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The usefulness of the measurement of amylin in the blood and pure pancreatic juice for the diagnosis of pancreatic cancer
April 2000
AGAA645
3279
3281
EXPRESSION OF INTERLEUKIN-8 CORRELATES WITH INVASIVENESS IN HUMAN PANCREATIC CANCER.
REACQUISITION OF P16/CDKN2 TUMOR SUPPRESSOR GENE EXPRESSION BY 5-AZA-2'DEOXYCYTIDINE AND CONCURRENT GROWTH INHIBITION OF NEUROENDOCRINE PANCREATIC TUMOR CELLS.
Yukio Kuwada, Akira Tsuchida, Tamito Sasaki, Kenji Matsubara, Shigeru Yamamoto, Yousuke Kawasaki, Kenji Morinaka, Hiroki Inoue, Yoshifumi Fujimoto, Masateru Murakami, Yasuhiko Kitadai, Goro Kajiyama, Hiroshima Univ Sch of Med, Hiroshima, Japan. Background Interleukin S (IL-S), a member of the CXC chemokine family, is a multifunctional cytokine shown to induce angiogenesis, haptotactic migration, and proliferation of certain cancer cells. IL-S is presumed to exert its action after binding to its specific receptors (IL-SRA and IL-SRB). Previously, we have reported that matrix metalloproteinase-9 (MMP-9) was increased by treatment with IL-S in gastric cancer cells. The aims of this study was to examine whether IL-S and IL-S receptors were expressed in human pancreatic cancer and to evaluate the role of IL-S on metastatic potential in pancreatic cancer. Methods The expression of IL-S, IL-SRA and IL-SRB in human pancreatic cancer cells (PANC-I, MIA PaCa-2, Capan-2) were examined by northern blot analysis and western blot analysis. The expressions of IL-S and its receptors were also investigated by immunohistochemistry in human pancreatic cancer tissues using specific antibody. Cytokine induced secretions of IL-S in supernatants of cultured medium from cells were investigated with ELISA (R&D). After treatment with various concentrations of recombinant IL-S (Sigma), the cell proliferations were assayed using Cell Counting Kit™ (Dojindo Labs., Japan). IL-S induced production and gelatinolytic activity of MMP-2 and MMP-9 were analyzed by western blot analysis and gelatin zymogram using conditioned mediums from each cell as samples. Results The expression of IL-S, IL-SRA and IL-SRB were detected in all investigated pancreatic cancer cells. Moreover, the secretion of IL-S was increased with cytokine stimuli. In addition, immunoreactive signals of IL-8 and its receptors were observed in cancer tissues.· Exogenous adding of IL-S did not induce proliferation of human pancreatic cancer cell lines. Surprisingly, the production and gelatinolytic activity ofMMP-2 and MMP-9 were increased by the treatment of recombinant IL-S in a dose-dependent manner. Conclusion These results suggest that IL-S regulate MMP activity and may play an important role in the invasiveness of human pancreatic cancer.
3280 THE USEFULNESS OF THE MEASUREMENT OF AMYLIN IN THE BLOOD AND PURE PANCREATIC JUICE FOR THE DIAGNOSIS OF PANCREATIC CANCER. Byeong Cheol Lim, Kyo Sang Yoo, Eun Taek Park, Dong Wan Seo, Sung Koo Lee, Myung Hwan Kim, Young II Min, Ji Yeon Kim, Kangnam Gen Hosp, Seoul, South Korea; Asan Med Ctr, Seoul, South Korea. Background/Aims: Insulin resistance and diabetes mellitus frequently occurs in patients with pancreatic cancer. Amylin,a hormonal factor secreted from pancreas f3-cell, is known to reduce insulin sensitivity in vivo and induce insulin resisitance and diabetes mellitus. We performed this study prospectively to inspect amylin concentration as a useful index for pancreatic cancer, which may be useful to differentiate pancreatic cancer from chronic pancreatitis. Methods: 63 patients admitted at Asan Medical Center in Korea from July, 1995 to June, 1999 were enrolled. The patients were divided as 6 groups according to pancreas state and the presence of diabetes mellitus as follows, group A: control group (n=S), B: Diabetes mellitus (n= 12), C: Chronic pancreatitis without DM (n= IS), D: Chronic pancreatitis with DM (n=S), E: Pancreatic cancer without DM (n= 11), F: Pancreatic cancer with DM (n=9). Amylin concentration was measured in the blood (n=63) and pure pancreatic juice (n=37) by radioimmunoassay. We analyzed data using Kruskal-Wallis test. Results: Amylin concentration in plasma was not significantly different between 6 groups. Amylin concentration in pure pancreatic juice was significantly higher in group C, D, E, F compared with group A or B (p<0.05). We grouped six groups into 3 groups accoding to pancreas state, i.e., group I: normal pancreas state (n=20), group 2: chronic pancreatitis (n=23), group 3: pancreatic cancer (n=20). Amylin concentration in plasma was significantly higher in group 3 compared with group 1 or 2 (p
Nikolaus Lubomierski, Michael Kersting, Ullrich Wulbrand, Detlev Bartsch, Rudolf Arnold, Babette Simon, Philipps Univ, Marburg, Germany. Introduction: Disruption of pS3 and Rb regulatory pathways affects deregulation of cell cycle control and apoptosis and is crucial in lumori~enesis. The INK4a-ARF locus encodes two proteins, p16INK4a and pl9 RF,that restrain cell growth by affecting the function of pRB and pS3, respectively. Mutational inactivation of pS3 and Rb are rare events in neuroendocrine gastroenteropancreatic tumors (NGT). Thus we investigated loss of INK4 cyclin-dependent kinase inhibitor CDKIs (pI6INK4a, pI9ARF, plS fNK4b and p IS fNK4<)expression in primary Tumors and neuroendocrine pancreatic cell lines. Methods: 37 tumors (9 gastrinomas, 10 insulinomas, 9 functionally inactive tumors(NFT), 9 small intestine carcinoids)and 2 human neuroendocrine pancreatic tumor cell lines ~BON, OGP) were analysed bv 32p_ labeled nested RT-PCR for plSINK b, &16mK4a, plS fNK4<, p19AR'F and p21c 1P ' . f3-actin served as control. p16 K4a mutation analysis was performed by SSCP and sequence analyis. The p16 INK4 DNA methylation status was determined by methylation-specific PCR (MSP). S-aza-2'deoxycytidine (DAC)was used for analysis of methylation. Growth kinetics were obtained by BrdU-labeling and cell counting. Results:Carcinoids and NFTs revealed loss in CDKIs expression in SO% and 33%,fespectively, but less frequently in gastrinomas and insulinomas with 22% and 30%.In gastrinomas and insulinomas there was preferential loss of piSINK4b and p IS fNK4< eXfJession rather than p16INK4a nor p19ARF .BON cells revealed loss of p16 K4a, piSINK4c and p19 ARF expression due to homozygous deletion, while QGP cells showed selective loss of pl6fNK4aexpression due to aberrant methylation of the p16fNK4a promotor region. Treatment with the methylation inhibitor DAC resulted in reacquisition of p16INK4a expression and concurrent inhibition of growth. Partial demethylation of CpG islands of pl6INK4a was confirmed by methylation-specific PCR. Conclusion:These data demonstrate that INK4 CDKIs are targets of inactivation in gastroenteropancreatic neuroendocrine tumors. Induction of growth inhibition can occur by dernethylation-dependent pathw~s in pancreatic neuroendocrine cells in which the expression of p16IN 4a is induced. Thus, DAC could prove a useful chemotherapeutic drug preventing or reversing p16INK4a inactivation.
3282 IDENTIFICATION OF THE CCK-C RECEPTOR IN HUMAN PANCREATIC CANCER CELLS AND TISSUES. Melissa Martenis, Jill P. Smith, Elizabeth Ballard, Michael F. Verderame, Ian S. Zagon, Penn State Univ Coli of Medicine, Hershey, PA. Human pancreatic cancer cells possess a CCK-like receptor that differs from the CCK-B receptor as being a spliced variant, and appears to function in gastrin-stimulated cell proliferation. Due to its association with cancer, the receptor has been called the CCK-C (cancer) receptor. A unique portion of this receptor, with no homology to CCK-A or CCK-B receptors, was utilized to raise antibodies in chickens. AIM: The purpose of this study was to evaluate the presence and cellular location of the CCK-C receptor in human pancreatic cancer cells and fresh tumors. METHODS: Human pancreatic cancer cell lines (BxPC-3, PANC-l, and MIA PaCa-2) were grown in cell culture and fresh tissues (normal and malignant pancreas) were obtained from the operating room. Cellular protein homogenates (SO mg) were subjected to SDS PAGE electrophoresis, transferred to nitrocellulose and hybridized with either the CCK-C antibody, the preimmune chicken antibody, or preaborbed antibody at a titer of 1:2,500 for 24 hrs. Blots were exposed to the secondary anti-chicken antibody (l: 10,000) for 2 hrs and immunoreactivity tested by chemiluminescence. Cells were grown directly on coverslips and fresh tissue was prepared in 10 /Lm sections, fixed and mounted and both samples subjected to immunohistochemistry with the CCK-C antibody or control sera. RESULTS: Western blotting with human pancreatic cancer cells and fresh pancreatic cancer, but not normal pancreas, revealed CCK-C immunoreactivity. Immunohistochemistry studies revealed CCK-C immunoreactivity associated with the plasma membrane in the pancreatic cancer cells and tissues. Preabsorbed controls and preimmune chicken antisera was negative for the CCK-C receptor. CONCLUSION: A novel CCK receptor has been identified in human pancreatic cancer cells and tumors but not in normal human pancreas. The location of this receptor is associated with the plasma membrane. Disclosure: This research was supported by a grant from the NIH, CAS0303 and a Student ADHF Research Fellowship Award.
AGAA645
3279
3281
EXPRESSION OF INTERLEUKIN-8 CORRELATES WITH INVASIVENESS IN HUMAN PANCREATIC CANCER.
REACQUISITION OF P16/CDKN2 TUMOR SUPPRESSOR GENE EXPRESSION BY 5-AZA-2'DEOXYCYTIDINE AND CONCURRENT GROWTH INHIBITION OF NEUROENDOCRINE PANCREATIC TUMOR CELLS.
Yukio Kuwada, Akira Tsuchida, Tamito Sasaki, Kenji Matsubara, Shigeru Yamamoto, Yousuke Kawasaki, Kenji Morinaka, Hiroki Inoue, Yoshifumi Fujimoto, Masateru Murakami, Yasuhiko Kitadai, Goro Kajiyama, Hiroshima Univ Sch of Med, Hiroshima, Japan. Background Interleukin S (IL-S), a member of the CXC chemokine family, is a multifunctional cytokine shown to induce angiogenesis, haptotactic migration, and proliferation of certain cancer cells. IL-S is presumed to exert its action after binding to its specific receptors (IL-SRA and IL-SRB). Previously, we have reported that matrix metalloproteinase-9 (MMP-9) was increased by treatment with IL-S in gastric cancer cells. The aims of this study was to examine whether IL-S and IL-S receptors were expressed in human pancreatic cancer and to evaluate the role of IL-S on metastatic potential in pancreatic cancer. Methods The expression of IL-S, IL-SRA and IL-SRB in human pancreatic cancer cells (PANC-I, MIA PaCa-2, Capan-2) were examined by northern blot analysis and western blot analysis. The expressions of IL-S and its receptors were also investigated by immunohistochemistry in human pancreatic cancer tissues using specific antibody. Cytokine induced secretions of IL-S in supernatants of cultured medium from cells were investigated with ELISA (R&D). After treatment with various concentrations of recombinant IL-S (Sigma), the cell proliferations were assayed using Cell Counting Kit™ (Dojindo Labs., Japan). IL-S induced production and gelatinolytic activity of MMP-2 and MMP-9 were analyzed by western blot analysis and gelatin zymogram using conditioned mediums from each cell as samples. Results The expression of IL-S, IL-SRA and IL-SRB were detected in all investigated pancreatic cancer cells. Moreover, the secretion of IL-S was increased with cytokine stimuli. In addition, immunoreactive signals of IL-8 and its receptors were observed in cancer tissues.· Exogenous adding of IL-S did not induce proliferation of human pancreatic cancer cell lines. Surprisingly, the production and gelatinolytic activity ofMMP-2 and MMP-9 were increased by the treatment of recombinant IL-S in a dose-dependent manner. Conclusion These results suggest that IL-S regulate MMP activity and may play an important role in the invasiveness of human pancreatic cancer.
3280 THE USEFULNESS OF THE MEASUREMENT OF AMYLIN IN THE BLOOD AND PURE PANCREATIC JUICE FOR THE DIAGNOSIS OF PANCREATIC CANCER. Byeong Cheol Lim, Kyo Sang Yoo, Eun Taek Park, Dong Wan Seo, Sung Koo Lee, Myung Hwan Kim, Young II Min, Ji Yeon Kim, Kangnam Gen Hosp, Seoul, South Korea; Asan Med Ctr, Seoul, South Korea. Background/Aims: Insulin resistance and diabetes mellitus frequently occurs in patients with pancreatic cancer. Amylin,a hormonal factor secreted from pancreas f3-cell, is known to reduce insulin sensitivity in vivo and induce insulin resisitance and diabetes mellitus. We performed this study prospectively to inspect amylin concentration as a useful index for pancreatic cancer, which may be useful to differentiate pancreatic cancer from chronic pancreatitis. Methods: 63 patients admitted at Asan Medical Center in Korea from July, 1995 to June, 1999 were enrolled. The patients were divided as 6 groups according to pancreas state and the presence of diabetes mellitus as follows, group A: control group (n=S), B: Diabetes mellitus (n= 12), C: Chronic pancreatitis without DM (n= IS), D: Chronic pancreatitis with DM (n=S), E: Pancreatic cancer without DM (n= 11), F: Pancreatic cancer with DM (n=9). Amylin concentration was measured in the blood (n=63) and pure pancreatic juice (n=37) by radioimmunoassay. We analyzed data using Kruskal-Wallis test. Results: Amylin concentration in plasma was not significantly different between 6 groups. Amylin concentration in pure pancreatic juice was significantly higher in group C, D, E, F compared with group A or B (p<0.05). We grouped six groups into 3 groups accoding to pancreas state, i.e., group I: normal pancreas state (n=20), group 2: chronic pancreatitis (n=23), group 3: pancreatic cancer (n=20). Amylin concentration in plasma was significantly higher in group 3 compared with group 1 or 2 (p
Nikolaus Lubomierski, Michael Kersting, Ullrich Wulbrand, Detlev Bartsch, Rudolf Arnold, Babette Simon, Philipps Univ, Marburg, Germany. Introduction: Disruption of pS3 and Rb regulatory pathways affects deregulation of cell cycle control and apoptosis and is crucial in lumori~enesis. The INK4a-ARF locus encodes two proteins, p16INK4a and pl9 RF,that restrain cell growth by affecting the function of pRB and pS3, respectively. Mutational inactivation of pS3 and Rb are rare events in neuroendocrine gastroenteropancreatic tumors (NGT). Thus we investigated loss of INK4 cyclin-dependent kinase inhibitor CDKIs (pI6INK4a, pI9ARF, plS fNK4b and p IS fNK4<)expression in primary Tumors and neuroendocrine pancreatic cell lines. Methods: 37 tumors (9 gastrinomas, 10 insulinomas, 9 functionally inactive tumors(NFT), 9 small intestine carcinoids)and 2 human neuroendocrine pancreatic tumor cell lines ~BON, OGP) were analysed bv 32p_ labeled nested RT-PCR for plSINK b, &16mK4a, plS fNK4<, p19AR'F and p21c 1P ' . f3-actin served as control. p16 K4a mutation analysis was performed by SSCP and sequence analyis. The p16 INK4 DNA methylation status was determined by methylation-specific PCR (MSP). S-aza-2'deoxycytidine (DAC)was used for analysis of methylation. Growth kinetics were obtained by BrdU-labeling and cell counting. Results:Carcinoids and NFTs revealed loss in CDKIs expression in SO% and 33%,fespectively, but less frequently in gastrinomas and insulinomas with 22% and 30%.In gastrinomas and insulinomas there was preferential loss of piSINK4b and p IS fNK4< eXfJession rather than p16INK4a nor p19ARF .BON cells revealed loss of p16 K4a, piSINK4c and p19 ARF expression due to homozygous deletion, while QGP cells showed selective loss of pl6fNK4aexpression due to aberrant methylation of the p16fNK4a promotor region. Treatment with the methylation inhibitor DAC resulted in reacquisition of p16INK4a expression and concurrent inhibition of growth. Partial demethylation of CpG islands of pl6INK4a was confirmed by methylation-specific PCR. Conclusion:These data demonstrate that INK4 CDKIs are targets of inactivation in gastroenteropancreatic neuroendocrine tumors. Induction of growth inhibition can occur by dernethylation-dependent pathw~s in pancreatic neuroendocrine cells in which the expression of p16IN 4a is induced. Thus, DAC could prove a useful chemotherapeutic drug preventing or reversing p16INK4a inactivation.
3282 IDENTIFICATION OF THE CCK-C RECEPTOR IN HUMAN PANCREATIC CANCER CELLS AND TISSUES. Melissa Martenis, Jill P. Smith, Elizabeth Ballard, Michael F. Verderame, Ian S. Zagon, Penn State Univ Coli of Medicine, Hershey, PA. Human pancreatic cancer cells possess a CCK-like receptor that differs from the CCK-B receptor as being a spliced variant, and appears to function in gastrin-stimulated cell proliferation. Due to its association with cancer, the receptor has been called the CCK-C (cancer) receptor. A unique portion of this receptor, with no homology to CCK-A or CCK-B receptors, was utilized to raise antibodies in chickens. AIM: The purpose of this study was to evaluate the presence and cellular location of the CCK-C receptor in human pancreatic cancer cells and fresh tumors. METHODS: Human pancreatic cancer cell lines (BxPC-3, PANC-l, and MIA PaCa-2) were grown in cell culture and fresh tissues (normal and malignant pancreas) were obtained from the operating room. Cellular protein homogenates (SO mg) were subjected to SDS PAGE electrophoresis, transferred to nitrocellulose and hybridized with either the CCK-C antibody, the preimmune chicken antibody, or preaborbed antibody at a titer of 1:2,500 for 24 hrs. Blots were exposed to the secondary anti-chicken antibody (l: 10,000) for 2 hrs and immunoreactivity tested by chemiluminescence. Cells were grown directly on coverslips and fresh tissue was prepared in 10 /Lm sections, fixed and mounted and both samples subjected to immunohistochemistry with the CCK-C antibody or control sera. RESULTS: Western blotting with human pancreatic cancer cells and fresh pancreatic cancer, but not normal pancreas, revealed CCK-C immunoreactivity. Immunohistochemistry studies revealed CCK-C immunoreactivity associated with the plasma membrane in the pancreatic cancer cells and tissues. Preabsorbed controls and preimmune chicken antisera was negative for the CCK-C receptor. CONCLUSION: A novel CCK receptor has been identified in human pancreatic cancer cells and tumors but not in normal human pancreas. The location of this receptor is associated with the plasma membrane. Disclosure: This research was supported by a grant from the NIH, CAS0303 and a Student ADHF Research Fellowship Award.