Influence of oncogenic mutants of the KIT receptor tyrosine kinase on its signal transduction pathways

reflects the actual infection status. Methods: FemtoLabH. pylori Cnx is a single-stepsandwichtype EnzymelmmunoAssay(EIA). It is based on monoclonal antibodies for capture and detection specific for defined H.pyloriantigens. The test was evaluatedon a set of 357 stool samples from symptomatic adults and 97 samples from eradicated patients. The H. pylori infection status of the patients was defined by the gold standards histology and/or 13Curea breath test (UBT). Results: The EIA correctly identified 141 of 148 H. pylori positive stool samples and 203 of 209 H. pylori negative stool samples. Specificity and sensitivity were 97% and 95% respectively.To show suitability for eradicationcontrol, samplesfrom 97patients were taken 4-6 weeks after eradication therapy. 94% sensitivity and 99% specificity were shown by using UBT as the referencetest. 17 of 18 H. pylori positive and 78 of 79 H.pylori negative samples were detected correctly. Discussion: We have shown that FemtoLab H. pylori Cnx is a very accurate and reliable test system for detecting H.pylori infections. It reaches specificity and sensitivity values equivalentto the gold standards histology and urea breath test. The EIA is well suited for monitoring eradicationtherapy. Thus FemtoLabH. pylori Cnx is useful for primary diagnosis, eradication control and the detection of reinfection.

HCT116+ ch2 cells and DNA MMR-proficient HCT116+ ch3 and SW480 cells and amplified by PCR surrounding the polyadeninetract of TGF[~RILWe extracted RNA, performed RTPCR, subcloned the RT-PCRproducts, and sequenced TGF~RIIat its polyadeninesequence and kinase domains. We performed a clonagenic assay on the HCT116 and HCT116+ch3 cells with and without 10 ng/ml of TGF/~ ligand. Results: HCT116+ch3 cells contain both the wild-type (A)lo and the mutant (A)9 sequencesof TGF~RII. With RT-PCR,the (A)10allele was present in 16 of 74 (22%) of clones obtained. In the (A)~oclones, there were no observed mutations in the kinase domain of TGF/3RII,except for a known polymorphism at codon 439 (alanineto valine). Clonagenicassaysusing T6F/3 ligand demonstratedno difference in growth when compared to control plates without ligand treatment. Conclusions: HCT116+ ch3 cells contain one wild type and two mutated copies of TGF/3RII,which is expressed in 22% of clones obtained, Despite its expression,there is no growth suppression with TGF/3ligand in clonagenicassays.The ligand unresponsivenessis not due to secondarymutation of TGF~RII at its kinasedomain. We suggest that perturbation of downstream signaling molecules might be involved to suppress TGF/3signaling in HCT116+ ch3 cells.

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Functional Impairment of Transforming Growth Factor-jB-Smad Signaling Pathway in Colorectal, Pancreatic, and Gastric, but not Hepatic Cancer Cell lines. Hideaki Ijichi, NaoyaKato, Tsuneo Ikenoue, Yuzo Mitsuno, Goichi Togo, Jun Kato, Yasushi Shiratori, Masao Omata, Univ of Tokyo, Tokyo Japan

LepUn Is A Growth Factor For Human Colon Cancer Ceils James C.H. Hardwick, Gijs R. Van Den Brink, Sander J.H. Van Deveuter, Maikel P. Peppelenboseh,AMC, Amsterdam Netherlands Background and aims: Obesity increases the risk of colon cancer whereas physical activity reduces the risk. Plasma levels of leptin rise in proportion to the level of obesity and are reduced by physical activity. Leptin acts as a growth factor for several ceil types and thus may provide a biological explanationfor the observed epidemiologisalrisk factors. We aimed to investigatewhether leptin is a growth factor for colon cancer ceils. Methods: The presence of the leptin receptor in colon cancer cell lines was assessed using RT-PCR,immunoblotting and binding studies, and its presence in human colonic tissue by immunohistochemistry. We assessed the effects of leptin in vitro on HT29 cells by assessing p42/44 MAP kinase phosphorylation, thymidine incorporation and cell numbers, and we assessedthe effects of intraperitoneal leptin injection on colonic proUferation in C57BL/6 mice by colonic BrdU incorporation. Results: The leptin receptor is expressedin colon cancer cell lines and human colonic tissue, as judged by three separatetechniques. Stimulation of HT29 ceils with leptin leads to phosphoryletionof p42/44 MAP kinaseand increasescolonic epithelial cell proliferation. A single injection of lOmO/kg leptin led to a highly significant (P
Background & Aims: The transforming growth factor-/?(TGF-/3)-Smad signaling pathway is considered to play an important role in human carcinogenesis.The aim of this study is to clarify the frequency and the mechanism of impairment of the TGF-/3-Smadsignaling pathway in human colorectal, pancreatic,gastric, and hepaticcancer cell lines using a functional assay. Methods: TGF-/3-Smadsignaling in 38 human cancer cell lines (11 colorectal, 9 pancreatic, 10 gastric, and 8 hepatic cancer cell lines) was examined by use of a luciferase reporter construct, p3TP-lux, which contains a major part of the promoter of the TGF-/?-inducible plasminogen activator inhibitor-I gene. To determine the inactivated signaling molecules in TGF-~ignal-deficient cells, analysis of the poly (A) stretch of the TGF-/~ojpe II receptor (T,BRll) gene in exon 3, Western blotting of Smad4 protein, and restoration of the pathway through the expression of T/~RI, T/3RII, Smad2, Smad3, or Smad4 protein (rescuing experiments) were performed. Results: A defect of p3TP-lux activation was observed in 20 cancer cell lines: 91% (10/11) of colorectal, 67% (6/9) of pancreatic,and 40% (4/10) of gastric, but none (0/8) of the hepatic cancer cell lines. In these TGF-~ignal-deficient cell lines, 70% (7/ 10) of colorectal cancer cells had inactivatedT/~RII, and 67% (4•6) of pancreaticcancer cells demonstrated inactivatedSmad4. One of 4 TGF-/3signal-deficientgastric cancer cell lines was consideredto have inactivatedT/3RI, but 75% (3/4) of them showed no apparent inactivated molecules. Conclusions:The TGF-.8-Smadsignaling pathwayis consideredto play a significant role as a tumor suppressor in carcinogenesisof the colon, pancreas, and stomach, but not the liver. The major inactivated molecule in the signaling pathway is revealedto be different among each cancer. T/3RII is the major inactivatedmoleculein colorectal cancers,and Smad4 in pancreatic cancers, but in gastric cancers, the molecule remains undetected,suggesting that there may be a novel mechanism underlying signal interruption in gastric cancers.

2511 Relationship Between Gastrin and EGF Receptor Lioands in a Panel of Human Colomctal Tumour Specimens as Assessed by Real Time PCR Daniel F. McWilliams, Susan A. Watson, Univ of Nottingham, Nottingham United Kingdom Introduction: Gastrin has been shown to up-regulate the genes encoding heparin binding EGF-likegrowth factor (HB-EGF)and amphiregulin in a rat gastric epithelial cell line (Miyazaki et al, Gastroenterology,113: 782-790, 1999). The aim of this study was to quantify gene expression of gastrin and HB-EGFin a series of human colorectal tumour specimens and assess the effect of introduction of an antisense gastrin construct on HB-EGFexpression of a human colorectal tumour cell line Methods: Human colorectal tumour specimens were collected immediately after surgica~ resection and the gene expression of gastrin, HB-EGF and amphiregulin measured by real time PCR. Total cellular RNA was extracted from tissue and reverse-transcribed.Real Time PCR was performed using the 5700 Sequence Detection System (PE Applied Biosystems). Each PCR was performed with a reaction buffer containing SYBR Green. The fluorescence of the SYBR Green dye bound to the PCR products was measured in real time and the cycle number recorded when the accumulated signal crossed an arbitrary threshold (Ct value). The relative gene expression (L~ACt) for each sample was determined using the formula 2~I~H~I~GF~t"I. The human colorectal tumour cell line, HCT116 was transfected with the pCR3.1 plasmid (Invitrogen) containing a gastrin anti-sense construct. HB-EGFgene expressionwas compared in the antisensecell line and HCT116cells transfected with a control plasmid by realtime PCR.Results:Therewas a significant correlation between gastrin and HB-EGF gene expression in colorectal turnouts (p
2508 G17DT Antibodies Inhibit Growth of a Human Pancreatic Tumour Growing in the Pancreas of Immonodeficient Mice Andrew D. Gilliam, Teresa M. Morris, Susan A. Watson, Univ of Nottingham, Nottingham United Kingdom Introduction: Gastrin is a known growth factor for pancreatic cancer. G17DTantibodies have been shown to be therapeutic in a number of gastrointestinal tumour systems. Aim: To evaluate the therapeutic effect of passively infused G17DT antibodies on the growth of a human tumour cell line transplanted into the pancreas of nude mice. Methods: The human pancreatic cell line, PAN1 was examined for gastrin and CCK2 receptor expression by real time PCR at the gene level, by use of the SYBR green dye, and by western blotting at the protein level, using antisera directed against the CCK-2 receptor and pmgastrin, respectively. Cells were injected into the body of the pancreas in immunodeficient mice (lx10~ in a 20/~1 volume). Rabbit G17DTantibodies were passively infused from day 1 until study termination (day 30). Control mice were treated with rabbit IgG, G17DT antibodies were also given to mice madehyper-gastrinaemicby co-administrationof lansoprazole(O.75mg/mouse).Results: PAN1 was shown to expressgastrin and CCK-2receptorsatthe geneand protein level. G17DT antibodies reduced mean tumour weight by 31% (p
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Influence of Oncogenic Mutants of the KIT Receptor Tyrosine Kinase on Its Signal TraosducUon Pathways. Koji Isozaki, Osaka Univ Graduate Sch of Medicine, OsakaJapan; Florence De Smedt, Christophe Erneux, IRiBHN, Faculty of Medicine, ULB, Brussels Belgium; Serge N. Schiffmann, Jean-Marie Vanderwinden, Nearophysiology,Faculty of Medicine, ULB, Brussels Belgium

Transforming Growth Factor ,8 (TGFjB) UnresponsivenessAfter Correcting Mutated TGFj8 Receptor II (TGF.BRII) by Chromosome 3 (ch3) Transfer in DNA Mismatch Repair (MMR) Restored HCT116+ ch3 Cells John M. Carethers, Univ of CA San Diego and San Diego VAMC, San Diego, CA; William M. Grady, Vanderbilt Univ, Nashville,TN; Betty L. Cabrera,Thu-Thao T. Pham, Univ of CA, San Diego, CA; Sanford Markowitz, CaseWestern ReserveUniv, Cleveland,OH; C. Richard Boland, Univ of CA San Diego and San Diego VAMC, San Diego, CA

Introduction: The c-kit proto-oncogeneencodesthe receptortyrosine kinase KIT,which ligand is the cytokine Stem Cell Factor (SCF). Oncogenic mutations(i.e, confering autonomous, SCF-independentactivation) affecting the juxtamembrane(JM) and phosphotransferese(PT) domains of KIT have been previously identified in gastrointestinal stromal tumors (GISTs) and in mast cell neoplasms.We identified a novel activating mutation resulting in a Lys-toGlu substitution at codon 642 of the ATP binding (BD) domain of KIT in a family with multiple GISTs and hyperpiesia of interstitial cells of Cajal (Isozaki et al. Am J Pathol 2000; 157: 1581).We hypothesizedthat the signal transduction pathways of KIT could be differentially affected by various oncogenic mutations. Materials & Methods: Wild type (WT) KIT and 8

Background&Aims: HCT116 colon cancer cells (which have only mutant hMLH1) have been previously corrected for microsatellite instability and defective DNA MMR by transfer of one copy of human chromosome3 containingthe hMLHI gene(locatedat 3p21). As a consequence of defective DNA MMR, both alleles of TGF~R/Iare mutated at its coding polyadeninetract in HCT116 cells. We hypothesizedthat chromosome 3 transfer would also restore TGF,8 responsiveness as one wild type copy of TGF~Ft//(located at 3p22) was also received by HCT116+ch3 cells. Methods: DNA was extracted from DNA MMR defective HCT116 and

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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

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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