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Gastric carcinogenesis in a murine model: Induction of adenocarcinoma and intestinal metaplasia by profound duodenogastric reflux
A768 AGA ABSTRACTS
4121 THE COMBINATION OF CYCLIN D1 OVEREXPRESSION AND P53 INACTIVATION IS CRITICAL FOR ORAL-ESOPHAGEAL CARCINOGENESIS IN TRANSGENIC MICE. Oliver G. Opitz, Yasir Suliman, Ben Rhoades, Norman E. Sharpless, Ani! K. Rustgi, Div of Gastroenterology, Univ of Pennsylvania, Philadelphia, PA; Div of Hematology/Oncology, MGH, Boston, MA. Introduction: The development and characterization of a genetic model of oral/esophageal squamous cell cancer is important in understanding the underlying molecular mechanisms of oral/esophageal carcinogenesis. C~ clin DI overexpression and p53 mutations are the most common genetic events in oral and esophageal squamous cell cancer. Having previously established the efficacy of the Epstein-Barr virus L2 promoter in targeting genes to the oral-esophageal epithelium, we have cross-bred L2-cyclin DI mice with p53 deficient mice to induce oral-esophageal malignant transformation. Methods: L2-cyclin DI transgenic mice, expressing high, moderate and low levels of cyclin DI, were cross-bred with p53 heterozygous deficient mice to generate cyclin Dl overexpressing/p53 heterozygous and homozygous deficient genotypes and their respective wild-type co~trols. Mice were genotyped using either southern blot or PCR based techniques. Histological analysis was done at different time points by HE staining. Results: Histological analysis was done at 3 months, 6 months and 12 months for cyclin Dl/p53 heterozygous deficient mice and at 4-5 months for cyclin Dl/p53 homozygous deficient mice. This analysis revealed a rapid induction of dysplasia in the oral-esophageal squamous epithelium with progression of the malignant phenotype in cyclin Dl/p53 heterozygous deficient mice. Cyclin Dl/p53 homozygous deficient animals revealed changes consistent with high grade dysplasia. Conclusions: The combination of cyclin D I overexpression and p53 inactivation plays a critical role in induction and progression of carcinogenesis in the oral-esophageal squamous epithelium.
4122 GASTRIC CARCINOGENESIS IN A MURINE MODEL: INDUCTION OF ADENOCARCINOMA AND INTESTINAL METAPLASIA BY PROFOUND DUODENOGASTRIC REFLUX, Ivete B. Prado, Fabio P. Lopasso, Paulo Kassab, Kyoshi Iriya, Antonio A. Laudanna, Med Sch, Univ of Sao Paulo, Sao Paulo, Brazil; Med Sch of Santo Amaro, Sao Paulo, Brazil. Introduction- Patients submitted to gastric resection for benign disease are at a high risk for later gastric tumor dev~lopment. Experimental models have been developed in order to study inflammatory and/or neoplasic changes in the stomach of animals submitted to alkaline reflux. Here we present macroscopic and histological study of gastric tumor induction in rats by surgical duodenogastric reflux with n? carcinoge~ added to diet. Material and Methods- Altogether, 42 male Wistar rats aging 16-20 weeks were randomized to undergo either split gastrojejunostomy (study group, 27 animals) or simple laparotomy and visceral manipulation (control group, 15 animals). The animals of the study group underwen~ a complete duodenogastric reflux: a loop of duodenum 1 em before Treitz ligament was anastomosed to the stomach at the greater curvature (figure 1). Animals from both groups were sacrificed at 12 months after surgery. Esophagus and afferent loop (study group) or duodenum (control) were sectioned and viscera removed en bloc (figure 2). Esophagus, stomach and duodenum were open, macroscopic findings noted (figure 3) and samples of gastric mucosa and gastrojejunal anastomosis were removed, fixed in formol and serial 5p,ID sections were haematoxylin-eosin s~ned. R;esults- ~ll animals with duodenogastric reflux showed macroscopic an~ microscopic J?ucosal inflammation (infiltrating inflammatory cells, capillary congestion and edema, figure 4). Among them, 16 animals (60%) showed areas of inte~ tinal metaplasia, with Paneth cells (figure 5) and 20 rats (74%) had gastn.c tubular adenocarcinoma, intestinal pattern (figure 6). All macro and rmcroscopic findings were always restricted to the duodenogastric anastomosis area or nearby. The animals of the control group did not show any macro or microscopic alteration in the studied samples. Conclusions- This study demonstrates that gastric adenocarcinoma may be induced in the rat at a great incidence by total duodenogastric reflux, without adding a~y carcinogen to diet. In addition, compared to other studies, it shows that 10 this model of tumor induction intestinal metaplasia is a frequent associated condition. Financial support by FAPESP
4123 THE EFFECT OF DIRECT CONTACT BETWEEN PLATELET AND TUMOR CELL IN METASTASIS OF MARIGNANT TUMOR. Keiichi Suzuki, Koichi Aiura, Masakazu Ueda, Masaki Kitajima, Dept of Surg, Sch of Medicine, Keio Univ, Tokyo, Japan. Background: There is increasing evidence that an interaction between tumor cells and platelet may contribute to the development of metastases. We comfirmed poorer prognosis of thrombocytosis in patients with pancreas cancer in our hospital. But the mechanism is still unclear. We studied the effect of direct contact between platelet and tumor cells to extravassel invasion. [Matrials and Methods] We performed Chemoinvasion assay with BIOCOAT MATRIGEL Invasion Chamber. We compared tumor cell invasion under with or without platelet. We used human pancreas cacncer, SW.l990, and human breast cancer, MCF 7, as tumor cells. And we used fibronectin as chemoatractant. We co-incubated tumor cells and platelet, tumor cells and activated platelet by thrombin and tumor cells only under
GASTROENTEROWGY Vol. 118, No.4
37 C,with 5% C02 atmosphere. After 24 hours incubation, we fixed and stained membranes of chambers in Hematoxylin-Eosin, and counted invated tumor cells to bottom of the membrane under 200x. Results: The average cell count of each group of SW.l990 was 26.91200x in tumor cells and platelet group, 58.21200x in tumor cells and activated platelet group and 3.3/200x in tumor cells only. And those of MCF 7 were 13.91200x, 20.61200x and 6.l/200x. Conclusion: Based upon these findings, we postulate that platelet activated extra vassel invasion of tumor cells. Direct contact between platelet and tumor cells may play an important role in this process of metastasis. We assume that tumor cells are stimulated in secretion of matrix metalloprotease such as MMP 9 under direct contact with platelet.
4124 REDUCED ANGIOGENESIS BY MARIMASTAT IN PERITONEAL DISSEMINATION MODEL OF GASTRIC CANCER. Norihito Wada, Yoshihide Otani, Tetsuro Kubota, Masaru Kimata, Akiko Minagawa, Koichiro Kumai, Kaori Kameyama, Yasunori Okada, Masaki Kitajima, Dept of Surg, Keio Univ, Tokyo, Japan; Dept of Pathology, Keio Univ, Tokyo, Japan. We have been investigating the role of matrix metalloproteinase (MMP) in the invasion and metastasis of gastric cancer, and revealed that peritoneal dissemination of gastric cancer is controlledby a MMP inhibitor, marimastat. The mechanisms of the down-regulation, however. are not clarified. We analyzed tumor angiogenesis by means ofanimal model of peritoneal dissemination treated with marimastat. A human gastric carcinoma cell line, TMK-I (I x 106cellslbody) was injected intraperitoneally into 2 groups of 5 BALB/cA scm mice (male, 5 weeks). Subsequently, 27 mglkg/day of marimastat (TA-2516, Tanabe Seiyaku Co., Japan) for treated group (M) or vehicle DMSO for control group (C) was administered for two weeks using subcutaneousmini-osmotic pumps, then the animals were sacrificed. Total weightof dissemination nodules was significantly low in the group M compared with the group C (0.16 g vs.0.40 g, P=0.026). Microvascular count of the group M was also significantly lower than that of the group C (20.5 vs.l5.3 vessels/mrrr', P=0.024). Maximum radius of microvessels in the dissemination nodule was 72.0 micrometerin M group and 15.3 in the C group, respectively (P=0.038). In the process of tumor angiogenesis, basement membrane of existing microvessels is degraded by proteases, chiefly by MMPs-2 and -9. Marimastat can inhibit the enzymes. Thereforeit may show the inhibitory effect for progression of peritoneal metastasis through reduction of angiogenesis by the inhibition of these gelatinases. We conclude that marimastat was associated with reduced tumor weight and microvasculature in peritoneal dissemination model.
4125 TGF-B PROMOTES EPITHELIAL-MESENCHYMAL TRANSITION AND INVASIVENESS VIA RHOB AND MAPK SIGNALING IN PANCREATIC CANCER CELL LINES. Michihiko Wada, Ryo Hosotani, Masayuki Imamura, Robert D. Beauchamp. Kyoto Univ, Kyoto. Japan; Vanderbilt Univ, Nashville. TN. [Purpose] Transforming growth factor-I3(TGF-I3) is a multifunctional growth factor that regulates cell proliferation, differentiation and extracellular matrix remodeling and is overexpressed in many human cancers. Previous reports suggested that TGF-l3can promote tumor invasion and angiogenesis in collaboration with oncogenic Ras. In this study, we investigated the mechanism of TGF-131-induced epithelial-mesenchymal transition (EMT) and invasiveness in pancreatic cancer cells. Methods: Two pancreatic cancer cell lines were used in this study (PANC-l and BxPC-3 cells). Wild type RhoB or RhoB mutant gene (dominant negative form: N19RhoB, constitutively active form: V14RhoB) was stably transfected into PANC-I cells. Motility and invasiveness by TGF-131 were measured by modified Boyden chamber assays. Levels of Rho G'I'Pases, E-Cadherin, ZO-l were monitored by, Western blot and Immunocytochemistry. We investigated the roles of RhoB function, MAPK and p38MAPK signaling in EMT. Results: TGF-131 induced rapid ERKII2 activation within 5-10 min in both PANC-I and BxPC-3 cells. TGF-131 also caused accumulation of RhoB and downregulation of ZO-I in both cell lines. More detailed studies were performed in PANC-l cells. TGF-131 decreased E-Cadherin levels and increased invasiveness in parental PANC-l cells, however, TGF-131 failed to decrease E-Cadherin and ZO-l expression and to increase invasiveness in cells expressing dominant negative RhoB. PANC-l cells expressing the constitutively active RhoB mutant exhibited downregulation of E-Cadherin. ZO-l and increased invasiveness even in the absence of TGF-131 treatment. The MEK inhibitor PD98059 suppressed TGf-Bl-induced EMT and prevented TGF-131-downregulation of E-Cadherin in parental PANC-I cells and cells expressing the constitutively active RhoB. The p38MAPK inhibitor SB203850 promoted TGF-I3Iinduced EMT and completely inhibited E-Cadherin expression. [Conclusions] This study suggests that TGF131 promotion of EMT and increased invasiveness occurs via RhoB and MAPK-dependent signaling pathway.
4121 THE COMBINATION OF CYCLIN D1 OVEREXPRESSION AND P53 INACTIVATION IS CRITICAL FOR ORAL-ESOPHAGEAL CARCINOGENESIS IN TRANSGENIC MICE. Oliver G. Opitz, Yasir Suliman, Ben Rhoades, Norman E. Sharpless, Ani! K. Rustgi, Div of Gastroenterology, Univ of Pennsylvania, Philadelphia, PA; Div of Hematology/Oncology, MGH, Boston, MA. Introduction: The development and characterization of a genetic model of oral/esophageal squamous cell cancer is important in understanding the underlying molecular mechanisms of oral/esophageal carcinogenesis. C~ clin DI overexpression and p53 mutations are the most common genetic events in oral and esophageal squamous cell cancer. Having previously established the efficacy of the Epstein-Barr virus L2 promoter in targeting genes to the oral-esophageal epithelium, we have cross-bred L2-cyclin DI mice with p53 deficient mice to induce oral-esophageal malignant transformation. Methods: L2-cyclin DI transgenic mice, expressing high, moderate and low levels of cyclin DI, were cross-bred with p53 heterozygous deficient mice to generate cyclin Dl overexpressing/p53 heterozygous and homozygous deficient genotypes and their respective wild-type co~trols. Mice were genotyped using either southern blot or PCR based techniques. Histological analysis was done at different time points by HE staining. Results: Histological analysis was done at 3 months, 6 months and 12 months for cyclin Dl/p53 heterozygous deficient mice and at 4-5 months for cyclin Dl/p53 homozygous deficient mice. This analysis revealed a rapid induction of dysplasia in the oral-esophageal squamous epithelium with progression of the malignant phenotype in cyclin Dl/p53 heterozygous deficient mice. Cyclin Dl/p53 homozygous deficient animals revealed changes consistent with high grade dysplasia. Conclusions: The combination of cyclin D I overexpression and p53 inactivation plays a critical role in induction and progression of carcinogenesis in the oral-esophageal squamous epithelium.
4122 GASTRIC CARCINOGENESIS IN A MURINE MODEL: INDUCTION OF ADENOCARCINOMA AND INTESTINAL METAPLASIA BY PROFOUND DUODENOGASTRIC REFLUX, Ivete B. Prado, Fabio P. Lopasso, Paulo Kassab, Kyoshi Iriya, Antonio A. Laudanna, Med Sch, Univ of Sao Paulo, Sao Paulo, Brazil; Med Sch of Santo Amaro, Sao Paulo, Brazil. Introduction- Patients submitted to gastric resection for benign disease are at a high risk for later gastric tumor dev~lopment. Experimental models have been developed in order to study inflammatory and/or neoplasic changes in the stomach of animals submitted to alkaline reflux. Here we present macroscopic and histological study of gastric tumor induction in rats by surgical duodenogastric reflux with n? carcinoge~ added to diet. Material and Methods- Altogether, 42 male Wistar rats aging 16-20 weeks were randomized to undergo either split gastrojejunostomy (study group, 27 animals) or simple laparotomy and visceral manipulation (control group, 15 animals). The animals of the study group underwen~ a complete duodenogastric reflux: a loop of duodenum 1 em before Treitz ligament was anastomosed to the stomach at the greater curvature (figure 1). Animals from both groups were sacrificed at 12 months after surgery. Esophagus and afferent loop (study group) or duodenum (control) were sectioned and viscera removed en bloc (figure 2). Esophagus, stomach and duodenum were open, macroscopic findings noted (figure 3) and samples of gastric mucosa and gastrojejunal anastomosis were removed, fixed in formol and serial 5p,ID sections were haematoxylin-eosin s~ned. R;esults- ~ll animals with duodenogastric reflux showed macroscopic an~ microscopic J?ucosal inflammation (infiltrating inflammatory cells, capillary congestion and edema, figure 4). Among them, 16 animals (60%) showed areas of inte~ tinal metaplasia, with Paneth cells (figure 5) and 20 rats (74%) had gastn.c tubular adenocarcinoma, intestinal pattern (figure 6). All macro and rmcroscopic findings were always restricted to the duodenogastric anastomosis area or nearby. The animals of the control group did not show any macro or microscopic alteration in the studied samples. Conclusions- This study demonstrates that gastric adenocarcinoma may be induced in the rat at a great incidence by total duodenogastric reflux, without adding a~y carcinogen to diet. In addition, compared to other studies, it shows that 10 this model of tumor induction intestinal metaplasia is a frequent associated condition. Financial support by FAPESP
4123 THE EFFECT OF DIRECT CONTACT BETWEEN PLATELET AND TUMOR CELL IN METASTASIS OF MARIGNANT TUMOR. Keiichi Suzuki, Koichi Aiura, Masakazu Ueda, Masaki Kitajima, Dept of Surg, Sch of Medicine, Keio Univ, Tokyo, Japan. Background: There is increasing evidence that an interaction between tumor cells and platelet may contribute to the development of metastases. We comfirmed poorer prognosis of thrombocytosis in patients with pancreas cancer in our hospital. But the mechanism is still unclear. We studied the effect of direct contact between platelet and tumor cells to extravassel invasion. [Matrials and Methods] We performed Chemoinvasion assay with BIOCOAT MATRIGEL Invasion Chamber. We compared tumor cell invasion under with or without platelet. We used human pancreas cacncer, SW.l990, and human breast cancer, MCF 7, as tumor cells. And we used fibronectin as chemoatractant. We co-incubated tumor cells and platelet, tumor cells and activated platelet by thrombin and tumor cells only under
GASTROENTEROWGY Vol. 118, No.4
37 C,with 5% C02 atmosphere. After 24 hours incubation, we fixed and stained membranes of chambers in Hematoxylin-Eosin, and counted invated tumor cells to bottom of the membrane under 200x. Results: The average cell count of each group of SW.l990 was 26.91200x in tumor cells and platelet group, 58.21200x in tumor cells and activated platelet group and 3.3/200x in tumor cells only. And those of MCF 7 were 13.91200x, 20.61200x and 6.l/200x. Conclusion: Based upon these findings, we postulate that platelet activated extra vassel invasion of tumor cells. Direct contact between platelet and tumor cells may play an important role in this process of metastasis. We assume that tumor cells are stimulated in secretion of matrix metalloprotease such as MMP 9 under direct contact with platelet.
4124 REDUCED ANGIOGENESIS BY MARIMASTAT IN PERITONEAL DISSEMINATION MODEL OF GASTRIC CANCER. Norihito Wada, Yoshihide Otani, Tetsuro Kubota, Masaru Kimata, Akiko Minagawa, Koichiro Kumai, Kaori Kameyama, Yasunori Okada, Masaki Kitajima, Dept of Surg, Keio Univ, Tokyo, Japan; Dept of Pathology, Keio Univ, Tokyo, Japan. We have been investigating the role of matrix metalloproteinase (MMP) in the invasion and metastasis of gastric cancer, and revealed that peritoneal dissemination of gastric cancer is controlledby a MMP inhibitor, marimastat. The mechanisms of the down-regulation, however. are not clarified. We analyzed tumor angiogenesis by means ofanimal model of peritoneal dissemination treated with marimastat. A human gastric carcinoma cell line, TMK-I (I x 106cellslbody) was injected intraperitoneally into 2 groups of 5 BALB/cA scm mice (male, 5 weeks). Subsequently, 27 mglkg/day of marimastat (TA-2516, Tanabe Seiyaku Co., Japan) for treated group (M) or vehicle DMSO for control group (C) was administered for two weeks using subcutaneousmini-osmotic pumps, then the animals were sacrificed. Total weightof dissemination nodules was significantly low in the group M compared with the group C (0.16 g vs.0.40 g, P=0.026). Microvascular count of the group M was also significantly lower than that of the group C (20.5 vs.l5.3 vessels/mrrr', P=0.024). Maximum radius of microvessels in the dissemination nodule was 72.0 micrometerin M group and 15.3 in the C group, respectively (P=0.038). In the process of tumor angiogenesis, basement membrane of existing microvessels is degraded by proteases, chiefly by MMPs-2 and -9. Marimastat can inhibit the enzymes. Thereforeit may show the inhibitory effect for progression of peritoneal metastasis through reduction of angiogenesis by the inhibition of these gelatinases. We conclude that marimastat was associated with reduced tumor weight and microvasculature in peritoneal dissemination model.
4125 TGF-B PROMOTES EPITHELIAL-MESENCHYMAL TRANSITION AND INVASIVENESS VIA RHOB AND MAPK SIGNALING IN PANCREATIC CANCER CELL LINES. Michihiko Wada, Ryo Hosotani, Masayuki Imamura, Robert D. Beauchamp. Kyoto Univ, Kyoto. Japan; Vanderbilt Univ, Nashville. TN. [Purpose] Transforming growth factor-I3(TGF-I3) is a multifunctional growth factor that regulates cell proliferation, differentiation and extracellular matrix remodeling and is overexpressed in many human cancers. Previous reports suggested that TGF-l3can promote tumor invasion and angiogenesis in collaboration with oncogenic Ras. In this study, we investigated the mechanism of TGF-131-induced epithelial-mesenchymal transition (EMT) and invasiveness in pancreatic cancer cells. Methods: Two pancreatic cancer cell lines were used in this study (PANC-l and BxPC-3 cells). Wild type RhoB or RhoB mutant gene (dominant negative form: N19RhoB, constitutively active form: V14RhoB) was stably transfected into PANC-I cells. Motility and invasiveness by TGF-131 were measured by modified Boyden chamber assays. Levels of Rho G'I'Pases, E-Cadherin, ZO-l were monitored by, Western blot and Immunocytochemistry. We investigated the roles of RhoB function, MAPK and p38MAPK signaling in EMT. Results: TGF-131 induced rapid ERKII2 activation within 5-10 min in both PANC-I and BxPC-3 cells. TGF-131 also caused accumulation of RhoB and downregulation of ZO-I in both cell lines. More detailed studies were performed in PANC-l cells. TGF-131 decreased E-Cadherin levels and increased invasiveness in parental PANC-l cells, however, TGF-131 failed to decrease E-Cadherin and ZO-l expression and to increase invasiveness in cells expressing dominant negative RhoB. PANC-l cells expressing the constitutively active RhoB mutant exhibited downregulation of E-Cadherin. ZO-l and increased invasiveness even in the absence of TGF-131 treatment. The MEK inhibitor PD98059 suppressed TGf-Bl-induced EMT and prevented TGF-131-downregulation of E-Cadherin in parental PANC-I cells and cells expressing the constitutively active RhoB. The p38MAPK inhibitor SB203850 promoted TGF-I3Iinduced EMT and completely inhibited E-Cadherin expression. [Conclusions] This study suggests that TGF131 promotion of EMT and increased invasiveness occurs via RhoB and MAPK-dependent signaling pathway.