corresponding non-tumorons tissues (P<0.01). In 17 of 24 (70.8%) HCC tissues, the promoter hypermethylation of either NQO1 or GSTP1 gene was observed. (Conclusion) These data indicate that the expression of NQO1 gene might be inappropriately suppressed by the promoter hypermethylation in HCC, as well as GSTP1 gene. Disruption of detoxifica- tion process mediated by NQO1 and GSTP1 may contribute to the pathogenesis of human HCC.

759

Regulation and Function of the Sonic Hedgehog (Shh) Signal Transduct ion Pathway in Isolated Gastric Parietal Cells Vinzenz Stepan, Saravanan Ramamoorthy, Hildegard Nitsche, Jung Park, Andrea Todisco

Shh plays an important role in gastric epithelial cell differentiation. We previously reported that incubation of punfied (>95%) canine gastric parietal cells in primary culture with EGF for 16 h has stimulatory effects on H+/K+-ATP-ase gene expression and on gastric acid production, through the activation of protein kinase Akt. In this study we explored if Shh mediates some of the actions of EGF in the parietal cells. EGF (10 nM) induced a six-fold increase in Shh expression, measured by western blots, after 5 h of incubation. This effect was inhibited by both the P13 kinase (P13K) inhibitor LY29042 (1 IzM) and by transduction of the parietal cells with an adenoviral vector expressing a dominant negative Akt gene, suggesting that EGF regulates Shh through PI3K- and Akt-dependem mechanisms. Shh induced H */K+ATP-ase gene transcription two-fold in panetal cells transfected with luciferase reporter plasmids containing 619 bases of the canine H+/K+-ATP-ase ~x-subuint gene pro- moter (H+/K+-luc). Since Gli transcription factors mediate the intmcellular actions of Shh, we co-transfected the parietal cells with the H+/K + -fuc plasmid together with one expressing the Gli2 protein. Gli2 induced H+/K+-luciferase activity five-fold. To explore if EGF induces H+/K+-ATP-ase gene transcription through the Shh signal transduction pathway, we co- transfected the parietal cells with the H+/K+-luc plasmid together with one expressing dominant negative Gli2. EGF induced H+/K+-luciferase activity two-fold, and dominant negative Gli2 inhibited this effect. Identical results were observed in the presence of the Shh signal transduction pathway inhibitor, cyclopamine (1 ~M). Transfection of the parietal cells with a dominant negative Akt gene inhibited EGF, but not Shh stimulation of H+/K +- ATP-ase-fuciferase activity, indicating that EGF but not Shh can signal through Akt. shh and EGF also induced by two-fold the transcriptional activity of an EGF response element (bases -162 to -156)-fuciferase reporter plasmid. We examined the role of Shh in gastric acid secretion. Pro-incubation of the cells for 16 h with either Shh or EGF enhanced histamine (100 gtM)-stimulated ~*C-aminopyrine uptake by 50%. Thus, EGF induces H+/K+ATP-ase gene transcription and it enhances secretagogne stimulated gastric acid production, through the sequential activation of the Akt and the Shh signal transduction pathways. This effect might represent an important mechanism regulating gastric epithelial cell differentiation.

760

Progastrin stimulates murine colonic mitosis after DNA damage via the cyclin Dl-cdk4 complex Penelope D. Ottewell, Alastair Watson, Timothy Wang, Graham Dockray, Mark Pritchard

Background and Aims: Transgenic mice which overexpress human progastrin (hGAS) are more susceptible to the induction of colonic aberrant crypt loci and adenomas by the chemical carcinogen azoxymethane than wild-type mice (FVB/N) and mice which overexpress amidated gasmn (INS-GAS) (Singh et.al. (2000) Am.J.Physiol. 277: G390-G399; Singh et.al. (2000) Gastroenterology 199: 162-70). We have previously shown that 4.5 hours following DNA damage by 8Gy ~/-radiation, colonic mitosis persists at significantly higher levels in hGAS compared with FVB/N or INS-GAS mice. We have now invesugated the molecular mechanisms responsible for this continued mitosis. Methods: Mice used were 10-12 week old liGAS and FVB/N. 4.5h following 8Gy "/-radiation colonic epithelial ceils were harvested using a modified Weiser technique. Expression of cell cycle regulatory genes was assessed using a GEArray TM pathway specific cDNA micruarray expression profiling system (Super- Array.corn). Protein expression of p21 w F~/c~P~, p27 K.p~, cdk6, cdk4, and cyclin D1 were analysed by Western blotting. Results: Gene array analysis showed increased abundance of cdk4, cdk6 and cyclin D1 mRNA in hGAS colonic epithelial cells compared to FVS/N 4.5h following 8 Gy ~,-radiation. Western blots confirmed that the abundance of cdk4 protein was significantly higher in hGAS colonic epithelium compared to FVB/N and did not change following ",/-radiation. In FVB/N mice, cyclin D1 protein expression was decreased in colonic epithelial cells 4.5h following 8Gy ',/-radiation. However, no change was observed in cycfin D1 protein expression in hGAS colonic epithelial cells after this treatment. No significant differences were observed between hGAS and FVB/N mice in the abundance of p27 K~p~, p21 w*F~P~ and cdk6 proteins in colonic epithelia. Conclusions: (1) High concentrations of circulating progastrln result in increased expression of cdk4 in murine colonic epithelia. (2) The persistent colonic epithelial mitosis found in hGAS mice after 'y-radiation is due to continued expression of cyclin D1.

761

Somatostatin Modulates Both Cyclooxygenase-2 Expression and Proliferation in Human Colon Cancer Cells Rocchina Colucci, Corrado Blandizzi, Maraia Tanini, Narcisa Ghisu, Maria Cristina Breschi, Mario Del Tacca

introduction. Among cyclooxygenase isoforms, COX-1 and COX-2, the latter is sigmficantly implicated in colorectal tumongenesis and cell proliferation. By contrast, somatostatin (SST) may negatively modulate colon cancer growth. This study investigates the effects of SST on COX-2 expression and proliferation in colon cancer cells, either under basal conditions or upon stimulation with gastrin-17 (G-17). Methods. Experiments were performed on human colon adenocarcinoma cell line HT29. Reverse transcription-polymerase chain reaction (RT- PCR) was used to assess mRNA expression of gastrin CCK-2 receptors, SST receptors (SSTR from 1 to 5), COX-1 and COX-2. Phosphorylation of MAP-kinase as well as COX-1 and

COX-2 protein expression were determined by westem blot analysis. Cell proliferative activity was estimated by direct cell count. Results. RT-PCR revealed the expression of CCK-2 receptors, SSTR-3, SSTR-4, SSTR-5, COX-1 and COX-2 transcripts under basal conditions. COX-1 and COX-2 protein expression was confirmed by western blot. SST (0.1-100 nM) significantly decreased the basal growth rate in HT29 cells (-41.3% at 1 riM), and concentra- tion-dependently inhibited the proliferative effects exerted by G-17 0.1 ~M (-55.2% at 1 nM). Western blot assay showed that COX-2, but not COX-l, expression was significantly enhanced by G-17. This stimulant action was prevented upon incubation of HT29 cells with PD-98059 (1-100 nM) or wortmannin (10-50 p,M), acting as inhihitors of MAP-kinase and PI3-kinase, respectively. SST reduced COX-2, but not COX-I, expression under basal conditions as well as after stimulation with G-17. Likewise, G-17 exerted a stimulant effect on MAP-kinase phosphorylation, which was fully counteracted by SST in a concentration- dependent manner. Conclusions. 1) SST can downregulate the expression of COX-2 in colon cancer cells, and such inhibitory effect may account for the antiproliferative action of this gastrointestinal peptide; 2) in colon cancer cells COX-2 expression is positively regulated by mitogenic transduction signals and this mechanism can be counteracted by inhibitory pathways induced by SST.

762

Vitamin D Inhibits CaCo-2 Cell Growth: Role of the Vitamin D Receptor and p21Waf l Merry J. Bolt, Anping C'hen, Dehra Stoiber, Yan C. Li, Marc Bissonnette, Michael D. Sitrin

Previous studies have shown that vitamin D (D) metabolites and analogs inhibit the growth of colon cancer-derived cell lines, and are potentially important chemopreventive agents. D secosteroids affect colunocytes by acting as liganda for the D receptor (VDR), a member of the nuclear steroid receptor superfamily, altering gene transcription. In addition, D com- pounds have rapid actions on signal transduction pathways in the colon that likely do not require the VDR, but may be mediated by a unique membrane receptor. To examine the role of the VDR in the anti-profiferative actions of D secosteroids, we prepared stable CaCo- 2 cell transfectants expressing the full length human VDR (hVDR) by G418 selection. The polyclonal population of hVDR transfectants had an 8-fold increase in VDR compared to empty vector (EV) tranfectants, assessed by Western immunoblotting. Cells were seeded at a density of 80,000 cells/well in 6-well culture plates, and media containing 100nM 1,25(OH)2 D3 (1,25D3), 100nM F6-D3, a non-calcemic vitamin D analog, or ethanol vehicle were changed daily. After 4 days (still pre-confluent), cells were harvested, counted, and boiled in SDS-containing buffer for analysis of cell cycle regulators by Western immunoblot- ting. 1,25D3 had no effect on the growth of EV transfectants, whereas F6-D3 had a modest inhibitory effect (8%). In contrast, 1,25D3 diminished the growth of hVDR transfectanti by 9%, and F6-D3 caused 26% growth inhibition (p<.O01). D compounds induced the expres- sion of p21Wafl, a key inhibitor of cell cycle progression, in a manner that paralleled their effects on growth. 1,25 D3 and F6-D3 increased p21Waf1 by 31% and 81%, respectively, in EV cells, versus 120% and 197%, respectively, in hVDR transfectants. In either cell type, no significant effects of either D compound on other cell cycle regulators, including p27Kipl, cyclin E, cyclin D1, or E-cadherin, were observed. Studies using monoclonal EV or hVDR transfectants confirmed the findings in the polyclonal cells; EV cells always had low levels of VDR and minimal responses to D secosteroids, whereas hVDR transfectants with high levels of VDR showed significant growth inhibition and p21Wafl expression in response to D compounds, F6-D3 > 1,25D3. Conclusions: The anti-profiferative effects of D compounds in CaCo-2 cells are mediated largely through the VDR, by induction of p21Waf1. The non- calcemic analog F6-D3 has more potent effects on cell growth and p21Waf1 expression than 1,25D3, and merits further investigation as a possible chemopreventive agent.

763

Caco-2 Cell Remodeling is Regulated by Transient Up-Regulation of Gastrin- Releasing Peptide (GRP) and its Receptor (GRPR) Sarah Glover, Kristin Keller, Richard V. Benya

Background: GRP/GRPR are normally--albeit transiently---expressed during intestinal devel- opment where they contribute to villus formation (Mech Dev 2002; 113: 121). When aberranfly re-expressed in colon cancer, these proteins act as murpbogens and critically regulate tumor cell differentiation (reviewed in Pepfldes 2001; 22: 689). Since Caco-2 cells are derived from a human colon cancer and are a model for the study of differentiation, the aim of this study was to determine if these cells express GRP/GRPR; and if so, elucidate their function when present. Methuds: Both Western blot analysis and immunohistochemistry using polyclonal antibodies were used to determine the expression of GRP and its receptor. Quantification of antibody-specific chromogen was determined at the cellular level as pre- viously described (] Histochem Cytochem 2003, in press). Remodeling was assessed after focal removal of confluent cells and following the behavior of those remaining by time-lapse photography in the presence or absence of the specific GRP antagonist D-Phe6(bombesin) methyl ester. Results: Confluent cells did not express GRP/GRPR. Disaggregation and plating at sub-confluent densities resulted in a rapid increase in GRP/GRP-R co-expression such that maximal levels were observed 72 hrs post-plating This was followed by a progressive disappearance of both proteins such that neither were detectable once cells achieved conflu- ence. Importantly, GRP/GRPR co-expressinn correlated with that of focal adhesion kinase (FAK), an enzyme that preliminary data indicates mediates the morpbogeinc properties of these proteins. Focal removal of cells from within a confluent monolayer resulted in a rapid up-regulation of GRP/GRPR within 6 hrs. This was assochated with an increase in cell size and migration into the gap such that the wound healed within 14 hr. In contrast, co- incubation with GRP antagonist significantly attenuated migration at the leading edge of the wound, with healing occurring in >40 hrs, but without affecting overall cell proliferation. Filling of the gap resulted in prompt down-regulation of GRP/GRPR. Conclusions: Caco-2 cells variably express GRP/GRPR as a function of confluence, and significantly contribute to cell migration as function of tissue remodeling without altering proliferation. The associated increase in FAK suggests that this enzyme may critically mediate the morphogenic properties of GRP and its receptor when both are aberrantly expressed in colon cancer.

AGA Abstracts A-100