in the maintenance of telomeres in pancreatic carcinogenesis. This work was supported by the Deutsche Krebshilfe.
• G1920 IMMUNOHISTOCHEMICAL CHA~CTERIZATION OF PANCREATIC TUMORS INDUCED BY DMBA IN RATS. R.E. Jimenez. K. Z'graggen, A.L. Warshaw, F. Graeme-Cook*, W. Hartwig, S. Kupa, J.A. Rivera, D.W. Rattner, C. Fernandez-del Castillo, Departments of Surgery and Pathology*, Massachusetts General Hospital and Harvard Medical School, Boston, MA Our laboratory has recently developed a model of pancreatic adenocarcinoma in rats (Surgery 1997;122:82-90). In the present study we report the expression pattern of ductal and acinar cell markers in these tumors. Methods: Pancreatic neoplasms were induced in Sprague-Dawley rats by implantation of dimethylbenzanthracene (DMBA) crystals in the head of the pancreas. Tumors were harvested 9 months after carcinogen exposure. Formalin-fixed paraffin embedded tissue sections and acetone-fixed frozen sections were prepared for both histology and immunohistochemistry. The following antibodies were used to analyze the tumors: anti-wide spectrum cytokeratin (polyclonal), anticytokeratin 19 (monoclonal), anti-cytokeratin 20 (monoclonal), and antichymotrypsin (monoclonal). Results: Ten representative tumors ranging in size from 7 to 65 mm were selected for the study, with normal pancreas used as control. Histology revealed moderatelydifferentiated mucinous ductal adenocarcinomas with frequent invasion of the duodenal wall. Immunohistochemistry of normal pancreatic tissue showed selective labelling of all pancreatic ducts (wide spectrum cytokeratin), large and intermediate sized ducts (cytokeratin 19), small and terminal ducts (cytokeratin 20), or acinar cells (chymotrypsin). Wide spectrum cytokeratin, cytokeratin 19, and cytokeratin 20 expression was strong in the cytoplasm of all cells comprising the neoplastic epithelium of the 10 tumors analyzed. No difference in expression of cytokeratin 19 and 20 could be identified within carcinomas. Areas of hyperplasia and high-grade dysplasia representing early tumor stages also showed unequivocal staining with anti-cytokeratin antibodies. No immunoreactivity to chymotrypsin was noted in any of the tumors studied. Conclusion: Pancreatic adenocarcinomas induced by DMBA in rats express markers consistent with a ductal phenotype. Small and large pancreatic duct markers are simultaneously expressed by these tumors. The
GASTROENTEROLOGYVol. 114, No. 4 pattern of cytokeratin expression reported is shared by human pancreatic carcinomas. The presence of ductal markers in early tumor stages suggests a duct cell origin for these neoplasms. G1921 GENE EXPRESSION OF HCGI3 IN PANCREATIC CANCER AND CLINICAL USEFULNESS OF URINARY GONADOTROPIN PEPTIDE (UGP). A. Jimi, Kurume, Fukuoka: A. Funakoshi, H. Wakasugi, T. Shimazoe, A. Kono, Fukuoka, Japan Urinary gonadotropin peptide (UGP), a catabolite of human chorionic gonadotropin ~-subunit (hCG[3), has been evaluated as a new tumor marker in pancreatic cancer patients. In this study we examined gene expression of and production of hCG[3 in the tumor tissue of surgically resected specimen and cultured pancreatic cancer cells results, and were compared with those of mutant p53 protein, a tumor suppresser gene product. Methods: UGP was measured by an EIA kit (ELNAS) using a specific antibody (B2102) for hCGI3 core fragment. Gene expression of hCGI3 was evaluated by the RTPCR followed by Southern blot hybridization. Mutant p53 protein was stained using a p53 antibody (Oncogene, USA). Three human pancreatic cancer cell lines (KP-1N, KP-2 and KP-3N: all duct cell type) were used for transplantation in nude mice subcutaneously. After two weeks urine was collected at any time and after 1 month tumor was excised. The prognosis of pancreatic cancer were evaluated by UGP and mutant p53. Results: The UGPpositive rates for detection of pancreatic cancer were higher for poorly differentiated carcinoma cells (67-100%) than well differentiated cell types (37.5%), although the mutant p53 protein-positive rates (40%) did not depend on carcinoma cell differentiation. The culture media showed high hCGI3 activity and urinary UGP was detected in tumor beating nude mice. Immunohistochemical testing of tumor tissue in surgically resected specimens and tumors transplanted into nude mice revealed that the cells contained hCGI3 and mutant p53 protein. Gene expression of hCGI3 was confirmed by the RT-PCR-Southern method. Mutant p53 expression revealed the malignant potencies of cancer cells. The abnormal high levels of UGP, but not mutant p53 expression indicated the poor prognosis ( < 6 months death) of the patients. Conclusion: These findings suggest that UGP is capable of serving as a noninvasive tumor marker that should be clinically useful in the diagnosis and the evaluation of prognosis of pancreatic cancer • G1922 SEVERITY PREDICTION BY INDIVIDUAL PROBABILITY INDEX OF COMPLICATIONS IN ACUTE PANCREATITIS. CD Johnsonl, S Phillips ~, SKC Toh l, M Mullee 2. University Surgical Uni0, Medical Statistics 2, Southampton General Hospital, Tremona Road, Southampton, UK SO 16 6YD. Multiple factor scoring systems categorize patients with AP as predicted mild or severe with an overall accuracy of 75 percent. The potential error of this categorisation is often overlooked. Obesity is a prognostic factor available at the time of admission. The aim of this study was twofold: (1) to integrate an objective assessment of obesity into the APACHE II system; (2) to develop a probability index to allow calculation of individual risk of complications. In an unselected series of 186 patients (Group 1), the components of APACHE II (age, acute physiology score (APS), chronic health points) body mass index (BMI), and other clinical data were used for stepwise logistic analysis to test association with subsequent complications. Age, BMI and APS showed significant associations. An equation was derived to calculate a probability index (PI) to give an individualized risk of complications. PI was calculated in a subsequent series (Group 2) of 88 patients with AP. Addition of obesity points (I=BMI 25-30, 2=BMI > 30) to APACHE II improved the predictive accuracy. Prospective evaluation of PI in Group 2 showed distribution of most patients to low ( < 0.3) or high ( > 0.6) risk groups. 10 (20%) of 51 patients with PI < 0.3 developed a complication, as did 18 (67%) of 27 patients with PI > 0.6. This study has shown the feasibility of combining clinical observation (obesity) with APACHE II scoring to improve its accuracy. New treatments require accurate assessment of risk of severe outcome, early in the course of AP. PI provides clinically useful individualised risk assessment. • G1923 DYNAMIN PARTICIPATES IN THE FORMATION OF CLATHRINAND NONCLATHRIN-COATED VESICLES FROM THE TRANSGOLGI NETWORK. S.M, Jones, J.R. Henley*, H. Cao*, K.E. Howell, and M.A. McNiven*, U. of CO School of Medicine, Denver, CO 80262 and *Dept. of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905 The dynamins comprise an expanding multigene family of 100-kD GTPases that support the scission of clathrin-coated vesicles from the plasma membrane during endocytosis. Whether dynamins participate in vesicle formation at other cellular compartments has not been tested directly, although recent studies have reported that dynamin is associated with the Golgi apparatus in acinar ceils and hepatocytes. Therefore, the GOAL of this study was to test for the participation of dynamin in the formation of nascent vesicles from the trans-Golgi network (TGN). To this end, we overexpressed
• G1920 IMMUNOHISTOCHEMICAL CHA~CTERIZATION OF PANCREATIC TUMORS INDUCED BY DMBA IN RATS. R.E. Jimenez. K. Z'graggen, A.L. Warshaw, F. Graeme-Cook*, W. Hartwig, S. Kupa, J.A. Rivera, D.W. Rattner, C. Fernandez-del Castillo, Departments of Surgery and Pathology*, Massachusetts General Hospital and Harvard Medical School, Boston, MA Our laboratory has recently developed a model of pancreatic adenocarcinoma in rats (Surgery 1997;122:82-90). In the present study we report the expression pattern of ductal and acinar cell markers in these tumors. Methods: Pancreatic neoplasms were induced in Sprague-Dawley rats by implantation of dimethylbenzanthracene (DMBA) crystals in the head of the pancreas. Tumors were harvested 9 months after carcinogen exposure. Formalin-fixed paraffin embedded tissue sections and acetone-fixed frozen sections were prepared for both histology and immunohistochemistry. The following antibodies were used to analyze the tumors: anti-wide spectrum cytokeratin (polyclonal), anticytokeratin 19 (monoclonal), anti-cytokeratin 20 (monoclonal), and antichymotrypsin (monoclonal). Results: Ten representative tumors ranging in size from 7 to 65 mm were selected for the study, with normal pancreas used as control. Histology revealed moderatelydifferentiated mucinous ductal adenocarcinomas with frequent invasion of the duodenal wall. Immunohistochemistry of normal pancreatic tissue showed selective labelling of all pancreatic ducts (wide spectrum cytokeratin), large and intermediate sized ducts (cytokeratin 19), small and terminal ducts (cytokeratin 20), or acinar cells (chymotrypsin). Wide spectrum cytokeratin, cytokeratin 19, and cytokeratin 20 expression was strong in the cytoplasm of all cells comprising the neoplastic epithelium of the 10 tumors analyzed. No difference in expression of cytokeratin 19 and 20 could be identified within carcinomas. Areas of hyperplasia and high-grade dysplasia representing early tumor stages also showed unequivocal staining with anti-cytokeratin antibodies. No immunoreactivity to chymotrypsin was noted in any of the tumors studied. Conclusion: Pancreatic adenocarcinomas induced by DMBA in rats express markers consistent with a ductal phenotype. Small and large pancreatic duct markers are simultaneously expressed by these tumors. The
GASTROENTEROLOGYVol. 114, No. 4 pattern of cytokeratin expression reported is shared by human pancreatic carcinomas. The presence of ductal markers in early tumor stages suggests a duct cell origin for these neoplasms. G1921 GENE EXPRESSION OF HCGI3 IN PANCREATIC CANCER AND CLINICAL USEFULNESS OF URINARY GONADOTROPIN PEPTIDE (UGP). A. Jimi, Kurume, Fukuoka: A. Funakoshi, H. Wakasugi, T. Shimazoe, A. Kono, Fukuoka, Japan Urinary gonadotropin peptide (UGP), a catabolite of human chorionic gonadotropin ~-subunit (hCG[3), has been evaluated as a new tumor marker in pancreatic cancer patients. In this study we examined gene expression of and production of hCG[3 in the tumor tissue of surgically resected specimen and cultured pancreatic cancer cells results, and were compared with those of mutant p53 protein, a tumor suppresser gene product. Methods: UGP was measured by an EIA kit (ELNAS) using a specific antibody (B2102) for hCGI3 core fragment. Gene expression of hCGI3 was evaluated by the RTPCR followed by Southern blot hybridization. Mutant p53 protein was stained using a p53 antibody (Oncogene, USA). Three human pancreatic cancer cell lines (KP-1N, KP-2 and KP-3N: all duct cell type) were used for transplantation in nude mice subcutaneously. After two weeks urine was collected at any time and after 1 month tumor was excised. The prognosis of pancreatic cancer were evaluated by UGP and mutant p53. Results: The UGPpositive rates for detection of pancreatic cancer were higher for poorly differentiated carcinoma cells (67-100%) than well differentiated cell types (37.5%), although the mutant p53 protein-positive rates (40%) did not depend on carcinoma cell differentiation. The culture media showed high hCGI3 activity and urinary UGP was detected in tumor beating nude mice. Immunohistochemical testing of tumor tissue in surgically resected specimens and tumors transplanted into nude mice revealed that the cells contained hCGI3 and mutant p53 protein. Gene expression of hCGI3 was confirmed by the RT-PCR-Southern method. Mutant p53 expression revealed the malignant potencies of cancer cells. The abnormal high levels of UGP, but not mutant p53 expression indicated the poor prognosis ( < 6 months death) of the patients. Conclusion: These findings suggest that UGP is capable of serving as a noninvasive tumor marker that should be clinically useful in the diagnosis and the evaluation of prognosis of pancreatic cancer • G1922 SEVERITY PREDICTION BY INDIVIDUAL PROBABILITY INDEX OF COMPLICATIONS IN ACUTE PANCREATITIS. CD Johnsonl, S Phillips ~, SKC Toh l, M Mullee 2. University Surgical Uni0, Medical Statistics 2, Southampton General Hospital, Tremona Road, Southampton, UK SO 16 6YD. Multiple factor scoring systems categorize patients with AP as predicted mild or severe with an overall accuracy of 75 percent. The potential error of this categorisation is often overlooked. Obesity is a prognostic factor available at the time of admission. The aim of this study was twofold: (1) to integrate an objective assessment of obesity into the APACHE II system; (2) to develop a probability index to allow calculation of individual risk of complications. In an unselected series of 186 patients (Group 1), the components of APACHE II (age, acute physiology score (APS), chronic health points) body mass index (BMI), and other clinical data were used for stepwise logistic analysis to test association with subsequent complications. Age, BMI and APS showed significant associations. An equation was derived to calculate a probability index (PI) to give an individualized risk of complications. PI was calculated in a subsequent series (Group 2) of 88 patients with AP. Addition of obesity points (I=BMI 25-30, 2=BMI > 30) to APACHE II improved the predictive accuracy. Prospective evaluation of PI in Group 2 showed distribution of most patients to low ( < 0.3) or high ( > 0.6) risk groups. 10 (20%) of 51 patients with PI < 0.3 developed a complication, as did 18 (67%) of 27 patients with PI > 0.6. This study has shown the feasibility of combining clinical observation (obesity) with APACHE II scoring to improve its accuracy. New treatments require accurate assessment of risk of severe outcome, early in the course of AP. PI provides clinically useful individualised risk assessment. • G1923 DYNAMIN PARTICIPATES IN THE FORMATION OF CLATHRINAND NONCLATHRIN-COATED VESICLES FROM THE TRANSGOLGI NETWORK. S.M, Jones, J.R. Henley*, H. Cao*, K.E. Howell, and M.A. McNiven*, U. of CO School of Medicine, Denver, CO 80262 and *Dept. of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905 The dynamins comprise an expanding multigene family of 100-kD GTPases that support the scission of clathrin-coated vesicles from the plasma membrane during endocytosis. Whether dynamins participate in vesicle formation at other cellular compartments has not been tested directly, although recent studies have reported that dynamin is associated with the Golgi apparatus in acinar ceils and hepatocytes. Therefore, the GOAL of this study was to test for the participation of dynamin in the formation of nascent vesicles from the trans-Golgi network (TGN). To this end, we overexpressed