research report

University of Leeds

2005/6


Faculty of Medicine and Health
Dean of the Faculty: Professor E.W. Hillhouse  

The Leeds Institute of Molecular Medicine
Director: Professor T. Rabbitts  

Academic Unit of Oncology & Haematology
Head of Academic Unit: Prof. P.J. Selby  


A study of the Prevalence of Occult B-cell Lymphoproliferative Disorders in the Healthy Population and in the Family Members of Patients  
Dr P. Hillmen, Dr A.S. Jack, Dr A.C. Rawstron and Dr M.S. Gilthorpe  

The surface protein expression profile of normal and neoplastic B-cells has been studied using a panel of antibodies identified from previous reports that showed differential expression either at the mRNA level from microarray studies, or directly from protein expression analysis.  Bone marrow from normal individuals, from patients with follicular lymphoma (FCL), diffuse large B-cell lymphoma, marginal zone lymphoma (MZL), mantle cell lymphoma, chronic lymphocytic leukaemia (CLL), monoclonal gammopathy of uncertain significance, myeloma, and 3 normal lymph nodes have been analysed.  Protein expression levels have been analysed with dChip software. Hierarchical clustering was used to group antigens, and different clusters were identified that could provide additive separation when combined. 66 cell surface markers have been assessed on samples from 54 B-cell leukaemia or lymphoma cases.  Disease specific profiles are evident for common B-cell disorders including CLL, FCL and MZL (Waldenstrom's macroglobulinaemia).  All disorders, with the exception of hairy cell leukaemia, down-regulate markers which are normally expressed on circulating B-cells and which are down-regulated on normal germinal centre B-cells.  Over 1000 normal individuals have been screened with antigen combinations which depend upon light chain restriction (i.e. relatively insensitive assays) and clonal non-CLL lymphoma cells are found in at least 1% of healthy adults.  The newly developed specific assays are being applied to normal individuals to establish the frequency of non-CLL monoclonal B-lymphocytosis.  In conclusion, the protein expression profile of neoplastic B-cells is different from that of normal circulating B-cells suggesting a common originating stage of differentiation for several apparently unrelated B-cell disorders.       


Faculty of Medicine and Health
Dean of the Faculty: Professor E.W. Hillhouse  

The Leeds Institute of Genetics, Health and Therapeutics (LIGHT)
Acting Director: Professor C.P. Wild  

Centre for Epidemiology and Biostatistics
Head of Centre : Professor C.P. Wild  

Genotoxic effects of binary exposures of carcinogens on skin cells  
Dr M.N. Routledge, Prof. E. Ingham and Prof. C.P. Wild  

UV radiation is the main risk factor in skin cancer but environmental chemical carcinogens such as benzo[a]pyrene (BP) can also contribute to skin carcinogenesis. We have studied the effects of combined exposure of different wavelengths of UV light with BP in primary human keratinocytes, using the comet assay. UVA in combination with BP induced a greater enhancement of DNA damage than UVB with BP. This suggests that UVA has the potential to play a bigger role in skin carcinogenesis than previously thought.  

Investigation of the risks of genetic damage associated with chromoendoscopy in Barrett's Oesophagus patients  
Dr L.J. Hardie and Prof. C.P. Wild  

Methylene Blue (MB) is currently used during chromoendoscopic examinations of the gastrointestinal tract to aid tissue visualisation.  In relation to the preneoplastic lesion, Barrett's Oesophagus (BE), MB-chromoendoscopy has been suggested to serve as a useful method for targeting biopsies to areas of dysplasia in the BE segment.  

Previous work by this group has shown that patients undergoing routine MB-chromoendoscopy demonstrate elevated levels of oxidative DNA damage in BE biopsy samples following this procedure. The photoexcitation of MB is thought to be responsible for this effect.  

The current grant aims to establish conditions which minimise DNA damage during methylene blue chromoendoscopy, without compromising visualisation of the tissue in clinical practice.  

Using cell lines we have mimicked chromoendoscopy conditions and measured DNA damage by the comet assay.  We have shown that the DNA damaging effect with MB is dose, light and time dependent. Furthermore, it is the red portion of endoscopic light that induces the DNA-damaging properties of MB, and inclusion of N-acetyl cysteine is partially protective.  Conditions which minimise DNA damage without compromising visualisation of tissue in vitro have now been established. A UK patent application was filed to protect this work, and has now proceeded to PCT.  

Validation of the optimised endoscopic conditions in the clinical setting is currently underway.  


The Leeds Institute of Molecular Medicine
Director: Professor T. Rabbitts  

Academic Unit of Molecular Medicine
Director: Professor D.T. Bonthron  

Functional characterisation of human EB1 homologues  
Dr J.M. Askham and Dr E.E. Morrison  

Mutations in APC are both involved in inheritable and spontaneous forms of colorectal cancer.  Resulting truncated APC proteins cannot interact with EB1, a microtubule-associated protein participating in the sub-cellular targeting of APC to microtubule tips.  Microtubule tip-associated APC at discrete sub-cellular locations appears to be crucial in defining cell polarity and in directed cell migration.  Hence, loss of the APC/EB1 interaction might be linked to the aberrant migration of mutant colonic epithelial cells as well as to chromosomal instability, common features of this type of cancer.  

Two EB1 homologues (EB3 and RP1) have been identified in human cells, but remain relatively uncharacterised.  We have demonstrated that all EB proteins interact with normal but not mutant APC. Live-cell video microscopy of GFP-tagged normal and chimeric EB proteins has shown that all exhibit a dynamic localisation to polymerising microtubule tips and that this requires EB protein dimerisation.  Furthermore, whilst EB1 and EB3 bind to the dynactin component p150^Glued, RP1 does not.  This interaction does not significantly contribute to the microtubule tip localisation of EB proteins but does mediate the EB1/3 centrosomal localisation.  

Additionally we have defined in APC a second binding site for EB proteins and demonstrated that interaction at both sites is required for a functional association between APC and EB proteins.  

Our data demonstrate the complexity of molecular interactions at the C-terminus of APC, all of which are lost in colorectal cancer cells.  This work has important implications for the understanding of the migratory defects and chromosomal instability commonly observed in colorectal cancer cells.  

Characterisation of OSC1, a novel tumour suppressor in oral squamous cell carcinoma and other cancers  
Dr S.M. Bell and Dr A.P. Jackson  

Loss of heterozygosity at 8p23 is a frequent event in Oral Squamous Cell Carcinomas (OSCC) and a number of other cancer types including breast cancer.  We have identified a large candidate tumour suppressor gene (OSC1) with a novel domain structure, which has homologies to proteins already implicated in carcinogenesis.  

We have developed OSC1 antibodies to investigate the distribution of OSC1 in normal and malignant tissue samples.  Three predominant OSC1 staining patterns have been identified, cell membrane staining in oral epithelial cells, on the apical membrane surface of breast duct epithelial cells and cytoplasmic cell staining.  In addition a perinuclear golgi body staining pattern has also occasionally been detected in colon samples. Reduced expression of OSC1 has been identified in 50% (26/52) of breast cancers. Interestingly down regulation of OSC1 was significantly associated with increasing tumor grade (p<0.0007).  A trend for loss of OSC1 expression was seen with the Nottingham Prognostic Index, with a high proportion of cancers with reduced OSC1 having a poor prognosis (p = 0.0923).  This study suggests that OSC1 plays a role in tumor differentiation and prognosis in breast cancers.  

SiRNA has been successfully used to investigate the biological effects of down regulation of OSC1 expression in two breast cancer cell lines. In the breast cancer cell line BT20 reduced OSC1 expression resulted in a decrease in adhesion to two extracellular matrix proteins fibronectin and vitronectin.  We hypothesize that OSC1 is a potential adhesion molecule due to its protein domain homology, cell surface localization and affect on adhesion.  

MCPH1, a potential predictor for response to cancer chemotherapy
Dr S.M. Bell and Dr V. Speirs  

Previously we have identified MCPH1, a DNA damage response protein involved in the regulation of the breast cancer tumour suppressor gene BRCA1, as the defective protein in one form of microcephaly.  Interestingly we found that reduced expression of MCPH1 causes premature chromosomal condensation (PCC) and a delay in chromosome decondensation after mitosis. PCC is a hallmark of mammalian cells that begin mitosis before completing DNA replication.  The PCC phenotype has been observed in a number of human cancers including gastric, breast, ovarian, brain, colon and leukemia.  The PCC phenomenon has been reported as an early indicator of relapse in human acute leukemia before other clinical signs.  

The MCPH1 locus (8p22-p23) is frequently deleted in many tumour types and this is associated with a poor prognosis and a reduced response to chemotherapy in breast cancer. Many chemotherapeutic agents such as taxanes (e.g. Taxol) and topoisomerase II poisons (e.g. etoposide) require a functional spindle checkpoint for the induction of apoptosis in cancer cells.  Our preliminary data indicates MCPH1 plays a role in resistance to chemotherapeutic agents such as Taxol through its involvement in the spindle checkpoint. We therefore hypothesise that, while germ line defects in MCPH1 cause microcephaly, somatic defects may confer resistance to chemotherapy.  Our preliminary immunohistochemistry data has identified reduced MCPH1 expression in 32% of breast cancers. These results are now being confirmed in a larger series of breast cancer samples using tissue micro arrays (TMAs) to investigate MCPH1 expression and patient survival and response to chemotherapy.  

Expression of oncogenic versions of c-myb and AML1 in in vitro differentiated mouse embryonic stem cells  Prof. C. Bonifer and Ms D. Clarke  

An important approach for advancing our understanding of how specific transcriptional regulation contributes to haemopoiesis has been to mutate transcription factor genes in mouse embryonic stem (ES) cells and to study subsequent effects on in vitro differentiation and target gene regulation.  In this work we concentrated on the role of normal and aberrant versions of c-Myb and AML1.  We have differentiated ES cells homozygous null for the c-myb locus and showed that definitive precursors accumulate in c-myb-/- embryoid bodies. We have also shown that c-myb has also a haploinsufficiency phenotype.  We could show that the timing of erythropoiesis in c-myb knockdown ES cells is deregulated, however, most erythroid marker gene expression was unaffected.  However, this work was on hold, because Ms Clarke went on maternity leave and has then been drawn to a close. A publication describing these results has been written. 

After she came back, Ms Clarke was instrumental in setting up two cell culture systems which are of vital importance for our department:  She established the Pax5 knock-out system and this led to two major publications in which the YCR was acknowledged.  She also performed the first experiments for the establishment of an in vitro differentiation system from ES cells which allows us to differentiate pure mesodermal cells and hematopoietic stem cell precursors.  This work will be the basis for future grant applications which will test the influence of the overexpression of leukaemic oncogenes on haemopoietic stem cell development.  

Epigenetic priming of the GM-CSF locus in acute myeloid leukaemia  
Dr. P. N. Cockerill  

GM-CSF is a myeloid cell growth factor. In T cells, monocytes, and mast cells, the GM-CSF gene is highly inducible, and is regulated by an inducible enhancer which undergoes chromatin remodelling to form an inducible DNaseI hypersensitive site (DHS). In acute myeloid leukaemia (AML), the enhancer typically exists as a constitutive DHS, and GM-CSF is often abnormally expressed as an autocrine growth factor.  

We have previously studied normal inducible mechanisms controlling the GM-CSF enhancer. We found that the enhancer occupies positioned nucleosomes in T cells and mast cells that become destabilised upon activation. The enhancer is activated via distinct mechanisms in T cells, where it is activated via composite NFAT/AP-1 binding sites, and in mast cells where the enhancer requires GATA and AP-1 elements.  

To study abnormal GM-CSF regulation in AML we purified blast cells or CD34 positive cells from 10 AML patients and measured GM-CSF expression. Unexpectedly, we found that most of the AML samples did not express GM-CSF in the absence of stimulation. We suggest, therefore, that the GM-CSF locus is regulated by paracrine mechanisms in AML. In this new grant we aim to test the hypothesis that specific mechanisms prime the locus at an epigenetic locus, and additional paracrine factors such as IL-1 are required for the transcriptional activation of the GM-CSF locus.  

Inhibition of Helicobacter pylori induced epithelial proliferative responses: a novel approach for cancer chemoprevention  
Dr. J.E. Crabtree, Prof. M.F. Dixon and Dr. P.A. Robinson  

H.pylori has been recently classified as a category I human carcinogen playing a causative role in the development of gastric cancer. Long-term infection of an in vivo model with H.pylori results in the development of gastric cancer. We have demonstrated that H. pylori stimulates epidemal growth factor (EGFR) transactivation in gastric epithelial cells through a 'triple membrane passing signal' (TMPS) cascade. H. pylori induced release of EGFR ligand, HB-EGF, is dependent on metalloprotease, EGFR and Mek-1 activities.  This signalling pathway is likely to lead to autocrine/paracrine signalling cascades promoting enhanced gastric epithelial cell proliferation and decreased apoptosis and ultimately neoplasia. To test the hypothesis that blocking the TMPS cascade will prevent H. pylori induced epithelial hyperproliferation and related gastric pathology, the effects of treatment with a specific EGFR-tyrosine kinase inhibitor (EKB-569) has been examined in a model system.  

EKB-569 treatment in H. pylori infected rodents resulted in a significant reduction (p < 0.05) in corpus atrophy and mucous metaplasia at 38 weeks post-infection. There was also a significant (p < 0.05) reduction in submucosal herniations with EKB-569 treatment. These pathological changes were associated with alterations in epithelial kinetics. In H. pylori infected groups, the gastric epithelial proliferation/apoptosis ratio in the untreated group was significantly greater (p<0.02) than in the EKB-569 treated group, where the ratio was comparable to uninfected controls. There was a marked increase in apoptotic subepithelial mononuclear cells in the EKB-569 treated infected animals, suggesting that mesenchymal-epithelial interactions have a role in mucous metaplasia and perturbations in epithelial kinetics. These studies demonstrate that bacterial induced transactivation of the EGFR has a key role in modulating epithelial kinetics in H. pylori infection.  

A novel transgenic model for analysis of Helicobacter pylori induced gastric carcinogenesis  
Dr J.E. Crabtree, Mr D.A. Brooke, Dr P.L. Coletta, Dr P.A. Robinson  

Helicobacter pylori has a major aetiological role in gastric carcinogenesis. We demonstrated using a model in vivo mutagenesis system that H. felis infection induces mutations in gastric tissue DNA. Long term infection with H. pylori in a model system results in gastric cancer and the pathological changes associated with H. pylori infection in the model are very similar to those observed in human infection. We are developing a new model mutagenesis system using the lambda/lacI transgene. Significant advancement has been made toward the development of this novel transgenic model.This model will be used to assess the mutagenicity of H. pylori in vivo.  The developed novel model will permit detailed analysis of gastric mutation frequency induced by specific H.pylori virulence factors, and the investigation of therapeutic strategies to reduce H. pylori induced genotoxicity. This model will allow analysis of the specific molecular mechanisms that regulate H. pylori induced gastric carcinogenesis in vivo.  

Towards a better definition of the human haemopoietic stem cell; surface marker expression and functional activity  
Dr E.A. de Wynter and Dr G.P. Cook  

The identification of human haemopoietic stem cells (HSC) has important implications for cancer treatment. We are examining two populations of stem cells: the 'SP' and CD133⁺/G₀ cells.  Although the ABCG2/Bcrp1 protein confers the SP (Side population) phenotype, our studies indicate that the ABCG2 marker is not specific to SP cells.  A proportion of lineage positive cells including NK cells, also express the protein.  However, we have confirmed that the majority of the cells in the SP 'tail' are CD34 negative.  The SP 'tail' has been divided into four regions based on Hoechst efflux activity.  Currently, we are examining each of these regions for functional stem cell activity.  

It is now clear that both CD34⁺ and CD34^- stem cells exist.  The CD133 marker identifies a subset of cells which contains both CD34⁺ and CD34^- cells.  Using cell cycle staining we have separated the CD133⁺ population into G₀ and G₁ cells.  The CD133⁺/G₀ cells are enriched in primitive stem cell activity in comparison to the CD133⁺ G₁ population.  In LTC-IC assays at week 5 and week 8, the CD133⁺/G₀ subset generated 100-fold more progenitor cells than the CD133⁺/G₁ subset.  Preliminary data in vivo indicate this stem cell subset is at least 6-8 fold more enriched in stem cells when compared to published data for CD34⁺/G₀ cells.  

Potassium channels as biomarkers of carcinogenesis in human colon  
Dr J.E. Linley, Dr M. Hunter and Prof. G.I. Sandle  

Colorectal cancer is a major cause of death in the UK and USA. Many start as adenomas, growing and becoming dysplastic, with an increasing risk of malignant change.  Dysplastic transformation is associated with depolarisation of cryptal colonocytes and a decrease in the concentration of intracellular Ca²⁺ (Cancer Res. 51:4492-4, 1991).  Potassium channels are a major determinant of the cell membrane potential and can be modulated by intracellular Ca²⁺.  Therefore we have extended our findings of altered K⁺ channel expression in colonic cancer to investigate the consequences of variations in cell Ca²⁺ on K⁺ channel activity.  

Using the perforated patch clamp technique, whole cell currents were measured from histologically normal human colonic crypts.  Our data show that raised intracellular Ca²⁺ stimulates the KCNN4 channel resulting in membrane hyperpolarisation.  Pharmacological inhibition of KCNN4 caused a large membrane depolarisation indicating that this channel is the predominant K⁺ channel involved in maintaining a hyperpolarised membrane potential. These data suggest that the depolarisation observed in colonic tumors may be due to altered Ca²⁺ regulation of KCNN4 channels, but this does not preclude changes in protein expression.  Currently we are investigating whether K⁺ channel protein levels change during malignant progression using immunocytochemistry.  If the changes in K⁺ channel expression observed in colon cancer are mirrored in adenomatous polyps, then these channels may provide a biomarker for malignant potential.  
Functional significance of altered prostaglandin-signalling in hormone resistant breast cancer  
Dr V. Speirs and Dr M. Cummings  

Resistance to endocrine therapy is a serious problem in the management of breast cancer. To address this, we applied microarray technology to an in vitro model of tamoxifen-resistance, developed in house from the breast cancer cell line MCF-7. Using Affymetrix U133A GeneChips, we identified 131 up-regulated genes and 156 down-regulated genes whose steady state expression levels differed by at least 3-fold between tamoxifen-resistant cells and TAM-sensitive controls (TAMr and TAMs, respectively). Several components of the prostaglandin-signalling pathway were differentially regulated in TAMr cells, including, increased mRNA expression of phospholipase A2 group III (PLA2G3), COX-1 and EP4 and decreased mRNA expression of EP3 and hydroxyprostaglandin dehydrogenase 15-NAD [HPGD]), which is responsible for prostaglandin inactivation. HPGD expression was downregulated in two independently derived TAMr MCF-7 cell lines, suggesting that HPGD downregulation may be a frequent mechanism involved in TAMr. Stable transfection of HPGD cDNA into TAMr cells resulted in significantly increased sensitivity to 4-hydroxy-tamoxifen compared to empty vector control cells, almost completely reversing the resistant phenotype. However, we were unable to detect PGE2 secretion in TAMs and TAMr cells, even in the presence of arachidonic acid, which is reported to stimulate prostaglandin synthesis. PGE2 is just one of many prostaglandins and it is possible that others may contribute to the resistant phenotype, since HPGD inactivates all prostaglandins. Indeed, preliminary data suggests that PGF2? production is greater in TAMr cells and is being explored further. Finally, tissue microarrays of TAMr and TAMs breast tumours have been constructed and are currently being used to determine immunohistochemical expression of HPGD and COX-1.  


Academic Unit of Oncology & Haematology
Head of Academic Unit: Prof. P.J. Selby  

Molecular epidemiology of DNA repair and bladder cancer  
Dr A.E. Kiltie and Prof. D.T. Bishop  

Cigarette smoking is a major risk factor for bladder cancer. Chemical carcinogens and reactive oxygen species in cigarette smoke damage DNA, so DNA repair capacity is likely to influence bladder cancer predisposition. DNA repair gene mutations are found in normal individuals at polymorphic frequency. We hypothesised that DNA repair mutations would be more frequent in cases, and would exert their influence by their interaction with cigarette smoking.  

We have undertaken a relatively large case-control study in bladder cancer (547 cases and 579 controls) for 14 XRCC1 single nucleotide polymorphisms (SNPs) and 23 XPC SNPs and shown a null result for three XPC SNPs in linkage disequilibrium, one of which was previously thought to be important in terms of bladder cancer risk in a smaller study, and another of which alters DNA repair function. Also, we found no association with increased bladder cancer risk for any of the XRCC1 SNPs.  

However, we found that the XPC Ala499Val SNP variant allele to be in strong linkage disequilibrium with two 3'UTR SNPs and all three were associated with increased bladder cancer risk. These findings have not previously been described in the literature. In addition, these three SNPs were associated with progression to muscle invasive disease. No gene-smoking interactions were observed but this will be tested for in XPC in a larger study of 1000 cases and 1000 controls.  

The influence of DNA double strand break signalling proteins on outcomes following radiotherapy for bladder cancer  
Dr A.E. Kiltie, Dr J. Bentley and Prof. M.A. Knowles  

Muscle invasive transitional cell carcinoma of the bladder has a 50% 5 year survival rate following radiotherapy or surgery. Accurate prediction of tumour radiosensitivity would allow rational patient selection for bladder conservation. DNA double strand break (DSB) repair capacity is an important influence on radiosensitivity; unrepaired DSBs are the lesions produced by radiotherapy which are lethal to cells. The DNA DSB damage response to ionising radiation involves the MRE11/RAD50/NBS1 complex activating the ATM kinase which initiates rapid phosphorylation of downstream effectors including H2AX, CHK2, p53, BRCA1 and NBS1 which are involved in checkpoint function and DNA repair. Phosphorylated H2AX is found at sites of DSB and is thought to act as an anchor to hold broken chromosome ends together for repair.  

The aim of this project is to test the hypothesis that reduced expression of the DNA DSB signalling proteins ATM, H2AX, MRE11, NBS1 and RAD50, as a result of genetic and/or epigenetic inactivation of their loci, are associated with increased tumour radiosensitivity and hence increased patient survival following radiotherapy for bladder cancer, due to aberrant DNA repair and hence increase cell killing by ionising radiation. Work is underway to assess protein expression levels using immunohistochemistry in a 90 patient training set and these findings shall subsequently be validated in an independent 100 patient tumour set. Tumours shall also be studied for loss of heterozygosity at the relevant loci using laser capture-microdissected samples, and cluster analysis will be used to identify the molecular 'signature' of tumours sensitive to radiotherapy.


Faculty of Biological Sciences
Dean of the Faculty: Professor A.J. Turner  

Assessing the role of human papillomavirus (HPV) oncoproteins in regulating cell-mediated immunity: implications for cervical cancer  
Dr G.E. Blair, Dr G.P. Cook and Dr G. Bottley  

Previous work from our laboratory has investigated the modulation of cell surface Major Histocompatability Complex class I (MHC class I) expression by the E7 oncoprotein from high risk Human Papillomavirus 16 (HPV16).  We utilized two complementary cell-based systems to induce or knock-down the E7 protein, and determined changes to MHC class I expression.  We have already shown that siRNA-mediated inhibition of E7 in HPV16-transformed cells results in significant down-regulation of MHC class I.  We have also shown that induction of HPV16 E7 using a tetracycline-repressor system results in a significant up-regulation of MHC class I.  This data taken together indicates that E7 from HPV16 actively down-regulates cell surface MHC class I.  

We have now extended this DATA (project) by considering the role of E7 from HPV18, the other major high risk HPV type.  Our results reveal that knockdown of HPV18 E7 also results in a significant down-regulation of MHC class I, confirming that this down-regulation is a property common to both high-risk HPV E7 molecules.  

We have recently shown that Human cells transformed with Adenovirus-12 have reduced MHC Class I on the cell surface and that this results in increased susceptibility to NK cells (1).  We have therefore examined the functional implications of MHC class I down-regulation by HPV E7 using a co-culture system with peripheral blood Natural Killer (NK) cells.  These co-culture experiments revealed that the cell surface changes in MHC class I levels mediated by E7 observed have a significant effect on susceptibility to NK cells, consistent with "the missing self hypothesis".  Thus E7-knockout cells that have increased MHC class I are more resistant to NK cell lysis.  Conversely, E7-induced cells that have reduced MHC class I are less resistant to NK cells.  

Data obtained to date reveal that E7 from high-risk HPV types is an immunomodulatory molecule, although more work needs to be undertaken to define the method of action and the role of E6, which is co-expressed in HPV transformed cells.  

Use of post-genomic technologies to discover new targets for colorectal cancer immunotherapy  
Dr P. Robinson, Dr S.E. Perry, Dr G.E. Blair, Dr G.P. Cook, Dr A.A. Melcher and Prof. P. Quirke  

Despite improvements in treatment, colorectal cancer remains a serious health problem and new treatments are required.  Immunotherapy offers the potential to increase the immune response to a tumour, aiding elimination.  Currently, the highly characterised tumour-associated antigens carcinoembryonic antigen (CEA) and epithelial cell adhesion molecule (Ep-CAM) are being employed in such strategies. With the view of increasing the scope of colorectal cancer immunotherapy, we have been investigating Cub Domain Containing Protein 1 (CDCP1) as a potential target for immunotherapy. This protein was described to be up-regulated in lung and colorectal carcinoma cell lines.  

We have shown that CDCP1 is up-regulated in colorectal cancer tissue and cell lines as well as in cervical carcinoma.  Comparison with the expression of CEA and Ep-CAM suggests that CDCP1 may indeed represent a useful target for therapy.  We have shown the existence of two splice variants of CDCP1 which give rise to two protein isoforms which localise to different cellular compartments.    

We are currently investigating the expression of the two isoforms of CDCP1 in colorectal cancer and evaluating the ability of antigen-presenting cells to prime cytotoxic T-cell responses against CDCP1 expressing tumours.  

Cellular proteins which regulate the latent-lytic switch in Kaposi's sarcoma associated herpesvirus  
Dr A Whitehouse  

Kaposi's sarcoma (KS) is a complex lesion characterised by endothelial proliferation, neoangiogenesis and inflammatory cell infiltration.  However, there is still some uncertainty regarding the nature of the proliferating cells and whether the lesions represent an exuberant hyperplasia or a neoplasm.  KS occurs in four well-defined clinical settings: classical, endemic, AIDS-associated, and transplant-related. Widespread HIV infection has now turned KS into an epidemic disease and a key concern is the major KS epidemic in Africa.  KS is now the most common adult tumour in parts of Africa.  Lytic replication plays an important role in the pathogenesis and spread of KSHV.  ORF 50 is the key protein which regulates viral reactivation and lytic gene expression.  It activates viral transcription by interacting with promoters containing a specific sequence motif.  However, the mechanism is not fully understood.  Recent analysis suggests that cellular proteins may direct or augment ORF 50 DNA binding.  We now have preliminary analysis to show that the HMG-A protein is one such cellular protein involved in ORF 50-mediated transactivation.  We have demonstrated that: (i) HMG-A proteins can enhance KSHV ORF 50 transactivation (ii) EMSAs have demonstrated that HMG-A protein can bind to KSHV promoters (iii) Transient expression of HMG-A in KSHV-latently infected cell lines leads to reactivation (iv) Ablation of endogenous HMG-A is essential for KSHV reactivation.  We are now assessing which lytic genes the HMG-A protein synergistically activates in the presence of ORF 50 and determine the mechanism of this activation.  This analysis may identify suitable antiviral targets for this important human pathogen.  

Regulation of the Miz-1 transcription factor  
Dr. S.C. Wright  

Many tumours are characterised by uncontrolled cellular proliferation and a block in differentiation.  We have shown that the overexpression of Myc in human tumours is associated with transcriptional repression of the differentiation-associated Mad4 gene. Myc mediates transcriptional repression of Mad4 by dimerisation with the multi-zinc finger protein Miz-1.  Myc:Miz-1 represses diverse target genes involved in growth arrest, differentiation and other physiological processes; overexpression of Myc in tumours thus leads to both de-regulated proliferation and a loss of differentiation potential.  We have characterised the mechanism whereby Miz-1 recognises diverse target genes and have identified additional novel proteins that interact with Miz-1.  This work will have relevance for the design of therapeutic strategies aimed at tumours associated with Myc overexpression.  

Transcriptional targets of mammalian Mad family proteins  
Dr S.C. Wright  

Myc and Mad family proteins are transcription factors that function as central regulators of cell proliferation, differentiation and apoptosis.  Myc promotes cell cycle progression; the overexpression of Myc in many human tumours results in uncontrolled cell division and a loss of differentiation.  The biological activity of Myc is normally antagonized by the Mad family proteins (Mad1, Mxi1, Mad3 and Mad4) that are thought to function as tumour suppressors.  The identification of transcriptional targets of Myc and Mad proteins is essential for the ultimate design of therapeutic strategies aimed at tumours associated with de-regulation of Myc or Mad.  We have used a combination of chromatin immunoprecipitation and genomic microarrays to identify genes that are bound and transcriptionally regulated by the various Mad family members in normal and tumour cells.   

Expression profiling of the NEP family of metalloproteinases in prostate cancer  
Dr B.A. Usmani and Prof. A.J. Turner  

The endothelin (ET) system in human prostate cancer (PC) is a novel therapeutic target since ET is elevated in men with metastatic PC.  

Alterations in the stroma profoundly influence tumour progression for example by increasing the availability of mitogenic peptides such as endothelin (ET-1).  ET-1 exerts both an autocrine (epithelial) and a paracrine (stromal) influence on growth. ET-1 is generated from its inactive precursor big-ET-1, by endothelin-converting enzyme (ECE-1), which exists as four distinct isoforms; ECE-1a, ECE-1b, ECE-1c and ECE-1d.  This study investigated the interaction between metastatic PC epithelial cells and stromal cells, both of which express ECE-1.  Previously we have reported that ECE-1 expressing stromal cells from benign and malignant prostate can greatly increase invasive potential and that specific inhibition of endogenous ECE-1 activity in these stromal cells can significantly reduce PC-3 invasion.  Our present data show siRNA duplexes targeted to ECE-1 reduced protein expression in stromal cells with a subsequent reduction in PC-3 invasion.  The addition of ET-1 to ECE-1 depleted stromal cells increased invasion but not to the level of control PC-3 cells suggesting a novel role for ECE-1 independent of ET-1 activation.  The effect of ECE-1 isoforms on cell invasion was examined and showed that transient over-expression of ECE-1c in PC-3 cells or stromal cells increased cell invasion significantly whereas, transient expression of ECE-1a into PC-3 cells or stromal cells reduced cell invasion.  Transient expression of ECE-1c in PNT1-a cells, a low invasive cell line which lacks ECE-1, dramatically increased invasion of these cells.  

The up-regulation of ECE-1 expression in metastatic cells highlights its role in cancer progression. Hence, siRNA technology has been used to suppress ECE-1 expression.  A synthetic siRNA duplex molecule has been cloned into a pSUPER-based plasmid and transfected into both epithelial (PC-3) cells and mouse stromal (STO) cells.  Diminished ECE-1 expression in transfected cells was assayed by western blotting and RT-PCR.  Initial data show ECE-1 level was decreased in transfected cells at 72 hour post-transfection by approximately 60%.  

We have already shown that each ECE isoform has a different subcellular localisation pattern and that the relative expression of each isoform can effect tumour cell invasion in vitro.  NEP, a molecule related to ECE inactivates a range of regulatory peptides and has been shown to be drastically down regulated in prostate cancer cells.  In addition to catalytic activity, NEP can exert biological effects through protein-protein interactions.  NEP has been shown to interact with p85 and p110ß, which are sub-units of the intracellular signalling molecule PI3 kinase.  The interaction of ECE with cellular proteins from prostate cancer cell lines of varying metastatic potential was investigated using SELDI-TOF mass spectrometry.  Two novel peaks at ~ 67 and ~99KDa were observed.  The proteins associated with these peaks have yet to be identified and the interaction with ECE confirmed using immuno-precipitation and western blotting.