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Gene array and knockout mouse analysis of VP-16 induced apoptosis in the murine small intestine
METHODS:Primary cultured gastric epithelial cells of rabbit were stimulated by 3-morpholinosydnonimine to produce peroxynitrite and by monochloramine, deoxycholic acid or by nitrite for 4 hours with or without the presence of nitric oxide. Plasma membrane damage was determined by releaseof LDH in the cell supernatantusing the formazan colorimetric method. Apoptosis was detected by flow cytometry. Apoptotic cells were quantified by staining with Annexin V-FITC and not staining by propidium iodide. Result: LDH release and apoptosis induced by the combination of 3-morpholinosydnonimineand nitric oxide were significantly greater than that produced by 3-morpholinosydnonimine alone (LDH release:15.5-+2.5 to 21.2±0.9%, apoptosis:65.1-+2.6 to 81.2-+2.7%, n = 5, p8 h. These data suggest that CUGBP2 binds to its target consensus sequencewithin the COX-2 3'UTR and stabilizes the transcript. Takentogether, these data suggest that CUGBP2may be involved in regulating apoptosis by stabilizing transcripts encoding a proapoptotic function. 3574 Fas Ligand Expressed in Colon Cancer Is Associated with Suppression of Activation and IL-2 Production in Tumor-infiltrating T-ceils Aileen Houston, Michael W. Bennett, Gerald C. O'Sullivan, Fergus Shanahan,Joe O'Connell, National Univ of Ireland, Cork Ireland Introduction: Fas Ligand (FasL/CD95L)has beenshown to mediateimmune privilege in human tumors by inducing Fas-mediatedapoptosis of tumor infiltrating lympbocytes. An additional immunosuppressiverolefor FasLhas emergedwith the discoverythat TCR-mediatedCa2+influx into T-cells, necessaryfor T-cell activation, is blocked by stimulation of Fas on these ceils. Aim: To investigate if FasL expression by colon cancer cells can, in addition to its apoptotic function, suppress activation of antitumor T-cell via stimulation of Fas. Methods: CD3+ peripheral blood T cells were purified using Ficoll gradient and human T cell enrichment columns. Jurkat T-cells and peripheral blood T-cells were co-cultured with SW620 (FasL÷) and HT29 (FasL-) colon carcinoma cell lines, during which the T-cells were activated by addition of PHA. IL-2 production was detectedusing RT-PCR,ELISAand immunofluorescence staining. In a panel of colon adenocarcinomas,expressionof FasL, CD3 and IL2 was localized immunohistochemically in consecutivetumor sections. Results: Stimulation of Fas on Jurkat T-cells and purified peripheralT-cells by FasLexpressedon SW620 cells preventedactivation of the T-cells, as demonstrated by loss of IL-2 production following PHA stimulation. IL-2 production in the suppressed T-cells could be restored by ionomycin, which bypassed the FasL-mediatedblockadeof Ca2+ influx. FasL-mediatedsuppressionof IL-2 production preceded the onset of T-cell apoptosis, and pronounced suppression was detectableafter only 2tir of co-culture with SW620 cells. FasL-specificantisenseoligonucleotidetreatment of the SW620 cells, which transiently inhibited FasL expression, protected the T-cells from inhibition of IL2 synthesis. In addition, FasL-neqativeHT29 cells did not inhibit PHA-inducedIL-2 production in co-cultured T-cells. In vivo, local expression of FasL in colon tumor nests was associated with a mean 2.5-fold decreasein the ratio of IL-2- to CD3-positivecells comparedto matched FasL-negative nests (n =6, p < 0.04). Conclusion: FasL expressed in colon cancer inhibits local antitumor immune responses in vivo by suppressing T cell activation and expression of IL-2.
3575 C09 Negatively Regulates Growth Signaling of the Gastrointestinal Cancer Cells Yoko Murayama, Yasuhisa Shinomura, Jun-lchiro Mlyagawa, Hitoshi Yoshida, Tatsuya Kiyohara, Yoshiji Miyazaki, GraduateSch of Medicine, Osaka Univ, Suita Japan; Shigeki Higashiyama, Sch of Allied Health Sci, Osaka Univ FacuHyof Med, Suita Japan; Yuji Matsuzawa, GraduateSch of Medicine, Osaka Univ, Suita Japan 6ackground: CD9 is a widely expressedcell surface antigen and a member of the tetraspanin superfamily, and has been shown to be involved in a variety of cellular activities such as migration, proliferation and adhesion, but the molecular mechanism by which it mediates such eventsis unclear.We demonstrateherethat anti-CD9 mAb ALB6 inhibits cell proliferation, reduces cell viability, and induces apoptosis in MKN-28 cells, a gastric cancer cell line. Methods: Human gastric carcinoma cell lines, MKN-28 and MKN-45 were cultured in RPMI1640 medium supplemented with 10% F-CSand human colon carcinoma cell lines, SW480, HT-29, CaCo2werecultured in DMEMsupplementedwith f 0 % FCS.The cell surface expression of C09, integrin subunit, ~3, a4, ~,/31 was analyzed by flow cytometry. Cell proliferation, viability and apoptosis were evaluatedby [3H] tbymidine uptake, cell number counts, trypan blue dye exclusion and aonexin-V staining. The association of CD9 with EGFR and/or/~1 intogrin was examinedby double-immunostainingand immunoprecipitation.Proteinsphophorylstion were analysedby Western blot analysis using anti-phosphotyrosineantibody (PY20) or anti-phospho-ERK1/2,JNK/SAPKor p38 MAP kinaseantibodies. Results: Oouble-immunofluorescent staining and immunoprecipitation indicatedthe formation of a complex composed of cDg--epidermalgrowth factor receptor (EGFR),as well as CD9-/31 integrin. Both complexes are located on the cell surface, at the cell-cell contact site. ALB6 induced dot or patch-like aggregation patterns of both CD9-EGFR and CD9-/~I integrin, and abrogated the growth stimulation of MKN-28 cells induced by EGFR ligands such as EGF and HB-EGF,while the tyrosine phosphoryfation of EGFR remained maffected. Analyses of the signaling molecules regulated by ALB6 revealedthat the tyrosine phosphoryletion of the Shc 45 kDa isoform, did not lead to Grb2 recruitment, the downregulation of the phosphorylation of ERK1/2, and a transient elevationin the level of phosphoryiationof JNK/SAPKand p38 MAP kinase. Conclusions: CD9 is involved in cell surface complex formation with EGFRand/~1 integrin which negatively regulates growth signaling of the gastrointestinal cancer cells via the modulation of EGFRsignaling.
Gene AnrayAnd KnockoutMeuse Analysis Of VP-16 Induced Apeptosis In The Mudne Small Intestine Dominic P. McMichaei, Andrea Davies, Emma Marshman, Penny D. Ottewell, John R. Jenkins, Aiastalr Jm Watson, Univ of Liverpool, Liverpool United Kingdom lutTeduction: As the epithelial cells of the small intestine move up the crypt to the villi they become progressively resistant to DNA damage-inducedapoptosis. VP-16 interferes with the breakage and religation activity of topoisomerase II resulting in the formation of cleavable complexes and double stranded DNA breaks. The genetic regulation of VP-15-induced apoptosis in the small intestine is currently unknown. Hypothesis: Etoposide induces apoptosis via a mechanism dependent on the tumour suppressor p53, the cyclin dependent kinase inhibitor p21 and the pro-apoptotic protein Bax. Methods: BDF wildtype mice, p53, p21 and Bax knockout mice and their witdtype iittermates were dosed with lOmg/kg etoposide by intraperitoneal injection (n = 4 for each). The small intestines were isolated over a series of time points and scored for apoptotic and mitotic bodies relative to position along the crypt/ villus axis by histological examination. Epithelial cells were harvested by a calcium chelation technique which largely excludes non-epithelial cells. Epithelial cell gene expression was analyzed using ClontechAtlas Mouse 1.2 Gene arrays three times using mRNA extracted 4.5 hours after etoposide treatment from p53 knockout mice and wildtype littermates. Results: At 4.5 hours after administration, stoposide induced apoptosis is p53 dependentand occurs maximally between cell positions 3 and 9 along the crypt/villus axis. No apoptosis occurred on the villi of any of the mouse strains studies. Furthermore, etoposide induced apoptosis is independentof p21 and Bax, both thought to lie downstream of p53 in the DNA damage induced pro-apoptotic pathway.Genearray analysisof wildtype and p53 knockout mice dosed with lOmg/kg etoposide shows 42 genes exhibiting a p53 dependentincrease in expression, including Defender Against Cell Death-t (11 fold increase _+ 1.6), Angiotensin Converting Enzyme (10 fold increase + 2.8) and MAP Kinase Kinase 3 (4 fold increase -+ 0.8); and 13 genes exhibiting a p53 dependentdecrease in expression, of particular note is bcl-2 (5 fold decrease ± 0.2), the c-abl protooncogene (2 told decrease -+ 0.44) and Calmodulin Kinase II (2.5 fold decrease± 0.065). Conclusions:Etoposideinducesapoptosis via a p53 dependent, p21/Sax independentpathway involving stimulation of the MAP kinase pathway and inhibition of Calmodulin Kinase II. Induction of the DefenderAgainst Cell Death-1 and inhibition of Bcl2 are likely to be a reaction against the induction of cell death.
~77 l~e Wild Type Gastrin Receptor But Not A Colon Cancer-Derived Gastrin Receptor Mutant Mediates A Pro-ApopteUcEffect Of Gastrin in Colorectal Cancer Ceils Frank Schmitz, Dept of Medicine I, St Josef-Hospital, Bochum Germany; Babette Reimann, Unlv of Kiel Germany; Alexander Arit, Karl H. Herzig, Univ of Kiel, Kiel Germany; Wolfgang E. Schmidt, Dept of Medicine I, St Joset-Hospital, Bochum Germany; Heiner Schaefer, Univ of Kiel, Kiel Germany 8eckgroond: Gastrin exhibits gruwth-promoting effects in some colorsctal cancer (crc) cell lines. The gastfin receptor (GR) structure appears to be a critical determinant for its growth factor action. Aim: To address the issue whether gastrin exhibits pro- or anti-apoptotic effects in crc cells which stably express the human GR wild type or a colon cancer-derived GR mutant cDNA. Methods: The human crc cell line Cold 320 was stably transtested with the human GR wild type cDNA (ColD 320 WT) or with an intracelluiar loop 3 mutant GR cDNA (ColD320 MUT). Affinitly for gastrin was determined by '~I-CCK-8 binding in stably transfected Cold 320 cells. Apoptosis was inducedby etoposide,doxorubicinor TNFa at various concentrations in the presence or absence of gastrin and quantitated by FITC-annexinV staining and
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3575 C09 Negatively Regulates Growth Signaling of the Gastrointestinal Cancer Cells Yoko Murayama, Yasuhisa Shinomura, Jun-lchiro Mlyagawa, Hitoshi Yoshida, Tatsuya Kiyohara, Yoshiji Miyazaki, GraduateSch of Medicine, Osaka Univ, Suita Japan; Shigeki Higashiyama, Sch of Allied Health Sci, Osaka Univ FacuHyof Med, Suita Japan; Yuji Matsuzawa, GraduateSch of Medicine, Osaka Univ, Suita Japan 6ackground: CD9 is a widely expressedcell surface antigen and a member of the tetraspanin superfamily, and has been shown to be involved in a variety of cellular activities such as migration, proliferation and adhesion, but the molecular mechanism by which it mediates such eventsis unclear.We demonstrateherethat anti-CD9 mAb ALB6 inhibits cell proliferation, reduces cell viability, and induces apoptosis in MKN-28 cells, a gastric cancer cell line. Methods: Human gastric carcinoma cell lines, MKN-28 and MKN-45 were cultured in RPMI1640 medium supplemented with 10% F-CSand human colon carcinoma cell lines, SW480, HT-29, CaCo2werecultured in DMEMsupplementedwith f 0 % FCS.The cell surface expression of C09, integrin subunit, ~3, a4, ~,/31 was analyzed by flow cytometry. Cell proliferation, viability and apoptosis were evaluatedby [3H] tbymidine uptake, cell number counts, trypan blue dye exclusion and aonexin-V staining. The association of CD9 with EGFR and/or/~1 intogrin was examinedby double-immunostainingand immunoprecipitation.Proteinsphophorylstion were analysedby Western blot analysis using anti-phosphotyrosineantibody (PY20) or anti-phospho-ERK1/2,JNK/SAPKor p38 MAP kinaseantibodies. Results: Oouble-immunofluorescent staining and immunoprecipitation indicatedthe formation of a complex composed of cDg--epidermalgrowth factor receptor (EGFR),as well as CD9-/31 integrin. Both complexes are located on the cell surface, at the cell-cell contact site. ALB6 induced dot or patch-like aggregation patterns of both CD9-EGFR and CD9-/~I integrin, and abrogated the growth stimulation of MKN-28 cells induced by EGFR ligands such as EGF and HB-EGF,while the tyrosine phosphoryfation of EGFR remained maffected. Analyses of the signaling molecules regulated by ALB6 revealedthat the tyrosine phosphoryletion of the Shc 45 kDa isoform, did not lead to Grb2 recruitment, the downregulation of the phosphorylation of ERK1/2, and a transient elevationin the level of phosphoryiationof JNK/SAPKand p38 MAP kinase. Conclusions: CD9 is involved in cell surface complex formation with EGFRand/~1 integrin which negatively regulates growth signaling of the gastrointestinal cancer cells via the modulation of EGFRsignaling.
Gene AnrayAnd KnockoutMeuse Analysis Of VP-16 Induced Apeptosis In The Mudne Small Intestine Dominic P. McMichaei, Andrea Davies, Emma Marshman, Penny D. Ottewell, John R. Jenkins, Aiastalr Jm Watson, Univ of Liverpool, Liverpool United Kingdom lutTeduction: As the epithelial cells of the small intestine move up the crypt to the villi they become progressively resistant to DNA damage-inducedapoptosis. VP-16 interferes with the breakage and religation activity of topoisomerase II resulting in the formation of cleavable complexes and double stranded DNA breaks. The genetic regulation of VP-15-induced apoptosis in the small intestine is currently unknown. Hypothesis: Etoposide induces apoptosis via a mechanism dependent on the tumour suppressor p53, the cyclin dependent kinase inhibitor p21 and the pro-apoptotic protein Bax. Methods: BDF wildtype mice, p53, p21 and Bax knockout mice and their witdtype iittermates were dosed with lOmg/kg etoposide by intraperitoneal injection (n = 4 for each). The small intestines were isolated over a series of time points and scored for apoptotic and mitotic bodies relative to position along the crypt/ villus axis by histological examination. Epithelial cells were harvested by a calcium chelation technique which largely excludes non-epithelial cells. Epithelial cell gene expression was analyzed using ClontechAtlas Mouse 1.2 Gene arrays three times using mRNA extracted 4.5 hours after etoposide treatment from p53 knockout mice and wildtype littermates. Results: At 4.5 hours after administration, stoposide induced apoptosis is p53 dependentand occurs maximally between cell positions 3 and 9 along the crypt/villus axis. No apoptosis occurred on the villi of any of the mouse strains studies. Furthermore, etoposide induced apoptosis is independentof p21 and Bax, both thought to lie downstream of p53 in the DNA damage induced pro-apoptotic pathway.Genearray analysisof wildtype and p53 knockout mice dosed with lOmg/kg etoposide shows 42 genes exhibiting a p53 dependentincrease in expression, including Defender Against Cell Death-t (11 fold increase _+ 1.6), Angiotensin Converting Enzyme (10 fold increase + 2.8) and MAP Kinase Kinase 3 (4 fold increase -+ 0.8); and 13 genes exhibiting a p53 dependentdecrease in expression, of particular note is bcl-2 (5 fold decrease ± 0.2), the c-abl protooncogene (2 told decrease -+ 0.44) and Calmodulin Kinase II (2.5 fold decrease± 0.065). Conclusions:Etoposideinducesapoptosis via a p53 dependent, p21/Sax independentpathway involving stimulation of the MAP kinase pathway and inhibition of Calmodulin Kinase II. Induction of the DefenderAgainst Cell Death-1 and inhibition of Bcl2 are likely to be a reaction against the induction of cell death.
~77 l~e Wild Type Gastrin Receptor But Not A Colon Cancer-Derived Gastrin Receptor Mutant Mediates A Pro-ApopteUcEffect Of Gastrin in Colorectal Cancer Ceils Frank Schmitz, Dept of Medicine I, St Josef-Hospital, Bochum Germany; Babette Reimann, Unlv of Kiel Germany; Alexander Arit, Karl H. Herzig, Univ of Kiel, Kiel Germany; Wolfgang E. Schmidt, Dept of Medicine I, St Joset-Hospital, Bochum Germany; Heiner Schaefer, Univ of Kiel, Kiel Germany 8eckgroond: Gastrin exhibits gruwth-promoting effects in some colorsctal cancer (crc) cell lines. The gastfin receptor (GR) structure appears to be a critical determinant for its growth factor action. Aim: To address the issue whether gastrin exhibits pro- or anti-apoptotic effects in crc cells which stably express the human GR wild type or a colon cancer-derived GR mutant cDNA. Methods: The human crc cell line Cold 320 was stably transtested with the human GR wild type cDNA (ColD 320 WT) or with an intracelluiar loop 3 mutant GR cDNA (ColD320 MUT). Affinitly for gastrin was determined by '~I-CCK-8 binding in stably transfected Cold 320 cells. Apoptosis was inducedby etoposide,doxorubicinor TNFa at various concentrations in the presence or absence of gastrin and quantitated by FITC-annexinV staining and
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