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Identification of the Hsrbc Tumor Suppressor Gene as a Novel Transcription Target of TNFα in Human Colon Epithelial Cells
Tu1730 The Order of Addition of Trastuzumab and Topoisomerase-1 Inhibitor (SN-38) Influences SN-38's Cytotoxic Effects on Gastric Cancer Cell Lines Positive or Negative for HER2 Expression Mihoko Yamade, Takahiro Uotani, Masafumi Nishino, Chise Kodaira, Mitsushige Sugimoto, Mutsuhiro Ikuma, Takahisa Furuta
Tu1728
Background/Aims: Some of gastric cancer cell lines express HER2, a member of the epidermal growth factor receptor family of protein tyrosine kinase growth factor receptors. Trastuzumab inhibits HER2 signaling, and recent clinical studies have demonstrated that this also results in an enhancement of the therapeutic efficacy of antineoplastic agents for treating gastric cancer. However, most antineoplastic agents, such as SN-38 (a topoisomerase-1 inhibitor), induce DNA breaks during replication or transcription. This raises the question of how the cell cycle delay induced by trastuzumab enhances the cytotoxicity of antineoplastic agents. Therefore, we studied the effects of trastuzumab on levels of DNA breaks induced by SN38 in gastric cancer cell lines positive or negative for HER2 expression. Methods: The gastric cancer-derived cell line, NCI-N87, strongly expresses HER2; whereas, HER2 expression is undetectable in a second cell line, designated MKN74. Cultures of both cell lines were exposed to SN-38 in the presence or absence of trastuzumab. Trastuzumab was added either prior to, or after SN-38, and the induction of gamma-H2AX was used as a marker for DNA double strand break (DSB) as determined by immunofluorescence and western blotting. The effects of trastuzumab on SN-38's cytotoxicity was measured by an MTT assay. Cell cycle progression in the presence of trastuzumab was determined by flow cytometry. In addition, the kinetics of synthesis of DNA repair proteins such as MRE11 and NBS1 were determined by immunofluorescence and western blotting. Results: SN-38 induced gammaH2AX in cultures of both NCI-N87 and MKN74. When trastuzumab was administered after SN-38, it increased gamma-H2AX levels over that induced by SN-38 alone. This resulted in enhanced SN-38's cytotoxicity for NCI-N87, but not MKN74. Accumulation of MRE11 and NBS1 at gamma-H2AX foci was attenuated when trastuzumab was added after SN-38. In contrast, pretreatment with trastuzumab decreased SN-38-induced gamma-H2AX levels in a dose dependent manner in NCI-N87, but not in MKN74. Trastuzumab induced Sphase delay only in NCI-N87. Conclusion: Trastuzumab appears to alter the function of DSB repair proteins, such as MRN, at SN-38-induced DSB sites in a HER2-dependent manner. This may account for SN-38's enhanced cytotoxicity in the presence of trastuzumab. In contrast, trastuzumab inhibits cell cycle progression, which would diminish the replicationdependent generation of DSBs by SN-38. Therefore, we conclude that the ability of trastuzumab to enhance SN-38's cytotoxicity depends on the order of administration of these two drugs.
Identification of the Hsrbc Tumor Suppressor Gene as a Novel Transcription Target of TNFα in Human Colon Epithelial Cells Hyo-Jong Kim hSRBC is a recently identified tumor suppressor located at the chromosomal region 11p15.4. hSRBC expression is frequently downregulated in many types of malignancies including colorectal cancer due to the aberrant promoter CpG sites hypermethylation. The hSRBC activation induces cell cycle arrest and apoptosis by enhancing the protein stability of p53. However, the molecular basis of hSRBC activation and its regulation has been poorly understood. To explore the implication of hSRBC abnormality in colorectal tumorigenesis and the molecular mechanism underlying its tumor suppressive function, the present study tested the possible regulation of hSRBC by TNFα.TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFa in a dose- and time-associated manner in various human colonic epithelial cells, including HCT15 and HT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα. TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFα in a dose- and time-associated manner in various human colonic epithelial cells, including HCT15 and HT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα.
Tu1731 Protein Tyrosine Kinase 6 Promotes STAT3 Activation and Colon Tumorigenesis In Vivo Jessica Gierut, Yu Zheng, Wenjun Bie, Angela L. Tyner
Tu1729 In a Xenograft Mouse Model, Bethanechol, a Muscarinic Receptor Agonist, Attenuates the Growth of Human Colon Cancer Cells Zhongsheng Peng, Jean-Pierre Raufman, Guofeng Xie
BACKGROUND & Aims: Protein Tyrosine Kinase 6 (PTK6) is an intracellular tyrosine kinase expressed in the normal gastrointestinal tract and skin, where its expression is associated with growth inhibition and differentiation. Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating a variety of cellular processes and persistent activation of STAT3 has been linked to development of colorectal cancers. It has been shown that PTK6 enhances activating tyrosine phosphorylation of STAT3 in cell lines of different origin. The aim of our study was to determine how PTK6 signaling contributes to colon carcinogenesis. METHODS: Ptk6 +/+ and Ptk6 -/- mice were treated with the colon carcinogen azoxymethane (AOM) alone or in combination with dextran sodium sulfate (DSS), a tumor promoter, and formation of premalignant aberrant crypt foci (ACF) and/or tumors was examined. Expression of PTK6 and activation of STAT3 was examined in mouse intestinal tissues and tumors, and in the human HCT116 colon cancer cell line with and without shRNA mediated knockdown of PTK6, using immunohistochemistry and/or immunoblotting. RESULTS: In contrast to the growth inhibiting functions of PTK6 in the normal intestinal epithelium, PTK6 promoted AOM induced tumorigenesis, and Ptk6 -/- mice were resistant to AOM and developed significantly less ACF and tumors. Increased apoptosis accompanied by increased compensatory proliferation and enhanced STAT3 activation and increased expression of STAT3 target genes was observed in the intestinal epithelium and intestinal tumors of wild type control mice treated with AOM, compared with Ptk6 -/- mice. In addition to enhancing STAT3 activation In Vivo, we found that PTK6 maintains increased basal STAT3 activity and promotes STAT3 activation in response to growth factors in the HCT116 colon cancer cell line. CONCLUSIONS: These data demonstrate that PTK6 plays an essential role in regulating STAT3 activity in the mouse colon and in human colon cancer cells. STAT3 activation has been correlated with development of colon tumors and PTK6 promotes colon tumorigenesis In Vivo.
The majority of human colon cancers over-express M3 muscarinic receptors (M3R). Previously, we showed that genetic ablation of M3R attenuates murine colon epithelial cell proliferation and neoplasia, indicating an important role for muscarinic receptors in intestinal tumorigenesis (Cancer Res 68:3573, 2008). To determine whether muscarinic receptor agonists affect tumor growth, we examined the effects of bethanechol, a muscarinic receptor agonist which does not cross the blood-brain barrier and is FDA-approved for treatment of urinary retention, on the growth of human colon cancer xenografts in nude mice. To generate human colon tumor implants, one million human H508 colon cancer cells that over-express M3R were injected subcutaneously into the flanks of 6-week-old male nude mice. Tumor size was measured weekly with calipers, and tumor volumes were calculated using the formula: tumor volume = (length ×width2)/2. At the end of the 2nd week, when tumors were measurable (about 70 mm3 in volume), the mice were randomly allocated to two groups; provided with drinking water that contained 400 μg/mL bethanechol chloride or drinking water alone. Mice were allowed to drink freely for 14 consecutive weeks and did not exhibit any notable side-effects. At the end of the 14th week, mice were euthanized and the tumors were excised and weighed; livers and lungs were examined by necropsy for metastases. From week 1 to week 8, tumor volumes were not significantly different in the 2 groups [mean ± SE in mm3; bethanechol group (N=10) vs. control group (N=8): 7th wk, 370.8 ± 89.0 vs. 387.6 ± 91.5; 8th wk, 515.8 ± 75.4 vs. 597.7 ± 109.1; all p > 0.05). Starting at the 9th week and persisting until the end of treatment, tumor volumes in the bethanechol-treated group decreased significantly compared with that of control mice (9th wk: 552.1 ± 114.1 vs. 721.0 ± 141.0; 10th wk: 614.3 ± 152.8 vs. 1170.0 ± 284.7; 11th wk: 663.0 ± 144.1 vs. 1321.0 ± 255.8 m; 12th wk: 835.3 ± 167.6 vs 1739.8 ± 228.8; 13th wk: 1002.2 ± 202.0 vs. 2046.1 ± 402.4; 14th wk: 1207.4 ± 234.1 vs. 2355.2 ± 385.6; all p < 0.05). Similarly, tumor weights decreased significantly in bethanechol-treated compared to control mice (mean ± SE: 0.88 ± 0.15 g vs. 1.74 ± 0.27 g, p < 0.05). No metastatic tumors were found in the livers and lungs in either group. These results indicate that bethanechol inhibits growth of human colon cancer xenografts in nude mice, and may be useful as a therapeutic agent for colon cancer. Although the results are surprising considering a slight pro-proliferative effect of bethanechol in H508 cells In Vitro, they are consistent with an earlier report that bethanechol inhibited pancreatic carcinogenesis in hamsters. This inhibitory effect of bethanechol on colon neoplasia is currently being investigated in other mouse models of intestinal carcinogenesis.
Tu1732 Polymorphisms in the GATA-Binding Sites of the Vitamin D Receptor Gene Affect Its Transcription in the Esophagus, and Are Associated With a Reduced Risk for Esophageal Disease Anouk van de Winkel, André Uitterlinden, Agnieszka M. Rygiel, Leon M. Moons, Pascal P. Arp, Joyce van Meurs, Kausilia K. Krishnadath, Ernst J. Kuipers, Luc J. van der Laan Background: Recent studies stress the role of vitamin D and its receptor (VDR) in complex diseases, including inflammatory bowel disease and colorectal cancer. The anti-proliferative and immune modulatory effects of VDR signalling are well recognized. Here we investigate the genetic variation of VDR and its functional implications in esophageal inflammation and malignancy. Aim: To determine single nucleotide polymorphisms (SNPs) in highly conserved non-coding regulatory regions of the VDR gene, and to study their association with risk for erosive esophagitis (EE), Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), as well as their function in regulating VDR gene expression. Methods: DNA was obtained from Caucasian subjects including patients with EE (n=307), BE (n=260), and EAC (n=
S-821
AGA Abstracts
AGA Abstracts
degree of LINE-1 hypomethylation is a unique feature of early-onset CRCs, and distinguishes them from older CRCs. Since LINE-1 hypomethylation is a surrogate marker for increased chromosomal instability, our data provide a novel and previously unrecognized explanation for some of the biological differences underpinning early-onset CRCs.
Tu1728
Background/Aims: Some of gastric cancer cell lines express HER2, a member of the epidermal growth factor receptor family of protein tyrosine kinase growth factor receptors. Trastuzumab inhibits HER2 signaling, and recent clinical studies have demonstrated that this also results in an enhancement of the therapeutic efficacy of antineoplastic agents for treating gastric cancer. However, most antineoplastic agents, such as SN-38 (a topoisomerase-1 inhibitor), induce DNA breaks during replication or transcription. This raises the question of how the cell cycle delay induced by trastuzumab enhances the cytotoxicity of antineoplastic agents. Therefore, we studied the effects of trastuzumab on levels of DNA breaks induced by SN38 in gastric cancer cell lines positive or negative for HER2 expression. Methods: The gastric cancer-derived cell line, NCI-N87, strongly expresses HER2; whereas, HER2 expression is undetectable in a second cell line, designated MKN74. Cultures of both cell lines were exposed to SN-38 in the presence or absence of trastuzumab. Trastuzumab was added either prior to, or after SN-38, and the induction of gamma-H2AX was used as a marker for DNA double strand break (DSB) as determined by immunofluorescence and western blotting. The effects of trastuzumab on SN-38's cytotoxicity was measured by an MTT assay. Cell cycle progression in the presence of trastuzumab was determined by flow cytometry. In addition, the kinetics of synthesis of DNA repair proteins such as MRE11 and NBS1 were determined by immunofluorescence and western blotting. Results: SN-38 induced gammaH2AX in cultures of both NCI-N87 and MKN74. When trastuzumab was administered after SN-38, it increased gamma-H2AX levels over that induced by SN-38 alone. This resulted in enhanced SN-38's cytotoxicity for NCI-N87, but not MKN74. Accumulation of MRE11 and NBS1 at gamma-H2AX foci was attenuated when trastuzumab was added after SN-38. In contrast, pretreatment with trastuzumab decreased SN-38-induced gamma-H2AX levels in a dose dependent manner in NCI-N87, but not in MKN74. Trastuzumab induced Sphase delay only in NCI-N87. Conclusion: Trastuzumab appears to alter the function of DSB repair proteins, such as MRN, at SN-38-induced DSB sites in a HER2-dependent manner. This may account for SN-38's enhanced cytotoxicity in the presence of trastuzumab. In contrast, trastuzumab inhibits cell cycle progression, which would diminish the replicationdependent generation of DSBs by SN-38. Therefore, we conclude that the ability of trastuzumab to enhance SN-38's cytotoxicity depends on the order of administration of these two drugs.
Identification of the Hsrbc Tumor Suppressor Gene as a Novel Transcription Target of TNFα in Human Colon Epithelial Cells Hyo-Jong Kim hSRBC is a recently identified tumor suppressor located at the chromosomal region 11p15.4. hSRBC expression is frequently downregulated in many types of malignancies including colorectal cancer due to the aberrant promoter CpG sites hypermethylation. The hSRBC activation induces cell cycle arrest and apoptosis by enhancing the protein stability of p53. However, the molecular basis of hSRBC activation and its regulation has been poorly understood. To explore the implication of hSRBC abnormality in colorectal tumorigenesis and the molecular mechanism underlying its tumor suppressive function, the present study tested the possible regulation of hSRBC by TNFα.TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFa in a dose- and time-associated manner in various human colonic epithelial cells, including HCT15 and HT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα. TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. It was found that mRNA expression of hSRBC is strongly activated by TNFα in a dose- and time-associated manner in various human colonic epithelial cells, including HCT15 and HT-29. An immunoblot assay also revealed that hSRBC protein expression is increased following TNFa treatment. TNFa induction of hSRBC was blocked by pretreatment of the cells with the NFkB inhibitor BAY11-7082 or transfection with a dominant negative mutant form of IkB, indicating that TNFα-induced hSRBC expression is mediated via the NFkB pathway. Flow cytometric analysis of sub-G1 phase using hSRBC-nonexpressing HCT15 cells revealed that ectopic expression of hSRBC greatly enhances apoptotic response to TNFα. Collectively, these data strongly suggest that hSRBC might be a novel transcription target of TNFα and play an important role in cellular response to TNFα.
Tu1731 Protein Tyrosine Kinase 6 Promotes STAT3 Activation and Colon Tumorigenesis In Vivo Jessica Gierut, Yu Zheng, Wenjun Bie, Angela L. Tyner
Tu1729 In a Xenograft Mouse Model, Bethanechol, a Muscarinic Receptor Agonist, Attenuates the Growth of Human Colon Cancer Cells Zhongsheng Peng, Jean-Pierre Raufman, Guofeng Xie
BACKGROUND & Aims: Protein Tyrosine Kinase 6 (PTK6) is an intracellular tyrosine kinase expressed in the normal gastrointestinal tract and skin, where its expression is associated with growth inhibition and differentiation. Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating a variety of cellular processes and persistent activation of STAT3 has been linked to development of colorectal cancers. It has been shown that PTK6 enhances activating tyrosine phosphorylation of STAT3 in cell lines of different origin. The aim of our study was to determine how PTK6 signaling contributes to colon carcinogenesis. METHODS: Ptk6 +/+ and Ptk6 -/- mice were treated with the colon carcinogen azoxymethane (AOM) alone or in combination with dextran sodium sulfate (DSS), a tumor promoter, and formation of premalignant aberrant crypt foci (ACF) and/or tumors was examined. Expression of PTK6 and activation of STAT3 was examined in mouse intestinal tissues and tumors, and in the human HCT116 colon cancer cell line with and without shRNA mediated knockdown of PTK6, using immunohistochemistry and/or immunoblotting. RESULTS: In contrast to the growth inhibiting functions of PTK6 in the normal intestinal epithelium, PTK6 promoted AOM induced tumorigenesis, and Ptk6 -/- mice were resistant to AOM and developed significantly less ACF and tumors. Increased apoptosis accompanied by increased compensatory proliferation and enhanced STAT3 activation and increased expression of STAT3 target genes was observed in the intestinal epithelium and intestinal tumors of wild type control mice treated with AOM, compared with Ptk6 -/- mice. In addition to enhancing STAT3 activation In Vivo, we found that PTK6 maintains increased basal STAT3 activity and promotes STAT3 activation in response to growth factors in the HCT116 colon cancer cell line. CONCLUSIONS: These data demonstrate that PTK6 plays an essential role in regulating STAT3 activity in the mouse colon and in human colon cancer cells. STAT3 activation has been correlated with development of colon tumors and PTK6 promotes colon tumorigenesis In Vivo.
The majority of human colon cancers over-express M3 muscarinic receptors (M3R). Previously, we showed that genetic ablation of M3R attenuates murine colon epithelial cell proliferation and neoplasia, indicating an important role for muscarinic receptors in intestinal tumorigenesis (Cancer Res 68:3573, 2008). To determine whether muscarinic receptor agonists affect tumor growth, we examined the effects of bethanechol, a muscarinic receptor agonist which does not cross the blood-brain barrier and is FDA-approved for treatment of urinary retention, on the growth of human colon cancer xenografts in nude mice. To generate human colon tumor implants, one million human H508 colon cancer cells that over-express M3R were injected subcutaneously into the flanks of 6-week-old male nude mice. Tumor size was measured weekly with calipers, and tumor volumes were calculated using the formula: tumor volume = (length ×width2)/2. At the end of the 2nd week, when tumors were measurable (about 70 mm3 in volume), the mice were randomly allocated to two groups; provided with drinking water that contained 400 μg/mL bethanechol chloride or drinking water alone. Mice were allowed to drink freely for 14 consecutive weeks and did not exhibit any notable side-effects. At the end of the 14th week, mice were euthanized and the tumors were excised and weighed; livers and lungs were examined by necropsy for metastases. From week 1 to week 8, tumor volumes were not significantly different in the 2 groups [mean ± SE in mm3; bethanechol group (N=10) vs. control group (N=8): 7th wk, 370.8 ± 89.0 vs. 387.6 ± 91.5; 8th wk, 515.8 ± 75.4 vs. 597.7 ± 109.1; all p > 0.05). Starting at the 9th week and persisting until the end of treatment, tumor volumes in the bethanechol-treated group decreased significantly compared with that of control mice (9th wk: 552.1 ± 114.1 vs. 721.0 ± 141.0; 10th wk: 614.3 ± 152.8 vs. 1170.0 ± 284.7; 11th wk: 663.0 ± 144.1 vs. 1321.0 ± 255.8 m; 12th wk: 835.3 ± 167.6 vs 1739.8 ± 228.8; 13th wk: 1002.2 ± 202.0 vs. 2046.1 ± 402.4; 14th wk: 1207.4 ± 234.1 vs. 2355.2 ± 385.6; all p < 0.05). Similarly, tumor weights decreased significantly in bethanechol-treated compared to control mice (mean ± SE: 0.88 ± 0.15 g vs. 1.74 ± 0.27 g, p < 0.05). No metastatic tumors were found in the livers and lungs in either group. These results indicate that bethanechol inhibits growth of human colon cancer xenografts in nude mice, and may be useful as a therapeutic agent for colon cancer. Although the results are surprising considering a slight pro-proliferative effect of bethanechol in H508 cells In Vitro, they are consistent with an earlier report that bethanechol inhibited pancreatic carcinogenesis in hamsters. This inhibitory effect of bethanechol on colon neoplasia is currently being investigated in other mouse models of intestinal carcinogenesis.
Tu1732 Polymorphisms in the GATA-Binding Sites of the Vitamin D Receptor Gene Affect Its Transcription in the Esophagus, and Are Associated With a Reduced Risk for Esophageal Disease Anouk van de Winkel, André Uitterlinden, Agnieszka M. Rygiel, Leon M. Moons, Pascal P. Arp, Joyce van Meurs, Kausilia K. Krishnadath, Ernst J. Kuipers, Luc J. van der Laan Background: Recent studies stress the role of vitamin D and its receptor (VDR) in complex diseases, including inflammatory bowel disease and colorectal cancer. The anti-proliferative and immune modulatory effects of VDR signalling are well recognized. Here we investigate the genetic variation of VDR and its functional implications in esophageal inflammation and malignancy. Aim: To determine single nucleotide polymorphisms (SNPs) in highly conserved non-coding regulatory regions of the VDR gene, and to study their association with risk for erosive esophagitis (EE), Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC), as well as their function in regulating VDR gene expression. Methods: DNA was obtained from Caucasian subjects including patients with EE (n=307), BE (n=260), and EAC (n=
S-821
AGA Abstracts
AGA Abstracts
degree of LINE-1 hypomethylation is a unique feature of early-onset CRCs, and distinguishes them from older CRCs. Since LINE-1 hypomethylation is a surrogate marker for increased chromosomal instability, our data provide a novel and previously unrecognized explanation for some of the biological differences underpinning early-onset CRCs.