research report

University of Leeds

2006/7

FACULTY OF MEDICINE AND HEALTH
Dean of the Faculty: Professor E.W. Hillhouse

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

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

Methylene blue (MB) dye is selectively absorbed by Barretts epithelium (BE), the recognised precursor lesion for oesophageal adenocarcinoma. In some clinical settings the dye is used to aid visualisation of this metaplastic lesion and target biopsy sampling to areas of dysplasia. We have previously demonstrated that patients undergoing MB-chromoendoscopy have elevated levels of oxidative DNA damage in BE biopsy samples following this procedure. White light generated by the endoscope is thought to be responsible for this effect, via photoexcitation of MB.

This study aims to identify conditions which minimise DNA damage during methylene blue chromoendoscopy, without compromising visualisation of the tissue in clinical practice.
Chromoendoscopy conditions have been simulated in the laboratory and DNA damage levels measured in MB exposed oesophageal cell lines. We have demonstrated that MB has DNA damaging effects which are dose, light and time dependent in oesophageal cells. Moreover, the red light portion of endoscopic light is responsible for the DNA-damaging properties of MB. Utilising filters which exclude only red light, we can minimise DNA damage during MB exposure without compromising visualisation of tissue in vitro. These results have recently been submitted for publication and we are currently in the process of translating these findings into the clinical setting.

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

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. We found that reduced expression of MCPH1 causes premature chromosomal condensation (PCC). PCC is a hallmark of mammalian cells that begin mitosis before completing DNA replication. Using time-lapse imaging we have identified further mitotic defects including slower mitotic progression displaying aberrant chromosomal congression and micronuclei formation in MCPH1 deficient cells. This mitotic phenotype suggests that loss of MCPH1 function in tumours could cause mitotic errors resulting in aneuploidy development.

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 data indicates MCPH1 plays a role in resistance to chemotherapeutic agents such as Taxol through its involvement in the spindle checkpoint. Our immunohistochemistry data has identified reduced MCPH1 expression in 32% of breast cancers, particularly in higher grade tumours. We therefore hypothesize that, while germ line defects in MCPH1 cause microcephaly, somatic defects may cause aneuploidy development and resistance to chemotherapy.

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

GM-CSF is a growth factor that controls many aspects of myeloid cell growth and function. The GM-CSF gene is regulated by an inducible enhancer. In normal cells the enhancer undergoes inducible chromatin remodelling to form an inducible DNaseI hypersensitive site (DHS) where the local nucleosome organisation has been disrupted. In acute myeloid leukaemia (AML) GM-CSF is often abnormally expressed as an autocrine growth factor, and the enhancer forms an abnormal constitutive disrupted chromatin structure.

To study abnormal GM-CSF regulation in AML we purified blast cells or CD34 positive cells from 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 study 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.

We have used mast cells as a model for studying GM-CSF enhancer regulation in normal myeloid cells. We have found that enhancer activation is accompanied by inducible disruption of three nucleosomes associated with a composite GATA/AP-1 site and 2 composite NFAT/AP-1 sites respectively, and recruitment of transcription factor complexes to these sites. Fine structure analysis of the enhancer in AML reveals that all three nucleosomes are abnormally disrupted in a constitutive fashion in many cases of AML. In these AMLs, GM-CSF expression is not truly constitutive, but can be induced via kinase signally pathways.

Targeting tumour antigens and adjuvants to exosomes; novel delivery vehicles for tumour vaccines
Dr G.P. Cook and Dr A.A. Melcher

This project (which began in July 2006) requires the cloning of tumour associated antigens (TAA) into plasmid vectors that will allow mammalian expression and targeting of the TAAs to exosomes (small vesicles released by most cell lines). The exosomes will then be isolated, exposed to DC cells which will process and present the TAAs in order to induce a T cell response towards the tumour.

Two model systems are being used: a human based model using a melanoma TAA (gp100) and a mouse based model using the well-defined SIINFEKL epitope derived from ovalbumin. The initial stage of this project has been accomplished. The gp100 melanoma TAA has been cloned into a GFP tagged fusion protein that has previously been shown to direct the protein to exosomes (our own unpublished work). This TAA has been cloned into two different sites in the fusion protein to investigate the ability of different fusions to be processed and presented by DC. Stable cell lines have been established in the human melanoma cell line Mel888. Similarly the SIINFEKL epitope has also been cloned into two sites of the targeting GFP fusion protein and stable mouse B16 cell lines established. We are currently investigating the levels of expression of these fusion proteins in exosomes. A plasmid that will potentially act as an adjuvant when co-expressed with the TAA-GFP fusion protein thus amplifying the T cell response has also been constructed.

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

Haemopoietic stem cells (HSC) provide long-term haemopoietic repopulation of all the blood cell lineages and are crucial in cancer treatment. While a single murine HSC with a defined phenotype (Tip-SP, CD34-c-Kit+Sca-1+Lin-) can reconstitute haemopoiesis in mice, human HSCs have not been isolated to this level of homogeneity. We have shown that using the CD133 marker and cell cycle staining, a population of human cells with the phenotype CD133+G0 can be isolated from cord blood. These cells exhibit the classic characteristics that define HSC populations including self-renewal and extensive proliferation capacity. The CD133+G0 cells also contain the highest frequency of stem cells reported to date as detected by long-term culture initiating cell (LTC-IC) in vitro assays. This indicated that CD133+G0 cells may be enriched in HSC activity.

We have now confirmed stem cell activity using in vivo assays in collaboration with Drs. D. Bonnet and D.Pearce (Cancer Research UK, London) and S. Howe and M. Blundell (Institute of Child Health, London). Only the CD133+G0 cells generated both lymphoid and myeloid engraftment and as few as 5,000 cells allowed reconstitution of the haemopoietic system in irradiated hosts. A more extensive assessment of the CD133+G0 phenotype revealed that a large proportion of the cells are CD34+CD38- and the majority also have high aldehyde dehydrogenase activity when compared to their CD133+G1 counterparts. This is a strong indication that stem cell activity resides almost exclusively in the CD133+G0 population. On this basis we conclude that in cord blood, CD133+G0 cells are a highly enriched stem cell population.

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 bladder cancer may be treated by cystectomy or radical radiotherapy with a 50% 5 year survival rate for both. Use of a predictive test of radiosensitivity in individual patients would allow rational treatment selection. Radiotherapy induces DNA damage in the form of double strand breaks (DSB), which initiates recruitment of DSB signalling proteins and repair via non-homologous end joining or homologous recombination.

We hypothesised that an absence of or lower expression of DSB signalling proteins would reduce cellular DSB repair capacity, leading to cell death and therefore increased radiosensitivity.

We have studied 92 muscle invasive tumours from patients who received radical radiotherapy from 1995-2000, with known clinical outcome data. Immuno-histochemical staining of formalin fixed paraffin embedded tumour sections was performed for ATM, Rad50, MRE11, NBS1, H2AX, gammaH2AX and Ki67. A combined staining score was given for each based on percentage positive cells and intensity of staining.

We found Ki67 expression to be a marker of proliferation in this cohort, but it was not correlated with patient survival or expression of any of the DSB signalling proteins. Rad50, ATM, NBS1 and gammaH2AX showed no correlation with either cause-specific or overall survival. However patients with low H2AX expression had improved survival (p=0.03) as did patients with high MRE expression (p=0.004). Work is ongoing to determine p53 status in these tumours and this will be correlated with clinical outcome and DSB signalling protein expression.

Potassium channels as biomarkers of carcinogenesis in human colon

Prof. G.I. Sandle, Dr M. Hunter and Prof. K. McLennan

Dysplastic/neoplastic transformation of cryptal colonocytes is associated with depolarisation. Since potassium channels are a major determinant of the cell membrane potential, we evaluated the expression and activity of apical BK and basolateral IK channels in healthy human colon and colonic cancer using immunohistochemistry, real-time PCR and patch clamp techniques.

Surface expression of BK and IK protein was undetectable or reduced, respectively, in the majority of tumours. BK mRNA was unchanged in cancer patients, suggesting abnormalities in translation or post-translation events in this group. Unlike healthy mucosa, IK protein was detected in the nuclear membrane, and mRNA levels were increased, suggesting continued synthesis but abnormal trafficking of IK channels in colon cancer. Stimulation of PKC (which is increased in colon cancer) inhibited IK channels, causing cell depolarization. Thus the cellular depolarisation that occurs during colonic carcinogenesis may be explained by our results.

We have additionally characterised the human colonic apical BK channel in biophysical and molecular terms for the first time, and shown it to be expressed along the entire crypt in both active and quiescent ulcerative colitis, a pre-malignant disease. By contrast, basolateral IK channel expression was significantly decreased in these sub-groups of colitics, as in colon cancer.

If the changes in BK and IK channel expression observed in colon cancer are mirrored in adenomatous polyps, then these channels may provide a biomarker of malignant potential.

Functional significance of altered prostaglandin-signalling in hormone resistant breast cancer

Dr V. Speirs and Dr M. Cummings

Building on previous microarray technology in which 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 and TAM-sensitive cell lines (TAMr and TAMs, respectively), this projects focused on components of the prostaglandin-signalling pathway which were differentially regulated in TAMr cells, in particular 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme responsible for biological inactivation of prostaglandins. We found that 15-PGDH was robustly and significantly downregulated in TAMr cells. Of note was the fact that this gene was also downregulated in an independently established TAMr cell line, where MCF7 cells were cultured in 100 nM 4-hydroxytamoxifen (4HT) for 24 months, suggesting that 15-PGDH downregulation may be a common mechanism in the acquisition of TAMr. We tested this hypothesis by establishing stable TAMr clones that overexpressed human 15-PGDH, which we showed to be functional using a 15-PGDH activity assay. Cell growth assays showed that 15-PGDH-transfectants were significantly sensitised to 4HT (P <0.01), compared to empty vector controls whilst their growth rate in the absence of 4HT was unchanged. Finally, immunohistochemical analysis of tissue microarrays constructed from TAMr (n=160) and TAMs (n=250) breast tumours showed reduced expression of 15-PDGH in the TAMr group.

FACULTY OF BIOLOGICAL SCIENCES
Dean of the Faculty: Professor A.J. Turner

Institute of Molecular and Cellular Biology
Director: Professor S.W. Homans

Use of post-genomic technologies to discover new targets for colorectal cancer immunotherapy

Dr P. Robinson, Dr A.A. Melcher, Prof. P. Quirke, Dr G.P. Cook and Dr G.E. Blair

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 well 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 a number of proteins including Cub Domain Containing Protein 1 (CDCP1) as potential targets for immunotherapy.

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 showed the existence of two splice variants of CDCP1 which give rise to two protein isoforms which localise to different cellular compartments. We also investigated the expression of the two isoforms of CDCP1 in colorectal cancer and normal colon tissues, as well as in a panel of colorectal cancer cell lines. This generated an interesting finding, namely that levels of CDCP1 mRNA varied between the cell lines tested and that some degree of correlation was found with that of CEA and Ep-CAM expression. This was further substantiated by analysing expression of the protein products using flow cytometry. A paper describing this work has been published in FEBS Letters.

We also investigated a novel method termed Nucleofection for delivery of potential immunotherapeutic genes to human dendritic cells (hDCs). This is a non-viral gene delivery system that relies on a proprietary formulation to deliver plasmid vectors to the nucleus of targeted cells and is marketed as providing high gene efficiency to hDCs. Initial unsatisfactory results generated by this method have led us to optimise the preparation of the cells and Nucleofection protocols but with very limited success.

We are also evaluating seven genes identified from microarray data derived by one of us (P. Quirke) that encoded potentially immunotherapeutic antigens and were found to be up-regulated by 1.8 to 2.3 fold in colorectal cancer tissues compared to normal colonic epithelium.

Assessing the role of human papillomavirus (HPV) oncoproteins in regulating cell-mediated immunity: implications for cervical cancer

Dr G. Bottley, Ms J. Jarvis, Dr G.P. Cook and Dr G.E. Blair

Previous work from this 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 expression of the E7 protein, and determined changes to MHC class I expression. We have already shown that siRNA-mediated inhibition of E7 in HPV16- and HPV18-transformed cells results in significant up-regulation of cell-surface MHC class I molecules. We have also shown that induction of HPV16 E7 using a tetracycline-repressor system results in a significant down-regulation of cell surface MHC class I molecules. In addition, analysis of total cell MHC class I protein by Western blotting has confirmed the results obtained by flow cytometry on cell-surface MHC class I. This data taken together indicates that E7 from both HPV16 and HPV18 actively down-regulates cell surface MHC class I.

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 surfaces 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 were more resistant to NK cell lysis. Conversely, E7-induced cells that have reduced MHC class I were 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. We are currently analyzing expression of components of the antigen presentation pathway in the cell-based systems described above.

Perturbation of b-catenin signalling by the hepatitis C virus NS5A protein: implications for development of hepatocellular carcinoma

Prof M. Harris

Hepatitis C virus is a significant cause of hepatocellular carcinoma, however the molecular mechanisms linking chronic viral infection to development of liver tumours are poorly understood.

We are investigating the role of the NS5A protein in this process. Our data show that NS5A activates the phosphatidylinositol 3-kinase (PI3K) pathway, leading to stabilisation of the proto-oncogene b-catenin.

We have carried out both transcription and protein stability assays to analyse the activation and stabilisation of b-catenin by NS5A. Experiments using a non-phosphorylatable b-catenin mutant (S45A) demonstrate that, as expected, the stability and activity of this protein is not affected by NS5A, although surprisingly S45A was activated by lithium chloride, an inhibitor of GSK-3b, the major b-catenin kinase. This result suggests a further level of complexity to b-catenin activation. Analysis of a panel of NS5A mutants revealed that a mutant previously shown not to bind the PI3K SH3 domain or activate intact PI3K in vivo, retains the ability to stabilise b-catenin. This intriguing result raises the possibility that NS5A might modulate b-catenin signalling via an alternative pathway.

To test this we are using co-immunoprecipation analysis to map the binding sites on NS5A for PI3K. If this study shows a lack of correlation between the ability to bind PI3K and the activation of b-catenin we will investigate both the Wnt pathway and also the possibility that NS5A might act further down the PI3K cascade for example by influencing the recently identified direct interaction between the PI3K effector, Akt, and b-catenin.

Expression profiling of the NEP family of metalloproteinases in prostate cancer

Dr B.A. Usmani and Prof. A.J. Turner

Cross-talk between tumour and stromal cells contributes to cancer invasion and metastasis. 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. They differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. This study investigated the interaction between metastatic PC epithelial cells and stromal cells, both of which express ECE-1, using Matrigel invasion chambers. Previously we have reported that ECE-1 is present in PC cell lines and primary tissue ex vivo. Using Matrigel invasion assays we have shown 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. Investigation into the role of the ECE-1 isoforms on cell invasion shows interesting interactions between the presence and absence of ECE-1c and/ or ECE-1a in either stromal and/or epithelial compartments. Transient over-expression of ECE-1c in PC-3 cells increased invasion by 20%. Interestingly, over-expression of ECE-1a decreased invasion by ~50%. Transient expression of ECE-1c increased invasion of non-invasive PNT1-a cells by ~150%. Co-transfection of ECE-1a with ECE-1c completely suppressed the effect of ECE-1c in PNT1-a cells. Transfection of both epithelial cells (PC-3) and stromal cells with ECE-1a further reduces the invasion of PC-3 through Matrigel. Furthermore transfection of stromal cells with ECE-1a counteracts the ECE-1c induced increase of PC-3 invasion. The ECE-1 isoforms may therefore be relevant targets for effective therapy for prostate and other cancers and warrants further investigation.

Tissue microarray (TMA) results have suggested that ECE and its related metalloproteinase NEP are useful indicators of tumour progression. This has been filed as a diagnostic patent and proof of concept is currently being investigated in a clinical model.

Cellular genes involved in Kaposis sarcoma associated reactivation and lytic replication

Dr A. Whitehouse, Dr J. Boyne, Dr J. McBride and Miss F. Gould

The etiological agent of Kaposis sarcoma, Kaposis sarcoma associated herpesvirus virus (KSHV), is the most recently identified human tumour virus. The presence of the virus has also been detected in a variety of lymphoproliferative disorders including primary effusion lymphoma and multicentric Castleman's disease. Like other herpesviruses, KSHV has two distinct forms of infection, latent persistence and lytic replication. Although latent persistence has been implicated in tumourigenesis, it is evident that reactivation and lytic replication plays an important part in the pathogenesis and spread of KSHV infection.

The KSHV Rta protein is the key gene product which regulates viral lytic gene expression. Analysis of the KSHV Rta protein has shown that it is produced as immediate early protein, capable of autoregulating its own expression and has the ability to activate transcription of various viral and cellular genes. It activates transcription directly following interactions with promoters containing a specific sequence motif, or indirectly by interactions with cellular transcription factors.

We are currently investigating the role of two cellular proteins which interact with Rta. Hey-1 is a cellular transcriptional repressor protein which interacts with KSHV Rta. The interaction is a particular intriguing one, as Hey-1 represses KSHV lytic promoters, therefore why would Rta interact with a transcriptional repressor protein, given its in transcriptional activation? However, we have demonstrated that KSHV Rta acts as an ubiquitin E3 ligase, targeting Hey-1 for proteasomal degradation to allow the initiation and completion of the lytic replication cycle. The second cellular protein KSHV Rta interacts with is HMG-A. We have shown HMG-A can synergistically enhance KSHV Rta transactivation of lytic promoters, by directly binding to these lytic promoters and enhancing KSHV Rta binding to these promoters. These results demonstrate that the recruitment of cellular genes by KSHV Rta are essential for KSHV reactivation and lytic gene expression.

Regulation of the Miz-1 transcription factor

Dr S.C. Wright

Many tumours are characterised by deregulation of the Myc transcription factor, resulting in 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. This repression is mediated by the interaction of Myc with the multi-zinc finger protein Miz-1. We have characterised the mechanism whereby Miz-1 recognises diverse target genes, and have identified additional novel proteins that interact with Miz-1. We are currently determining the structure of Miz-1 in association with its interacting partners. This work will have relevance for the design of cancer therapeutic strategies aimed at disrupting the interaction of Miz-1 with its co-regulators.

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 members of the Myc/Max/Mad network in normal and tumour cells.