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336 A GENETIC ANALYSIS OF COELIAC DISEASE
S. Popat, N. Hearle, S. Bevan, G.K.T. Holmes, P.D. Howdle, L. Hogberg, C.P. Braegger, D. O'Donoghue, K. Falth-Magnusson, H. Jenkins, S. Johnston, N.P. Kennedy, P. Kumar, R.F.A. Logan, M.N. Marsh, C.J. Mulder, K. Sjoberg, L. Stenhammar, J.R.F. Walters, D.P. Jewell, R.S. Houlston. Section of Cancer Genetics, Institute of Cancer Research, Sutton, UK
Background and Aims: A genetic susceptibility to coeliac disease is well recognised. Although a strong association is seen between HLA DQ2 and coeliac disease, this does not entirely account for the observed familial risk. In order to assess the contribution of HLA to coeliac disease and to identify non-HLA linked coeliac disease susceptibility genes, 3 complimentary strategies were adopted.
Methods: (1) Allele sharing across HLA was calculated by non-parametric linkage analysis. (2) A genome-wide linkage search for non-HLA linked susceptibility loci was performed on 24 multiplex families using ∼240 microsatellite markers. (3) Analysis of candidate genes was undertaken by linkage, allelic association, and/or direct mutational analysis. Candidate loci tested were TGM2 (encoding tissue transglutaminase) and CTLA4-CD28 (on chromosome 2q33, implicated in a number of autoimmune diseases).
Results: (1) The HLA locus only accounts for ∼40% of the familial risk of coeliac disease. (2) In addition to linkage to HLA, there was evidence of linkage to chromosomes 19p13.3 (p=0.02) and 4p14 (p=0.03). No significant linkage was observed at candidate regions proposed in other reported linkage searches. (3) Mutational analysis of TGM2 did not show any disease causing mutations. Analysis of the CTLA4-CD28 gene region showed evidence for linkage (p=0.004) and association (p=0.039). Pooling these findings with published analyses through a meta-analysis showed significant evidence for linkage (p=0.0008) and association (p=0.0006). Mutational analysis of both CTLA4 and CD28 did not show any disease-causing mutations
Conclusions: Non-HLA gene(s) are likely to be a stronger determinant of disease susceptibility than HLA. Sequence variation in genes centromeric to CTLA4 confer an increased risk of coeliac disease, but are unlikely to account for all of the non-HLA linked inherited susceptibility.
337 ROLE OF TRANSCRIPTION FACTOR NF-κB IN CYCLOXYGENASE-2 PROMOTER INDUCTION BY HELICOBACTER PYLORI IN ENDOTHELIAL CELLS
P.A. Corcoran, D.J. Fitzgerald, K.M. Sheehan, J.C. Atherton, F.E. Murray, M.F. Byrne. Depts of Gastroenterology and Clinical Pharmacology, Beaumont Hospital/Royal College of Surgeons in Ireland, Dublin, Ireland
Several studies have demonstrated that H. pylori induces COX-2 in gastric mucosa. We have described regulation of the COX-2 promoter by H. pylori in epithelial and endothelial cell models but the promoter elements involved remain unknown. H. pylori induces the translocation of NF-κB. NF-κB mediates the induction of COX-2 in response to cytokines and free radicals. The aim of this study was to determine the role of two NF-κB sites on the COX-2 promoter in H. pylori induction.
A parent 5' flanking DNA fragment (-891/+9) and its NF-κB deletion mutants of the human COX-2 gene were constructed into a promoterless luciferase expression vector pGL3. A proximal NF-κB site mutant (-222 to -213), a distal NF-κB site mutant (-447 to -438), and a double NF-κB mutant were used. Transfected cells BPAEC (vascular endothelial cells) were incubated for 24 hours with live H. pylori (strain 60190, tox++, cagA+). Firefly and renilla luciferase activities were measured.
Mutation of either or both of the NF-κB sites on the COX-2 promoter at −222 and −447 base pairs from the transcriptional start site reduced basal COX-2 promoter activity (normalised luciferase activity 0.26±0.03 v 0.19±0.02, parent v double mutant construct, p<0.05). H. pylori induced COX-2 promoter activity with all constructs (normalised luciferase activity 0.53±0.09 v 0.26±0.03, parent construct with H. pylori v control medium, p<0.05) but this induction was unaffected by deletion of either or both of the NF-κB sites.
H. pylori induces COX-2 in vascular endothelial cells via gene induction. The resultant increased generation of endothelial cell prostacyclin may play a role in modulating mucosal blood flow, platelet function and inflammatory cell infiltration in response to H. pylori infection and may play a role in development of gastric cancers. However, this induction of COX-2 by H. pylori does not involve the NF-κB sites on the proximal portion of the promoter.
338 P53 MUTATIONS IN OESOPAHGEAL ADENOCARCINOMA ARE COMMON AND ARE ASSOCIATED WITH DISEASE RESPONSE
S.D. Oglesby, E. Warbrick, D.A. Johnston, J.F. Dillon, A.J. Munro, T.R. Hupp, A.M. Thompson. Department of Surgery & Molecular Oncology, University of Dundee, Dundee DD1 9SY, UK
Introduction: p53 is a key regulator of cellular response to radiation. Until recently, sequencing has been the gold standard for identifying inactivating mutations. However, sequencing can perform poorly when used to analyse material with a mixture of cell types such as tumour biopsies. The functional yeast assay (FYA) is a relatively new method of evaluating p53 mutations and outperforms sequencing when used on biopsy material. Duddy et al (J Mol Diagn 2000) found that sequencing p53 exons 5–8 identified only 54% of mutations detected by FYA. We sought to identify the frequency of mutations in adenocarcinoma of the oesophagus and to correlate mutational status with patient survival.
Methods: Tumour biopsies were obtained from 10 consenting patients with oesophageal adenocarcinoma who were to receive radiotherapy. The biopsies were assessed for p53 mutations using the FYA. Patient survival was compared with mutation data.
Results: 9 out of 10 tumours contained mutant p53. One patient has had a long post treatment survival (currently 23 months) the longest survivor of the remaining 9 patients was 17 months, median 9 months. The surviving patient was found to have no evidence of disease at endoscopy 22 months after treatment. Mutational analysis showed a His273 p53 mutation in this patients tumour.
Discussion: Although small, this cohort has a higher p53 mutation frequency than previously reported. The one long term survivor has a mutation which has been extensively studied in vitro. Restoration of functional activity of this mutant is possible through a variety of mechanisms including interaction with small peptides and antibodies. It is likely that similar mechanisms occur in vivo. Given that the presence of a mutation is very common, this factor alone is unlikely to predict response, indeed the precise nature of the mutation is likely to be more important. We have demonstrated this in our small cohort.
339 DOWNREGULATION OF α2β1 INTEGRIN PRECEDES THE INDUCTION OF APOPTOSIS BY BUTYRATE IN COLORECTAL CANCER CELL LINES
A. Buda, M.A. Jepson1, D. Martines2, M. Pignatelli (introduced by M. Pignatelli).
Department of Pathology and Microbiology, 1 Department of Biochemistry, University of Bristol, Bristol, UK; 2 Department of Surgical and Gastroenterological Sciences, University of Padua, Italy
Background: Integrins mediate cell-matrix adhesion and regulate growth and cell survival. In colonic epithelial cells α2β1 integrin controls glandular differentiation and proliferation. Butyrate stimulates differentiation and induces apoptosis in vitro.
Aim: We investigate whether butyrate induction of apoptosis was associated with perturbation of integrin-mediated cell matrix adhesion.
Methods: Four colonic cancer cell lines (SW 1222, HT29, SW620, LS174T) were studied. Adhesion to extracellular matrix proteins was investigated by a cell-matrix adhesion assay. Expression and cellular localization of α2β1 integrin were studied in adherent cells after treatment with 4 mmol/L butyrate by FACS analysis and confocal mcroscopy. Apoptosis was assessed by Annexin V binding. A selective COX-2 inhibitor (NS-398) was also used as a control.
Results: Butyrate decreased the attachment to type I collagen in HT-29 (p=0.004) and SW620 (p=0.003) cells and type I (p=0.01) and IV (p=0.03) collagen in LS174T cells. The decreased cell attachment was associated with downregulation of α2β1 and increase in apoptosis in adherent cells (SW620 9.6±0.5 vs. 3.5±0.2, p=0.000; HT29 6.1±0.4 vs. 2.3±0.1, p=0.01; LS174T 9.3±0.6 vs. 4.3±0.3, p=0.003). No changes in α2β1 expression and matrix adhesion were seen in SW1222 cells, which were also found less sensitive to the butyrate-induction of apoptosis (2.4±0.2 vs. 3.3±0.4 p=0.123). Treatment with NS-398 increased apoptosis (5.9±0.08 vs. 2.06±0.14, p=0.000) without affecting integrin expression and cellular localization. Downregulation of α2β1 integrin occurred in viable cells and preceded the detection of apoptosis.
Conclusion: Cell detachment and apoptosis induced by butyrate are associated with downregulation of expression and functional activity of α2β1 integrin. Perturbation of cell matrix adhesion may be a novel mechanism by which butyrate induces apoptosis in colorectal cancer cells.
340 HIGH LEVELS OF MICROSATELLITE INSTABILITY (MSI-H) IN HYPERPLASTIC POLYPOSIS (HPP) ASSOCIATED COLORECTAL CANCER (CRC) PROVIDES EVIDENCE FOR THE SERRATED CRC PATHWAY
M. Lockett, W. Smale, J. Lloyd, K. Pack, S. Burke, I. Talbot, I. Tomlinson, W. Atkin.
ICRF CRC Unit, St. Mark's Hospital, Harrow, Middlesex, UK
Background: MSI-H occurs in 10–15% sporadic CRC. The precursor is unknown but may be the hyperplastic polyp (HP) (serrated CRC pathway). In HPP there are multiple, large HPs and an increased CRC risk. If CRC in HPP showed MSI and the MSI-H phenotype (proximal, multiple, CRCs in females with mucinous, undifferentiated histology and a prominent lymphocyte infiltrate), this would support the serrated CRC pathway.
Aim: To describe the phenotype and microsatellite (MS) status of CRC in HPP to provide evidence for the serrated CRC pathway.
Methods: HPP patients were identified after a national call for cases and using the UK flexible sigmoidoscopy screening trial database. Clinical and histological features were described. Paraffin-embedded tissue samples were microdissected and DNA was extracted. MSI analysis was performed by PCR at Bat25, Bat 26, MycL1, D2S123, APC, D15S221 and D17S250 and compared to genomic or normal tissue DNA. MS status was defined as follows: MSI-H ≥3 unstable (≥1 mononucleotide marker); MSI-Low (MSI-L) 1–2 unstable; MS stable (MSS) 0 unstable. Tissue samples were analysed by immunohistochemistry for mismatch repair proteinshMLH1, hMSH2 and hMSH6.
Results: 42 HPP patients were identified. 32 CRCs occurred in 18 patients (multiple CRCs in 6). 72% of the 18 CRC patients were male (median age 63 years male, 52 years female). None had a family history of CRC. 69% of CRCs were proximal to the splenic flexure, differentiation was poor in 31%, moderate in 47% and well in 9%. 22% showed mucinous or signet ring cell histology. 19% had a Crohn's-like or conspicuous lymphocyte infiltrate. 6/22 (27%) CRCs were MSI-H and were proximal and showed hMLH1 loss. 5 (23%) were MSI-L and 50% MSS.
Conclusions: Both MSI and chromosomal instability pathways are important in HPP carcinogenesis. There is an increased prevalence of MSI CRC in HPP. This suggests that the HP is the precursor of MSI CRC in HPP. This supports the theory that the HP may also be the precursor of sporadic MSI-H CRC.
341 PROGASTRIN STIMULATES MURINE COLONIC EPITHELIAL MITOSIS AFTER DNA DAMAGE
P.D. Ottewell1, A.J.M. Watson1, T.C. Wang2, G.J. Dockray3, D.M. Pritchard1.
Departments of 1 Medicine and 3 Physiology, University of Liverpool, UK; 2 University of Massachusetts Medical Center, Worcester, MA, USA
Background and Aims: The non-amidated precursors of gastrin stimulate proliferation of colonic epithelial cells. Transgenic mice which overexpress human progastrin (hGAS) are more susceptible to the induction of colonic aberrant crypt foci and adenomas by the chemical carcinogen azoxymethane than wild type mice (FVB) and mice which overexpress amidated gastrin (INS-GAS). We have investigated in murine intestinal epithelium in vivo whether alterations in the regulation of apoptosis or mitosis following DNA damage contribute to the procarcinogenic effects of progastrin.
Methods: Apoptosis and mitosis were assessed on a cell positional basis by light microscopic assessment of small intestinal and colonic crypts from 10–12 week mice, following 1 or 8 Gy γ-irradiation. Mice analysed were progastrin overexspressing (hGAS), gastrin overexpressing (INS-GAS) and gastrin-knockout (GAS-KO) along with their wild-type counterparts (FVB and C57BL/6). p21WAF1/CIP1 expression was analysed by immunohistochemistry and Western blotting using a rabbit polyclonal primary antibody.
Results: 4.5h following 1Gy and 8Gy γ-radiation, apoptosis was induced to similar levels in the small intestinal and colonic crypts of all mice. Colonic mitosis was inhibited to almost undetectable levels by 8Gy γ-radiation in wild-type, GAS-KO and INS-GAS mice. However, significant colonic mitosis persisted in hGAS mice 4.5h following 8Gy γ-radiation. hGAS mice also showed increased length of colonic crypts compared to FVB and INS-GAS. γ-irradiation caused induction of p21WAF1/CIP1 to similar levels in hGAS and FVB colonic epithelium.
Conclusions: (1) 4.5h after DNA damage by 8Gy γ-radiation, mice with elevated progastrin exhibit significantly higher levels of colonic mitosis than wild-type or gastrin overexpressing mice. (2) Progastrin induced stimulation of mitosis following DNA damage may account for its procarcinogenic properties.
342 NSAIDS INHIBIT β-OXIDATION OF PALMITATE AND ARACHIDONATE IN HCT-116 CELLS
M.J. Garle1, B. Middleton1, C.J. Hawkey 2.1School of Biomedical Sciences,2Division of Gastroenterology, University Hospital Nottingham, UK
Introduction: Non-steroidal anti-inflammatory drugs (sulindac derivatives in particular) are useful for prevention of colon cancer. Earlier reports describe inhibition of palmitate β-oxidation by NSAIDS and demonstrate that elevated cellular arachidonate levels cause apoptosis1.
Methods: We hypothesised that inhibition of β-oxidation of arachidonate would lead to arachidonate accumulation and possibly cell death. So we determined effects of NSAIDS on palmitate and arachidonate β-oxidation and cell viability. HCT-116 cells were grown to confluence on 24-well cluster plates and were incubated over 2 hours in DPBS with 3H-palmitate or 3H-arachidonate (100μM, at 42 and 48 dpm/pmole respectively) in the presence of defatted BSA (1.2mg/ml). β-Oxidation rates were measured by 3H-water production. NSAIDS including indomethacin, sulindac sulphoxide, sulindac sulphide and ibuprofen (0–1000μM) and a palmitoyl carnitine transferase-1 inhibitor (etomoxir 10μM) were incubated in DPBS containing defatted albumin for 45 min prior to 2hr incubation of chemicals with 3H-palmitate or 3H-arachidonate. Effects of NSAIDS on cell death was determined over 48 hr in serum free DMEM, using resazurin reduction as a viability marker. Rates of 3H-palmitate or 3H-arachidonate β-oxidation were 152 +/- 5.0 and 21 +/- 2.0 pmoles/min/mg protein respectively. Both activities were reduced by etomoxir (10μM) to less than 10% of control values.
Results and Conclusions: Effects of NSAIDS on β-oxidation and cell viability are shown in the table below. Ibuprofen caused an unexpected 2-fold stimulation of 3H-arachidonate β-oxidation at concentrations up to 500mM. Results represent mean +/- sem of IC50 (μM), (n=4). See table.
1.Chan TA. et al (1998) PNAS (USA);95:681–686
343 A ROLE FOR VON WILLEBRAND FACTOR IN HELICOBACTER PYLORI-INDUCED PLATELET AGGREGATION
P.A. Corcoran, S. Kerrigan, D. Cox, J.C. Atherton, D.J. Fitzgerald, F.E. Murray, M.F. Byrne. Depts of Clinical Pharmacology/Gastroenterology, Beaumont Hospital/RCSI, Dublin, Ireland
Human and animal data have demonstrated platelet aggregates in gastric vasculature in association with H. pylori infection. We have previously shown that certain strains of H. pylori induce platelet aggregation via the platelet glycoprotein GPIb. The GPIb receptor mediates adhesion to von Willebrand factor under high shear. We sought to further characterise this interaction and investigate the role of plasma factors such as von Willebrand factor.
An aggregating and non-aggregating strain of H. pylori (60190 and J104) were grown under standard conditions. 50μl of bacterial suspension (4x109 CFU/ml) were added to 50μl of polyclonal anti-vWF antibody or a monoclonal anti-vWF antibody and incubated for 30 minutes. The bacteria were then centrifuged and the pellet re-suspended in PBS and FITC-labelled anti-mouse secondary antibodies for 30 minutes at 37°C. Stained bacteria were analysed on a FACSCalibur flow cytometer. H. pylori strain 60190 bound anti-vWF antibody (mean fluorescent intensity: polyclonal anti-vWF 324±75 and 11±4 for control; monoclonal anti-vWF 52±4 and 10±1 for control). However, the non-aggregating strain J104 showed reduced binding of the polyclonal anti-vWF antibody (strain J104: Geometric mean 62±19; strain 60190: Geometric mean 223±23, p<0.005, n=3, t-test). In addition, H. pylori 60190 failed to induce aggregation of washed platelets (5.5±3.9% aggregation, n=3). However, H. pylori pre-incubated in platelet poor plasma induced platelet aggregation (28±6.4% aggregation, n=3). The polyclonal anti-vWF antibody inhibited this platelet aggregation (95±2% inhibition, n=3). Plasma immunoglobulin plays a role in this interaction.
H. pylori induces platelet aggregation in a strain specific manner and plasma factors, notably von Willebrand factor, are involved in the interaction with platelet GPIb. Induction of platelet aggregation by H. pylori may be a critical early event in the inflammatory cascade in gastrointestinal pathogenesis and may also explain the putative link with cardiovascular events.
344 EXPRESSION OF THE HUMAN INTESTINAL EPITHELIAL CALCIUM TRANSPORTER ECaC2/CaT1
N.F. Barley, J.O. Hayat, A. Bhattacharjee, R. Jeffrey, R. Poulsom, J.R.F. Walters. Gastroenterology Section, Imperial College, Hammersmith Campus and ICRF, London, UK
The intestinal absorption of dietary calcium is essential for the development and maintenance of bone mineralisation and the prevention of osteoporosis. Understanding of the molecular mechanisms of calcium absorption by intestine and kidney has been strengthened with the recent identification of the apical membrane calcium channels, ECaC1 (also known as CaT2) and ECaC2 (CaT1). We previously showed that there is considerable individual variability in the duodenal expression of ECaC2/CaT1 transcripts in humans and have now performed further studies to attempt to identify the mechanisms responsible for this.
Specific riboprobes were synthesised from non-conserved regions in the 3′-UTR of the human ECAC1 and ECAC2 genes and used for Northern blotting and in situ hybridisation. Only ECaC2/CaT1 was detectable in duodenum, although ECaC1/CaT2 was found in kidney. The presence of ECaC2/CaT1 transcripts were also confirmed in placenta and prostate.
Sequence differences in ECaC2/CaT1 mRNA have been reported at a number of sites and were further investigated. Two sites were confirmed to be single nucleotide polymorphisms; one of which resulted in a coding change. The different allelic forms were relatively common and were all expressed in duodenum. No relationship was found between these haplotypes and the level of RNA expression.
To be able to investigate the control of gene expression, we determined the nucleotide sequence of 939bp of the ECAC2 gene upstream from the translation initiation site, comprising the 5'-UTR and the promoter. No classical TATA-box is present, but a number of other potential transcription factor binding sites can be identified, some of which are conserved in the mouse gene. A consensus vitamin D-response element in the mouse gene is deleted in the human sequence.
These studies into the mechanisms governing expression of ECAC2 gene-products in human duodenum will enable progress in the understanding of the molecular factors affecting calcium absorption.
345 APOPTOTIC PATHWAYS OF DETACHMENT INDUCED CELL DEATH IN ISOLATED HUMAN ENTEROCYTES
I. Daniels, R.G. Long. Medical Research Centre, City Hospital, Hucknall Road, Nottingham NG5 1PB, UK
Objectives: the primary culture of intestinal enterocytes has been an elusive goal for many years. The reasons that enterocytes fail to survive in culture are complex, but the single most important factor is that cells die from detachment-induced cell death (DICD) when stripped from the underlying lamina propria. Understanding the mechanisms that result in this form of apoptosis may provide insights into the long-term primary culture of human enterocytes.
Methods: Human duodenal biopsies were isolated from consenting patients presenting with irritable bowel syndrome and iron deficient anaemia. All biopsies showed normal histology. Whole biopsies were incubated for 4 hours at 4oC in cell disassociation fluid (Matrisperse), after which time they were shaken vigorously to free the enterocytes from the lamina propria. Both the enterocyte fraction and the lamina propria were washed and assessed for purity using Western blotting. Fractions were incubated at 37oC in serum free media for various times. Apoptosis was assessed by assaying surface expression of phosphatidylserine (PS) using flow cytometery and by DNA laddering. Activation of the apoptotic proteins poly(ADP-ribose) polyimerase (PARP), BH3 interacting domain death agonist (BID), caspase 3, caspase 8 and caspase 9 were assayed by Western blotting.
Results: Matrisperse treatment of human duodenal biopsies produces a pure, non-apoptotic population of enterocytes. Isolated enterocytes undergo rapid DICD as assessed by surface expression of PS and DNA laddering. DICD is independent of de-novo protein synthesis, is evident within 30 mins of detachment and is accompanied by the activation of caspase 3, caspase 8 and caspase 9. In support of these observations cleavage products of the geneomic surveillance protein PARP and the caspase 8 substrate BID were detected. DICD can be delayed but not halted by the addition of the broad spectrum caspase inhibitor (Boc-D-FMK) and the selective caspase 8 inhibitor ( Z-IETD-FMK).
346 THE IMPACT OF ALGINATE BIOPOLYMERS AND EPIDERMAL GROWTH FACTOR ON FLUID PHASE ENDOCYTOSIS
P. McPherson1, P.W. Dettmar2, P.E. Ross1. 1Gastroenterology Research Laboratory, Ninewells Hospital and Medical School, Dundee, DD1 9SY;2Reckitt Benckiser Healthcare (UK) Ltd, Dansom Lane, Hull, UK
Background: There has been a dramatic rise in oesophageal disease over the past thirty years. Gastroesophageal reflux disease (GORD) is a debilitating condition that may lead to Barretts' Oesophagus, a precurser to oesophageal adenocarcinoma. Acid suppression therapy has been the preferred method of treatment in the shape of H2 antagonists and proton pump inhibitors (PPI).
There is evidence to suggest that epidermal growth factor (EGF), and its receptor (EGFr), play an important role in tissue repair, cell proliferation and migration. Alginate biopolymers are widely used in the treatment of gastroesophageal reflux disease (GORD) and impart cytoprotective biological effects. In this study we have investigated the impact on fluid phase endocytosis (FPE) of alginates and EGF.
We have used four oesophageal cell lines, 2 squamous cell carcinomas and 2 adenocarcinomas. Cells were incubated with fluorescent microspheres or EGF or alginate or combinations. Incubation time was one hour. Analyses was carried out by flow cytometry and confocal microscopy.
Results: All alginates upregulate FPE significantly. EGF upregulates FPE. EGF and alginate added together upregulate FPE. Alginate H120L has the greatest impact on FPE. All batches of H120L have a similar impact on FPE.
Conclusions: The results presented here indicate that we are gradually unravelling the properties of alginate biopolymers and how these properties impact on FPE. This in turn may lead to improvemnets in medications aimed at the treatment of oesophageal disease.
347 GLYCINE-EXTENDED GASTRIN CAN PROMOTE AN INCREASE IN PRO AND ACTIVE MMP-2 EXPRESSION AT THE PROTEIN LEVEL IN CELLS
R. Asher-Dean, S.A. Evans, D.F. McWilliams, S.A. Watson. Cancer Studies Unit, Dept of Surgery, QMC, Nottingham NG7 2UH, UK
Background and Aims: Over expression of various matrix metalloproteinases (MMPs) has previously been speculated to correlate with tumour progression in a variety of cancers. The aim of this study was to investigate whether GlyG17, a hormone known to be significantly expressed in colorectal cancers, has any effect on the expression of MMP-2 and -9 in a mouse fibroblast cell line transfected with two forms of the gastrin receptor CCK-2.
Methods: Gelatin zymography and real-time PCR were used to investigate the protein and gene expression of MMP-2 and -9 in four cell lines with or without the stimulation of GlyG17 (10–7M) and or the addition of 3 different CCK-2 receptor antagonists (YM022, JB95008 and JMV1155). The cell lines investigated were the human fibrosarcoma HT1080 transfected with MT1-MMP, and the mouse fibroblast NIH 3T3 transfected with classical CCK-2 or truncated CCK-2 receptors. Matrigel invasion chambers were used to assess the invasiveness of the cells with G17-Gly stimulation.
Results: The human fibrosarcoma cell line transfected with MT1-MMP produced a significant increase in pro and active MMP-2 (p<0.05) and proMMP-9 (p<0.05) at the protein level, following stimulation by GlyG17. Only the truncated CCK-2 receptor NIH 3T3 transfectants had a significant increase (p<0.05) in proMMP-2. None of the cell lines tested had a significant change in gene expression for the MMPs tested. JB95008 was the only CCK-2 receptor antagonist that significantly reduced GlyG17 increased MMP-2 expression (p<0.05). With the stimulation of GlyG17, only the HT1080 MT1-MMP transfectants had a significant (p<0.05) increase in their matrigel invasion.
Conclusion: The stimulation of cells by GlyG17 has been shown to be cell line specific but not receptor specific. GlyG17 has the ability to increase protein levels of pro and active MMP-2 and proMMP-9. GlyG17 increases the invasiveness of cells that already express active MMP-2 but has no effect on cells that express only the pro form. The blocking of the CCK-2 receptor reduces GlyG17 stimulation of MMP-2 expression.
348 A STUDY INTO THE RELATIONSHIP BETWEEN EPIDERMAL GROWTH FACTOR, ITS RECEPTOR AND GANGLIOSIDES GM1 AND GM3
P. McPherson1, G. Scobie2, P.W. Dettmar2, P.E. Ross1.1Gastroenterology Research Laboratory, Ninewells Hospital and Medical School, Dundee, DD1 9SY;2Reckitt Benckiser Healthcare (UK) Ltd, Dansom Lane, Hull, UK
Introduction: Endocytosis is a process whereby mammalian cells take up extracellular material by a variety of different mechanisms. In this investigation we look specifically at receptor mediated endocytosis (RME). Gangliosides are necessary for the efficient functioning of this process. We look at the relationship between EGFr and gangliosides GM1 and GM3 in four oesophageal cell lines, and the impact of gangliosides on RME.
Background: EGF is a 6 kd polypeptide that has a role in tissue repair, cell proliferation, ulcer healing and cell migration. Many gangliosides have been identified on the basis of their carbohydrate structure that is mainly expressed on the outer surface of the plasma membrane. Gangliosides act like receptors for some viruses, bacteria and bacterial toxins allowing passage into the cell.
Methods: Four oesophageal cell lines were used in this study, two squamous cell carcinomas and two adenocarcinomas cell lines. Cells were incubated with fluorescent labels, harvested for FACScan™ analyses or slide fixed for confocal microscopy. Immunogold technique was employed to analyse the plasma membrane location of EGFr and GM1 and GM3 by electron microscopy.
Results: Confocal analyses indicates colocalisation of EGFr and ganglioside on the cell membrane. FACScan® analyses indicates cell lines with greater expression of EGFr also have greater amounts of ganglioside. Immunogold electron microscopy images indicate colocalisation of EGFr and ganglioside. Increased levels of EGFr are present in squamous cell carcinoma cell line than that of adenocarcinomas. Greater amounts of ganglioside are present in squamous cell carcinoma cell line than that of adenocarcinomas. GM3 inhibits the mode of action of EGF in squamous cell carcinoma cell lines but not in adenocarcinoma cell lines.
Conclusion: These results provide us with information on the location of both EGFr and gangliosides on the cell membrane. Their location suggests that a co-operative relationship exist between them. This evidence indicates that gangliosides are directly involved in the signalling mechanism that is induced when EGF binds to EGFr, and that GM3 acts as a monitor for the binding of EGF to EGFr.
349 VARIABLE EXPRESSION OF SEP70, A NOVEL SQUAMOUS EPITHELIAL STRESS PROTEIN, IN BARRETT'S METAPLASIA
H.H. Dalziel, J.P. Cotton, T.R. Hupp, J.F. Dillon. GI Research Laboratories, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK
Introduction: The human oesophageal squamous epithelium is exposed to environmental extremes of heat and low extracellular pH. These stressors are thought to be predisposing factors for metaplastic change to Barrett's epithelium, a precursor of oesophageal adenocarcinoma. In many human and animal epithelial cells such stressors activate classical heat shock protein responses. This laboratory has previously demonstrated, in human oesophageal squamous epithelium ex-vivo, that HSP70 is down-regulated and that SEP70 is up-regulated in response to thermal, ethanol, low pH and glycopaenic stress. Further study of this novel observation will help to elucidate the involvement of molecular chaperones in the control of mechanisms such as p53 pathways and their role in the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. The present study investigates expression of SEP70 and HSP70 in paired normal and Barrett's tissue.
Methods: Pinch biopsies of normal squamous and Barrett's metaplastic epithelium were obtained from patients undergoing upper GI endoscopy for investigation of reflux symptoms. Western blot analysis, using mouse monoclonal antibodies to HSP70 and SEP70, was performed on tissue lysates prepared using urea or detergent lysis buffers.
Results: Identification of tissue as squamous cells or Barrett's was confirmed by detection of Barrett's specific hAG-2 protein. HSP70 was present in squamous epithelium. It was, however, variably expressed in Barrett's samples. SEP70 was expressed in normal squamous epithelium but in Barrett's tissue urea lysates demonstrated a laddering of SEP70 immunoreactivity to different molecular weights.
Conclusion: The laddering of SEP70 immunoreactivity may be due to detection of sub-cytoplasmic pools. This could reflect selective targeting for ubiquitinisation as part of the control of heat shock protein responses in this tissue. Alternatively, it may be due to a generalised dysregulation of proteosomal degradation pathways in Barrett's metaplasia. Further study of this phenomenon will clarify the dynamics of the novel heat shock protein response in this tissue.
350 SODIUM BUTYRATE DOWNREGULATES IGF-BINDING PROTEIN-3 EXPRESSION IN THE ABSENCE OF DE NOVO PROTEIN SYNTHESIS
N.R.J. White, P. Mulligan, I.R. Sanderson. Adult and Paediatric Gastroenterology, St Bartholomew's Hospital, London EC1A 7BE, UK
Butyrate, the pleitropic by-product of bacterial fermentation, has known effects on histone acetylation. The upregulation of genes by alteration of nucleosome-DNA interactions via the inhibition of histone deacetylases is a well understood mechanism by which butyrate directly upregulates gene expression. IGFBP-3 is constitutively secreted by intestinal epithelial cells and its transcription is downregulated by butyrate. The aim of this study was to determine whether the inhibition of IGFBP-3 by butyrate was due to an upregulation of an inhibitor of IGFBP-3 transcription or whether it was due to a direct effect at the IGFBP-3 gene.
Methods: Caco-2 cell lines were cultured in complete medium (10%FCS) and resuspended in serum-free media 24 hours prior to the addition of 5mM butyrate in the presence or absence of cycloheximide (CHX) for 6, 12, 24, 36 and 48 hours. IGFBP-2 and -3 mRNA transcripts were analysed by Southern blotting of semi-quantitative RT-PCR amplicons, IGFBP3 protein was assessed by Western blotting.
Results: Incubation of Caco-2 cells with butyrate resulted in decreased secretion and mRNA expression of IGFBP-3. To examine if the inhibitory effect of butyrate on IGFBP3 was dependent on de novo protein synthesis, Caco-2 cells were stimulated with butyrate in the presence and absence of cycloheximide (CHX). At doses of up to 10μM, CHX did not affect the butyrate-induced down-regulation of IGFBP-3. Butyrate caused an up-regulation of IGFBP-2 mRNA and this effect was again not altered by the protein synthesis-blocking effects of CHX. We verified that 10μM CHX inhibited protein synthesis by interrupting IGFBP-3 expression.
Conclusion: Our data indicate that the modulatory effects of butyrate on IGFBP-2 and -3 are independent of de novo protein synthesis. Therefore, butyrate's effects are not through the synthesis of a repressor of IGFBP-3. This suggests that butyrate has direct inhibitory effects which may involve the modification of the acetylation status of proteins in the nucleus.
351 ALGINATES ENHANCE THE EARLY RESPONSES OF MUCOSAL REPAIR BY STIMULATING MIGRATION IN VITRO
E.M. Dunne1, R. Del Buono1, P.W. Dettmar2, I.G. Jolliffe2, M. Pignatelli1.1Department of Pathology and Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD;2Reckitt Benckiser Healthcare, Dansom Lane, Hull, UK
Background: Alginates are a group of naturally occurring polysaccharides found in seaweed. Alginates are composed of blocks of two uronic acids, β- mannuronic acid (M) and 1–4 linked α-L guluronic acid (G) the proportion and distribution of which determines the molecule's chemical and physical properties. Previous in vivo experiments have shown that one M- rich alginate H120L protects the stomach of rats against ulceration while a G- rich alginate, LFR5/60 does not. It is thought that the trend in G- and M- make up of the alginates may be important in influencing this protective activity.
Aim: The ability of alginates to enhance early responses in mucosal repair using an in vitro model of cellular migration.
Methods: Three alginates, H120L (low fraction G-residues), LFR5/60 (high fraction G-residues) and Poly M (95% M-residues) were tested for their ability to stimulate migration of two human oesophageal and gastric cell lines. Epidermal Growth Factor (EGF) and Bovine Serum Albumin (BSA) were used as controls.
Results: All cell lines migrated in response to H120L and EGF. Poly M stimulated a weaker migratory response than H120L, no migration occurred in response to LFR5/60.
Conclusion: H120L caused stronger migration in all cell lines compared to LFR5/60 suggesting that M monomers are important in inducing migration in gastrointestinal cell lines. G blocks may also play a role as Poly M (only M blocks) causes migration but to a much lesser degree than H120L. Thus the composition of alginates may influence the migratory response and aid in the restitution process.
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