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Decreased membrane distribution of e-cadherin results in poor cell-cell adhesion in a highly invasive pancreatic adenocarcinoma, PC-1
April 1998
Gastrointestinal Oncology A667
• G2751 LIPID PEROXIDE INDUCES REDOX IMBALANCE AND OXIDATIVE DNA DAMAGE IN HUMAN COLONIC CACO-2 CELLS. C.A. Rhoads, N. Sieber, T.Y. Aw. Department of Molecular and Cellular Physiology, Lousiana State University Medical Center, Shreveport, LA 71130-3932.
• G2753 THE HUMAN GASTROINTESTINAL TRACT IS A RESERVOIR FOR JC VIRUS INFECTION. Luigi Ricciardiello*, Pradeep Ramamirtham*, Ann Randolph, Luigi Laghi, James V. Neel and C. Richard Boland. UCSD and VAMC, San Diego, CA, and Univ of Michigan, Ann Arbor, MI.
Peroxidized lipids are among a variety of natural mutagens and carcinogens in the human diet that can initiate degenerative processes via generation of oxygen radicals. The molecular events in the pathogenesis of intestinal disorders and cancer are unresolved, but an underlying mechanism may involve cellular redox imbalance and oxidative DNA damage. The objective of this study is to determine the effect of subtoxic levels of lipid peroxides (LOOH) on intestinal redox status and DNA damage using a human colonic carcinoma cell line, CaCo-2. Method. CaCo-2 cells were cultured to confluency and incubated with 0 - 50 }aM LOOH for 24h. Cellular redox status was determined by the GSI-I/GSSG ratio. Formation of the DNA adduct, 8-hydroxy guanosine (8-OHG) was used as an index of oxidative DNA damage. Cell viabillity was determined by trypan blue exclusion. Results.. Exposure of CaCo-2 cells to low doses of LOOH (0 - 10 }aM) caused dose-dependent increases in oxidative DNA damage. Maximal 8-OHG formation occurred at 5 }aM LOOH as compared to control (6.3 ± 1.2 vs 2.4 ± 0.7 8-OHG/105 2-deoxyguanine residues). Importantly, the extent of DNA damage paralleled the oxidation of cellular GSH and induction of oxidative stress (GSH/GSSG ratio = 20, vs control = 60). At these low peroxide concentrations, cells were > 90% viable. However, at higher LOOH levels (20 and 50 }aM) substantial cell death occurred by apoptosis (50% and 100%, respectively). Pretreatment of CaCo-2 cells with buthionine sulfoximine, an inhibitor of GSH synthesis, resulted in an enhancement of LOOH-induced 8-OHG formation, indicating a direct correlation between decreased cellular thiol with increased oxidative damage to DNA. Conclusion. Collectively, the findings show that intestinal redox imbalance and oxidative DNA damage are associated with mild lipid peroxide stress while apoptotic cell death occurs at higher oxidative challenge. These results provide insights into lipid peroxide-mediated dismption of cellular redox balance and enhancement of oxidative damage to DNA that have important implications for genesis of intestinal disorders. This research is funded by NIH grants DK-44510 and DK-43785.
JC virus (JCV), the etiologic agent of PML, like other polyomaviruses is an oncogenic double-stranded circular DNA virus, which has been found in brain tumors and recently in colon cancers and normal colonic tissues. Its oncogenic potential is mediated by a transforming protein, T antigen (Tag). JCV has been associated with aneuploid "rogue" lymphocytes, and induces aneuploidy when infected into human embryonic tissues. We have previously demonstrated that 100% of our colon cancer specimens harbors JCV, compared with 75%of normal colonic tissues. Furthermore, colorectal tumors harbored significantly more viral copies than did normal tissues. The mode of transmission of this virus is still unclear and we have hypothesized that the GI tract may be a reservoir for this virus. Methods: In 33 patients undergoing upper and lower endoscopy for other indications at the San Diego VAMC, we collected tissue samples from throughout the GI tract. DNA was extracted from 132 samples and prior to PCR analysis subjected to topoisomerase I treatment to relax the supercoiled DNA which facilitates detection. PCR was designed to amplify the 522 base pair target within the region coding for the Tit antigen. Hybridization with Southern blot analysis, and direct sequencing were used to confirm the identity of the PCR products. Results: 70% of patients harbored JCV sequences, equally distributed throughout the GI tract. 12/16 (75%) of patients undergoing colonoscopy and 11/17 (65%) undergoing upper endoscopy were positive for the virus. The rate of infection for each portion of the GI tract was as follows: esophagus 6/15 (40%), gastric body 7/17 (42%), gastric antrum 3/17 (17%), duodenum 7/17 (42%), cecum 6/15 (40%), transverse colon 8/16 (50%), sigmoid colon 6/16 (37.5%), and rectum 9/16 (56.3%). All 7 patients with coexisting adenomas or carcinomas were positive carded the virus in non-involved tissue. No patients that were negative for JCV displayed any neoplastic lesions. Conclusion: JCV infection can be found throughout the gastrointestinal tract. This indicates that the gastrointestinal tract is a reservoir for the virus, and suggests that the near universal infection of humans with this virus is due to a fecal-oral route. *Both authors contributed equally to this work
• G2752 DECREASED MEMBRANE DISTRIBUTION OF E-CADIIERIN RESULTS IN POOR CELL-CELL ADHESION IN A HIGHLY INVASIVE PANCREATIC ADENOCARCINOMA, PC-1. G. Rhodes S. Yang, J. Marts, T: Howard, Indiana University Medical Center. Indianapolis, Indiana, IN. Introduction: The E-cadherin-catenin complex serves as an essential mediator of compact cell-cell adhesion in epithelial cells and as a suppressor of tumor cell invasion. The PC-1 cell line is derived from an invasive pancreatic adenocarcinoma in hamsters. We hypothesized that its invasive behavior was due to an abnormal expression or function of the E-cadhetin-catenin complex. Methods: Cell aggregation assays were conducted in HEPES buffered saline solution to determine the extent of cell clustering at 15 minute intervals during a 60 minute period. Aggregation indices (AI) were determined by dividing the total number of cluster particles at a given time t by the total cell count at time 0. An index that approaches 1.0 demonstrates poor cell-cell adhesion. Student's t tests were used to evaluate the data. Immunoblots of both cell lysates and immunoprecipitates of E-cadherin complexes were also conducted along with immunofluorescence studies. Results: PC-1 (AI=0.96) demonstrated poor cell-cell adhesion compared to HT-29 controls (AI=0.70), p<0.05. E-cadherin and its associated catenins including ct, [3, ? and pl20-cas were expressed at normal levels, compared to MDCK controls, both in cell lysates and E-cadherin immunoprecipitates. Immunofluorescence showed a decreased membrane distribution of E-cadherin in PC-1 cells compared to MDCK controls. Herbimycin A treatment (tyrosine kinase inhibitor) increased the membrane distribution of E-cadherin, via immunofluorescence, and significantly improved the aggregation index to 0.78, p<0.05. Conclusion: There is no apparent abnormality in gene expression of the E-cadherin-catenin complex in PC-l, but an increased tyrosine phosphorylated state results in decreased membrane distribution of E-cadherin, poor cell-cell adhesion, and an invasive phenotype.
• G2754 AND FAS LIGAND EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA. D.A. Rigberg, J. Centeno, F.S. Kim, M.A. Cole, K. Swanson, M. Maggard, D.W. McFadden, Department of General Surgery, UCLA, Los Angeles, California.
FAS
Fas (Apo-1), a member of the tumor necrosis factor family of receptors, induces programmed cell death (apoptosis) after binding Fas ligand (FasL). Recent evidence suggests tumor cells may use the Fas/FasL interaction to evade the host immune response or kill immune cells. Fas/FasL expression has not been previously reported in esophageal cancers. We hypothesized that expression of Fas would render esophageal cancer cells susceptible to Fas antibody and that irradiation of the cells would increase Fas expression. METHODS: Two human esophageal squamous cell carcinoma lines, KYSE 150 and 410, were exposed to increasing doses of radiation. RNA and protein were extracted. RT-PCR was used to detect mRNA for both Fas and FasL. Fas protein was quantitated by ELISA and confirmed by Westem analysis. Cells were grown in 96-well plates and exposed to increasing doses of Fas monoclonal antibody (maximum 0.05 }ag/ml)± cycloheximide for 6 to 48 hours. Cell viability was assessed by MTT assay. Student's t-test was used to compare protein expression. RESULTS: Both lines expressed Fas and FasL mRNA and protein. Protein levels are shown below in FAS units/ml ± SD: Cell line control KYSE 150 5.0±0.14 KYSE410 4.5±0.01 * p < 0.05 versus controls
3 Gy 8.8±0.04* 4.6±0.04
6 Gy 10.2±0.14" 5.2±0.13"
No alteration in cell growth was detected for antibody treated groups. CONCLUSIONS: Herein, we report for the first time that both Fas and FasL are expressed in esophageal cancer. In addition, we have shown that Fas levels are significantly increased in response to irradiation. However, cells were resistant to treatment with Fas antibody. Further studies are planned to clarify the roles of these mediators in esophageal cancer.
Gastrointestinal Oncology A667
• G2751 LIPID PEROXIDE INDUCES REDOX IMBALANCE AND OXIDATIVE DNA DAMAGE IN HUMAN COLONIC CACO-2 CELLS. C.A. Rhoads, N. Sieber, T.Y. Aw. Department of Molecular and Cellular Physiology, Lousiana State University Medical Center, Shreveport, LA 71130-3932.
• G2753 THE HUMAN GASTROINTESTINAL TRACT IS A RESERVOIR FOR JC VIRUS INFECTION. Luigi Ricciardiello*, Pradeep Ramamirtham*, Ann Randolph, Luigi Laghi, James V. Neel and C. Richard Boland. UCSD and VAMC, San Diego, CA, and Univ of Michigan, Ann Arbor, MI.
Peroxidized lipids are among a variety of natural mutagens and carcinogens in the human diet that can initiate degenerative processes via generation of oxygen radicals. The molecular events in the pathogenesis of intestinal disorders and cancer are unresolved, but an underlying mechanism may involve cellular redox imbalance and oxidative DNA damage. The objective of this study is to determine the effect of subtoxic levels of lipid peroxides (LOOH) on intestinal redox status and DNA damage using a human colonic carcinoma cell line, CaCo-2. Method. CaCo-2 cells were cultured to confluency and incubated with 0 - 50 }aM LOOH for 24h. Cellular redox status was determined by the GSI-I/GSSG ratio. Formation of the DNA adduct, 8-hydroxy guanosine (8-OHG) was used as an index of oxidative DNA damage. Cell viabillity was determined by trypan blue exclusion. Results.. Exposure of CaCo-2 cells to low doses of LOOH (0 - 10 }aM) caused dose-dependent increases in oxidative DNA damage. Maximal 8-OHG formation occurred at 5 }aM LOOH as compared to control (6.3 ± 1.2 vs 2.4 ± 0.7 8-OHG/105 2-deoxyguanine residues). Importantly, the extent of DNA damage paralleled the oxidation of cellular GSH and induction of oxidative stress (GSH/GSSG ratio = 20, vs control = 60). At these low peroxide concentrations, cells were > 90% viable. However, at higher LOOH levels (20 and 50 }aM) substantial cell death occurred by apoptosis (50% and 100%, respectively). Pretreatment of CaCo-2 cells with buthionine sulfoximine, an inhibitor of GSH synthesis, resulted in an enhancement of LOOH-induced 8-OHG formation, indicating a direct correlation between decreased cellular thiol with increased oxidative damage to DNA. Conclusion. Collectively, the findings show that intestinal redox imbalance and oxidative DNA damage are associated with mild lipid peroxide stress while apoptotic cell death occurs at higher oxidative challenge. These results provide insights into lipid peroxide-mediated dismption of cellular redox balance and enhancement of oxidative damage to DNA that have important implications for genesis of intestinal disorders. This research is funded by NIH grants DK-44510 and DK-43785.
JC virus (JCV), the etiologic agent of PML, like other polyomaviruses is an oncogenic double-stranded circular DNA virus, which has been found in brain tumors and recently in colon cancers and normal colonic tissues. Its oncogenic potential is mediated by a transforming protein, T antigen (Tag). JCV has been associated with aneuploid "rogue" lymphocytes, and induces aneuploidy when infected into human embryonic tissues. We have previously demonstrated that 100% of our colon cancer specimens harbors JCV, compared with 75%of normal colonic tissues. Furthermore, colorectal tumors harbored significantly more viral copies than did normal tissues. The mode of transmission of this virus is still unclear and we have hypothesized that the GI tract may be a reservoir for this virus. Methods: In 33 patients undergoing upper and lower endoscopy for other indications at the San Diego VAMC, we collected tissue samples from throughout the GI tract. DNA was extracted from 132 samples and prior to PCR analysis subjected to topoisomerase I treatment to relax the supercoiled DNA which facilitates detection. PCR was designed to amplify the 522 base pair target within the region coding for the Tit antigen. Hybridization with Southern blot analysis, and direct sequencing were used to confirm the identity of the PCR products. Results: 70% of patients harbored JCV sequences, equally distributed throughout the GI tract. 12/16 (75%) of patients undergoing colonoscopy and 11/17 (65%) undergoing upper endoscopy were positive for the virus. The rate of infection for each portion of the GI tract was as follows: esophagus 6/15 (40%), gastric body 7/17 (42%), gastric antrum 3/17 (17%), duodenum 7/17 (42%), cecum 6/15 (40%), transverse colon 8/16 (50%), sigmoid colon 6/16 (37.5%), and rectum 9/16 (56.3%). All 7 patients with coexisting adenomas or carcinomas were positive carded the virus in non-involved tissue. No patients that were negative for JCV displayed any neoplastic lesions. Conclusion: JCV infection can be found throughout the gastrointestinal tract. This indicates that the gastrointestinal tract is a reservoir for the virus, and suggests that the near universal infection of humans with this virus is due to a fecal-oral route. *Both authors contributed equally to this work
• G2752 DECREASED MEMBRANE DISTRIBUTION OF E-CADIIERIN RESULTS IN POOR CELL-CELL ADHESION IN A HIGHLY INVASIVE PANCREATIC ADENOCARCINOMA, PC-1. G. Rhodes S. Yang, J. Marts, T: Howard, Indiana University Medical Center. Indianapolis, Indiana, IN. Introduction: The E-cadherin-catenin complex serves as an essential mediator of compact cell-cell adhesion in epithelial cells and as a suppressor of tumor cell invasion. The PC-1 cell line is derived from an invasive pancreatic adenocarcinoma in hamsters. We hypothesized that its invasive behavior was due to an abnormal expression or function of the E-cadhetin-catenin complex. Methods: Cell aggregation assays were conducted in HEPES buffered saline solution to determine the extent of cell clustering at 15 minute intervals during a 60 minute period. Aggregation indices (AI) were determined by dividing the total number of cluster particles at a given time t by the total cell count at time 0. An index that approaches 1.0 demonstrates poor cell-cell adhesion. Student's t tests were used to evaluate the data. Immunoblots of both cell lysates and immunoprecipitates of E-cadherin complexes were also conducted along with immunofluorescence studies. Results: PC-1 (AI=0.96) demonstrated poor cell-cell adhesion compared to HT-29 controls (AI=0.70), p<0.05. E-cadherin and its associated catenins including ct, [3, ? and pl20-cas were expressed at normal levels, compared to MDCK controls, both in cell lysates and E-cadherin immunoprecipitates. Immunofluorescence showed a decreased membrane distribution of E-cadherin in PC-1 cells compared to MDCK controls. Herbimycin A treatment (tyrosine kinase inhibitor) increased the membrane distribution of E-cadherin, via immunofluorescence, and significantly improved the aggregation index to 0.78, p<0.05. Conclusion: There is no apparent abnormality in gene expression of the E-cadherin-catenin complex in PC-l, but an increased tyrosine phosphorylated state results in decreased membrane distribution of E-cadherin, poor cell-cell adhesion, and an invasive phenotype.
• G2754 AND FAS LIGAND EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA. D.A. Rigberg, J. Centeno, F.S. Kim, M.A. Cole, K. Swanson, M. Maggard, D.W. McFadden, Department of General Surgery, UCLA, Los Angeles, California.
FAS
Fas (Apo-1), a member of the tumor necrosis factor family of receptors, induces programmed cell death (apoptosis) after binding Fas ligand (FasL). Recent evidence suggests tumor cells may use the Fas/FasL interaction to evade the host immune response or kill immune cells. Fas/FasL expression has not been previously reported in esophageal cancers. We hypothesized that expression of Fas would render esophageal cancer cells susceptible to Fas antibody and that irradiation of the cells would increase Fas expression. METHODS: Two human esophageal squamous cell carcinoma lines, KYSE 150 and 410, were exposed to increasing doses of radiation. RNA and protein were extracted. RT-PCR was used to detect mRNA for both Fas and FasL. Fas protein was quantitated by ELISA and confirmed by Westem analysis. Cells were grown in 96-well plates and exposed to increasing doses of Fas monoclonal antibody (maximum 0.05 }ag/ml)± cycloheximide for 6 to 48 hours. Cell viability was assessed by MTT assay. Student's t-test was used to compare protein expression. RESULTS: Both lines expressed Fas and FasL mRNA and protein. Protein levels are shown below in FAS units/ml ± SD: Cell line control KYSE 150 5.0±0.14 KYSE410 4.5±0.01 * p < 0.05 versus controls
3 Gy 8.8±0.04* 4.6±0.04
6 Gy 10.2±0.14" 5.2±0.13"
No alteration in cell growth was detected for antibody treated groups. CONCLUSIONS: Herein, we report for the first time that both Fas and FasL are expressed in esophageal cancer. In addition, we have shown that Fas levels are significantly increased in response to irradiation. However, cells were resistant to treatment with Fas antibody. Further studies are planned to clarify the roles of these mediators in esophageal cancer.