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Neoplasia and cell/molecular biology free papers 124–139

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124 BONE MARROW DERIVATION OF PERICRYPTAL MYOFIBROBLASTS IN THE MOUSE AND HUMAN SMALL INTESTINE AND COLON

S.J. Forbes1,2, T. Hunt1, M. Brittan1, R. Jeffery1, R. Poulsom1, K. Hodivala-Dilke1, M.R. Alison1,3 , N.A. Wright1,4. 1Histopathology Unit, Imperial Cancer Research Fund, London; 2Department of Medicine, and 3Department of Histopathology, Imperial College School of Medicine; 4Department of Histopathology, Barts and the London, Queen Mary's School of Medicine and Dentistry, London, UK

Background: The intestinal sub-epithelial myofibroblasts (ISEMF) are found in the lamina propria of the intestine under the epithelial cells, and are of critical importance in epithelial-mesenchymal interactions. It has been suggested that the origin of ISEMF might be from the neural crest, or locally from mesenchymal stem cells sited in the muscularis mucosae.

Aims/Methods: In order to establish whether extra-intestinal cells contribute to the turnover and repair of gastrointestinal tissues we studied: (i) the colons and small intestines of female mice that had received whole body irradiation followed by a male bone marrow transplant, (ii) gastrointestinal biopsies from female patients that had received a male bone marrow transplant and then developed graft versus host disease. In situ hybridisation for the Y-chromosomes was combined with immunohistochemistry to define the phenotype of these cells of donor (bone marrow) origin.

Results: In female mouse recipients of male bone marrow grafts we observed clusters of Y-chromosomes positive/ alpha-smooth muscle actin positive myofibroblasts. While few of these were present at 7 days after bone marrow transplantation, they were numerous at 14 days and by 6 weeks, whole columns of pericryptal myofibroblasts could be seen surrounding crypts in both the small intestine and colon. These columns appeared to extend into the villi in the small intestine. In the human intestine we confirmed that the bone marrow-derived cells within the intestine exhibited a myofibroblast phenotype.

Conclusion: Our data suggests that the bone marrow contributes to the regeneration of intestinal myofibroblasts after damage. This axis of gut regeneration may have therapeutic potential.

125 PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE REGULATES THE HISTIDINE DECARBOXYLASE PROMOTER VIA DUAL SIGNALLING MECHANISMS AND A DISTINCT RESPONSE ELEMENT

J.T. McLaughlin1, N.F. Sinclair, R. Collucci, R. Raychowdhury, T.C. Wang, T.J. Koh (introduced by Q Aziz1). 1GI Sciences, Hope Hospital, Salford; Division of Digestive Diseases and Nutrition, University of Massachusetts Medical School, Worcester MA, USA

Background: The gastric enterochromaffin-like cell has recently been shown to be under the influence of neural pituitary adenylate cyclase-activating polypeptide (PACAP). Its histidine decarboxylase gene promoter is known to be regulated by gastrin, and we have explored the effects of PACAP on this regulatory sequence and the signalling pathways responsible.

Methods: 1.8kB of the histidine decarboxylase promoter was ligated into a luciferase promoter-reporter construct and transiently transfected into PC12 cells, either wild type or stable transfectants expressing the gastrin-CCKB receptor. The effects of PACAP and gastrin on promoter activity were assessed via a series of 5' deletions and other appropriately derived constructs. Classical signalling pathway inhibitors were also used (H7 to block PKC, H89 to block PKA).

Results: Promoter activitation by PACAP was found to be dose-dependent and temporally biphasic, with an initial peak occurring at 4–6 hours (5-fold over basal, p<0.01), falling to a trough at 16 hours, and then rising to a secondary peak at 24 hours (4-fold over basal, p<0.01). The initial peak was shown by inhibitor studies to depend upon activation of both adenyl cyclase and phospholipase C-dependent pathways, while the second peak was entirely PKA-dependent. Deletional analysis, block mutation and electrophoretic mobility shift assays demonstrated a PACAP-response element (PRE) at –177 to –170 (CCTGGAGG), wholly necessary for the effects of PACAP but not gastrin. Gastrin also exhibited key differences in the timing (small peak at 6 hours, major peak at 16 hours) and magnitude (maximally 3.5-fold) of its effects.

Conclusions: The data show that discrete neural and endocrine pathways impinging on histaminergic cells can exhibit different patterns of post-receptor signalling and promoter element activation. As previously described, the PACAP receptor is able to activate both PKA- and PKC-dependent pathways. A newly characterised PRE in the promoter is pivotal in PACAP's effect on the HDC promoter, and quite distinct from the gastrin response-elements which have been localised to the +1-+48 region of the first nontranslated exon.

JMc supported by MRC Travelling Fellowship and DDF Senior Fellowship.

126 IDENTIFICATION BY GENE ARRAY OF PLASMINOGEN ACTIVATOR INHIBITOR-2 (PAI-2) AS A NOVEL TARGET OF GASTRIN IN HYPERGASTRINAEMIA

A. Varro, E. Hemers, D. Archer, A. Pagliocca, C. Haigh, S. Ahmed, R. Dimaline, G.J. Dockray. Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, UK

Background and Aim: Gastrin controls acid secretion and the organisation of the gastric mucosa. Some gastrin-regulated events involve changes in gene expression. We sought to identify major, new, gastrin-regulated genes using a gene array.

Methods: A cancer gene array was probed with samples from AGS-cells expressing the gastrin-CCKB receptor stimulated with gastrin. The expression of gastrin-regulated genes was characterised by Western blot and ELISA in tissue and blood of hypergastrinaemic patients. Gene expression was studied using promoter-luciferase reporter constructs.

Results: The gene array revealed PAI-2 as a major, previously unknown, gastrin-regulated gene in AGS cells. The relevance was confirmed by showing significantly (p<0.05, t test) elevated PAI-2 in plasma of hypergastrinemic patients (pernicious anaemia (PA): plasma gastrin, 1.20±0.16 nM, plasma PAI-2, 1.4±0.3ng/ml, n = 9; multiple endocrine neoplasia type 1 (MEN-1), gastrin, 0.53±0.09 nM, PAI-2, 2.0± 0.2 ng/ml, n=4; controls: gastrin, 0.03±0.01nM; PAI-2, 0.6±0.2ng/ml, n=9). Moreover, PAI-2 was detected as a strong band in Western blots of gastric biopsies of 8 of 9 PA patients, 5 of 7 MEN-1 patients, but was at or below the limit of detection in 9 of 10 controls. To examine cellular control mechanisms we transiently transfected AGS cells with 2.34kb of the PAI-2 promoter in a luciferase reporter construct; gastrin (1nM) increased expression 14.0±1.3 fold over control (p<0.05). Pharmacological agents and dominant negative vectors indicated that responses were mediated partly via protein kinase C, RhoA, and the transcription factors CREB and AP1. Over-expression of the tumour suppressor menin (which is mutated in MEN-1) significantly inhibited gastrin-stimulated PAI-2 expression (p<0.05).

Conclusions: PAI-2 is a novel, gastrin-regulated gene. PAI-2 is thought to inhibit apoptosis and extracellular proteolysis, so the data suggest a novel potential mediator of gastrin-stimulated changges in epithelial organisation.

127 PARACRINE COX-2-MEDIATED SIGNALLING BY MACROPHAGES PROMOTES TUMORIGENIC PROGRESSION OF INTESTINAL EPITHELIAL CELLS.

C.W.S. Ko, K.S. Chapple, G. Hawcroft, P.L. Coletta, A.F. Markham, M.A. Hull. Molecular Medicine Unit, University of Leeds, St James's University Hospital, Leeds, UK

Cyclooxygenase-2 (Cox-2) plays an important role during the early stages of intestinal tumorigenesis. Cox-2 is expressed predominantly by stromal macrophages in murine and human intestinal adenomas. This implies the existence of paracrine COX-2-mediated signalling between stromal macrophages and intestinal epithelial cells.

In vitro models were developed incorporating a murine macrophage cell line (RAW264.7) and a non-transformed rat intestinal epithelial cell line (IEC-6). Functional Cox-2 expression was up-regulated in RAW264.7 cells activated by γ-interferon and lipopolysaccharide. The effect of activated RAW264.7 cells on IEC-6 cell phenotype was tested in macrophage-conditioned medium (MCM) and co-culture experiments.

In the presence of activated MCM (AMCM), IEC-6 cells reproducibly (n=5) developed `fibroblastoid' morphology with decreased contact inhibition and cell overgrowth. Similar findings were demonstrated using the co-culture model. AMCM-cultured IEC-6 cells developed a stable, homogeneous `transformed' phenotype by approximately 60 days. Incubation with non-activated MCM (NMCM) was not associated with significant changes compared with control IEC-6 cells. AMCM-cultured IEC-6 cells maintained cytokeratin expression but expressed decreased membranous E-cadherin, decreased TGFβRII (associated with resistance to TGFβ1) and increased Cox-2 as well as PAI-1 compared with control and NMCM-cultured IEC-6 cells. AMCM-cultured IEC-6 cells exhibited anchorage-independent growth in soft agar and basement membrane matrix but were non-tumorigenic in nude mice. The presence of the selective Cox-2 inhibitor SC236 (Pharmacia) during (but not after) RAW264.7 cell activation inhibited AMCM-induced IEC-6 cell changes.

Activated macrophages promote phenotypic change of IEC-6 intestinal epithelial cells (compatible with tumorigenic progression) via a paracrine Cox-2-dependent mechanism. These models provide direct in vitro evidence for Cox-2-mediated macrophage-intestinal epithelial cell signalling during the early stages of intestinal tumorigenesis.

128 AN N-TERMINALLY TRUNCATED CYTOPLASMIC FORM OF OXYGEN-REGULATED PROTEIN 150 (ORP150), THE MAJOR INTRACELLULAR LIGAND FOR THE ANTI-PROLIFERATIVE MUSHROOM LECTIN, IS ESSENTIAL FOR NUCLEAR LOCALISATION SEQUENCE (NLS)-DEPENDENT NUCLEAR PROTEIN IMPORT IN HUMAN INTESTINAL CANCER CELLS

L.G. Yu, J.M. Rhodes. Department of Medicine, University of Liverpool, Liverpool L69 3GA, UK

The classical NLS-dependent nuclear import system, which mediates import of large nuclear proteins, is fundamentally important for maintaining nuclear function. Our previous studies have shown that the inhibition of cell proliferation of mushroom Agaricus bisporus lectin (ABL) (Cancer Res 1993;53:4627) is linked to its internalisation and selective blockade of NLS-dependent nuclear protein import (J Biol Chem 1999;274:4890). One of the major intracellular ABL-binding ligands is an N-terminally truncated cytoplasmic form of Orp150 [Gastroenterology 2001;120(suppl1):3579]. Orp150 is a stress-related protein and is up regulated in tumours and highly expressed in cancer cell lines. In this study we investigated the role of Orp150 in nuclear protein import.

Nuclear protein import was performed in digitonin semi-permeabilized human colorectal cancer HT29 and gastric cancer AGS cells using a fluorescein-conjugated synthetic NLS peptide/bovine albumin complex (NLS-BSA-FITC) as a transport marker. It was found that introduction of an anti-Orp150 antibody, but not other irrelevant antibodies, into the transport system resulted in 57% and 48% reduction of nuclear accumulation of NLS in HT29 and AGS cells respectively. Removal of cytosolic Orp150 from the transport system by immunoabsorption caused over 40% reduction of NLS nuclear accumulation. The ras-related nuclear transport factor Ran was identified in the Orp150 immunoprecipitate. Orp150 was also identified by immunoblotting in the immunoprecipitates of Ran but not in the immunoprecipitates of other Ran-associated proteins (Ran-BP1, RCC1, Ran-GAP1 and NTF2).

This result suggests that the truncated cytoplasmic Orp150 has a crucial role in NLS-dependent nuclear protein import probably by direct interaction with Ran.

129 CONTIGUOUS HYPERPLASTIC POLYP (HP), SERRATED ADENOMA (SA) AND COLORECTAL CANCER (CRC): PATHWAYS AND TIMING

M. Lockett1, V. Johnson1, W. Smale1, S. Burke1, E. Jaeger2, J. Lloyd1, I. Talbot1, I. Tomlinson2, W. Atkin1. 1ICRF CRC Unit, St. Mark's Hospital, Harrow, Middlesex, UK; 2Molecular & Population Genetics, Lincoln's Inn Field, London, UK

Background: HPs may progress to CRC via the SA (serrated CRC pathway). In hyperplastic polyposis (HPP) this occurs by microsatellite instability (MSI) and chromosomal instability (CI). In sporadic CRC, the serrated CRC pathway has been suggested to lead to proximal CRC with high levels of microsatellite instability (MSI-H).

Aims: To investigate the heterogeneity of MSI and K-ras and p53 mutations in contiguous SA/CRC and contiguous HP/SA to help understand the timings and genetic mechanisms driving the serrated pathway.

Methods: Paraffin-embedded tissue blocks with contiguous SA/CRC or contiguous HP/SA were selected. Using a haematoxylin-eosin stained section as a reference, tissue components were identified, micro-dissected and DNA was extracted. MSI analysis was done by PCR at Bat 25 and Bat 26 and by immunohistochemistry for the mismatch repair proteins hMLH1 and hMSH2. DNA samples were analysed for point mutations in codons 12 and 13 of the K-ras gene using minisequencing. Abnormal p53 accumulation was identified using immunohistochemistry.

Results: 11 contiguous SA/CRC and 2 contiguous HP/SA were analysed. 10/11 SA/CRC samples were MS stable. MSI was seen in both SA & CRC components in 1/11. The CRC but not the SA showed loss of hMLH1. K-ras mutations were detected in 4/11 CRCs. 3/4 contiguous SAs had the same K-ras mutation (1/4 PCR failure). The remaining 7 SA/CRCs were wild type. P53 results were available in 10. All 10 CRCs and 5/10 contiguous SAs showed accumulation of mutant p53. Neither of the two HP/SAs showed MSI or K-ras mutations.

Conclusions: All contiguous SA/CRC or HP/SA were concordant in MS and K-ras status. This supports the serrated CRC pathway. K-ras mutation appears to be an early event whereas p53 over-expression (and presumably mutation) a late event. MSI was rare in this predominantly distal tumour series.

130 TELOMERASE EXPRESSION IN BARRETT'S, OESOPHAGEAL AND GASTRIC ADENOCARCINOMA

J. Barclay, B. Usselmann, D. Aldulaimi, A. Morris, M. Karteris, E. Hillhouse, C.U. Nwokolo. University Hospitals Coventry and Warwickshire and Dept of Biological Sciences, University of Warwick, Coventry, UK

Ribonucleoprotein enzyme telomerase is increased in most cancers and is present in small quantities in gastrointestinal epithelia. Telomerase is involved in carcinogenesis but the contribution of the gene encoding its catalytic subunit (hTERT) to the regulation of telomerase activity is unclear. We assessed hTERT expression and telomerase activity in Barrett's, oesophageal and gastric adenocarcinoma.

Methods: hTERT mRNA was quantitated using real-time RT-PCR and telomerase activity measured by the TRAP assay in the following: paired samples from Barrett's (n=16) and adjacent cardia, paired samples from gastric (n=15) and oesophageal (n=21) adenocarcinoma and adjacent macroscopically normal tissue.

Results: (median expressed as arbitrary units) In Barrett's and adjacent cardia, telomerase activity was 20 and 11 respectively, p = 0.28 and hTERT mRNA was 3.6 and 2.3 respectively, p =0.12. There was no significant difference. In gastric adenocarcinoma, compared to adjacent normal tissue, telomerase activity was increased significantly from 0 to 16, p = 0.01 and hTERT mRNA was increased also from 2.2 to 7.1, p =0.008. In oesophageal adenocarcinoma, compared to adjacent normal tissue, telomerase activity was increased significantly from 5 to 229, p < 0.0001 but hTERT mRNA was not significantly different, 1.7 and 2.5, p =0.48. Comparing oesophageal cancer and Barrett's, telomerase activity was 229 and 20 respectively, p = 0.001 but hTERT mRNA was not significantly different 2.5 vs. 3.6.

Conclusions: Telomerase enzyme activity was increased in cancers but not Barrett's suggesting that increased enzyme activity occurs late in oesophageal carcinogenesis. In gastric adenocarcinoma, increased hTERT expression was associated with increased enzyme activity suggesting a modest regulatory role of hTERT in this cancer. However, in general, hTERT expression correlated poorly with telomerase activity confirming the complexity of telomerase regulation in malignant and benign tissue of the human foregut.

131 TNF-α IN BARRETT'S OESOPHAGUS

C. Tselepis1, I. Perry1, D. Wakelin1, R. Hardy1, S.J. Darnton2, R. Harrison3, J.A.Z. Jankowski1,3. 1Epithelial Laboratory, Department of Medicine, University of Birmingham, B15 2TH, UK; 2Oesophageal Research Laboratory, Birmingham Heartlands Hospital B9 SS; 3Department of Pathology, University of Birmingham, B15 2TT, UK

Barrett's metaplasia of the oesophagus (BM) is an early lesion in the progression from oesophageal inflammation, through dysplasia to the development of Barrett's adenocarcinoma (BA). Previous work indicates that BM and BA are associated with reduced E-cadherin expression and increased cytoplasmic/nuclear pools of its associated protein β-catenin. β-catenin participates in Wnt signalling and activates oncogene transcription by complexing with T-cell factors (TCF).

Since we have previously shown that TNF-α can down-regulate E-cadherin expression we have assessed TNF-α expression in Barrett's oesophagus and examine if TNF-α can promote β-catenin mediated transcription of oncogenes in a gastrointestinal model system.

Epithelial expression of TNF-α was determined by immunohistochemistry and Western blot analysis of oesophageal tissue. β-catenin mediated transcription was assessed in TNF-α stimulated cell lines using the TOPFLASH reporter system. C-myc expression was assessed by real time PCR.

Epithelial expression of TNF-α ?increases with the metaplasia-dysplasia-carcinoma sequence. In an intestinal cell model TNF-α induces c-myc expression, which is mediated through β-catenin regulated transcription, independent of NF-κB activation.

In summary TNF-α is up regulated in the progression of Barrett's Oesophagus. β-catenin mediated transcription of c-myc is a pathway whereby elevated levels of TNF-α may lead to oncogene transcription in gastrointestinal epithelia.

132 INCREASED EXPRESSION OF THE SRC, MET AND ERB-B2 KINASES IN THE PROGRESSION OF BARRETTS METAPLASIA TO ADENOCARCINOMA

M.R. Anderson, J.A. Jankowski, D.C. Rowlands, C. Tselepis. The Epithelial Laboratory, University of Birmingham, Edgbaston, UK

Background: The development of oesophageal adenocarcinoma is characterised by progression along the Barretts metaplasia – dysplasia – carcinoma sequence. It is known that the growth factor TGFα and its receptor, EGFR show increased expression along this sequence. Receptor activation leads to phosphorylation of kinase substrates involved in signal transduction pathways. This may promote cell proliferation and invasion. Further tyrosine kinase receptors may also be involved in dysplastic progression and could provide therapeutic targets. ErbB2, src and met are three tyrosine kinases that may be influenced by such growth factors.

Aims: To investigate the expression of erbB2, src and met along the metaplasia – dysplasia – carcinoma sequence.

Methods: Routine immunohistochemistry staining was carried out on paraffin sections from specimens of normal squamous oesophagus, Barretts metaplasia, dysplastic Barretts and oesophageal adenocarcinoma. Staining was scored semi-quantitatively. Western blotting was carried out on biopsy samples of normal oesophagus, Barretts metaplasia and oesophageal adenocarcinoma.

Results: Up-regulation of met was seen along the sequence with strong membranous staining seen in 0/10 normal oesophagus, 4/10 Barretts, 5/10 dysplasias and 7/10 carcinomas. Src is ubiquitously expressed but strong membranous staining was seen in only 3/10 Barretts and 6/10 carcinomas. ErbB2 showed reduced expression in Barretts compared with normal oesophagus, but then showed strong membranous staining in 3/9 dysplasias and 4/10 carcinomas. Western blotting confirmed these patterns of altered expression.

Conclusions: The increased expression of met is an early step in the metaplasia – dysplasia – carcinoma sequence. The increased expression of src and erbB2 appear to be later steps in the sequence, akin to EGFR. The altered expression of met, erbB2 and src suggests that whilst there may be some redundancy in tyrosine kinase signaling, one of these could provide a mechanism that promotes the progression from metaplasia to carcinoma, and may be a potential therapeutic target.

133 VARIATIONS IN CYTOKINE EXPRESSION IN THE MALIGNANT PROGRESSION OF BARRETT'S OESOPHAGUS AND FOLLOWING PHOTODYNAMIC THERAPY

P. Sirieix2, T.K.L. Wong1, L.B. Lovat1, R.C. Fitzgerald2. 1National Medical Laser Centre, Department of Surgery, Royal Free and University College School of Medicine, London; 2Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge CB2 2XZ, UK

Background: Oesophagitis has a Th1 cytokine profile in contrast to the Th2 profile (IL-4, IL-10 with low levels of TGF-β) of Barrett's oesophagus (BO). Cytokines influence neoplastic progression via alterations in immune surveillance however this is poorly studied in BO. Patients with high-grade dysplasia in BO can be treated endoscopically by photodynamic therapy (PDT). Post-PDT, the regenerating epithelium is ideally squamous, but BO may persist.

Aims: 1. To compare the cytokine levels in the malignant progression of BO 2. To determine the effect of PDT on cytokine expression in BO.

Methods: Competitive RT-PCR was used to assess the cytokine profile of OE33 (adenocarcinoma cell line) and OE21 (squamous cell carcinoma line) cells and of biopsies from normal squamous oesophagus (NO, n=10), non-dysplastic BO (n=50), and Barrett's adenocarcinoma (AC, n=5). For PDT patients with high grade dysplasia (n=5), cytokines were determined by semi-quantitative PCR up to 2 months after PDT compared with the pre-PDT biopsies.

Results: TGFβ, IL-1β and IL-8 expression is increased in OE33 cells compared to OE21 (p<0.05, p<0.05 and p<0.005 respectively). In AC biopsies, IL-4 levels are increased (16 fold increase cf. NO, p<0.05) as well as IL-1β levels (>50 fold increase cf. NO and BO, p<0.05). TGFβ expression is decreased in BO (cf. NO p<0.05), but increases again in AC to squamous mucosal levels (8 fold difference, p<0.05). Following PDT IL-10, IL-8, IL-1β and KGF were increased in BO at least 3-fold 24 hours after therapy. 2 months later these cytokine levels reverted to baseline. TGF-β levels in BO were unaffected by PDT. Neo-squamous epithelium had a cytokine profile that was intermediate between NO and BO. The cytokines levels in NO were unaffected by PDT

Conclusions: Cytokine expression is altered in BO neoplasia and post-PDT. Whether, the cytokine profile has a causal role in the determination of the cell phenotype post-PDT merits further study.

134 LUMINAL NITROSATION POTENTIAL FOLLOWING NITRATE INGESTION IS MAXIMAL AT THE GO JUNCTION

H. Suzuki, K. Iijima, A. Moriya, V. Fyfe, K.E.L. McColl. Dept of Medicine & Therapeutics, Western Infirmary, Glasgow, UK

Background: Acidification of nitrite in the presence of nitrosatable chemicals produces potentially carcinogenic N-nitrosocompounds. The reaction is catalysed by thiocyanate (SCN) and inhibited by ascorbic acid (AA). Saliva contains a high concentration of nitrite (NO2), derived from dietary nitrate (NO3), and swallowed saliva is the main source of NO2 entering the acid stomach.

Aim: To determine if there are regional variations in the nitrosating potential in the upper GI tract.

Methods: We used a validated technique involving microdialysis probes to simultaneously measure the chemicals relevant to nitrosation in the oesophagus, cardia, proximal stomach and distal stomach of 15 healthy volunteers before and following ingestion of 2mmol nitrate (equivalent to a salad portion).

Results: The NO3 ingestion increased median saliva NO2 from 37μM to 215μM and distal oesophageal NO2 from 29μM to182μM (p<0.01 each). Within the acid stomach, the NO2 concentration progressively decreased and AA concentration progressively increased with distance distal to the GO junction producing the highest acidic NO2/AA ratio right at the GO junction. Results following NO3 ingestion are shown in table (values are medians).

Abstract 134

Conclusions: 1) Nitrosation within the acid secreting stomach will be maximal at the GO Junction. 2) Dietary nitrate may be involved in the aetiology of mutagenesis and carcinogenesis at this site.

135 SUPPRESSION OF PROLIFERATION AND INDUCTION OF APOPTOSIS IN HUMAN OESOPHAGEAL ADENOCARCINOMA CELLS BY NATURAL AND SYNTHETIC COX-2 INHIBITORS

E. Cheong1,2, K. Ivory1, M. Parker1, M. Rhodes2, I.T. Johnson1. 1Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA; 2Norfolk and Norwich University Hospital, Colney, Norwich NR4 7UA, UK

Background: Adenocarcinoma arising from Barrett's oesophagus is the most rapidly increasing cancer in the west. Epidemiological studies suggest that use of NSAIDs is associated with up to 90% decreased risk of developing oesophageal cancer. The main biochemical target for NSAIDs is cyclooxygenase and the isoenzyme COX-2 is up-regulated in oesophageal carcinogenesis. The protective effect of NSAIDs may result from inhibition of COX-2. Synthetic COX-2 inhibitors suppress enzyme activity, but the food-borne flavonoid quercetin also suppresses COX-2 mRNA expression. We examined the effects of 3 commercially available COX-2 inhibitors (NS-398, nimesulide and niflumic acid) and quercetin on cell proliferation and apoptosis in the poorly differentiated human oesophageal adenocarcinoma cell line OE-33.

Methods: Cell viability after treatment with COX-2 inhibitors for 24, 48 and 72 h was assessed 0n 96-well plates using a neutral-red dye technique. Changes in the relative numbers of adherent and floating cells were assessed in culture-flasks, and apoptotic cells among the floaters were identified using ethidium bromide and acridine orange staining under fluorescent microscopy. Flow cytometric analysis of attached and floating populations was used to quantify apoptotic cells and to examine the effects of the agents on the cell cycle.

Results: Western blot analysis confirmed COX-2 expression in the OE-33 cell line. The selective COX-2 inhibitors and quercetin suppressed OE-33 cells proliferation in a dose- and time-dependent manner, and increased the fraction of floating cells in the population. The majority of the floating cells were identified as apoptotic. The antiproliferative effects induced by quercetin were significantly greater, and the number of floating cells was significantly higher after quercetin treatment compared with the selective COX-2 inhibitors (p<0.05). Cell cycle analyses revealed that quercetin blocked the cell in S phase while the selective COX-2 inhibitors (NS-398 and Niflumic acid) blocked the cell in G0/G1 interphase.

Conclusion: Selective COX-2 inhibitors are able to suppress proliferation and induce apoptosis in human oesophageal adenocarcinoma cells in vitro. However, quercetin, a food borne COX-2 inhibitor, has an even greater inhibitory effect on these cells, and is a potent inducer of apoptosis and cell-cycle arrest.

136 ANGIOGENIC POTENTIAL OF HUVEC CELLS IS INCREASED BY AMIDATED AND GLYCINE-EXTENDED GASTRIN-17

P.A. Clarke, S. Evans, D. McWilliams, S.A. Watson. Cancer Studies Unit, University of Nottingham, Nottingham, UK

Introduction: Gastrin peptides directly and indirectly promote the growth of malignant cells. Gastrin modulates expression of heparin binding EGF (HB-EGF), which may play a role in angiogenesis (Miyizaki et al, 1999). The aim of these studies was to determine whether gastrin modifies endothelial vessel formation of human umbilical vein endothelial (HUVEC) cells in in vitro culture.

Methods: HUVEC cells were grown in a mixed culture system with irradiated feeder cells in 24-well plates. Amidated human gastrin-17 (G17) and glycine extended G17 were added to the cultures at concentrations of 10nM. Vascular endothelial growth factor (VEGF, 2ng/ml) and suramin (20μM) were used as positive and negative controls (n=2 wells were set up for each condition), respectively. Media supplementation took place on days 4 and 7. At day 8 the cultures were fixed with ethanol and stained using a CD31 monoclonal antibody. Image analysis was used to quantify tubule node formation.

Results: The mean nodes assessed for 2 separate experiments are shown in the table.

Abstract 136

Conclusion: G17 and GlyG17 are able to induce an angiogenic response in HUVEC cells which is equal in magnitude to that induced with VEGF.

137 GASTRIC ADENOMATOUS POLYPS DEMONSTRATE ACCUMULATION OF MUTANT P53

G.V. Smith, R. Feakins, A. Ballinger. Adult and Paediatric Gastroenterology and Experimental Pathology, Barts and the London, Queen Mary's School of Medicine, London, UK

Gastric adenomas are a rare finding at endoscopy, occurring at 1 in 3000 of endoscopies. They are associated with the development of gastric cancers mirroring the adenoma-carcinoma neoplastic pathway in the colon however in the stomach they account for a small proportion of cancers. P53 gene mutations are found in between 40 and 50% of all gastric cancers and occur relatively late in the neoplastic cascade.

Aim: To assess the degree of p53 mutation by detecting accumulation of clone DO-7 type mutated p53 in gastric adenomatous polyps.

Method: 15 Sequential archived paraffin blocks taken from gastric resections and endoscopic biopsies of gastric adenomas were analysed by immunohistochemistry using a monoclonal antibody raised against p53 clone DO-7 protein (Dako). An avidin-biotin bridge and DAB detection system were employed (Vector). Positive controls from an oesophageal carcinoma (Dako) and negative controls of 15 normal aged matched gastric sections and replacement of the primary antibody with serum from the host animal were employed. 15 sections from unselected gastric adenocarcinomas were also analysed. Sections were counterstained with haematoxylin and assessed for the presence or absence of mutated p53 accumulation.

Results: All 15 adenomas exhibited p53 accumulation, indicated by nuclear staining, compared with none of the control specimens (see table 1).

Abstract 137

Discussion: Mutated p53 accumulation is strongly associated with gastric adenomata, its presence being detectable in a far greater proportion than in gastric carcinomas and normal gastric tissue. This pattern of p53 expression more closely matches that seen in the adenoma-carcinoma pathway in the colon than the more common metaplasia-dysplasia-carcinoma pathway that occurs in the stomach.

138 DETECTION OF TELOMERASE ACTIVITY IN CIRCULATING COLORECTAL TUMOUR CELLS

L. Titu, J. Greenman, V. Jordison, J.R.T. Monson, R.L. Loveday. Academic Surgical Unit, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, UK

Background: Despite an apparently curative resection, up to 10% of Dukes A and 25% of Dukes B colorectal cancer patients relapse and die of metastatic disease within 5 years. This indicates that the metastatic process may already have been established prior to, or at the time of resection, and that the detection of tumour cells in the circulation at this time may have important prognostic or diagnostic implications.

Methods: Thirty-six sequentially presenting patients undergoing surgery for colorectal cancer and ten healthy controls were included in the study. Patient blood samples were taken pre-operatively, and 1 and 7 days post-operatively. Peripheral blood mononuclear cells (PBMC) were obtained from whole blood by Ficoll-Hypaque density gradient centrifugation. Circulating epithelial cells were then isolated from PBMC by incubating with the epithelial specific antibody BerEP4 conjugated to magnetic beads. Cells were harvested using a magnetic field, lysed and protein content determined and standardised. Telomerase activity was detected in lysates using TeloTAGGG Telomerase PCR ELISA (Roche, UK).

Results: We identified 12 (33%) patients who were positive for circulating tumour cells (CTC) pre-operatively, indicating that the method described may have some value as a diagnostic tool. Sixteen patients (44%) were negative for CTC pre-operatively but positive post-operatively, suggesting that surgical manipulation had resulted in the introduction of tumour cells into the circulation. For 11 (67%) of these patients the CTC were still detectable at 7 days after surgery. The presence of telomerase positive CTC did not correlate with the cancer's stage. None of the healthy controls exhibited telomerase activity in epithelial cells.

Conclusions: The method described represents a simple and specific method for the detection of CTC in colorectal cancer patients. The detection of telomerase activity in CTC may have prognostic implications independent of currently established staging systems and the longer term follow up of these patients will assess this.

139 CORRELATION BETWEEN UPTAKE OF LABELLED ANTI-CCKB/GASTRIN RECEPTOR ANTIBODIES AND THE OCCURRENCE OF APOPTOSIS IN HEPATOMA CELL LINES

M. Stubbs, K. Khan, K. Savage, S. Grimes, D. Michaeli, A.P. Dhillon, S.A. Watson, M.E. Caplin. Royal Free and University College Medical School, Pond Street, London NW3 2PF, UK

Background: It has been reported that administration of an anti-CCKB/gastrin receptor (CCK-BR) antibody to mice bearing human xenograft tumours results in increased apoptosis and necrosis (Watson et al., 2000, Cancer Res. 60: 5902–5907). We have previously found that cell lines exposed to an antibody raised against a peptide corresponding to residues 5–21 of the amino terminus of the CCK-BR display endocytosis of the antibody into the cytoplasm and nucleus.

Aim: To assess whether the endocytosis of this anti-CCK-BR antibody correlates with the occurrence of apoptosis in these same cell lines.

Methods: The anti-CCK-BR antibody was labelled with Alexa Fluor 488 dye (Molecular Probes, USA). HepG2 (human hepatocyte carcinoma), PLC/PRF/5 (human liver hepatoma), MCA RH 7777(rat hepatoma), HTC (rat hepatoma) and WRL 68 (human liver embryonic) cells were exposed to the labelled antibody at 20 μg/ml for 1 hour at 37°C. The cells were fixed and subsequently costained for apoptosis using an immuno-fluorescent rhodamine assay (ApopTag Red kit, Intergen, USA). Cells were imaged using a fluorescent microscope with filters for the Alexa Fluor 488 and rhodamine fluorescence.

Results: In all five cell lines uptake of the labelled anti-CCK-BR antibody was correlated with apoptosis.

Conclusions: Here, we demonstrate a direct relationship between the uptake of the antibody and cell death (apoptosis). This observation has important implications in the treatment of CCK-BR positive tumours including hepatomas where there are limited therapeutic options.

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