familial adenomatous polyposis patients and advanced colonic carcinoma. Methods: An avidin- biotin complex technique with double immunolabeling was used to identity COX-2-producing cells in archival formalin fixed, paraiffn-embedded human tissue sections. Results: COX-2 expression was essentially absent in the normal mucosa and in hyperplastic polyps, except for areas with mucosal ulceration and granulation tissue. In these cases, it was localized to the periluminal region of the lamina propria of the mucosa. Double immunolabeling showed that, in polyps, the cells expressing COX-2 were alpha smooth muscle actin positive stromal cells (myofibroblasts). Stromal COX-2 expression was found in 100% of patients in the sporadic adenoma group (n=24), 78% of familial polyposis patients (n=9) and 62% of patients with frankly invasive adenocarcinoma (n=37). In invasive adenocarcinoma COX-2 expression was also observed in endothelial cells and epithelium of the malignant lesions. Conclusions: This study indicates that COX-2 expression in stromal myofibroblasts may play an important permissive role early in the process of colorectal carcinogenesis. These results support the hypothesis that myofibroblasts may be important target cells for NSAID-mediated chemoprevention of colorectal cancer. (Supported by the DK55783-01)

865

Establishment and Characterization of a New Pancreatic Tumor Cell Line from Intraductal Papillary Mucinous Tumor(IPMT) Seung Woo Park, Yonsei Univ Coil of Medicine, Seoul South Korea; Ji-Eun Lee, Jung-Hee Lee, Yule Shin, Yonsei Univ Medical Research Ctr, Seoul South Korea; Jun Pyo Chung, Jae Bock Chung, Si Young Song, Yonsei Univ Coil of Medicine, Seoul South Korea

BACKGROUND: IPMT is characterized by mucin secretion and resultant pancreatic duct dilation. IPMT has all spectrums from adenoma to invasive carcinoma, and shows far less tendency to invasion than ductai adenocarcinoma. However the genetic differences are largely unknown. Though many human pancreatic carcinoma cell lines have been established, there are no known cell lines established from IPMT. AIM: To establish and characterize cell lines from IPMT, METHODS: Stable cell lines were established from a typical IPMT (borderline malignancy) by minsing, sieving, and cultivation in DMEM, and two colonies(COL1, 7) were selected according to the cell and colony morphology. RESULTS: Individual cell and colony morphology showed some differences between the two cell lines. Whereas COL1 showed several cyto- plasmic projection and scattered growth pattern, COL7 grew in close contact between cells and formed well-defined colonies. Both cell lines maintained initial morphology during over 40 passages. Population doubling times were 30.7 & 32.9 for COL1 and 7, respectively, and plating efficiency was over 90%. On Western blot, both lines expressed cytokeratin 19, a ductal marker, and were positive for Mucl and Muc5a but negative for Muc2, and also strongly expressed TGFa and amphiregulin. Though both lines did not form colonies in soft agar, they formed solid tumors both in nude and SCID mice. On karyotyping, both cell lines showed aneuploidy. On mutation analysis, both lines were heterozygous mutant for K-ras (codon 12, GGTSGAT), and analyses for p53, p16, and DPC4 are under investigation. CONCLUSION: These cell lines can be a model for studying IPMT and we are going to investigate the genetic differences between IPMT and ductal adenocarcinoma.

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866

Quantification of Frameshifl Mutations at a (CA)13 Micresatellite with a Novel, Enhanced Green Fluorescence Protein (EGFP) Based Plasmid. Christoph Gasche, Christina L. Chang, Jennifer Rhees, Christian Arnold, C. Richard Boland, Dept of Medicine and Cancer Ctr, UCSD and VAMC, San Diego, CA

The mutator pathway in MSI + tumors takes advantage of the selective inactivation of genes that harbor exonic microsatellites (MS's) such as TGF/~-RII, IGF2R or BAX. Aim: To develop a model for quantifying mutation rates at defined MS's in mismatch repair deficient (MMR) cells. Methods: The EGFP cDNA was retrieved by digestion of pEGFP-N3, and a new Pmel- Ascl site was created immediately after the EGFP start codon by PCR mutaganesis. The PCR product was cloned into plREShyg2 [Clontech] resulting in the plasmid plREShyg2-EGFP. Ligation of an MS construct [i.e. (CA)~3, which was generated through synthetic oligonucleo- tides] into the PmeI-Ascl-digested plREShyg2-EGFP produced the plasmid plREShyg2-EGFP/ CA13 and shifted the EGFP reading frame into a + 2 position. Plasmid DNA was isolated and the sequence was confirmed. HCT116 (MMR-) and HCT116 + ch3 (MMR +) were transfected with plREShyg2-EGFP/CA13 and single cell clones were selected with hygromycin B. The number of genomic integration sites was analyzed by Southern blot with EGFP as a probe. Frameshift mutations that restored the EGFP reading frame allowed EGFP expression and detection of cells by fluorescence microscopy or FACS. Results: After selection, at least three clones with single integration sites were identified from each cell line. Six weeks after transfection, a subpopulation in the HCT116 clones was fluorescence positive [mutant fraction (MF): 1.3-1.9%] but none in the HCT116 + chr3 clones. Genomic DNA was isolated and MS PCR of the transfected EGFP gene showed a 2-bp frameshift in the MF. Single cells from the MF were sorted by FACS, grown, and DNA was isolated from small colonies. The EGFP insert was amplified by PCR and MS mutations [(CA)~3,(cA)~2] were confirmed by DNA sequencing. Non-fluorescent cells were sorted by FACS (1000 cells per well), cultured over 2 weeks,, and the MF was determined by FACS at several time points. The MF increased linearly with the number of generations (r=0.9992). The mutation rate, calculated for each clone from the slope of the MF versus generations, was 5.0 xlO 4 mutations/(CA)Jgeneration (range 3.6 xlO -4 to 5.9 x10"4). Conclusions: Spontaneous frameshiff mutations at a (CA)13 MS in HCT116 are mostly losses of one CA-repeat and occur at a rate of 0.0005 mutations/(CA)~/generation. Our model is suitable for studying mutation rates at different MS's and for screening drugs,

which might alter the mutation frequency, and thus may interfere with the mutator pathway of MSI+ tumors.

867

Chromosomal vs Micreaateilite Instability in Colorectal Cancers: Evidence for Previously Unrecognized Pathways Ajay Geel, Christian N. Arnold, Dong Kyung Chang, Luigi Ricciardiello, Dharam P. Chauhan, Dept of Medicine and Cancer Ctr, UCSD, La Jolla, CA; John M. Carethers, Dept of Medicine and Cancer Ctr, UCSD and VAMC, La Jolla, CA; Linda Wasserman, Cancer Ctr, UCSD, La Jolla, CA; Carolyn Compton, Dept of Pathology, McGill Univ, Montreal Canada; Donna Niedzwiecki, Duke Univ, Durham, NC; Robert J. Mayer, Dana-Farber Cancer Institute, Boston, MA; Monica M. Bertagnolli, Brigham and Women's Hosp, Boston, MA; C. Richard Boland, Dept of Medicine and Cancer Ctr, UCSD and VAMC, La Jolla, CA

Background: Colorectal carcinogenesis can be classified according to two mechanisms of genomic instability: chromosomal instability (CIN) or microsatellite instability (MSI). CIN is characterized by aneuploidy and loss of heterozygosity (LOH) preferentially involving chromo- somes 5q, 18q and 17p. MSI is characterized by the accumulation of somatic alterations in microsatellite sequences caused by a defect in the DNA mismatch repair system. Aims: We tested the hypothesis that all colon cancers are associated with either MSI or CIN, and asked whether there is any overlap between both mechanisms, and whether some tumors might lack both of these mechanisms. Methods: We examined 243 paired normal colonic mucosa and colorectal tumors obtained from the Cancer and Leukemia Gmup-B (CALGB) protocol 9865 and UCSD Cancer Center. A panel of 5 Bethesda conference recommended markers were used to study MSI status of these samples. Six additional markers, including five dinucleotide repeat markers (D3S1029, D17S201, D18S64, D18S474, D18S69) and a tetra- nucleotide repeat marker (MYCL1) were used to study LOH and MSI events in all the tumors. DNA was extracted from paraffin embedded tissue sections, amplified by PCR using radiola- beled primers and electrophoresed on polyacrylamide gels. Tumors with instability in at least two markers of the recommended panel were classified as MSI-H, whereas others were classified as MSI-L (instability at one locus) or MSS (no instability). LOH was determined by loss of one allele in the tumor at any locus. Results: Of all 243 tumors, 17.3% were MSI-H, 16.9% tumors showed MSI-L and the remainder were MSS (65.4%). We identified 114 tumors (46.9%) with an LOH event at one or more locus of the nine markers studied. The frequency of LOH events was most common at chromosome 18q, followed by 17p and 5q. Additionally, 83 tumors (34.2%) demonstrated neither MSI nor LOH at any of the eleven markers tested, while 38 tumors (15.6%) showed both MSI and LOH phenotypes. Interestingly, 68.3% (28/ 41) of MSI-L tumors also had overlap with LOH. Conclusions: Although it is assumed that the MSI and LOH pathways are mutually exclusive, we observed a significantly high degree of overlap between these mechanisms. More importantly, we provide substantive evidence that there is an additional subset of colorectai tumors that are not associated with either of the currently recognized forms of genomic instability, suggesting that additional mechanisms of tumor development may be operative in the colon.

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Functional Aspects of Gastric Infection with Helicobacter pylori in Mongolian Gerbils at Early Stage of Adenocarcinoma Formation Tomasz Brzozowski, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Peter C. Konturek, Dept of Medicine, Univ of Edangen-Nuremberg, Erlangen Germany; Jerzy Stachura, Dept of Pathology, Univ Sch of Medicine, Cracow Poland; EIzbieta Karczewska, Wladyslaw Bielanski, Slawomir Kwiecien, Piotr Pierzchalski, Stanislaw J. Konturek, Dept of Physiology, Univ Sch of Medicine, Cracow Poland

Background: Gastric Helicobacter pylori (Hp) infection in Mongolian gerbils is an established experimental model to assess the long-term associacion between Hp-infection and gastric carcinogenesis but functional aspects in the stomach such as influence of this infection on gastric secretion, gastric blood flow (GBF) and gastrin-somatostatin link have not been studied extensively. Methods: We studied the effects of intragastric inoculation of Mongolian gerbils with Hp strain (cagA+ vacA+,5xlO 6 CFU/ml) that had been isolated from a patient with gastric ulcer vs administration of vehicle (saline) in gerbils equipped with or without gastric fistula at 2, 4, 12, 30 weeks upon Hp inoculation. The morphological changes in glandular mucosa were assessed by histology, the viable Hp analyzed by rapid urease test and density of Hp-colonization was evaluated by counting of the number colonies per plate. Gastric blood flow (GBF) was measured by H2.gas clearance technique and plasma gastrin and gastric luminal somatostatin were determined by RIA. Results: The gastric Hp infection was detected in all animals by histology, Hp culture and rapid urease test. Gastric acid output in non- infected gerbils averaged 38_+4 p~mol/h and this was reduced by over 50% immediately after Hp inoculation and persisted all the time intervals tested. Early lesions were seen already 4 weeks upon Hp-inoculation and consisted of chronic gastritis with increased mucosal foldings and elongated interfoveolar ridges and formation of multiple lymphoid follicle in the gastric mucosa. Typical adematous hyperplasia with cellular atypia was observed together with increased mitotic activity and apoptotic bodies formation, while lamina propria was reduced leaving dilated gastric gland situated "back-to-back". The GBF in Hp-infected gerbils was significantly lower than that in vehicle-controls and this decrease in GBF remained constant until the end of observation period. Hp-infection was accompanied by an increase in plasma gastrin from 38 pM before to about 300 pM and the significant fall in gastric somatostatin contents observed at the end of observation. Conclusion: Hp-infection in Mongolian gerbils in early stages before adanocarcinoma formation mimics that observed in human gastric mucosa and is associated with typical functional pathology such as suppression of gastric secretion and impairment of both, gastric mucosal microcirculation and gastdn-somato- statin link.

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