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Infliximab exerts anti-inflammatory capacity in IBD by induction of apoptosis in monocytes
GASTROENTEROLOGY Vol. 118, No.4
A664 AGA ABSTRACTS
3630
3632
INFLIXIMAB EXERTS ANTI-INFLAMMATORY CAPACITY IN ran BY INDUCTION OF APOPTOSIS IN MONOCYTES. Michael Schmidt, Norbert Luegering, Andreas Luegering, Klaus SchulzeOsthoff, Hans Gerd Pauels, Wolfram Domschke, Torsten Kucharzik, Dept of Medicine B, Muenster, Germany; Div of Immunology and Cell Biology, Muenster, Germany; Institute of Immunology, Muenster, Germany. Background: Activation of monocytes and macrophages plays a major role during the inflammatory process of lED. Infliximab is known to be a potent down-regulator of intestinal inflammation in steroid refractory ffiD-patients. However, the exact anti-inflammatory mechanisms of infliximab are not totally understood. Aims: The aim of this in vitro study was to examine the influence of infliximab on monocyte apoptosis from patients with ffiD. Methods: Peripheral blood monocytes were isolated from patients with active lED by using density gradient centrifugation methods with Ficoll and Percoll. Monocytes with a purity >90% were cultured for different times in RPMI medium. Apoptosis was determined with annexin-V staining by FACS analysis, by transmission electron microscopy (TEM), by DNA laddering and by the TDNEL method. Necrosis was excluded by propidium-iodide staining. CD95 and CD95L were determined by FACS analysis, while Caspase 8, Cytochrome C and PARP were measured by Western blots. Results: We were able to show that infJiximab induces apoptosis in IBD monocytes in a dose and time dependent manner. Induction of apoptosis could be demonstrated by four independent methods (annexin-V staining, TUNEL, TEM and DNA laddering). Infliximabinduced apoptosis required the activation of proteases of the caspase family, since a peptide caspase inhibitor attenuated cell death. In addition, the proteolytic activation of caspase-8 followed by a cleavage of PARP into the p85 fragment was observed. After treatment with infJiximab mitochondrial release of cytochrome C could be demonstrated by immunoblot. CD95 and CD95-L expression on monocytes was not involved in infliximab induced apoptosis. Conclusion: This is the first report demonstrating that infliximab induces apoptosis in peripheral monocytes. Induction of programmed cell death in ffiD-monocytes seems to be mediated by a FaslFasL-independent mitochondrial cytochrome C release followed by an activation of caspase signaling. The anti-inflammatory properties of infliximab in steroid refractory lED-patients may therefore be explained, at least in part, by an increased rate of apoptosis in activated monocytes.
INTERACTIVE REGULATION OF HUMAN MISMATCH REPAIR (MMR) SYSTEM COMPONENTS IN MMR DEFICIENT CELLS. Dong K. Chang, Luigi Ricciardiello, Ajay Goel, Christina L. Chang, C. Richard Boland, DCSD, La Jolla, CA; DCSD, and VAMC, La Jolla, CA.
3631 A NOVEL FUNCTION FOR THE TUMOR SUPPRESSOR PI6INK4A: INDUCTION OF ANOIKIS VIA UPREGULATION OF THE AsH I FmRONECTIN RECEPTOR. Thomas Plath, Katharina Detjen, Martina Welzel, Zofia von Marschall, Katrin Farwig, Michael Schimer, Bertram Wiedenmann, Stefan Rosewicz, Charite, Virchow-Klinikum, Berlin, Germany; Schering AG, Berlin, Germany. Background: The tumor suppressor ~ene pl61NK4a is frequently inactivated during human carcinogenesis. pl61 K4a inhibits the kinase activity of the CDK4-6/cyclin D complexes and subsequent phosphorylation of critical substrates necessary for transit through the G I phase of the cell cycle. Recent studies suggested that control of the G)/S boundary might not be the sole biological function of p161NK4a. We therefore hypothesized that p l6lNK4a might influence hitherto unknown critical features of a malignant epithelial phenotype, such as anchorage-dependent apoptosis (anoikis). Methods: Full length human pl6INK4a cDNA was stably transfected into the representative human pancreatic cell line Capan-l. Effects of pl6INK4a reexpression were investigated by colony formation assays and tumor growth in nude mice. To examine anoikis in vitro cells were cultured on polyHEME coated dishes. Apoptosis was evidenced by cell cycle distribution. DNA-laddering, PARP-cleavage and TUNEL staining. Integrin expression was analysed by flow cytornerry, Immunoprecipitation and Northern blot analysis. Results: Reexpression of the tumor suppressor p 16INK4a restitutes anoikis in human pancreatic Capan-l cells. Anoikis of pl6lNK4a transfected cells was evidenced by 4-5 fold inhibition of anchorage independent growth, complete loss of tumorgenicity due to early apoptotic cell death of xenotransplanted tumors and 5-6 fold increase of apoptosis upon loss of anchorage compared to control cells. p16INK4a induced anoikis was mediated by selective, pretranslational 3-5 fold upregulation of the as integrin chain of the a s{31 fibronectin receptor and was completely abolished by supplementation with soluble fibronectin and neutral ising as antibodies, whereas laminin was ineffective. Conclusion: These data suggest a novel functional interference between a cell cycle regulating tumor suppressor gene and membrane bound integrins, thus regulating a hallmark feature of an epithelial transformed phenotype: susceptibility to anoikis.
The DNA MMR system is made up of a series of evolutionarily conserved proteins homologous to the bacterial MutS and MutL counterparts. MutS homologs heterodimerize (hMSH2+hMSH3 or hMSH6) to make up two different DNA mismatch recognition complexes. Similarly, MutL homologs heterodimerize (hMLHl+hPMS2 or hPMSI) to form complexes that are thought to be involved in signalling a DNA repair response. We hypothesized that when one component of the MMR system is defective, the relationships among all of the proteins might change. Methods: DNA MMR-defective colon cancer cell lines (with known MMR mutations) were compared with MMR-proficient cells. Absolute amounts of MMR transcripts were measured using a competitive quantitative RT-PCR, andeach MMR protein was compared using western analysis standardized using purified MMR proteins. Results: hMSH2-mutated LoVo cells did not express detectable hMSH2, hMSH3, or hMSH6 proteins, although hMSH3 and hMSH6 mRNAs were expressed, and up-regulated. Similarly, hMLHI-mutated HCTll6 did not express any detectable hPMS2 protein although its mRNA was expressed. When hMLHI was restored by stable chromosome transfer in HCTl16+chr3 cells, hPMS2 protein was reexpressed without a change in its mRNA level. In SW48, which lacks hMLHI expression due to promoter methylation, hPMS2 protein was also nearly undetectable, however there was there was robust expression of its transcript. In hMSH6-mutated DLDI and HCTl5 cells, hMSH3 protein expression was increased. In these cells, hMSH3 mRNA was increased 2-3 fold in comparison with MMR proficient cells, which suggests that transcriptional up-regulation may contribute to this compensation. Conclusions: The human MMR system is regulated transcriptionally and posttranslationally. hMSH2 and hMLHI are key components of DNA MMR, and these proteins are relatively stable. Stability of the hMSH3 and hMSH6 proteins appears to depend upon the presence of hMSH2, and likewise, the stability of the hPMS2 protein on hMLHI. When hMSH6 is defective, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. In addition to the functional redundancy between hMSH3 and hMSH6, this compensatory regulation might explain why there is an attenuated familial cancer phenotype in patients with germline mutations in the hMSH6 gene.
3633 REARRANGEMENTS OF JC VIRUS MAD-l PROMOTER REGION ARE FREQUENT EVENTS IN INFECTED COLON CANCERS BUT NOT IN MATCHED NORMAL TISSUES. Luigi Ricciardiello, Dong K. Chang, Luigi A. Laghi, Ajay Goel, Christina L. Chang, C. Richard Boland, DC San Diego, La Jolla. CA; DC San Diego and VAMC, La Jolla, CA.
IC Virus is a member of the polyomavirus family, which encodes for a class of highly conserved proteins named T antigen capable of inducing aneuploidy in cultured cells. We have previously isolated ICV T antigen DNA variants from colon cancers and matched normal GI tissues, suggesting that ICV might be correlated to the development of colon cancer. The viral replication and transforming activity is highly dependent upon its regulatory region, which includes promoters for T antigen, the capsid and the agno proteins. The regulatory region of the JCV Mad-l strain contains two 98 base pair repeats that have been found highly rearranged in brain tumors. In this study we evaluated differences in the regulatory region, in colon cancers and normal epithelium, which could explain differences in viral replication and transforming activity in the colon. Methods: Eight pairs of matched normal/colon cancer DNA samples previously found positive for T antigen, underwent Topoisomerase I treatment prior to PCR amplification designed to amplify the whole ICV regulatory region. PCR products were hybridized with a specific probe and underwent further cycling with nested primers. Nested PCR products were cloned and, after screening colonies by PCR, clones of the expected size and those showing band shifts were sequenced in both directions. Results: 285 clones were screened (157 normals, 128 tumors). From all colon cancer tissues, 1-4 clones were characterized by duplication or deletion of the 98 bp tandem repeat, while in one normal tissue sample, one clone out of 20 displayed duplication of the 98 bp tandem repeat (11157 in normals vs 22/128 in
A664 AGA ABSTRACTS
3630
3632
INFLIXIMAB EXERTS ANTI-INFLAMMATORY CAPACITY IN ran BY INDUCTION OF APOPTOSIS IN MONOCYTES. Michael Schmidt, Norbert Luegering, Andreas Luegering, Klaus SchulzeOsthoff, Hans Gerd Pauels, Wolfram Domschke, Torsten Kucharzik, Dept of Medicine B, Muenster, Germany; Div of Immunology and Cell Biology, Muenster, Germany; Institute of Immunology, Muenster, Germany. Background: Activation of monocytes and macrophages plays a major role during the inflammatory process of lED. Infliximab is known to be a potent down-regulator of intestinal inflammation in steroid refractory ffiD-patients. However, the exact anti-inflammatory mechanisms of infliximab are not totally understood. Aims: The aim of this in vitro study was to examine the influence of infliximab on monocyte apoptosis from patients with ffiD. Methods: Peripheral blood monocytes were isolated from patients with active lED by using density gradient centrifugation methods with Ficoll and Percoll. Monocytes with a purity >90% were cultured for different times in RPMI medium. Apoptosis was determined with annexin-V staining by FACS analysis, by transmission electron microscopy (TEM), by DNA laddering and by the TDNEL method. Necrosis was excluded by propidium-iodide staining. CD95 and CD95L were determined by FACS analysis, while Caspase 8, Cytochrome C and PARP were measured by Western blots. Results: We were able to show that infJiximab induces apoptosis in IBD monocytes in a dose and time dependent manner. Induction of apoptosis could be demonstrated by four independent methods (annexin-V staining, TUNEL, TEM and DNA laddering). Infliximabinduced apoptosis required the activation of proteases of the caspase family, since a peptide caspase inhibitor attenuated cell death. In addition, the proteolytic activation of caspase-8 followed by a cleavage of PARP into the p85 fragment was observed. After treatment with infJiximab mitochondrial release of cytochrome C could be demonstrated by immunoblot. CD95 and CD95-L expression on monocytes was not involved in infliximab induced apoptosis. Conclusion: This is the first report demonstrating that infliximab induces apoptosis in peripheral monocytes. Induction of programmed cell death in ffiD-monocytes seems to be mediated by a FaslFasL-independent mitochondrial cytochrome C release followed by an activation of caspase signaling. The anti-inflammatory properties of infliximab in steroid refractory lED-patients may therefore be explained, at least in part, by an increased rate of apoptosis in activated monocytes.
INTERACTIVE REGULATION OF HUMAN MISMATCH REPAIR (MMR) SYSTEM COMPONENTS IN MMR DEFICIENT CELLS. Dong K. Chang, Luigi Ricciardiello, Ajay Goel, Christina L. Chang, C. Richard Boland, DCSD, La Jolla, CA; DCSD, and VAMC, La Jolla, CA.
3631 A NOVEL FUNCTION FOR THE TUMOR SUPPRESSOR PI6INK4A: INDUCTION OF ANOIKIS VIA UPREGULATION OF THE AsH I FmRONECTIN RECEPTOR. Thomas Plath, Katharina Detjen, Martina Welzel, Zofia von Marschall, Katrin Farwig, Michael Schimer, Bertram Wiedenmann, Stefan Rosewicz, Charite, Virchow-Klinikum, Berlin, Germany; Schering AG, Berlin, Germany. Background: The tumor suppressor ~ene pl61NK4a is frequently inactivated during human carcinogenesis. pl61 K4a inhibits the kinase activity of the CDK4-6/cyclin D complexes and subsequent phosphorylation of critical substrates necessary for transit through the G I phase of the cell cycle. Recent studies suggested that control of the G)/S boundary might not be the sole biological function of p161NK4a. We therefore hypothesized that p l6lNK4a might influence hitherto unknown critical features of a malignant epithelial phenotype, such as anchorage-dependent apoptosis (anoikis). Methods: Full length human pl6INK4a cDNA was stably transfected into the representative human pancreatic cell line Capan-l. Effects of pl6INK4a reexpression were investigated by colony formation assays and tumor growth in nude mice. To examine anoikis in vitro cells were cultured on polyHEME coated dishes. Apoptosis was evidenced by cell cycle distribution. DNA-laddering, PARP-cleavage and TUNEL staining. Integrin expression was analysed by flow cytornerry, Immunoprecipitation and Northern blot analysis. Results: Reexpression of the tumor suppressor p 16INK4a restitutes anoikis in human pancreatic Capan-l cells. Anoikis of pl6lNK4a transfected cells was evidenced by 4-5 fold inhibition of anchorage independent growth, complete loss of tumorgenicity due to early apoptotic cell death of xenotransplanted tumors and 5-6 fold increase of apoptosis upon loss of anchorage compared to control cells. p16INK4a induced anoikis was mediated by selective, pretranslational 3-5 fold upregulation of the as integrin chain of the a s{31 fibronectin receptor and was completely abolished by supplementation with soluble fibronectin and neutral ising as antibodies, whereas laminin was ineffective. Conclusion: These data suggest a novel functional interference between a cell cycle regulating tumor suppressor gene and membrane bound integrins, thus regulating a hallmark feature of an epithelial transformed phenotype: susceptibility to anoikis.
The DNA MMR system is made up of a series of evolutionarily conserved proteins homologous to the bacterial MutS and MutL counterparts. MutS homologs heterodimerize (hMSH2+hMSH3 or hMSH6) to make up two different DNA mismatch recognition complexes. Similarly, MutL homologs heterodimerize (hMLHl+hPMS2 or hPMSI) to form complexes that are thought to be involved in signalling a DNA repair response. We hypothesized that when one component of the MMR system is defective, the relationships among all of the proteins might change. Methods: DNA MMR-defective colon cancer cell lines (with known MMR mutations) were compared with MMR-proficient cells. Absolute amounts of MMR transcripts were measured using a competitive quantitative RT-PCR, andeach MMR protein was compared using western analysis standardized using purified MMR proteins. Results: hMSH2-mutated LoVo cells did not express detectable hMSH2, hMSH3, or hMSH6 proteins, although hMSH3 and hMSH6 mRNAs were expressed, and up-regulated. Similarly, hMLHI-mutated HCTll6 did not express any detectable hPMS2 protein although its mRNA was expressed. When hMLHI was restored by stable chromosome transfer in HCTl16+chr3 cells, hPMS2 protein was reexpressed without a change in its mRNA level. In SW48, which lacks hMLHI expression due to promoter methylation, hPMS2 protein was also nearly undetectable, however there was there was robust expression of its transcript. In hMSH6-mutated DLDI and HCTl5 cells, hMSH3 protein expression was increased. In these cells, hMSH3 mRNA was increased 2-3 fold in comparison with MMR proficient cells, which suggests that transcriptional up-regulation may contribute to this compensation. Conclusions: The human MMR system is regulated transcriptionally and posttranslationally. hMSH2 and hMLHI are key components of DNA MMR, and these proteins are relatively stable. Stability of the hMSH3 and hMSH6 proteins appears to depend upon the presence of hMSH2, and likewise, the stability of the hPMS2 protein on hMLHI. When hMSH6 is defective, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. In addition to the functional redundancy between hMSH3 and hMSH6, this compensatory regulation might explain why there is an attenuated familial cancer phenotype in patients with germline mutations in the hMSH6 gene.
3633 REARRANGEMENTS OF JC VIRUS MAD-l PROMOTER REGION ARE FREQUENT EVENTS IN INFECTED COLON CANCERS BUT NOT IN MATCHED NORMAL TISSUES. Luigi Ricciardiello, Dong K. Chang, Luigi A. Laghi, Ajay Goel, Christina L. Chang, C. Richard Boland, DC San Diego, La Jolla. CA; DC San Diego and VAMC, La Jolla, CA.
IC Virus is a member of the polyomavirus family, which encodes for a class of highly conserved proteins named T antigen capable of inducing aneuploidy in cultured cells. We have previously isolated ICV T antigen DNA variants from colon cancers and matched normal GI tissues, suggesting that ICV might be correlated to the development of colon cancer. The viral replication and transforming activity is highly dependent upon its regulatory region, which includes promoters for T antigen, the capsid and the agno proteins. The regulatory region of the JCV Mad-l strain contains two 98 base pair repeats that have been found highly rearranged in brain tumors. In this study we evaluated differences in the regulatory region, in colon cancers and normal epithelium, which could explain differences in viral replication and transforming activity in the colon. Methods: Eight pairs of matched normal/colon cancer DNA samples previously found positive for T antigen, underwent Topoisomerase I treatment prior to PCR amplification designed to amplify the whole ICV regulatory region. PCR products were hybridized with a specific probe and underwent further cycling with nested primers. Nested PCR products were cloned and, after screening colonies by PCR, clones of the expected size and those showing band shifts were sequenced in both directions. Results: 285 clones were screened (157 normals, 128 tumors). From all colon cancer tissues, 1-4 clones were characterized by duplication or deletion of the 98 bp tandem repeat, while in one normal tissue sample, one clone out of 20 displayed duplication of the 98 bp tandem repeat (11157 in normals vs 22/128 in