- Email: [email protected]
Temporal relationship between induction of mutant Ha-Ras and upregulation of EGF receptor ligands and cyclooxygenase-2 in rat intestinal epithelial cells
different oncogenic mutants (JM, PT, BE))were stably expressed into BaJF3routine cell IJnes Their influence on cell proliferation (assessed by MTT colorimetric assay) and chemotaxis were investigated. Protein kinase B (PKB) activity was measured using a specific peptide and 32PATP.The effect of compound STI 571 (Novartis), an mhibttor of KIT phosphorylation, was also evaluated. Results: Native BaJF3 cells proliferated only in an IL-3 dependent manner whilst Ba/F3 with WT KIT responded to both IL-3 and SCF. BaJF3cells with the oncogenic mutants proliferated autonomously, although at different rates. STI571 (0.1 - lOp.M) did not alter the IL-3-dependent proliferation whilst it suppressed KIT-dependent response in a dosedependent manner.In Ba/F3 with WT KIT, basal PKB activity was low and increased strongly upon stimulation by SCF. In contrast, for the various oncogenic mutants, the low basal PKB activity did not increase upon stimulation by SCF. Conversely, the chemotactic response to SCF of the oncogneic mutants was similar to WT KIT. Conclusion: Our results indicate that oncogenic mutations of KIT differentially affect various biological responses. The identification of the respective signaling pathways involved is currently under way.
cyclooxygenase 1 and 2 mRNA and protein, as well as for the EP family of PGE2receptors, we sought to define the biology of PGE~in normat and tumor-adjacent human gastric mucosa. A consistent and structured expression of cyclooxygenaseand PGE2receptor immunoreactive prote=n on mucosal cells in normal stomach was uncovered: cyclooxygenase 1 and PGE2 receptor EP4were expressed on mucosal CD3- T lymphocytes, and prostanoid receptors EP2, EP3 and EP4,on gastric epithelium lining gastric pits. In situ hybridization with COX cDNA's confirmed these findings, and neither COX 2-specific mRNA nor protein was detected in normal gastric tissue. In contrast, in mucosa adjacent to an invasive gastric adenocarcinoma, expression of cyclooxygenase and EP receptors was altered: cyclooxygenase 1 in T lymphocytes now surrounded nests of tumor cells and, at times, penetrated them. Densitometry showed these tumor-adlacent T cells had modest levels of COX 1 immunoreactive protein (relative intensity, 3.2). Cyclooxygenase 2 was newly expressed but limited (<25% of the total number of cyclonxygenasa-posifive cells) in tumor-adjacent mucosa. Further, CD3+ mononuclear cells, adjacent to tumor, expressed prostanoid receptor EP4 in substantial amounts (relative intensity, 8.0), but cells with this receptor were not evident in the tumor itself. Thus, our studies suggest that synthetic machinery and receptors for PGE2are newly expressed by T cells in gastric mucosa at its boundary with tumor cells, and may play a central role in prostanoid-driven tumorigenesis of this tissue.
2513 TNF-a induces/]-catenin / TCF Mediated Transcription: Oppo=degAstions of MAP1( and NFKB Pathways. tan Perry, Chris Tselepis, Birmingham Univ, Birmingham United Kingdom; Brian T. Cooper, Tariq H. Iqbal, City Hosp, Birmingham United Kingdom; Janusz A.Z. Jankowsld, Birmingham Univ, Birmingham United Kingdom
2516 The PACAPType I Receptor (PACl) Is Expressed and Is Coupled to Signaling Cascades in Human Colonic Tumors Pstdzia M. Germane, CURE/DigestiveDiseases Research Ctr, Dept of Med, UCLA, Los Angeles, CA; Sang Le, CURE: Digest Diseases Research Ctr, Los Angeles, CA; Robert Fan, VA Greater Los Angeles Heaithcare System, Los Angeles, CA; Joseph R. Pisegna, Div GI VA GLAHS and Dept Medicine UCLA, Los Angeles, CA
Neoplasia and epithelial remodelling are both features of chronic inflammation. We have previously shown that TNF-
2514 Prostaglandin Ez Increases Growth and Invasivonessof Colorectal Carcinoma Cells Hongmiao Sheng, Jinyi Shag, Raymond N. Dubois, Vanderbilt Univ Medical Ctr, Nashville, TN Evidence shows that numerous non-steroidal antiinftammatory drugs (NSAIDs) which share the property of inhibiting the cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes delay or prevent colorectal carcinogenesis, suggesting that these agents may be exerting part of their protective effect through inhibition of the COX enzymes. Cumulative evidence shows an increased expression of COX-2 and an increased production of PGE2in colorectal cancers; hence PGE2may contribute to the malignant potential of colorectal epithelial cells. Methods: The LS-174 human colon cancer cell line was maintained in McCoy's 5A medium containing 10% fetal bovine serum. Cells were grown in Matrigel and the colony size was measured. LS-174 cells were xenografted in nude mice and then the mice were treated with vehicle or Misoprostol (a PGE analog, 30 p.g/kg for 17 days). Results: Xenngrafted LS-174 tumors remained moderately well differentiated, consisting of closely packed glands with focal mucin production and a rim of fibrous tissue of variable thickness. In contrast, much more prominent invasion of the capsule and surrounding tissues was observed in the Misoprostol-treated animals. Satellite nodules of infiltrating tumor cells were clearly present within the capsule and adjacent connective tissue following Misoprostol treatment. PGE2 exerted a growthstimulatory effect on LS-174 cells grown in Matrigel. The size of LS-174 colonies in Matrigel increasedfollowing PGE2treatment in a dose-dependent manner. Treatment with 10 nM PGE2 resulted in optimal stimulation of LS-174 cell growth, causing a 120% increase in colony diameter. Treatment with PGE2also caused a significant change in the morphology of the LS-174 colonies. When grown on plastic culture dishes, LS-174 cells form in "non-spreading" round clumps. Addition of 10 nM PGE~leads to a rapid change in phenotype, which includes increased spreading of cells. PGE2treatment results in protruding actin filaments from the cell periphery in the form of microspikes and an increase in the number of stress fibers. The oncogenic PI3K/Akt pathway was activated by PGE treatment. Inhibition of PI3K activity abolished PGE2induced growth stimulation and cell spreading. Conclusions: PGE2enhances the proliferation and invasion of colorectal carcinoma cells via activation of major intracellular signal transduction pathways such as PI3K/Akt.
2515 Prostanoids in Human Gastric Cancer. Peal-tumor Increase in Cox-l* T lymphocyies with Receptorsfor PGEz. Vivian Takatuji, Rosana Cosine, James K. Roche, Univ of Virginia, Charlottesville, VA Recent evidence suggests that prostanoids are an important participant in the pathobioJogy of gastric adenocarcinoma, but the location and identity of cells in normal or cancerous gastric mucosa able to synthesize and/or bind specific prostanoids is not clear. Using probes for
A-494
Background: The PACAPType I (PAC,) receptor is a G protein coupled receptor that stimulates the activation of both adenytyl cyclase (AC) and Jnositol phosphates (IP). PAC1 is expressed and stimulates the growth of lung and breast cancer cell lines and PACAP stimulates the induction of the immediate early genes and tumor growth. To date the expression of PAC1 and influence on the growth of colon cancer cell lines has not been determined. Purpose: To determine the expression and pharmacology of PAC1 expressed on human colon cancer cells. Methods: The expression of PAC1 on tumoral cell lines was determined using immunohistochemistry(IH), RT-PCR, fluorescence microscopy and FACS analysis. Polyclonal antibodies to the PAC1 protein were used to identify receptor expression on isolated cells and in paraffinembedded tissue sections. Confocal microscopy and FACSanalysis was used to characterize receptor expression on the cell surface using both the anti-PAC1 and the fluorescent PACAP ligand, Ruor-PACAP. PACAPstimulation of adenylyl cyclase (AC) and total inosoitol phosphates (IP) were performed by chromtography. Results: PAC~is expressedon human adenocarcinoma as determined by IH and RT-PCR. FACS analysis using the anti-PAC1 antibody and FluorPACAP identified expression of PAC1 on the HCT8 human adenocarcinoma cell line. RTPCR using primers specific for the PAC1 confirmed the expression of PAC1 (hop variant predominant). Confocal microscopy and Fluor-PACAP demonstrated cell surface expression and internalization occurred rapidly (75% at 10 minutes of ligand exposure). Similar findings were observed using the polyclonal anti-PAC1 antibody. PACAP-27 elevated cAMP and IP levels in a dose-dependent manner in both the HCT8 and a control rat brain tumor cell line (FlO) cells with a haft-maximal (EC50) stimulation of approximately 1 nM. Conclusions: These data indicate that human colon cancer cells such as HCT8appear to express biologically active PAC1 receptors. PACAP hormone is capable of stimulating intracellular signaling pathways in human colonic tumors suggesting a role in tumor growth stimulation in these cell lines leading to signal transduction pathways coupled to AC and IP. The identification of biologically active PAC1 receptors on colonic tumors may have important implications for regulating their growth in humans.
2517 Temporal Relationship between Induction of Mutant Ha-Ras and Upregulntion of EGF Receptor Ligands and Cyclonxygenace-2in Rat Intestinal Epithelial Cells Min Lu, Robert J. Coffey Jr., Vanderbilt Univ, Nashville, TN; Daniel R. Beauchamp BACKGROUND Enhanced expression of epidermal growth factor receptor (EGFR) ligands (transforming growth factor~[TGF~], amphiregulin [AR], heparin-binding EGF-like growth factor [HB-EGF]) and Cyclooxygenase-2 (COX-2) has been observed in activated Ha- and KRas transformed rat intestinal epithelial cells (RIE-1). This study was designed to better understand the temporal relationship between expression of EGFRligands and COX-2 in HaRas inducible RIE-1 cells. METHOD: RIE-t cells were transfected with a Ha-Ras Val-12 cDNA expression vector that is under the transcriptional control of the Lac operon, and is inducible with IPTG. Using this system and a specific EGFRtyrosine kinase inhibitor (EKI-785), the temporal relationship between steady state mRNA expression of TGF~, AR, and COX-2 after induction of mutant Ha-Ras was examined by northern blot analysis. RESULTS: Ras protein increased 1-2 hr after IPTG administration. TGF~and AR mRNA began to increase 2-4 hr after IPTGtreatment, and this was followed by COX-2 expression 2-4 hr later. EKI-785 (2/~M) had no effect on the early (2-4hr) expression of TGF
cyclooxygenase 1 and 2 mRNA and protein, as well as for the EP family of PGE2receptors, we sought to define the biology of PGE~in normat and tumor-adjacent human gastric mucosa. A consistent and structured expression of cyclooxygenaseand PGE2receptor immunoreactive prote=n on mucosal cells in normal stomach was uncovered: cyclooxygenase 1 and PGE2 receptor EP4were expressed on mucosal CD3- T lymphocytes, and prostanoid receptors EP2, EP3 and EP4,on gastric epithelium lining gastric pits. In situ hybridization with COX cDNA's confirmed these findings, and neither COX 2-specific mRNA nor protein was detected in normal gastric tissue. In contrast, in mucosa adjacent to an invasive gastric adenocarcinoma, expression of cyclooxygenase and EP receptors was altered: cyclooxygenase 1 in T lymphocytes now surrounded nests of tumor cells and, at times, penetrated them. Densitometry showed these tumor-adlacent T cells had modest levels of COX 1 immunoreactive protein (relative intensity, 3.2). Cyclooxygenase 2 was newly expressed but limited (<25% of the total number of cyclonxygenasa-posifive cells) in tumor-adjacent mucosa. Further, CD3+ mononuclear cells, adjacent to tumor, expressed prostanoid receptor EP4 in substantial amounts (relative intensity, 8.0), but cells with this receptor were not evident in the tumor itself. Thus, our studies suggest that synthetic machinery and receptors for PGE2are newly expressed by T cells in gastric mucosa at its boundary with tumor cells, and may play a central role in prostanoid-driven tumorigenesis of this tissue.
2513 TNF-a induces/]-catenin / TCF Mediated Transcription: Oppo=degAstions of MAP1( and NFKB Pathways. tan Perry, Chris Tselepis, Birmingham Univ, Birmingham United Kingdom; Brian T. Cooper, Tariq H. Iqbal, City Hosp, Birmingham United Kingdom; Janusz A.Z. Jankowsld, Birmingham Univ, Birmingham United Kingdom
2516 The PACAPType I Receptor (PACl) Is Expressed and Is Coupled to Signaling Cascades in Human Colonic Tumors Pstdzia M. Germane, CURE/DigestiveDiseases Research Ctr, Dept of Med, UCLA, Los Angeles, CA; Sang Le, CURE: Digest Diseases Research Ctr, Los Angeles, CA; Robert Fan, VA Greater Los Angeles Heaithcare System, Los Angeles, CA; Joseph R. Pisegna, Div GI VA GLAHS and Dept Medicine UCLA, Los Angeles, CA
Neoplasia and epithelial remodelling are both features of chronic inflammation. We have previously shown that TNF-
2514 Prostaglandin Ez Increases Growth and Invasivonessof Colorectal Carcinoma Cells Hongmiao Sheng, Jinyi Shag, Raymond N. Dubois, Vanderbilt Univ Medical Ctr, Nashville, TN Evidence shows that numerous non-steroidal antiinftammatory drugs (NSAIDs) which share the property of inhibiting the cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes delay or prevent colorectal carcinogenesis, suggesting that these agents may be exerting part of their protective effect through inhibition of the COX enzymes. Cumulative evidence shows an increased expression of COX-2 and an increased production of PGE2in colorectal cancers; hence PGE2may contribute to the malignant potential of colorectal epithelial cells. Methods: The LS-174 human colon cancer cell line was maintained in McCoy's 5A medium containing 10% fetal bovine serum. Cells were grown in Matrigel and the colony size was measured. LS-174 cells were xenografted in nude mice and then the mice were treated with vehicle or Misoprostol (a PGE analog, 30 p.g/kg for 17 days). Results: Xenngrafted LS-174 tumors remained moderately well differentiated, consisting of closely packed glands with focal mucin production and a rim of fibrous tissue of variable thickness. In contrast, much more prominent invasion of the capsule and surrounding tissues was observed in the Misoprostol-treated animals. Satellite nodules of infiltrating tumor cells were clearly present within the capsule and adjacent connective tissue following Misoprostol treatment. PGE2 exerted a growthstimulatory effect on LS-174 cells grown in Matrigel. The size of LS-174 colonies in Matrigel increasedfollowing PGE2treatment in a dose-dependent manner. Treatment with 10 nM PGE2 resulted in optimal stimulation of LS-174 cell growth, causing a 120% increase in colony diameter. Treatment with PGE2also caused a significant change in the morphology of the LS-174 colonies. When grown on plastic culture dishes, LS-174 cells form in "non-spreading" round clumps. Addition of 10 nM PGE~leads to a rapid change in phenotype, which includes increased spreading of cells. PGE2treatment results in protruding actin filaments from the cell periphery in the form of microspikes and an increase in the number of stress fibers. The oncogenic PI3K/Akt pathway was activated by PGE treatment. Inhibition of PI3K activity abolished PGE2induced growth stimulation and cell spreading. Conclusions: PGE2enhances the proliferation and invasion of colorectal carcinoma cells via activation of major intracellular signal transduction pathways such as PI3K/Akt.
2515 Prostanoids in Human Gastric Cancer. Peal-tumor Increase in Cox-l* T lymphocyies with Receptorsfor PGEz. Vivian Takatuji, Rosana Cosine, James K. Roche, Univ of Virginia, Charlottesville, VA Recent evidence suggests that prostanoids are an important participant in the pathobioJogy of gastric adenocarcinoma, but the location and identity of cells in normal or cancerous gastric mucosa able to synthesize and/or bind specific prostanoids is not clear. Using probes for
A-494
Background: The PACAPType I (PAC,) receptor is a G protein coupled receptor that stimulates the activation of both adenytyl cyclase (AC) and Jnositol phosphates (IP). PAC1 is expressed and stimulates the growth of lung and breast cancer cell lines and PACAP stimulates the induction of the immediate early genes and tumor growth. To date the expression of PAC1 and influence on the growth of colon cancer cell lines has not been determined. Purpose: To determine the expression and pharmacology of PAC1 expressed on human colon cancer cells. Methods: The expression of PAC1 on tumoral cell lines was determined using immunohistochemistry(IH), RT-PCR, fluorescence microscopy and FACS analysis. Polyclonal antibodies to the PAC1 protein were used to identify receptor expression on isolated cells and in paraffinembedded tissue sections. Confocal microscopy and FACSanalysis was used to characterize receptor expression on the cell surface using both the anti-PAC1 and the fluorescent PACAP ligand, Ruor-PACAP. PACAPstimulation of adenylyl cyclase (AC) and total inosoitol phosphates (IP) were performed by chromtography. Results: PAC~is expressedon human adenocarcinoma as determined by IH and RT-PCR. FACS analysis using the anti-PAC1 antibody and FluorPACAP identified expression of PAC1 on the HCT8 human adenocarcinoma cell line. RTPCR using primers specific for the PAC1 confirmed the expression of PAC1 (hop variant predominant). Confocal microscopy and Fluor-PACAP demonstrated cell surface expression and internalization occurred rapidly (75% at 10 minutes of ligand exposure). Similar findings were observed using the polyclonal anti-PAC1 antibody. PACAP-27 elevated cAMP and IP levels in a dose-dependent manner in both the HCT8 and a control rat brain tumor cell line (FlO) cells with a haft-maximal (EC50) stimulation of approximately 1 nM. Conclusions: These data indicate that human colon cancer cells such as HCT8appear to express biologically active PAC1 receptors. PACAP hormone is capable of stimulating intracellular signaling pathways in human colonic tumors suggesting a role in tumor growth stimulation in these cell lines leading to signal transduction pathways coupled to AC and IP. The identification of biologically active PAC1 receptors on colonic tumors may have important implications for regulating their growth in humans.
2517 Temporal Relationship between Induction of Mutant Ha-Ras and Upregulntion of EGF Receptor Ligands and Cyclonxygenace-2in Rat Intestinal Epithelial Cells Min Lu, Robert J. Coffey Jr., Vanderbilt Univ, Nashville, TN; Daniel R. Beauchamp BACKGROUND Enhanced expression of epidermal growth factor receptor (EGFR) ligands (transforming growth factor~[TGF~], amphiregulin [AR], heparin-binding EGF-like growth factor [HB-EGF]) and Cyclooxygenase-2 (COX-2) has been observed in activated Ha- and KRas transformed rat intestinal epithelial cells (RIE-1). This study was designed to better understand the temporal relationship between expression of EGFRligands and COX-2 in HaRas inducible RIE-1 cells. METHOD: RIE-t cells were transfected with a Ha-Ras Val-12 cDNA expression vector that is under the transcriptional control of the Lac operon, and is inducible with IPTG. Using this system and a specific EGFRtyrosine kinase inhibitor (EKI-785), the temporal relationship between steady state mRNA expression of TGF~, AR, and COX-2 after induction of mutant Ha-Ras was examined by northern blot analysis. RESULTS: Ras protein increased 1-2 hr after IPTG administration. TGF~and AR mRNA began to increase 2-4 hr after IPTGtreatment, and this was followed by COX-2 expression 2-4 hr later. EKI-785 (2/~M) had no effect on the early (2-4hr) expression of TGF