Expression of PPAR gamma and RXR alpha in tissue and cell lines of gastric carcinoma

in odL-6DKO mice, TNFR2expression,proliferation,and NF-KBactivationof CECwas markedly reduced compared to TCR,~ KO mice. oENFR2 mice also showed significantly less severe colitis comparedto TCRo~KO mice. Increasedapoptosis in the cultured CEC of ~TNFR2 DKO mice was observed comparedto TCR,~KO mice by quantitative ELISA method. Conclusions: Expression of TNFR2 is consistently increased on CEC in both murine colitis models as well as IBD patients. The TNFR2 expression is upregulated by a cooperative effect of cytokines including TNFot, IL-6 and IL-1/~. Functionally, TNFR2 mediates both increased proliferation and suppression of apoptosis in CEC.TNFR2 plays a critical role in inflammation associated alteration in the intestinal epithelium.

of colorectal carcinomas. In the present study, we sought to determine the biological effects of activation of PPAR.yinK-Ras-transformed intestinal epithelial ceils. Methods: Rat intestinal epithelial (IEC-6) cells with an inducible activated K-Rasv'~2cDNA (referred as to IEC-iK-Ras cells) were generatedby using LacSwitch eukaryoticexpressionsystem. IPTGat a concentration of 5 mM was used to induce the expression of mutated K-Ras. Results: Morphological transformation of IEC-iK-Ras ceils was observed between 48-72 h following IPTG treatment. BRL 49653 treated IEC-iK-Rascells displayed a less transformed morphology and were less fibroblostold, retained cell-cell contact inhibition, and lower cell density. Treatmentwith BRL 49653 inhibited the growth of transformed IEC-iK-Ras cells in a concentration-dedendent manner. Addition of 10 #M of BRL 49653 reduced the cell number by 55.3% and 20 #M of BRL 49653 inhibited cell growth by 82%. Anchorage-independentgrowth assay showed that treatment with BRL 49653 slightly reduced the number of colonies but greatly reduced the size of colonies that were formed by IEC-iK-Rascells in soft agarosein the presenceof IPTG. Row cytometric analysis indicated that treatment with BRL 49653 resulted in accumulation of cells in G1 phase of the cell cycle and reduced entry to S phase in IEC-iK-Ras cells. Induction of K-Ras elevatedthe levels of both cyclin D1 mRNA and protein that were closely coincided ~ the levels of the Ras. Addition of BRL 49653 inhibited Ras induction of cyclin D1 in a concentration-dependentmanner. Ectopic expression of a stable cyclin D1 mutant (cyclin D1/I'286A) partially abolishedthe growth-inhibitory effect of BRL-49653. Conclusions: Activation of PPAR~,by specific ligand, BRL 49653, results in G1 growth arrest of K-Ras transformed intestinal epithelial cells. Inhibition of Ras induced expression of cyclin D1 significantly contributes to the anti-neoplastic effect of BRL 49653.

313 CEA (Carcinoambtyonic Antigen) Shows Identity to gp180 and Can Modulate Mecesal Immune Responses. Mee S. Park, Kai Becker, Lloyd Mayer, Mount Sinai Sch of Medicine, New York, NY BACKGROUND:We have previously reported that the monoclonal antibody 69 reactive 180 kD glycoprotain gp180 activates the CD8 associated tyrosine kinase Ick when T cells were co-cultured with gp180. We also reportedthe interaction of gp180 with COld and CD8. Partial sequence obtained from analysis of MAb Bg-purified gp180 showed homology wh'h the GPIanchored 180 kD glycoprotain CEA,which led us to characterizethe role of CEA in immune modulation. METHODS:We transfectad 293T, the retrovirus packaging cell line Phoenix, human melanoma ceil line FO-1, and CDld-transfected FO-t derivative FO-1D5 with a CEAIRES-GFP bicistronic construct. Vector-transfected 293T, Phoenix, FO-1, and FO-1D5 were used as controls. The 293T/CEA, FO-1/CEA, and FO-1DS/CEA were PIPLC (phosphatidyl inositol specific phospholipaseC)-treated to generate CEA-containingsupernatant. The CEA supernatants and the CEAtransfectants were used for adsorption experiments,tyrosine Idnase assays, and whole cell-based extracelluiar protein-protein intera~on assays, which took advantage of the dual expression of CEA and GFP. Western blots were used for adsorption studies and tyrosine kinase pbosphoryiation studies. Cell panning methods were used for whole cell-based interaction studies. RESULTS:All CEA-transfectantswere stained by MAb B9. CEA was adsorbed by the human CD8 a chain-transfected murine T cell hybridoma 368 but not by CD4-transfected 364. FD-lO5 bound to CEA supematant-ceateddishes 3 fold greater than FO-1 cells. Also, Phoenix/CEAbound to FO-1D5 but not to an FO-1 monolayer. PIPLC treatment of FO-1D5/CEAreleased not only CEA but also a small fraction of CDld, and PIPLC supernatant from FO-1D5/CEAenhanced Ick phospborylation in PB T cells to a greaterextentthan supernatantsfrom FO-1/CEAcells. CONCLUSIONS:Thesedata demonstrate that CEA shows identity with gp180 in terms of 89 reactivity, binding to CD8, and binding to CDld. PIPLC supernatant from CEA and CDld co-transfestants showed enhanced Ick phosphoryiation,similar to what was previously observedwith gp180 purified from T84 cells. The epitope recognizedby MAb 89 appearsto be critical to the interaction between CO8 and gp180 (CEA). However,this epitope may not be a CEA specific epitope as we have previously reported that MAb 89 reactive material is present on the basoiateral surface of the IEC as well (non GPI-anchored). There may be post-translational modifications to CEA subfamily members, which imbue immunoreguiatory properties.

316 PPAR,yand/3-catonln Interact to Regulate COX-2 Expression induced by Insulin in HT-2fl Colon Cancer Cells. Shaun G. Weller, William E. Karnes ,Jr., Mayo Clin, Rochester, MN; ,John A. Copland, Univ of Texas Medical Branch, Galveston,IX; Jason D. Morrow, Vanderbilt Univ Medical Ctr, Nashville, TN; RebeccaChinery, Mayo Clio, Rochester, MN BACKGROUND:Epidemiological, animal and in vitro studies indicate that hyperinsulinemia (Type 2 diabetesor SyndromeX) or elevatedinsulin-like growth tactor-1 (IGF-I: acromegaly) are associatedwith increased risk for colorectal cancer (CRC). We hypothesizedthat chronic hyperinsulioemia promotes colorectal carcinogenesisby activating IGF-1 receptors (IGF-1R) which in turn, activate downstream target genes important in transformation. AIMS: To determine the effects of insulin on COX-2, PPAR% E-cadherinand ,8-catenin expression and activity in human CRC HT-29 cells. METHODS:Effects of insulin (0-10/~M) on COX-2, PPAR,y and E-cadherinexpression in HT-29 cells were determined by Western analysis. Changes in COX-2 activity were determined by prostaglandin analysis of conditioned medium using gas chromatograpby/negativeion chemical ionization mass spectrometry. Insulin effects on the transcriptionalactivity of TCF/~-cataninand PPAR-ywereassayedafter 24 hours with luciferase reporters containing TCE(TOPFLASH)and PPAR.yresponseelements.Specificity was verified using dominant-negative(dn) TCF-4or E-cadherintyro-tail (ECCT),and dnPPAR,,/,respectively. involvementof IGF-1Rwas determinedusing the selectiveIGF-1R antagonist mlR3. RESULTS: Insulin decreasedE-cadherin protein levels beginning at 0.1p,M, and increased protein levels of COX-2 and PPAR~ beginning at I~M. Changes in E-cadherin were suppressed by a-IR3. Induction of COX-2protein by insulin increasedprostagiandinproduction, and was suppressed by ECCT, dnTcf-4 and the PPAR-y agonist trogiltazone. The PPAR~, reporter was dosedependently induced as concentrations of insulin exceeded 1/LM. In contrast, TOPFLASH reporter activity was induced by 0.1pM insulin but was quenched by higher concentrations, and was blocked by ECCT or dnTcf-4. PPAR.y dose-dependentlysuppressed induction of TOPFLASHactivity by/3-catanin and ~-catenin dose-dependentlysuppressedinduction of the PPAR~, reporter by PPAR-~.CONCLUSIONS: Insulin induces COX-2 expression through a mechanism that involves E-cadherin suppression and promotion of/3-catenin-induced transcription. PPAR~,functionally interacts with/3-catenin to suppress transcription. We speculate that hyperinsulinemiaor activation of IGF-1R promotes CRCthrough induction of genes,such as COX-2, by ~catenin/TCF. If PPAR~/agonistssuppress transcription of/3-catenin/TCFtarget genes in vivo, they may suppress colorectal carcinogenesis.

314 Expression of PPAR gamma and RXR alpha in Tissue and Cell Lines of Gastric Carcinoma Kaichun Wu, Liping Yao, Hanging Wu, Zhiguo Liu, Ling Li, Taidong Oiao, Daiming Fan, Dept of Gastroenterology,Xijing Hosp, Xi'an People'S Rep Of China Peroxisome proliferator-activated receptor(PPAR)gammafunctions as a ligand-dependent transcription factor in associationwith retinoic acid receptor(RXR)alphawhich plays important roles in differentiation of many cell types including malignancies.The aim of our study is to investigate the expression of PPAR gamma and RXR alpha in gastric carcinoma tissue and cell lines. Methods Immunohistochemistry was adopted to examinethe expression of PPAR gamma and RXR alpha in specimensfrom 53 patients with gastric carcinoma, 18 with gastric mucosal atypic hyperplasia,30 with chronic atrophic gastritis and 31 with chronic superficial gastritis. Immunocytochemistry,RT-PCRand Western blotting were used to detect expression of PPAR gamma and RXR alpha in gastric cancer cell lines. Rmm~ There was a marked increase in expression of PPAR gamma(pO.05). Expression of PPAR gamma and RXR alpha was also seen in most of the gastric cancer cell lines by immunocytochemistpj and Western blotting, except SGC7901 cell line demonstrated no PPARgamma but high RXR alpha content. Semiquantitative RT-PCR showed mRNA expression of PPAR gamma in all gastric cancer ceil lines among which MKN45 presentedwith highestamount of PPARgammamRNA.Conclusion There was an overexpressionof PPARgamma and RXR alpha in gastric carcinoma, indicating the two nuclear hormone receptors might play an independent or synergetic role in the developmentand progress of gastric carcinoma.

317 Pharmacologically Inactivating Mutations in the GRPR Gene Increase in Frequency as Human Colon Cancers De-Differentiate. Maria S. Tretiakova, Sarah C. Glover, Richard V. Benya, Univ of Florida, Gainesville,FL Background: Epithelialcells lining the mature colon do not normally express gestrin-releasing peptide receptors (GRPR). In contrast, we have shown that when aberrantly expressed in functional form in colon cancer, this protein acts as a morphogen where it causes tumor cells to adopt a better-differentiated phenotype (Cell Growth Diff 2000; 11: 385). Although GRPR mRNA is ubiquitously expressed by all human colon cancer cell lines studied, it is invariably mutated such that non-functional protein is often produced (Mol Pharmacol 2000; 58: 601). Since colon cancersare heterogeneouslydifferentiated,the aim of this study was to identify and characterizemutations in the GRPRgeneas a function of tumor cell differentiation. Methods: 17 cancers (3 Dukes A, 5B, 5C, 4D) containing 26 separate regions of distinct differentiation were randomly selected from our GI Tumor Database. We performed Laser Capture Microdissectionto isolateall cells of defined differentiation within each colon cancer, extractedthe genomic DNA,and subjectedit to automatedsequencing.Mutationswere studied in CHO-K1cells transiently transfected with cDNA containingthe re-createdalteration. Results: Overall 40 different mutations were identified. The GRPRgene was mutated in all malignant cells, as well as in non-malignant cells immediately across the tumor margin. All mutations were silent in non-malignant ceils. As tumor caJis de-differentiated, the proportion of silent mutations progressively decreased while overall mutation frequency increased (1.3_+0.9, 3.5_+0.9 and 6.1 _+1.5 separatemutations per well, moderateand poorly differentiatedtumor regions respectively).The GRPRgenein all poorly differentiatedtumor cells containedreceptorinactivating mutations (R72stop, E175K, $211P, $214P, L278P); while well and moderately differentiated tumor cells never contained inactivating mutations. Multiple mutations were identified in many different cancers (G125A, 0149H); with most mutations occurring in 2 distinct regions within the 2nd exon. Conclusions: The GRPR gene is increasingly mutated,

315 Activation of PPAR,,/InhibitsK-Ras-mediatod Transformation of Intestinal Epithelial Cells Jinyi Shag, Hongmiao Sheng, Raymond N. Dubois, Vanderbilt Univ Medical Ctr, Nashville, TN Peroxisome pmliferator-activated receptor ~,(PPAR-/) is a member of the nuclear receptor superfamily of ligand-activatedtranscription factors. Many chemical agents including thiazolidinedones(TZDs), rosiglitazone(BRL 49653), and pioglitazonecan bind and activate PPAR~,. Recently, PPAR-~ligandswere shown to inhibit the growth of a variety of transformed cells. Mutations of K-Ras occur in a number of malignant lesions including a significant number

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