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2 activation and stimulates cell proliferation via a metalloproteinase- and Tgfα-dependent pathway
growth factor, the interaction between c-Met protein and DCP was assessed. DCP bound to c-Met protein and activated STAT (signal transducers and activatiors of transcription) signaling pathway. Thus DCP may be a novel autocrine mitogen of hepatoma cells. These results suggest that maintaining serum DCP at a low level might be an effective new therapy for HCC.
M973
Inhibition of Cell Growth by a beta-catenin Responsive Element Hadas Dvory-Sobol, Talia Kunik, Dina Kazanov, Olga Kolker, Meirav Rozenblat, Efrat Cohen-Noyman, Ludmila Strier, Nadir Arber Background: Many colon cancers harbor mutations in the APC/beta-catenin pathway, leading to actwation of downstream genes with beta-catenin/T-cell factor (Tci)- responsive promoters. The ultimate therapy of cancer is by targeting neoplastic cells without damaging normal cells. Aim: To selectively kill beta catenin transformed cells by up-regulation of lethal genes using a mutated signal transduction pathway. Materials & Methods: A Xbal fragment contains the TOP/FOP-cFos sequence was cut out from the TOP/FOP flash plasmids (a gift from Hans Clevers) and then cloned to the pGL3-basic vector (Promega), into the Nhel site. The resultant plasmid was designated as pGL3-TOP/FOP. Then, the Luc gene was cut out from this plasmid by Hindlll-Xbal digestion and a cloning site linker was inserted instead of the Luc gene to make the plasmid pGL3-TOP/FOP-HX. The TOP/FOP-HX was used to construct 3 separate genes: Bax, caspase 8 and PKG. The new plasmids were designated as TOP/ FOP-Bax, TOP/FOP-Cas8, and TOP/FOP-PKG. Exponentially growing SW 480 cells, with increased expression of beta~catenin, were transiently transfected with these constructs and then treated for 48 and 72hr with different dosages of a specific COX 2 inhibitor (Celecoxib, 0-50uM). Results: The growth of SW 480 cells was not hampered by the transfection with the FOP plasmids. The TOP-PKG and TOP-Bax plasmids inhibited cell growth by approximatly 20-35%, respectively, while the plasmid TOP-cas8 did not inhibit cell growth. The growth inhibition was associated with induction of apoptosis.Celecoxib inhibited the growth of SW480 ceils The addition of celecoxib to the TOP-Bax/cas8/PKG transfected cells, augmented the growth inhibition in a time and dose dependent manner. Conclusions: 1. This approach may be used to develop therapies to target tumor cells that have defects in the Wnt/beta-catenin/Tcf signal transduction pathway. 2. Selective over-expression of some pro-apoptotic genes, in beta-catenin transformed cells increase their sensitivity to celeco:db. 3. In this model Bax and PKG but not caspase 8 could serve as potential targets, to selectively induce turner cell death.
M976
The Her Tyrosine Kinase Inhibitor CilO33 Modulates Apoptosis and Radiosensitivity in Bile Duct Carcinoma Cell Line Masateru Murakami, Tamito Sasaki BACKGROUNDS. Over the last decade, considerable evidence has implicated HER receptor tyrosine kinase family in the development and progression of a variety of human tumors. CI 1033 is a quinazoline-based HER family tyrosine kinase inhibitor that is currently being evaluated as a potential anticancer agent. However, the expression of these receptors and effectiveness of CI1033 in bile duct carcinoma has not been analyzed. We examined the expression of this receptor, and effects of 0 1 0 3 3 on proliferation, apoptosis, and radiosensitivity in human bile duct carcinoma. METHODS. We used the human bile duct carcinoma cell line (HuCCT-1), and surgically resected bile duct tissue specimens. HER expression was detected by Immunohistochemical staining, and Western blotting. For proliferation assay, cells were treated with various concentration of Cl1033 for up to 8 days, then we counted by hemacytometer with trypan blue dye exclusion at indicated time intervals. Apoptosis induced by CI1033 was analyzed by flow cytometry using AnnexinV-EGFP and Propidium Iodide(H) staining. And radiosensinvity enhanced by CI1033 was examined by clonogenic survival. RESULTS. The expression of HER protein was detected in both cell lines and tissues by lmmunohistochemical staining and Western blotting using specific antibodies. Cell growth was significantly inhibited by CI1033 in a dose and time dependent manner. Moreover, we found that CI1033 exposure induced apoptosis by flow cytometry, and reduced colony formation with radiation by clonogenic survival. CONCLUSIONS. This may be the first study to examine the effect of CI1033 on human bile duct carcinoma. These results suggested that HER play an important role in maintaining the growth of human bile duct carcinoma, and C11033 may be an useful agent against the growth of bile duct carcinoma.
M974
Carbachol Induces ERK1/2 Activation and Stimulates Cell Proliferation Via a Metalloproteinase- and Tgfa-Dependent Pathway James Hemon, Susanne Lindqvist, Alyson Prior, Micheal Rhodes, William Stebbings, Gillian Murphy, Mark Williams
M977
Co-Targeting Integrin Adhesion Receptors and Epidermal Growth Factor Receptors as a Strategy for Cancer Therapy Hyun Soo Kim, Michael G. Brattain, Zhen Fan
Activation of MAP kinase family members ERK1/2 has been demonstrated to play a key role in intestinal cell proliferation and differentiation. A body of evidence suggests that G protein coupled receptor calcium signals transactive the EGFR-MAP kinase pathway via a metalloproteinase (MP)-mediated shedding of membrane bound TGFA(e.g. McCole et al., JBC, 2002, Heruon et aL, Gastroenterology, 2002)PURPOSE: To pharmacologicallycharacterise the MP activity implicated in muscarinic acetylcholine receptor-induced ERK1/2 activation and study the role of this pathway in regulating human colonic epithelial cell proliferation. METHODS: HT29 cells were cultured in DMEM supplemented with 5% FCS (5% CO2/ 37oC). On reaching 30% confluence cells were stimulated by carbachol in the presence or absence of cell signaling inhibitors. ERK 1/2 activation was assessed by immunoblotting and/or immunocytochemical techniques based on the use of an anti-phospho-ERK1/2 antibody. Cell proliferation was assessed by 3H thymidine incorporation. RESULTS: Carbachol (100 muM) stimulated ERK1/2 in the cytoplasm and nucleus of HT29 cells, maximal stimulation was noted at 1 hour and persisted for at least 24 hours. Carbachol induced ERK activation was reduced by 50% in the presence of BB94 (5 muM ) (metanoproteinase inhibffor), TIMP 3 (0.5 muM) (Tissue Inhibitor of Metalloproteinase ), anti-TGFA(10ng/ml) and tyrophostin (1 muM) (EGFR kinase inhibitor). No inhibition was seen in the presence of T1MP 1 and 2 (0.5 muM). Carbachol induced a 40% increase in HT29 cell 3H tymidine incorporation. This effect was abrogated by BB94 (5 muM), TIMP 3 (0.5 muM), antiTGFA(10ng/ml), and tyrophostin (1 muM). CONCLUSIONS: Carbachol induces ERK1/2 activation and stimulates cell proliferation xaa a metalloproteinase- and TGFA-dependent pathway.
Integrin adhesion receptors and epidermal growth factor (EGF) receptor family both play important roles in cellular adhesion and migration. Although integrin and EGF receptor can independently propagate intracellular signals, the signals activated by extracellular mat~x (ECM) and EGF may provide synergism in the initiation and progression of tumorigenesis and metastasis. This may justify a novel strategy by co-targeting the two pathways for cancer therapy. In our current study, we used cell adhesion assays to evaluate this strategy in human colon cancer cells. Treatment of GEO human colon cancer cells with exogenous EGF stimulated cell adhesion to both collagen (CN) and fibronectin (FN) in a fold of 1.55 and 1.45 respectively. We also examined the cell adhesion of FET human colon cancer cells transfected with transforming growth factor-alpha (TGF-a). Compared with control vectortransfected cells (EET-neo), the cells expressing TGF-a (FET6a26x) showed an increased adhesion to both CN and FN, with a factor of 2.76 and 1.75 respectively. Exposure of GEO cells to 20 nM anti-EGF receptor monoclonal antibody (mAb) 225 for 48 hours caused a 37% reduction of cell adhesion of GEO cells to CN and a 50% such reduction to FN. Treatment of FET-neo cells with mAb 225 reduced cell adhesion to CN (23% reduction) and to FN (20% reduction). Interestingly, mAb 225 did not inhibit the adhesion of FET6a26x cells to CN or FN We also examined the effect of a synthetic integrin-binding motif peptide of ECM, Arg-Gly-Asp (RGD), on inhibiting the adhesion of GEO and FET cells to CN or FN. We found an RGD dose-dependent inhibition of the cells to the ECM A combination treatment of mAb 225 and RGD resulted in additive effect on inhibiting the adhesion of both cell lines to CN or FN. Our results indicate that a combined therapy with anti-EGF receptor mAb 225 or RGD peptide may offer an enhanced effect on inhibiting cancer cell adhesion, migration and metastasis, and may therefore be a promising new approach for cancer treatment.
M975 Des~-Carboxy Prothrombin is an Autocrine Mitogen for Hepatocellular Carcinoma Mayumi Suzuki, Hidenori Shiraha, Tatsuya Fujikawa, Nobuypki Takaoka, Akinobu Takaki, Yutaka Nakanishi, Naoki Ueda, Kazuko Koike, Kohsaku Sakaguchi, Yasnshi Shiratori
M978
Localization Of Human Equilibrative Nucleoside Transporter-1 (HENT1) Protein in Normal Human Gastrointestinal Tissues Clarence K. W. Wong, Lawrence D. Jewell, Laith Dabbagh, Stephen A. Baldwin, James D. Young, John R. Mackey, Carol E. Cass
Des-'y-carboxy prothromhin (DCP) is a well-recognized tumor marker for hepatocelhilar carcinoma (HCC). In DCP, some of the 10 "y-Gla residues, which are present from the Nterminal, are not transformed and are remained as Ghi residues. DCP is produced because HCC cells have a diminished capacity to cathoxylate prothrombin. Portal venous invasion (PVI) of HCC seriously affects the prognosis of the patient. It has been reported that serum DCP level is significantly correlate with the development of PVI (Koike Y. et al. Cancer;91:561 9 2001). Moreover Ki-67 labeling index, a marker for cell proliferation, was significantly correlate with tissue DCP expression level in HCC. However, its fine molecular mechanism is not yet known. This study was performed under the hypothesis that DCP might be a stimulant of cell proliferation and that autocrine production of DCP might result in poor prognosis of HCC. We purified DCP and normal prothrombin from conditioned media of hepatoma cell line PLC/FRF/5 using high performance liquid chromatography. Purified DCP was utilized in the following experiments. Cell proliferative activity was assessed by H 3tbymidine incorporation. DCP stimulated cell proliferation of hepatoma cell line Hep3B in a dose dependent manner (194-fold at a concentration of 20 ng/ml), while control prothrombin stimulated cell proliferation only 1.50-fold at a concentration of 20 ng/ml. DCP also enhances the cell proliferation of other human hepatoma cell lines Hu-7. Moreover DCP stimulated the cell proliferative activity of human colon cancer cell line HT-29, which does not produce DCP by itself. Since DCP has two kringle domains which are similar to that of hepatocyte
Background/Aim: Nucleosides and nucleoside analogue drugs often require cellular uptake by nucleoside transport (NT) proteins to reach intracefiular targets. Localization of NT proteins in the gastrointestinal tract is vital in the understanding of drug absorption as well as drug accumulation in target tissues. Previous studies have identified hENT1 presence functionally, but direct cellular localization studies have not been performed. This study aimed to characterize the cellular distribution of hENT1 in human gastrointestinal tissues. Methods: Gastrointestinal tissues were endoscopically or surgically obtained, fixed in formalin, and paraffin embedded. Sections of normal esophagus, stomach, duodenum, jejunum, ileum, colon, liver and pancreas were incubated with murine anti-hENT1 monoclonal antibodies (mAb) obtained by immunization of mice with spedfic synthetic hENT1 peptides. Sections were then washed and stained with goat anti-monse immunoperoxidase dextran conjugate. Immunohistochemical staining was assessed according to intensity and specificity for immunoperoxidase stain. An expert pathologist reviewed all slides. Results: Samples incubated with anti-hENT1 mAb revealed specific hENT1 staining dependent on the anatomic location throughout the gastrointestinal tract, hENT1 was prominently localized on the basolateral but not the apical surface in gastric, small intestine and colonic epithelial cells.
A-285
AGA Abstracts
M973
Inhibition of Cell Growth by a beta-catenin Responsive Element Hadas Dvory-Sobol, Talia Kunik, Dina Kazanov, Olga Kolker, Meirav Rozenblat, Efrat Cohen-Noyman, Ludmila Strier, Nadir Arber Background: Many colon cancers harbor mutations in the APC/beta-catenin pathway, leading to actwation of downstream genes with beta-catenin/T-cell factor (Tci)- responsive promoters. The ultimate therapy of cancer is by targeting neoplastic cells without damaging normal cells. Aim: To selectively kill beta catenin transformed cells by up-regulation of lethal genes using a mutated signal transduction pathway. Materials & Methods: A Xbal fragment contains the TOP/FOP-cFos sequence was cut out from the TOP/FOP flash plasmids (a gift from Hans Clevers) and then cloned to the pGL3-basic vector (Promega), into the Nhel site. The resultant plasmid was designated as pGL3-TOP/FOP. Then, the Luc gene was cut out from this plasmid by Hindlll-Xbal digestion and a cloning site linker was inserted instead of the Luc gene to make the plasmid pGL3-TOP/FOP-HX. The TOP/FOP-HX was used to construct 3 separate genes: Bax, caspase 8 and PKG. The new plasmids were designated as TOP/ FOP-Bax, TOP/FOP-Cas8, and TOP/FOP-PKG. Exponentially growing SW 480 cells, with increased expression of beta~catenin, were transiently transfected with these constructs and then treated for 48 and 72hr with different dosages of a specific COX 2 inhibitor (Celecoxib, 0-50uM). Results: The growth of SW 480 cells was not hampered by the transfection with the FOP plasmids. The TOP-PKG and TOP-Bax plasmids inhibited cell growth by approximatly 20-35%, respectively, while the plasmid TOP-cas8 did not inhibit cell growth. The growth inhibition was associated with induction of apoptosis.Celecoxib inhibited the growth of SW480 ceils The addition of celecoxib to the TOP-Bax/cas8/PKG transfected cells, augmented the growth inhibition in a time and dose dependent manner. Conclusions: 1. This approach may be used to develop therapies to target tumor cells that have defects in the Wnt/beta-catenin/Tcf signal transduction pathway. 2. Selective over-expression of some pro-apoptotic genes, in beta-catenin transformed cells increase their sensitivity to celeco:db. 3. In this model Bax and PKG but not caspase 8 could serve as potential targets, to selectively induce turner cell death.
M976
The Her Tyrosine Kinase Inhibitor CilO33 Modulates Apoptosis and Radiosensitivity in Bile Duct Carcinoma Cell Line Masateru Murakami, Tamito Sasaki BACKGROUNDS. Over the last decade, considerable evidence has implicated HER receptor tyrosine kinase family in the development and progression of a variety of human tumors. CI 1033 is a quinazoline-based HER family tyrosine kinase inhibitor that is currently being evaluated as a potential anticancer agent. However, the expression of these receptors and effectiveness of CI1033 in bile duct carcinoma has not been analyzed. We examined the expression of this receptor, and effects of 0 1 0 3 3 on proliferation, apoptosis, and radiosensitivity in human bile duct carcinoma. METHODS. We used the human bile duct carcinoma cell line (HuCCT-1), and surgically resected bile duct tissue specimens. HER expression was detected by Immunohistochemical staining, and Western blotting. For proliferation assay, cells were treated with various concentration of Cl1033 for up to 8 days, then we counted by hemacytometer with trypan blue dye exclusion at indicated time intervals. Apoptosis induced by CI1033 was analyzed by flow cytometry using AnnexinV-EGFP and Propidium Iodide(H) staining. And radiosensinvity enhanced by CI1033 was examined by clonogenic survival. RESULTS. The expression of HER protein was detected in both cell lines and tissues by lmmunohistochemical staining and Western blotting using specific antibodies. Cell growth was significantly inhibited by CI1033 in a dose and time dependent manner. Moreover, we found that CI1033 exposure induced apoptosis by flow cytometry, and reduced colony formation with radiation by clonogenic survival. CONCLUSIONS. This may be the first study to examine the effect of CI1033 on human bile duct carcinoma. These results suggested that HER play an important role in maintaining the growth of human bile duct carcinoma, and C11033 may be an useful agent against the growth of bile duct carcinoma.
M974
Carbachol Induces ERK1/2 Activation and Stimulates Cell Proliferation Via a Metalloproteinase- and Tgfa-Dependent Pathway James Hemon, Susanne Lindqvist, Alyson Prior, Micheal Rhodes, William Stebbings, Gillian Murphy, Mark Williams
M977
Co-Targeting Integrin Adhesion Receptors and Epidermal Growth Factor Receptors as a Strategy for Cancer Therapy Hyun Soo Kim, Michael G. Brattain, Zhen Fan
Activation of MAP kinase family members ERK1/2 has been demonstrated to play a key role in intestinal cell proliferation and differentiation. A body of evidence suggests that G protein coupled receptor calcium signals transactive the EGFR-MAP kinase pathway via a metalloproteinase (MP)-mediated shedding of membrane bound TGFA(e.g. McCole et al., JBC, 2002, Heruon et aL, Gastroenterology, 2002)PURPOSE: To pharmacologicallycharacterise the MP activity implicated in muscarinic acetylcholine receptor-induced ERK1/2 activation and study the role of this pathway in regulating human colonic epithelial cell proliferation. METHODS: HT29 cells were cultured in DMEM supplemented with 5% FCS (5% CO2/ 37oC). On reaching 30% confluence cells were stimulated by carbachol in the presence or absence of cell signaling inhibitors. ERK 1/2 activation was assessed by immunoblotting and/or immunocytochemical techniques based on the use of an anti-phospho-ERK1/2 antibody. Cell proliferation was assessed by 3H thymidine incorporation. RESULTS: Carbachol (100 muM) stimulated ERK1/2 in the cytoplasm and nucleus of HT29 cells, maximal stimulation was noted at 1 hour and persisted for at least 24 hours. Carbachol induced ERK activation was reduced by 50% in the presence of BB94 (5 muM ) (metanoproteinase inhibffor), TIMP 3 (0.5 muM) (Tissue Inhibitor of Metalloproteinase ), anti-TGFA(10ng/ml) and tyrophostin (1 muM) (EGFR kinase inhibitor). No inhibition was seen in the presence of T1MP 1 and 2 (0.5 muM). Carbachol induced a 40% increase in HT29 cell 3H tymidine incorporation. This effect was abrogated by BB94 (5 muM), TIMP 3 (0.5 muM), antiTGFA(10ng/ml), and tyrophostin (1 muM). CONCLUSIONS: Carbachol induces ERK1/2 activation and stimulates cell proliferation xaa a metalloproteinase- and TGFA-dependent pathway.
Integrin adhesion receptors and epidermal growth factor (EGF) receptor family both play important roles in cellular adhesion and migration. Although integrin and EGF receptor can independently propagate intracellular signals, the signals activated by extracellular mat~x (ECM) and EGF may provide synergism in the initiation and progression of tumorigenesis and metastasis. This may justify a novel strategy by co-targeting the two pathways for cancer therapy. In our current study, we used cell adhesion assays to evaluate this strategy in human colon cancer cells. Treatment of GEO human colon cancer cells with exogenous EGF stimulated cell adhesion to both collagen (CN) and fibronectin (FN) in a fold of 1.55 and 1.45 respectively. We also examined the cell adhesion of FET human colon cancer cells transfected with transforming growth factor-alpha (TGF-a). Compared with control vectortransfected cells (EET-neo), the cells expressing TGF-a (FET6a26x) showed an increased adhesion to both CN and FN, with a factor of 2.76 and 1.75 respectively. Exposure of GEO cells to 20 nM anti-EGF receptor monoclonal antibody (mAb) 225 for 48 hours caused a 37% reduction of cell adhesion of GEO cells to CN and a 50% such reduction to FN. Treatment of FET-neo cells with mAb 225 reduced cell adhesion to CN (23% reduction) and to FN (20% reduction). Interestingly, mAb 225 did not inhibit the adhesion of FET6a26x cells to CN or FN We also examined the effect of a synthetic integrin-binding motif peptide of ECM, Arg-Gly-Asp (RGD), on inhibiting the adhesion of GEO and FET cells to CN or FN. We found an RGD dose-dependent inhibition of the cells to the ECM A combination treatment of mAb 225 and RGD resulted in additive effect on inhibiting the adhesion of both cell lines to CN or FN. Our results indicate that a combined therapy with anti-EGF receptor mAb 225 or RGD peptide may offer an enhanced effect on inhibiting cancer cell adhesion, migration and metastasis, and may therefore be a promising new approach for cancer treatment.
M975 Des~-Carboxy Prothrombin is an Autocrine Mitogen for Hepatocellular Carcinoma Mayumi Suzuki, Hidenori Shiraha, Tatsuya Fujikawa, Nobuypki Takaoka, Akinobu Takaki, Yutaka Nakanishi, Naoki Ueda, Kazuko Koike, Kohsaku Sakaguchi, Yasnshi Shiratori
M978
Localization Of Human Equilibrative Nucleoside Transporter-1 (HENT1) Protein in Normal Human Gastrointestinal Tissues Clarence K. W. Wong, Lawrence D. Jewell, Laith Dabbagh, Stephen A. Baldwin, James D. Young, John R. Mackey, Carol E. Cass
Des-'y-carboxy prothromhin (DCP) is a well-recognized tumor marker for hepatocelhilar carcinoma (HCC). In DCP, some of the 10 "y-Gla residues, which are present from the Nterminal, are not transformed and are remained as Ghi residues. DCP is produced because HCC cells have a diminished capacity to cathoxylate prothrombin. Portal venous invasion (PVI) of HCC seriously affects the prognosis of the patient. It has been reported that serum DCP level is significantly correlate with the development of PVI (Koike Y. et al. Cancer;91:561 9 2001). Moreover Ki-67 labeling index, a marker for cell proliferation, was significantly correlate with tissue DCP expression level in HCC. However, its fine molecular mechanism is not yet known. This study was performed under the hypothesis that DCP might be a stimulant of cell proliferation and that autocrine production of DCP might result in poor prognosis of HCC. We purified DCP and normal prothrombin from conditioned media of hepatoma cell line PLC/FRF/5 using high performance liquid chromatography. Purified DCP was utilized in the following experiments. Cell proliferative activity was assessed by H 3tbymidine incorporation. DCP stimulated cell proliferation of hepatoma cell line Hep3B in a dose dependent manner (194-fold at a concentration of 20 ng/ml), while control prothrombin stimulated cell proliferation only 1.50-fold at a concentration of 20 ng/ml. DCP also enhances the cell proliferation of other human hepatoma cell lines Hu-7. Moreover DCP stimulated the cell proliferative activity of human colon cancer cell line HT-29, which does not produce DCP by itself. Since DCP has two kringle domains which are similar to that of hepatocyte
Background/Aim: Nucleosides and nucleoside analogue drugs often require cellular uptake by nucleoside transport (NT) proteins to reach intracefiular targets. Localization of NT proteins in the gastrointestinal tract is vital in the understanding of drug absorption as well as drug accumulation in target tissues. Previous studies have identified hENT1 presence functionally, but direct cellular localization studies have not been performed. This study aimed to characterize the cellular distribution of hENT1 in human gastrointestinal tissues. Methods: Gastrointestinal tissues were endoscopically or surgically obtained, fixed in formalin, and paraffin embedded. Sections of normal esophagus, stomach, duodenum, jejunum, ileum, colon, liver and pancreas were incubated with murine anti-hENT1 monoclonal antibodies (mAb) obtained by immunization of mice with spedfic synthetic hENT1 peptides. Sections were then washed and stained with goat anti-monse immunoperoxidase dextran conjugate. Immunohistochemical staining was assessed according to intensity and specificity for immunoperoxidase stain. An expert pathologist reviewed all slides. Results: Samples incubated with anti-hENT1 mAb revealed specific hENT1 staining dependent on the anatomic location throughout the gastrointestinal tract, hENT1 was prominently localized on the basolateral but not the apical surface in gastric, small intestine and colonic epithelial cells.
A-285
AGA Abstracts