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255. THE IMPACT OF ALGINATE AND EPIDERMAL GROWTH FACTOR ON ENDOCYTOSIS—A STUDY IN FOUR OESOPHAGEAL CELL LINES
  1. M.F. Byrne,
  2. P.A. Corcoran,
  3. J.C. Atherton,
  4. D.J. Fitzgerald,
  5. F.E. Murray
  1. M.F. Byrne,
  2. J. Murphy,
  3. P.A. Corcoran,
  4. D.J. Fitzgerald,
  5. F.E. Murray
  1. M.F. Byrne,
  2. P.A. Corcoran,
  3. J.C. Atherton,
  4. D.J. Fitzgerald,
  5. F.E. Murray
  1. M.F. Byrne,
  2. P.A. Corcoran,
  3. S.W. Kerrigan,
  4. J.C. Atherton,
  5. D.J. Fitzgerald,
  6. D. Cox,
  7. F.E. Murray
  1. N. Parnell,
  2. S. Martucci,
  3. Y. Daniel,
  4. J. Ellis,
  5. N. O'Reilly,
  6. S. Rosen-Bronson,
  7. P. Ciclitira
  1. Depts of Gastroenterology and Haematology, St Thomas's Hospital, London, UK
  1. B.C. McKaig,
  2. Y.R. Mahida
  1. Division of Gastroenterology, University Hospital, Nottingham, UK
  1. S.A. Weaver,
  2. K.M. Patel,
  3. K.L. Wright,
  4. D.A.F. Robertson,
  5. S.G. Ward
  1. Dept of Pharmacy and Pharmacology, Dept of Medical Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK
  1. K.S. Chapple,
  2. N. Scott11-1,
  3. P.J. Guillou11-2,
  4. P.L. Coletta,
  5. M.A. Hull
  1. Molecular Medicine Unit,11-1Dept of Pathology and 11-2Academic Unit of Surgery, University of Leeds, St James's University Hospital, Leeds, UK
  1. S.A. Bustin,
  2. S.-R. Li,
  3. N.S. Williams,
  4. S. Dorudi
  1. Academic Dept Surgery, St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK
  1. N. Andrews,
  2. L.-G. Yu,
  3. I. Grierson,
  4. J.M. Rhodes
  1. Dept of Medicine, University of Liverpool, Liverpool L69 3GA, UK
  1. M.N. Woodward,
  2. S.E. Kenny,
  3. C.R. Vaillant,
  4. D.A. Lloyd,
  5. D.H. Edgar
  1. Institute of Child Health, Alder Hey Children's Hospital and Dept of Human Anatomy and Cell Biology, University of Liverpool, UK
  1. P. McPherson,
  2. P. Dettmar15-1,
  3. N. McCullough,
  4. P. Ross
  1. Gastroenterology Research Laboratories, Molecular and Cellular Pathology, University of Dundee, Dundee, DD19SY; 15-1Reckitt Benckiser, Dansom Lane, Hull HU8 7DS, UK

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There is considerable evidence to suggest that epidermal growth factor (EGF) and its receptor (EGFr) play an important role in tissue repair, cell proliferation and migration. EGF also inhibits acid production and imparts a cytoprotective mechanism against refluxed gastric contents entering the oesophagus.

Alginate biopolymers are widely used in the treatment of Gastrooesophageal Reflux Disease (GORD). Like EGF they have cytoprotective biological effects although their mechanism(s) of action have not been specifically identified. Alginate biopolymers up-regulate endocytosis, although this activity is not strictly dependent on the interaction of EGF with its receptor. There is, however, evidence of a co-operative relationship.

The action of EGF is due to the ligand binding to its receptor, EGFr, resulting in its activation, generating membrane signalling and subsequent internalisation of EGF and the receptor. Glycolipids are involved in the transport of proteins in the endocytic pathway. We have evidence to suggest that glycolipids such as gangliosides GM1 and GM3 may participate in the regulation of EGF/EGFr mediated endocytosis. It is known that GM3inhibits EGF.

In our study we have examined four cell lines derived from oesophageal carcinomas (2 squamous cell carcinomas and 2 adenocarcinomas). Direct immunofluorescence protocols with confocal microscopy and FACScan® techniques have been employed to observe the relationship between EGF, alginate, EGFr and gangliosides.

Results: Alginate up-regulates fluid phase endocytosis. EGF up-regulates fluid phase endocytosis. EGF and alginate up regulates fluid phase endocytosis. Alginate does not up-regulate receptor mediated endocytosis.

256. CO-CULTURE OF HUMAN SQUAMOUS OESOPHAGEAL AND FIBROBLAST CELL LINES IN ACTIVATION OF PROMMP-2 RESULTING IN A DOWN REGULATION OF INTEGRIN αVβ3 EXPRESSION AND MMP-2, MT1-MMP EXPRESSION

Background and aims: Over expression of various matrix metalloproteinases (MMPs) and decreased expression of tissue inhibitors of metalloproteinases (TIMPs) have previously been speculated to correlate with tumour progression in a variety of cancers. The aim of this study was to investigate MT1-MMP and αVβ3 interactions in the activation of MMPs in human oesophageal cancer cell lines during co-culture with human fibroblasts.

Methods: Gelatin zymography, western blotting and real-time PCR were used to investigate the protein and gene expression of MMP-2, -9, MT1-MMP, TIMP1 and TIMP2 in cell lines: OE19, OE33 (adenocarcinomas) and OE21 (squamous carcinoma). Immunoprecipitation was used to investigate levels of integrin αVβ3 by western blotting. Contact and non-contact co-culture experiments were undertaken involving the tumour cells and the human fibroblast line, 46 Br.1G1.

Results: The squamous oesophageal carcinoma (OE21) produced significant 14 fold greater levels of MMP-2 and -9 mRNA (p<0.01) compared with that of the adenocarcinoma oesophageal cell lines (OE19, 33). This correlated with increase of levels of proMMP-2 (2.5 fold) and proMMP-9 (2 fold) at the protein level. Integrin αVβ3 protein expression was also exclusively expressed in OE21 cells. Contact co-culture of OE21 and fibroblasts produced a significant decrease of MMP-2 and MT1-MMP (p<0.01) and an increase of active MMP-2 (12 fold) at the protein level. Integrin αVβ3 expression decreased with contact co-culture.

Conclusion: Higher levels of MMPs at the gene and protein level has been shown in a squamous oesophageal carcinoma compared with oesophageal adenocarcinomas. Down regulation of integrin αVβ3, MMP2 and MT1-MMP at the gene level may be correlated with activation of proMMP-2 and occurs only in the tumour cells when in contact co-culture with fibroblasts.

257. EFFECT OF DIFFERENT STRAINS OF HELICOBACTER PYLORI ON CYCLOOXYGENASE-2 PROMOTER ACTIVITY—VAC A AND CAG A NOT INVOLVED

Several studies have demonstrated that H. pylori induces COX-2 in gastric mucosa and suggested that this may explain the increased risk of gastric cancer withH. pylori infection. We have previously described regulation of the COX-2 gene by H. pylori at a transcriptional level. It has been suggested that certain strains of H. pylori are more virulent than others for induction of peptic ulcer disease or gastric cancer. We thus examined the effect of different strains ofH. pylori on COX-2 induction at the promoter level.

COX-2 promoter activity was measured in a cancer cell line of gastric origin (AGS) using transient transfections with 5' deletion constructs of the COX-2 promoter driving luciferase reporter gene expression. Eight fragments were used ranging in size from –50 to –1840 base pairs upstream of the transcriptional start site. Transfected cells were incubated for 8 hours with four strains ofH. pylori, two vacA and cagA positive (60190 and M99) and two vacA and cagA negative (Tx30a and 87203) (2x108 CFU/ml). Results were corrected for transfection efficiency.

COX-2 promoter activity was induced by all H. pylori strains used. In untreated and treated cells, there appeared to be a positive regulatory element between –50 and –80 bp upstream from the transcriptional start site, a region that contains a cAMP response element (CRE). With constructs larger than 161 base pairs, there was increased expression of the COX-2 promoter in cells treated with H. pylori compared to uninfected cells. The increased expression corresponded to the inclusion of an NF-κB binding site at –222 base pairs. The overall pattern of COX-2 promoter induction was similar with all the strains used, irrespective of Cag and Vac status of the organism.

These data confirm that H. pylori regulates COX-2 at a transcriptional level and that the NF-κB site on the COX-2 promoter at –232 bp may be critical in this regulation. However, CagA and VacA do not play a significant part in COX-2 induction.

258. HELICOBACTER PYLORI INCREASES ENDOTHELIAL SELECTIN AND CELLULAR ADHESION MOLECULE EXPRESSION AND NEUTROPHIL ADHESION

There is growing evidence that H. pylori exerts some of its effects on the stomach by actions on the gastric vasculature. We have shown upregulation of both isoforms of COX in a vascular endothelial model with H. pylori and described a mechanism for H. pylori associated platelet aggregation. In this study, we examined the effect of H. pylori on the expression of adhesion molecules and subsequent leukocyte-endothelial interactions.

Human umbilical vein endothelial cells (HUVEC) were grown to confluence in 96 well plates, serum starved overnight and then exposed to control medium, interleukin (IL) 1β (100U/ml) or H. pylori M99 for 18 hours. HUVEC were examined for adhesion molecule expression by standard ELISA using monoclonal antibodies against P-selectin, E-selectin, VCAM-1 and ICAM-1 (vascular or intracellular adhesion molecules). Binding of polymorphonuclear cells (PMN) to HUVEC was carried out on cells pretreated in the same way on 24 well plates. PMN were allowed to adhere for 20–30 mins, non-adherent cells removed and adherent cells measured using a myeloperoxidase assay.

There were 46% (± 14%) and 36% (± 10%) increases in the absorbance readings for P-selectin and VCAM-1 respectively inH. pylori exposed HUVEC compared to controls. There were smaller increases in relation toH. pylori exposure for E-selectin and ICAM-1. There was a 2–3 fold increase in PMN adhesion to IL-1β treated HUVEC and an even more pronounced 5–7 fold increase on theH. pylori treated cells.

H. pylori causes upregulation of adhesion molecules in endothelial cells with resultant increased neutrophil adhesion. Upregulation of P-selectin is also known to be important inH. pylori activation of platelets. Increased white cell adhesion may play a role in the pathogenesis ofH. pylori induced ulcer disease.

259. TRANSCRIPTIONAL REGULATION OF THE “CONSTITUTIVE” CYCLOOXGENASE-1 GENE BY HELICOBACTER PYLORI

Although COX-1 is often referred to as a constitutively expressed gene, we have previously described induction of COX-1 as well as COX-2 by H. pylori suggesting that COX-1 may contribute to inflammatory responses. We have also shown transcriptional regulation of the COX-2 gene by H. pylori using promoter constructs. The aim of this study was to determine if H. pylori exerts a regulatory effect on the COX-1 promoter.

COX-1 promoter activity was measured in a gastric cancer cell line (AGS) using transient transfections with a 5' deletion construct of the COX-1 promoter driving luciferase reporter gene expression. The COX-1 promoter differs from that of COX-2 in that it contains no TATA box but has several transcriptional start sites. We used a 2075-base pair fragment (-2095 to -21 relative to the translation start codon). Transfected cells were incubated for 8 hours with liveH. pylori (strain 60190, tox++,cagA+) at a concentration of 2x108 bacteria per ml.

H. pylori caused an increase of mean 31.3 % (+/- 6.9 %) in COX-1 promoter activity with this construct compared with uninfected cells. Of note, this region of the COX-1 promoter contains two Sp1 binding sites known to activate basal gene transcription.

This is the first report of regulation of the COX-1 gene byH. pylori at the promoter level. We speculate that previously described “housekeeping” promoter binding sites such as Sp1 may be important in this regulation. This work strongly supports the suggestion that H. pylori exerts its effects on the stomach via induction of COX-1 as well as COX-2.

260. HELICOBACTER PYLORI, OXIDATIVE DAMAGE AND GASTRIC CARCINOGENSIS

Background: Mutation of the p53 tumour suppressor gene is the most common genetic alteration in human cancers and has been implicated as a key factor in early gastric carcinogenesis. In addition, Helicobacter pylori (HP), is a class 1 carcinogen whose presence is associated with cancer of the mid or distal stomach. We hypothesised that HP may bring about its carcinogenic effect by promoting reactive oxygen species.

Aim: To investigate this relationship in anin vitro system, using hydrogen peroxide as a generator of reactive oxygen species.

Method: A transformed human gastric cell line was dosed with 0μM, 100μM and 300μM of hydrogen peroxide to mimic oxidative damage. Following DNA extraction the restriction site mutation (RSM) assay was employed to analyse mutations occurring in 8 restriction enzyme sites of the human p53 gene, exons 5–8. Each restriction site was analysed in triplicate for each dose range.

Results: Mutations were only found in the Msp1 restriction site (hotspot 248) and were mainly GC to AT transitions (60%). This experimental data implicates the 5-hydroxy-cytosine adduct in these mutations and not the 8-hyroxy-guanosine adduct which is the most common cause of oxidative damage. This finding is not surprising as 8-hydroxy-guanosine is only weakly mutagenic and easily repaired whereas 5-hyroxy-cytosine is highly mutagenic.

Conclusion: This experiment shows that the RSM assay can be used as a detection method for oxidative damage in early gastric cancer. It will now be applied to patient biopsy samples (normal, gastritis and intestinal metaplasia), in order to elucidate what mutations occur in potentially pre-cancerous gastric tissue.

261. MECHANISMS OF H. PYLORI INDUCED PLATELET AGGREGATION—COSIGNALLING BETWEEN PLATELET GPIb AND FcγRIIa BUT NO ROLE FOR PLATELET ACTIVATING FACTOR

Human and animal data have demonstratedH.pylori induced platelet aggregation in gastric vasculature. Formation of platelet aggregates may contribute to the pathogenesis of H.pylori associated gastric injury and may also explain the link with cardiovascular events. Recent work has suggested that induction of platelet aggregation by H. pylori is due to secretion of platelet activating factor (PAF) or PAF like substances byH. pylori. We have previously described a mechanism for induction of platelet aggregation byH. pylori which is platelet glycoprotein (GP) Ib dependent. The aim of this study was to further define the signalling pathways of this mechanism and also to investigate the role of PAF.

50 μl of bacterial suspension (H. pylori60190 4x109 CFU/ml) were added to 450 μl of platelet rich plasma. Platelet aggregation was measured by changes in light transmission. In a separate experiment, CHO β-IX cells were transiently transfected with GPIbα (interaction site of GPIb). FITC labelled mock and transfected CHO cells were then incubated with labelled H. pylori strain 60190.

H. pylori strain 60190 induced platelet aggregation (mean 62% ± 2%). Using confocal microscopy we found that CHO β-IX cells transfected with GPIbα boundH. pylori 60190 whereas no binding occurred in mock transfected cells confirming the role of GPIb. Also,H. pylori- induced aggregation of platelets via GPIb is dependent on signalling through the platelet bound FcγRIIa receptor as FcγRIIa blocking antibody (CD 16/32)(10μg/ml) inhibited H. pylori induced platelet aggregation by 99±0.4% (p=0.0001). However, PAF is not involved as PAF antagonist (1μm) had no effect on H. pylori induced platelet aggregation but completely inhibited PAF induced platelet aggregation.

These data confirm that H. pylori induces platelet aggregation by initial activation of the platelet via GPIbα involving cosignalling with the platelet FcγRIIa receptor. This mechanism is independent of PAF. These data support the suggestion thatH. pylori induced platelet microthrombi in gastric vasculature may be an important early event in gastric injury.

262. DIFFERENTIAL EXPRESSION OF RAS ISOFORMS IN NORMAL HUMAN PANCREAS: AN IN VIVO IMMUNO-HISTOCHEMICAL ANALYSIS

Background: Ras monomeric GTPases play a vital role in cellular signalling pathways by linking cell surface receptors and integrins to intracellular machinery and thus influencing proliferative and apoptotic events. Ki-Ras in its mutated oncogenic form is known to occur commonly in various pancreatic pathologies but wild-type Ras isoforms (Ha-, Ki-, and N-) have not been studied in normal human pancreas.

Methods: Archival formalin-fixed, paraffin embedded specimens of normal pancreas were cut and consecutive 4μm sections were processed and incubated (overnight at 4° C) with pan-Ras and isoform-specific Ras monoclonal antibody (mAb) with appropriate positive and negative controls. Detection by immuno-histochemistry involved using a modified polymer system. Two independent observers carried out semi-quantitative analysis.

Results: Pancreatic ductal cells expressed Ha-Ras in the cytoplasm, with Ki-Ras in the apical region and N-Ras (50% of cases) in a supra-nuclear distribution. The acinar cell complex showed mild staining with pan-Ras, nuclear staining with Ki-Ras and supra-nuclear distribution of N-Ras (50% of cases). The cells of the Islets of Langerhans showed marked staining with pan-Ras, Ha- and Ki-Ras mAb but mild staining with N-Ras mAb. Fibroblasts showed staining with pan-Ras and Ki-Ras mAb, but no staining with Ha- or N-Ras mAb.

Conclusions: This work suggests that the Ras isoforms have distinct and separate cellular functions. Understanding these functions in detail is an essential step if Ras is to be targeted in the pancreas by gene therapy.

263. TISSUE TRANSGLUTAMINASE INCREASES THE BINDING OF A-GLIADIN 31–49, A COELIAC TOXIC EPITOPE, AND A-GLIADIN 51–70, TO HLA-DQ8

Introduction: Presentation of gliadin peptides in association with DQ2 or DQ8 to gluten specific small intestinal T-cells is thought to be one of the key pathogenetic mechanisms in coeliac disease (CD).Tissue transglutaminase (tTG) enhances binding of certain gliadin petides to DQ2 and DQ8 by deamidation of specific glutamine residues. A-gliadin 31–49 (31–49) has been shown by us to have in vivo andin vitro coeliac toxic activity, and to bind to DQ2 and DQ8.However there is recent evidence for one or two dominant DQ2 restricted epitopes within A-gliadin 56–75 with tTG deamidation of Q65 being important for T-cell recognition. Here we assess binding of 31–49 and A-gliadin 51–70 (51–70), containing the putautive dominant epitopes, to DQ8,with and without tTG.

Methods: Binding of biotinylated peptides was assessed with murine fibroblast transfectants expressing only human DQ8. Following incubation double staining with avidin D-FITC was undertaken before FACS analysis of live cells. Mean FL-1 flourescence, the measure of binding, was adjusted for class II surface expression using a monoclonal to DQ8. For tTG (guinea pig liver) assays, peptides were incubated with tTG at 37oC for 2 hours before addition to cells.

Results: The mean level of binding of 51–70 to DQ8 compared to 31–49 was 128.3%. tTG treatment enhanced the binding of both peptides, by a factor of approximately 3.5 for 51–70 and 1.5 for 31–49.

Discussion: It is not known whether DQ2 and DQ8, both strongly associated with CD, share a similar repetoire of gliadin T-cell epitopes. The binding motifs of these two molecules are similar in that both have a propensity for accepting negatively charged residues, which can be generated by the action of tTG. Here a peptide similar to peptides thought to be dominant CD epitopes binds to DQ8 with higher affinity than a peptide with in vivo CD activity (whether this is true specifically in DQ8+ patients is not known). tTG enhances this binding.It is suggested that 51–70 may be involved in disease pathogenesis in DQ8+ and DQ2+ CD. It is possible that these class II heterodimers share the same, possibly narrow, repetoire of gliadin epitopes.

264. DIFFERENTIAL EFFECTS OF TRANSFORMING GROWTH FACTOR (TGF) β ISOFORMS ON THE EXPRESSION OF TISSUE INHIBITOR OF METALLOPROTEINASE –1 (TIMP-1) AND MATRIX METALLOPROTEIANSE-3 (MMP-3) BY INTESTINAL MYOFIBROBLASTS (IMFs): IMPLICATIONS FOR STRICTURE FORMATION IN CROHN'S DISEASE

Background and aims: We have previously shown that, compared to normal and ulcerative colitis IMFs, Crohn's disease IMFs constitutively release more TGFβ-2 and less TGFβ-3. TIMP-1 and MMP-3 are expressed in mucosal tissue with active inflammatory bowel disease. In this study, we have investigated the effect of recombinant isoforms of TGF-β1, TGF-β2 and TGF-β3 on the expression of MMP-3 and its inhibitor TIMP-1, by primary human IMFs.

Methods: Monolayers of primary normal colonic IMFs (n=10) were cultured with rTGF-β1, rTGF-β2 or rTGF-β3 (5ng/ml) or control medium for 24 h. Concentrations of MMP-3 and TIMP-1 in supernatant samples were determined by ELISA. Results are expressed as median (range) of TIMP-1: MMP-3 ratios.

Results: Compared to control medium [14.8 (9.4—18.4)], rTGF-β1 [17.0 (14.7—22.9)] and rTGF-β2 [16.4 (11.4—18.7)] treated IMFs, the TIMP-1: MMP-3 ratios were significantly lower in rTGF-β3 [12.1 (6.9—15.2)] treated IMFs (p<0.046, p<0.005 & p<0.011, respectively). TIMP-1: MMP-3 ratios between control and rTGF-β1 treated cells did not reach statistical significance (p=0.059).

Conclusions: In contrast to rTGF-β1 and rTGF-β2, rTGF-β3 significantly reduced the expression of TIMP-1 relative to MMP-3 in IMFs. Greater activity of MMP-3, in the presence of TGF-β3, may inhibit excessive deposition of fibrous tissue by breaking down components of the extra cellular matrix. Thus, reduced constitutive expression of TGF-β3 by Crohn's disease IMFs may lead to excessive deposition of fibrous tissue and stricture formation.

265. COX2 EXPRESSION AND ACTIVITY IS DEPENDENT ON P38 STRESS ACTIVATED PROTEIN KINASE IN COLONIC EPITHELIAL CELLS

Introduction: COX2 is up-regulated in Inflammatory Bowel Disease (IBD) and in Colorectal Carcinoma (CRC). Indeed it has been proposed that the increased expression in IBD may be the reason that these patients are at increased risk of CRC. Therefore understanding the regulation of COX2 is essential in these conditions. The p38 stress activated protein kinase (SAPKinase) has been shown to play a role in the induction of COX2 in other systems although this has never been demonstrated in the colon.

Aim: To investigate the role of p38 in COX2 expression and activity in colonic epithelial cells.

Methods: COX2 expression and activity were assessed in the HT-29 colonic epithelial cell line. COX2 mRNA expression was assessed using northern analysis. Functional activity of COX2 was assessed by quantifying PGE2 production using an ELISA.

Results: TNFα (100ng/ml) and IL-1α (10ng/ml) were independently used to induce COX2 mRNA in HT-29 cells. For both cytokines this induction of COX2 was partially inhibited by pre-incubation with the specific p38 SAPKinase inhibitor SB203580 in a concentration dependent manner (0.3–30μM). To assess whether this effect was reflected on COX2 functional activity, TNFα induced PGE2 production was assessed at 72hours. In the presence of SB203580 (3 and 10μM) there was >90% inhibition of PGE2production, a more marked effect than that seen on COX2 mRNA.

Conclusions: This work demonstrates for the first time that specific p38 SAPKinase inhibition inhibits cytokine induced COX2 expression and activity in colonic epithelial cells. It also implies that activation of p38 by TNFα and IL-1α plays an important role in the induction of COX2 in the colonic epithelium. Therefore p38 inhibition may provide a useful therapeutic target to inhibit COX2 in the future.

266. CYCLOOXYGENASE-2 AND ANGIOGENESIS IN HUMAN SPORADIC COLORECTAL ADENOMAS

Background: We have previously reported that cyclooxygenase (COX)-2 is expressed predominantly by interstitial macrophages in human sporadic colorectal adenomas. In this study, superficial areas containing COX-2-positive macrophages were often noted to be very vascular. A putative role for COX-2 during intestinal tumorigenesis is via promotion of angiogenesis. Therefore, we investigated the association between microvessel density (MVD), COX-2 expression and several clinicopathological correlates in human sporadic colorectal adenomas.

Methods: CD31 immunohistochemistry (IHC) was performed on a series of human sporadic colorectal adenomas (n=37) for which we had previously determined COX-2 expression levels by IHC. The mean MVD from 3 high power field (x400) views of vascular “hotspots” in both superficial and deep interstitial areas was assessed by two independent observers blinded to adenoma origin and COX-2 expression level. MVD was correlated with adenoma characteristics, including COX-2 expression level (scored 0–3).

Results: Superficial MVD was increased in COX-2-positive adenomas (median [IQR] 35 [18–45]) compared with COX-2-negative adenomas (13 [7–30]; P=0.037, Mann-Whitney U test). There was a non-significant trend towards increasing superficial MVD with increasing COX-2 expression level (P=0.08, one way ANOVA). However, multiple linear regression analysis of clinicopathological data, including COX-2 level, demonstrated that adenoma size (P=0.007) was the only significant predictor of MVD. No relationship was evident between MVD and the COX-2 expression level in deep interstitial areas of adenomas.

Conclusion: COX-2 protein expression by superficial interstitial cells in human sporadic colorectal adenomas is associated with increased angiogenesis. However, in this series, adenoma size was a stronger predictor of increased MVD in superficial areas of adenomas. Promotion of angiogenesis may play an important role during early stages of intestinal tumorigenesis in humans.

267. HUMAN COLORECTAL CANCER IS ASSOCIATED WITH DOWN REGULATION OF THE Ca2+-ACTIVATED CHLORIDE CHANNELS CLCA1 and CLCA2

Background: The role of ion channels in carcinogenesis and tumour progression remains unclear. CLCA1 and 2 are members of a novel Ca2+-dependent chloride channel family that are expressed mainly in the digestive tract. Both share significant sequence identity with a dual-function cell adhesion/chloride channel molecule and CLCA2 is a tumour suppressor in breast cancer.

Methods: Suppression subtractive hybridisation, reverse northern dot blot analysis andin silico cloning resulted in the identification of mRNAs differentially expressed between paired normal colonic epithelium and carcinoma. One was CLCA1 and its transcription, as well as that of CLCA2, was quantitated by real-time RT-PCR analysis in a further 44 paired normal and carcinoma samples.

Results: CLCA1 and CLCA2 mRNA was detected in 44 and 38 paired samples, with no difference in mRNA copy numbers in nine and four paired samples, respectively. CLCA1 mRNA levels were significantly increased in one and decreased in 35 tumours, with median copy numbers reduced from 3.2x107 to 5.5x105 per μg RNA (p<0.00001, Mann Whitney U-test). CLCA2 was up regulated in two and down regulated in 32 tumours with its copy number reduced from 2.7x106 to 1.3x105 per μg RNA (p<0.001). Transcription of CLCA1, but not of CLCA2, was significantly associated with that of c-myc in normal tissue (R=0.82, p<0.0001, Spearman rank correlation), with deregulation observed in the tumour samples (R=0.36, p<0.015). Transcription of both mRNAs was virtually absent from three colorectal cancer cell lines, T84, HT29 and Caco2 (<1copy/cell).

Conclusion: Our results show that reduced CLCA1/2 transcription is associated with colorectal tumorigenesis and suggest that their gene products may function as tumour suppressors in colorectal cancer.

268. INVESTIGATION OF THE SUBCELLULAR LOCALIZATION OF THE GROWTH INHIBITORY MUSHROOM LECTIN AND ITS INTRACELLULAR BINDING LIGAND Grp170 BY ELECTRON MICROSCOPY IN HT29 HUMAN COLON CANCER CELLS

Increased cell surface expression of the Thomsen-Friedenreich antigen (TF, Galβ1–3GalNAcα-) is common in malignant and hyperplastic epithelia. Our previous studies have shown that a TF antigen-binding lectin from the common edible mushroomAgaricus bisporus (ABL) produces marked reversible inhibition of epithelial cell proliferation (Cancer Res1993;53:4627) and this effect is linked to its inhibition of nuclear localization sequence (NLS)-dependent nuclear protein import after internalization (J Biol Chem 1999;274:4890). Our more recent studies have revealed that glucose regulated protein 170 (Grp170), which we have shown expresses sialyl-TF antigen, is the major intracellular ABL ligand. The purpose of this study is to investigate the subcellular localization of ABL and Grp170 using electron microscopy, and to see whether either protein co-localizes with the known cytoplasmic factors required for NLS-dependent nuclear protein import.

HT29 human colon cancer cells were incubated with FITC-ABL, for 24h in serum-free DMEM. The cells were then fixed in 2.5% glutaraldehyde, dehydrated and embedded and left to harden in resin for 24h. FITC-ABL treated and untreated sections were immunolabelled for ABL and Grp170 for 1 hour and then incubated with a secondary 10nm gold for 1h. Examination by electron microscopy revealed significant amounts of both ABL and Grp170 in the cytoplasm after 24h. ER localization of Grp170 was also observed. Interestingly, both ABL and Grp170 were observed co-localised around the nuclear pores. Ran, one of the essential cytoplasm factors required in NLS-dependent nuclear protein import, was also observed in the vicinity of nuclear pores. These results suggest that Grp170, a highly divergent Hsp70-like protein, may be directly involved in NLS-dependent nuclear protein import. Further studies are needed to assess the functional importance of its glycosylation with sialylTF and to assess its potential as a target for cancer therapy.

269. ENDOTHELIN-3 BLOCKADE PROMOTES PREMATURE NEURONAL DIFFERENTIATION IN AN ORGAN CULTURE MODEL OF HIRSCHSPRUNG DISEASE

Aims: Pharmacological blockade of entothelin-3 (ET3) in an embryonic mouse gut culture system results in incomplete migration of enteric neural crest cells (NCC) along the gut and thus distal colonic aganglionosis. We aimed to determine the mechanisms controlling migration by analysing effects of endothelin blockade on NCC proliferation, differentiation, and apoptosis.

Methods: Gut was dissected from day 11.5 mouse embryos and transferred into culture with or without BQ788, a specific blocker for ET3's receptor (the ETB receptor). NCC proliferation and apoptosis were determined by BrdU incorporation and TUNEL reactions respectively after 24h culture. NCC differentiation was determined using nitric oxide synthase (NOS) immunoreactivity after 24–72h culture.

Results: ET3 blockade had no effect on either proliferation or apoptosis of enteric NCC. After 72h, numerous NOS-positive neurons were identified in gut cultured with BQ788, but not in controls, and this was associated with distal colonic aganglionosis.

Conclusions: ET3 prevents enteric NCC differentiation rather than influencing proliferation or apoptosis. Thus, in the absence of ET3, migrating enteric NCC differentiate prematurely into non-migratory neurons and distal aganglionosis (Hirschsprung disease) results.

270. EPIDERMAL GROWTH FACTOR, ITS RECEPTOR AND THE ROLE OF GANGLIOSIDES GM1 AND GM3

Endocytosis is a process whereby eukaryotic cells take up extracellular material by a variety of different mechanisms. These endocytic functions are involved in the regulation of cell surface receptor expression, maintenance of cell polarity, cholesterol homeostasis and a host of other physiological processes. In this investigation we look specifically at receptor mediated endocytosis (RME), a pathway used for the internalisation of ligand/receptor complexes, in this case epidermal growth factor (EGF) and its receptor (EGFr). Gangliosides are necessary for the efficient functioning of this process and this study looks at the relationship between EGFr and gangliosides GM1 and GM3 in four oesophageal cell lines, and the impact of gangliosides on RME. We have also quantified the amounts of EGFr and gangliosides in the cell lines.

Background: EGF binds tightly to its receptor, a 175 kd single transmembrane protein, on the cell surface. Once bound, activation of specific protein kinases takes place inducing a number of cellular responses. Many gangliosides have been identified on the basis of their carbohydrate structure that is mainly expressed on the outer surface of the plasma membrane. They play an essential role in a variety of cellular responses such as cell adhesion, signal transduction, cell growth and differentiation.

Methods: Four oesophageal cell lines were investigated in tissue culture, two squamous cell carcinoma (SCC) and two adenocarcinoma (AC) cell lines. Cells were incubated with fluorescent labels, harvested for FACScan® analyses or slide fixed for confocal microscopy.

Results: Co-localisation of EGFr and ganglioside on the cell membrane; cell lines with greater expression of EGFr also have greater amounts of ganglioside; increased levels of EGFr are present in SCC compared to that seen in AC; greater amounts of ganglioside are present in SCC than AC; GM3 inhibits the mode of action of EGF in SCC but not in AC.

Conclusion: These results provide us with information on the location of both EGFr and gangliosides on the cell membrane. Their co-localisation suggests that a co-operative relationship exists between them. This evidence indicates that gangliosides are directly involved in the signalling mechanism that is induced when EGF binds to EGFr, a tenet supported by the inhibitory aspect of GM3 on one effect of EGF.

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    BMJ Publishing Group Ltd and British Society of Gastroenterology