899

Ureedeoxycholic Acid and an Analogue of Vitamin D inhibit COX-2 Expression in AOM-induced Colonic Tumors and in HCA-7 Cells: Roles of C/EBPfland VEGF Sharad Khare, Ramesh K. Wall, Sonia R. Cerda, John A. Hart, Michael D. Sitrin, Thomas A. Brasitus, Marc Bissonnette, Univ of Chicago, Chicago, IL

BACKGROUND: We previously demonstrated that the bile acid, ursodeoxycholic acid (UDCA), and an analogue of 1,25(0H)~ vitamin D3 (FED3), inhibit cydooxygensse-2 (COX-2) mRNA and protein expression in azoxymethane (AOM)-induced colonic tumors. We have now extended these studies by investigating the effects of these chemopreventive agents on several transcription factors implicated in the regulation of COX-2 gene expression. We have also examined AOi-inducad tumors for the effects of UDCA and FeD3 on VEGF, an angioganic factor and downstream target of COX-2, important in tumor progression. To further dissect the molecular mechanisms by which UOCA and FeD3 inhibit COX-2 expression, we have examined the effects of these agents on basal and COX-2 stimulated expression by the tumor promoting deoxycholic acid (DC) in HCA-7 ceils, derived from a human colon carcinoma~ METHODS: Tumors were induced by AOM in rats fad standard chow (AIN-76A); or chow supplemented with 0.4% UDCA, or with Fe-D3 (2.5 nmol/kg chow). After 32 weeks, rats were sacrificed and tumors harvested. A section of each tumor was used to characterize tumor histology. A separate portion was homogenized and proteins extracted in Laemmli buffer. HCA-7 cells were treated with DC alone, or with UDCA, or FED3, and cells ware then lysod in RIPA buffer. Proteins from cellular and tumor lysatss were separated by SOS-PAGE Expression levels of C/EBPs, VEGF and COX-2 were measured by qua,i;;,,~;ve Western bluffing. RESULTS: In AOM-induced tumors, compared to normal colon, OEBP-,8 and VEGF expression levels increased 8_+2-fold, and 20_+4-fold respectively (p <0.05). In contrast, OEBP-a and - 8 were not changed in these tumors. In tumors from rats supplemented with UOCA or Fs- D3, we observed a 60 - 10% decrease in the expression of C/EBP-p;, in these tumors, moreover, F6-D3, caused a 50-+20% decrease in VEGF, compared to carcinogen-induced tumors from unsupplemented animals (p<O.05). In HCA-7 cells, UDCA (100/AWl) and F=-D3 (100 nM) significantly inhibited basal COX-2 expression by 50% (p<O.05). In these cells DC (50 pM, 24 hrs) caused a 9_+2 . fold induction of COX-2 (p<O.05). Fe-D3 also inhibited DC-ctJmuluted COX-2 expression by 55% (p<O.05). CONCLUSIONS: UDCA and Fe'D3 significantly inhibited COX-2 expression in both AOM-induced colonic tumors and HCA-7 cells, indicuttng that these agents may exert their chemopreventive effects, at least in part, by radudng COX-2 expression during tumorigenesis.

900

Deoxycholic (DGC) Acid-induced Expression Of Cyctomqfgonu~2 (COX-2) in Human Colonic Fibrohlaats Shazia Rafiq, Yingting Zhu, Peter Lance, SUNY Buffalo, Buffalo, NY

Secondary bile acids, such as DOC, may contribute to colorectal carcinogenesis, with which COX-2 up-regulation has been causally linked. We previously demonstrated that pro-inflemma- tory cytokines elicited major increases of prosteglandin E2 (PGE2) production and COX-2 expression in human colorectal fibroblast strains in vitro. In the present study, we hypothesized that bile salts might also induce flbrobiast COX-2 expression. METHODS: Human colonic CCD-18Co fibroblasts were cultured to confluence in medium supplemented with 10% fetal bovine serum (FBS), then shifted to medium containing 1% FBS for 72 h prior to harvesting. Selected cultures were treated with 0.3 mM DOC or cholic acid, a primary bile acid. PGE~ production, and COX-2 mRNA and protein levels, respectively, were assessed by radioimmuno- assay, and Northern and Western analysis. RESULTS: DOC caused 18- to 31-fold increases compared to control in PGE2 levels, depending on the duration of exposure, over a 48-h period (see table). Profound dose- and time-relatad inductions of COX-2 mRNA and protein expression occurred in DOC-treatad cultures that were consistent with the increases in PGE2 synthesis seen under the same conditions. In marked contrast, cholic acid caused neither any increase in PGE2 synthesis nor up-regulation of COX-2 mRNA or protein expression. CONCLUSIONS: Our findings demonstrate that a secondary bile acid can potently induce COX- 2 expression in colorectal fibrohlasts in vitro. These results suggest a possible mechanism for colorectal COX-2 induction in vivo. The complete failure of a primary bile acid to induce COX-2 expression is consistent with the evidence that secondary but not primary bile acids are linked causally to human colorsctal neoplasia.

Induction of PGE2 Synthesis by DOC (0.3 rnM)

Treatment (hours) Fold (x) Inczrease in PGE= ~ 6 18x t2 29 × 24 31 × 48 21 x

901

Method For And Characterization Of Proteim Isolated By Later Capture Miorodissection From Formalin-Fixed, Paraffin-Embedded Tumors. Sarah C. Glover, Univ of Illinois, Chicago, IL; Kristina A. Matkowskyj, Richard V. Banya, Univ of Florida, Gainesville, FL

Background: Formalin-fixed, paraffin-embedded tumors archived by Departments of Pathology represent a valuable yet relatively unexplored research resource. Laser capture microdissection (LCM) is a new technique that allows for individual cells to be isolated and extracted from larger sheets of tissue. However, methods for isolating proteins from cells removed by LCM from formalin-fixed tissues do not currently exist, largely because the proteins in tissues so stored are extensively cross-linked. Given the importance of proteomlos in the post-genomic era, the aim of this study was to devise a technique for rapidly extracting proteins from cells isolated by LCM from formalin-fixed, paraffin-embedded tissues. Methods: Human colon cancer cell lines were formalin-fixed and paraffin-embedded using an identical process us is used for surgical pathology specimens. 5 previously resected human colon cancer paraffin

blocks were randomly selected from our (31 Tumor Database. All tissues were sectioned to a thickness of 4 ,urn for study. LCM was performed using an Acturus PixCell II microscope with built-in 810 nm low-heat IR laser. Since dyes traditionally used to permit microscopic analysis such as eosin cross-link proteins, tissues were stained with 0.1% methyl green, a DNA cross-linking agent. Results: We determined that 3 critical steps were necessary to increase detectable yields: 1), extract proteins in the presence of multiple protease inhibitors including chymostatin, pepstatin A, aprotinin, elupeptin, and PMSF; 2), concentrate proteins by precipitating in TCA/acetone; and 3), using EDTA (1 mM final concentration) when re- suspending the proteins post precipitation. Using this approach, we found that 1 ~ protein could be isolated per 2,000 cells removed by LCM, with similar amounts isolated from archived specimens as for formalin-fixed cell lines. There was no difference in the amount of protein isolated from cells cultured in vitro from those that were formalin-fixed. However, -1g-fold more protein needed to be loaded to be visualized by SDS-PAGE. Whereas proteins sized from 36 to 94 kDa were readily identified from cultured cells, formalin-fixation and paraffin embedding resulted in proteins predominately between MW 36 to 60 kDa. Conclusions: Isolation of proteins from formalin-fixed, paraffin-embedded tissues is technically possible. However, the fixation process dramatically reduces the amount and spectrum of big-avail- able protein.

I.mlle Sonls Expression 4n'ay Analysis Of A Colon Cancer Progression Model Reveals Altmml Regulation Of 1251 Genes Including The OownragufaUon Of Traneglntondonse. Sergio Hue~a, David Heber, UCLA Ctr for Human Nutrition, Los Angeles, CA; Julio Licinio, Ma-Li Wong, Jonas Hannestad, UCLA, Los Angeles, CA; Edward H. Livingston, VA Greater Los Angeles and UCLA, Los Angeles, CA

Background/Objectives: The progression of colon cancer from adenoma to metastatic cancer involves numerous genetic changes. Expression for various molecules is up or downregulated when cells acquire metastatic potential. Large scale micrnarrsy expression analysis facilitates simultaneous examination of numerous genes. We utilized a cell line system for cancer in a large-s~ expression microarray to determine which genes are expressed or repressed as colon cancer tmosi*dons from primary to metastatic cancer. Methods. Cell lines SW480 (primary colon cancer) and SW620 (metastatic lesion from the same patient) were obtained from the ATCC and grown under standard conditions. Microarray analysis was performed with the HG-U95A chipset on an Affymetrix ® reader. Total RNA was extracted from the cell lines using an RNAUSy" isolation Kit. Synthesis of cDNA, biotin labeling, digestion, and hybridization was performed and the housekeeping genes GAPDH and beta-actin used quanti- fate the RNA extraction. Results: Of the 9,600 genes on the chip 1251 had a greater than 2- fold difference in expression between the cell lines. Most genes lost expression in the metastatic cell line relative to the primary tumor. Three ware systematic losses of apoptosis mediators and homoobox genes. The adhesion molecules CD44, integrins alpha 3,6,7 and beta 5 lost expression in the metastatic cell line. Intagrin IN and ICAM2 were upregulated in the metastatic cells. Expression of the angingenesis inducer VEGF was increased in the metastatic cell lines as was muclo-3 and galdctin. Transglutaminase was expressed at 83% of control in the primary tumor cell line and fell 30x to 2% of control in the metastatic cells. Conclusions: Microarray analysis for gene expression is a powerful toll that facilitates simultaneous analysis of entire molecular systems. Genes not previously recognized as mediators of phenotypic change may be recognized. Using this technology we identified significant expression of transglutaminass, a mediator of cell-matrix adhesion and apoptosis that accounts for metastatic behavior in prostate cancer. As occurs with prostate, transglutaminase expression was mark- edly attenuated in metastatic colon cancer cells.

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~HTa Reonptm, Immunomactivity in Human and Rat Intestines Dania Reiche, Jutta Hahn, Wen Jiang, Univ of Tuebingen, Tuebingen Germany; David Grundy, Univ of Sheffield, Sheffield United Kingdom; Ronald H. Stead, McMaster Univ, Hanitton Canada

BACKGROUND: Serotonin (5HT) is released from enterochromatfin (EC) cells upon mucoeal stimulation; and both extrinsic and intrinsic afferent neurones respond to 5HT by activation of lonntropic 5HT3 receptors (5HT3R). However, the distribution of 5HT3R in neuronal and non-neuronal cells in the intestine has not been documented. METHODS: 5HT3R-immunoreac- tivlty (-IR) was localized using a polyclonal anti-5HT3R (Oncogene, Calbiochem, Bad Sudan, Germany), and a biotin-streptavidin detection system (DAKO, Hamburg, Germany). Paraffin sections from human and rat small intestines (fixed in neutral buffered formalin [NBF] or acetic acid - alcohol [AA]) were employed. Absorption with synthetic 5HT3R peptide (Oncogene; lO0#g/mL) abolished staining with the specific antiserum. 5HT was immunostained using a monnclonal anti-SHT (DAKO). RESULTS: Rat: 5HT3R-IR was observed in both NBF and AA fixed tissues. Small intestinal entemendocrine (EE)-Iike cells expressed strong 5HT3R-IR. The 5HT3R-IR EE-like cells were fewer in number than the 5HT-IR EE cells; and these appeared to be different populations. In the lamina pmpria, 5HT3R-IR nerve fibres formed a basket- work pattern around the bases of the crypts. Some submucosal ganglion cells exhibited 5HT3R-IR and strong staining of 5HT3R-IR varicosities was detected in both submucosal and myenteric ganglia. However, myantaric neumnes expressing 5HT3R-IR were absent or rare. In some slides, intra-epitbellal leukocytes were also stained. Human: NBF-fixed mucosal/ submucosal samples of small (n = 5) and large (n = 20) bowel consistently expressed 5HT3R- IR in EE cells. These were present in variable numbers and appeared to be different to the 5HT-IR cells. Some submucosal ganglion cells and varicosities in the ganglia were also stained. DISCUSSION: In the rat small intestine, vagal nerve fibres have been shown to penetrate the gut wall, form intrsganglionic laminar endings (IGLEs) and project to the mucosa. Also, nodose ganglion neumnes are known to express 5HT3R and electrophysilogical studies have shown the activation of intestinal vagal afferents through 5-HT3R receptors. Our findings of strongly labelled varicose nerve fibres in both plexuses and in the lamina propria provides micro-anatomical support for the physiological data. Furthermore, the presence of intrinsic afferents in human and rat is suggested by the presence of 5-HT3R-IR submucosal nerve cells. The present study further suggests that 5HT3R-IR EE cells differ from those containing 5HT.

A-170