research report

University of Leeds

2003/4

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

School of Medicine
Dean of the School: Professor E.W. Hillhouse

Academic Unit of Epidemiology and Health Services Research
Head of Academic Unit: Professor C.P. Wild

The role of free radical induced DNA damage during the evolution of oesophageal adenocarcinoma.
Dr L.J. Hardie, Mr G.W.B. Clark, Prof C.P. Wild

The incidence of oesophageal adenocarcinoma (OA) is increasing rapidly in the Western world and is associated with a poor prognosis. OA develops from a pre-malignant condition known as Barrett's oesophagus (BE) in which the normal squamous epithelium of the oesophagus is replaced by a specialised intestinal epithelium. Gastro-oesophageal reflux (GOR) is a major risk factor for both BE and OA however no molecular mechanism has been established to explain this association. This study has investigated the association between reflux, DNA damage and the evolution of OA. We have optimised and validated the standard and Fpg-modified comet assay for use on endoscopic biopsies and measured levels of DNA damage in control, BE and OA patient groups. We have demonstrated increased levels of DNA damage in Barrett's mucosa of BE and OA patients compared to matched normal squamous and gastric mucosa with the highest levels of DNA damage measured in Barrett's mucosa of OA patients. In addition we have demonstrated that BE patients with DNA damage in the highest quartile had an increased risk of developing OA. Smoking was associated with higher levels of DNA damage, as was reporting of current reflux symptoms in the control group, current use of non-steroidal anti-inflammatory drugs/aspirin were associated with a reduction in DNA damage. Collectively data from this study imply a condition of genetic instability in the pre-malignant Barrett's mucosa which may affect the likelihood of progression to OA.

Genotoxic effects of binary carcinogen exposure in DNA and skin cells.
Dr M.N. Routledge, Prof. E. Ingham, Prof. C.P. Wild

Environmental carcinogens such as UV radiation or mutagenic chemicals in diesel fumes are usually studied as isolated exposures. However, in everyday life we are exposed to combinations of such carcinogens and interactions between such carcinogens may be important in the carcinogenic process. We have previously shown that the UV exposure of DNA damaged by a chemical carcinogen, benzo[a]pyrene (BP), causes enhanced mutagenicity compared to the individual exposures. We are now investigating whether similar effects are seen in skin cells as UV radiation is a known cause of skin cancer. Both skin cell lines and primary skin cells obtained from donors are being used in these studies. To date we have shown that exposure of the skin cells to BP followed by UV irradiation leads to enhanced levels of DNA damage as measured by the comet assay, which detects induced single strand breaks in the DNA of single cells. We are currently adapting the supF mutation assay that we have used in previous studies so that we can measure the induction of mutations in the skin cells exposed to the binary combination of UV and BP.

Identification of Barrett's Oesophagus patients at high risk of progression to oesophageal adenocarcinoma using molecular markers.
Prof. D. Forman, Dr M. Scott, Prof C.P. Wild

Oesophageal adenocarcinoma (OA) develops from a pre-neoplastic condition known as Barrett's oesophagus (BE). This change is accompanied by a series of molecular alterations in cell cycle regulatory genes. These alterations may identify BE patients at particularly high risk of disease progression. OA cases arising in a Northern Ireland prospective cohort of BE patients were identified and matched with controls from within the cohort, who had not developed OA. Twenty-two BE patients developed OA; one patient developed an oesophageal adenosquamous carcinoma, a further six patients developed undifferentiated or unspecified carcinomas of the oesophagus and six developed high grade dysplasia, giving a total of 35 cases. Histology blocks were retrieved from the time of diagnosis and immunohistochemical staining performed for cyclin D1, p53, ¥â-catenin and Cox-2. The odds of positive staining for cyclin D1 was not raised in cases compared to controls. Moderate elevation in the odds of positive staining for Cox-2 or ¥â-catenin was seen in cases (especially when restricted to definite adenocarcinomas) but statistical significance was not reached. Compared to controls, the odds ratio for biopsies from patients who developed incident oesophageal adenocarcinoma showing diffuse or intense staining for p53 was substantially elevated at 8.38 (95% CI, 1.86 to 37.7). This suggests that positive staining for p53 in biopsies of BE patients is associated with an increased risk of progression to OA.

Academic Unit of General Surgery, Medicine and Anaesthesia
Head of Academic Unit: Prof. P.D. Howdle

Tumour vascularity, cellular proliferation and the role of angiopoietins in colorectal cancer.
Dr D. Burke, Mr J. Uddin, Dr P. Jones, Prof. P. Quirke

Tumour angiogenesis is an important process for tumour progression and is regulated by angiogenic proteins secreted by the malignant cells. Angiopoietins are recently identified angiogenic proteins that act upon a distinct endothelial cell receptor -Tie2. Tumour angiogenesis plays an important role in colorectal cancer development, but the mechanism by which this occurs is unclear. The relationship between tumour angiogenesis, Angiopoietin and Tie2 expression, and epithelial cell proliferation may be vital in understanding tumour biology but is not well understood.

Immunohistochemical studies have been performed on greater than 600 colorectal tumour and matched normal specimens from a large, national clinical trial. These studies quantify expression of angiopoietins-1 and -2, Tie2, tumour vascularity, and cellular proliferation in these specimens. Results demonstrate a significant increase in Angiopoietin 2 in tumour compared to normal tissue. This is associated with an increase in tumour cellular proliferation and microvessel density in Angiopoietin 2 positive tumours. This is the largest study to date of Angiopoietins in solid malignancies. The data suggest a role for Angiopoietin 2 in the progression of colorectal cancer, and particularly in the switch to an angiogenic state. The angiopoietins may represent potential targets for anti-angiogenic therapy.

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

Functional characterisation of human EBI homologues.
Dr J.M. Askham, Dr E.E. Morrison

EB1 is a microtubule-associated protein which interacts with the APC tumour suppressor protein. The interaction participates 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. APC mutations commonly associated with colorectal cancer result in a loss of the APC/EB1 interaction, which 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. Furthermore, whilst EB1 and EB3 bind to the dynactin component p150Glued, RP1 does not. Having previously shown that p150Glued regulates APC/EB1 binding, this new data suggests the RP1/APC interaction is not regulated by p150Glued. Live-cell video microscopy of GFP-tagged normal and chimeric EB proteins has demonstrated that all three associate with polymerising microtubule tips with varying affinities, and has enabled identification of the protein domains responsible for this microtubule association. Additionally, identification of multiple EB protein-binding regions in APC and demonstration that APC binds multiple EB proteins has raised the possibility that APC promotes microtubule stability by cross-linking adjacent microtubule-bound EB proteins.

The new data demonstrates the complexity of molecular interactions involving the C-terminus of APC, all of which are lost in colorectal cancer cells. This work has important implications for the understanding of both 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 such as breast cancer. We have identified a large candidate gene (OSC1) with a novel domain structure, which has homologies to proteins already implicated in carcinogenesis. Additionally, multiple intragenic homozygous deletions have been demonstrated in OSCC cell lines.

OSC1 expression is being investigated at both the RNA and protein level in oral samples and a range of tissue types. At least 8 different transcripts and 5 novel exons have been identified so far, and their expression pattern in normal and malignant samples is being analysed. Two peptide antibodies for the N-terminus and C-terminus of OSC1 have been commercially produced, their specificity for OSC1 has been confirmed by ELIZA, immunofluorescence co-localisation and Western blotting using OSC1-GFP constructs. These OSC1 antibodies are now being used to determine protein expression in normal oral and breast musosa, premalignant lesions and tumour specimens. We are currently establishing short interfering RNA (siRNA) technology to investigate the biological effect of knock down of OSC1 expression in breast cancer cell lines using cell proliferation, cell survival and invasion assays.

Characterisation of this very large and complex tumour suppressor gene OSC1 will increase our understanding of the mechanisms involved in carcinogenesis in oral squamous cell carcinomas and other malignancies.

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

A useful 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 concentrate 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. In the last year we have also shown that c-myb has also a haploinsufficiency phenotype. We are now in the process of generating ES cells which carry an oncogenic version of c-myb by gene targeting.

A translocation that fuses the AML1 to the ETO gene generates a chimaeric protein with leukaemogenic properties. Mice and ES cells, which carry the human AML1-ETO fusion protein knocked into the wild-type AML1 locus die at day E12. In order to study the effect of AML-ETO expression of haemopoiesis, we have examined the differentiation properties of knock-in ES cells. Our experiments indicate that in contrast to the c-myb knock-out myelopoiesis in AML1-ETO ES cells is greatly diminished and it was not possible to yield enough cells to study the effect of AML1-ETO on target gene expression. We therefore decided to study human cells carrying the translocation and could for the first time demonstrate how AML1-ETO deregulates target genes. We could show that AML1-ETO acts as a repressor of gene expression; however, this repression is not permanent and should be amenable to therapeutic intervention.

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

GM-CSF is also often abnormally expressed as an autocrine growth factor in acute myeloid leukaemia (AML) and juvenile myelomonocytic leukaemia.

We are investigating mechanisms that contribute to the normal inducible regulation of the GM-CSF gene in white blood cells, and to the abnormal constitutive activation of the GM-CSF gene in AML. We have previously shown that GM-CSF expression in myeloid cells is dependent upon an inducible transcriptional enhancer that undergoes chromatin remodelling to form a DNaseI hypersensitive site (DHS) when the GM-CSF gene is activated. We have shown that the enhancer is activated via 2 NFAT binding sites which are themselves sufficient to direct the formation of an inducible DHS. Furthermore, activation of the enhancer by NFAT is associated with increased accessibility to nucleases such as DNaseI, micrococcal nuclease and restriction enzymes. This increased accessibility accounts for the inducible binding of many transcription factors, such as Sp1 and AML1, to the enhancer. Activation of the enhancer is also associated with extensive mobilisation of the nucleosomes across the enhancer, indicating that the enhancer region is creating an open chromatin conformation spanning the GM-CSF locus. In parallel studies we are also assaying changes in chromatin structure that occur in AML cells.

Tumour initiation and progression in the ApcMin mouse model of familial adenomatous polyposis.
Dr P.L. Coletta, Dr C. Bonifer and Dr M.A. Hull

The Adenomatus polyposis coli tumour suppressor gene is mutated in the hereditary form of colorectal cancer, familial adenomatous polyposis (FAP) and in the majority of sporadic colorectal cancers. It is also mutated in a number of mouse models of FAP including the ApcMinmouse. We have previously shown that the inducible form of cyclooxygenase, Cox-2, is upregulated in macrophages early in this pathway in the ApcMin mouse

In addition to the changes in macrophage gene expression in ApcMin/+ mice, we have also show that there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition, ApcMin/+ mice show parallel depletion of splenic NK cells, immature B cells and B progenitor cells in bone marrow due to complete loss of IL-7 dependent B cell progenitors. Using bone marrow transplantation experiments into wild-type recipients, we have shown that the capacity of transplanted ApcMin/+ bone marrow cells for T and B cell development appears normal. In contrast, although the ApcMin/+ bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow, transplanted ApcMin/+ animals subsequently underwent lymphodepletion. CFU-F colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in bone marrow of ApcMin/+ mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of FAP.

Inhibition of Helicobacter pylori induced epithelial proliferative responses: a novel approach for cancer chemoprevention.
Dr J.E. Crabtree, Prof. M.F. Dixon, 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 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. We hypothesise that blocking this TMPS cascade will prevent H. pylori induced epithelial hyperproliferation associated with malignant transformation of gastric epithelial cells. This hypothesis is being tested in a model system using a specific EGFR-tyrosine kinase inhibitor. The effects of the inhibitor on H. pylori induced changes in gastric pathology, epithelial cell proliferation, apoptosis, activation of downstream signalling pathways and gene expression is being examined. To investigate the importance of H. pylori induced EGF receptor signalling in the model system, ADAMs genes, EGF receptor (EGFR), EGF ligands and COX2 have been characterised in the model. Long term infection with H. pylori is associated with marked increases in gastric expression of ADAM17, COX-2 and EGFR, demonstrating the importance of this signalling pathway in the model.

Further defining the role of estrogen receptor b in breast cancer: using variant isoforms to predict hormone response.
Dr V. Speirs, Dr D.J. Scott

The role of ERb in breast pathobiology still remains unclear. However, it is becoming apparent that the overall estrogenic/antiestrogenic response of a given cell or tissue appears to be dependent on the expression, not only of wild type ERb but also variant isoforms. Despite the presence of these variants in breast cancer, surprisingly little is known about their biological role. The aim of this project is to identify the expression and function of ERb variants in tamoxifen (TAM) resistance. This is currently on-going using tissue microarrays prepared from consented archival breast specimens. We have additionally developed a TAM-r clone of the MCF-7 cell line through continuous culture with 0.1mM TAM. Real time PCR analysis was used to determine the expression levels of ERa, ERa3D, ERa7D, ERb1, ERb2/cx, ERb5, ERb5D in TAM-r cells compared with TAM-sensitive (TAM-s) cells. Although no significant difference in ERa and b and their variant isoforms was observed, subsequent microarray analysis (in collaboration with Dr Indra Poola, Howard University, Washington DC) has revealed several candidate genes that may provide an insight into the mechanism of TAM-r. The functional significance of these is currently under study.

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, Dr M.S. Gilthorpe

The work to date on this project has focused on the development of specific flow cytometric assays to identify the various sub-types of lymphoproliferative disorders. We have analysed the surface protein expression profile of normal and neoplastic B-cells with a panel of 66 antibodies identified from previous reports that showed differential expression either at the mRNA level from microarray studies, or directly from protein expression analysis. Three samples each of bone marrow from normal individuals, from patients with follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, mantle cell lymphoma, chronic lymphocytic leukaemia, monoclonal gammopathy of uncertain significance, and myeloma, and 3 normal lymph nodes have been analysed. Protein expression levels have been analysed with dChip software. The software incorporates a 'compare samples' function that identifies the proteins that are most differentially expressed between cell populations. Hierarchical clustering was then used to group antigens, and antigens from different clusters were identified that could provide additive separation when combined. Antigens with the least power to differentiate normal and neoplastic B-cells were excluded from further analysis.

We have now commenced the second stage of assessing 39 antigens on a larger series of cases. To date we have assessed this panel on a further 26 cases, including 7 cases of follicular lymphoma, 5 marginal zone lymphoma, 3 mantle cell lymphoma, 3 diffuse large cell lymphoma, 1 Burkitts, and 1 Hairy cell leukaemia. This round of analysis will permit the development of specific assays to differentiate these disorders from normal B-cells and from each other. These assays will then be applied to normal individuals and family members of patients with lymphoproliferative disorders.

A molecular genetic analysis of DNA repair and bladder cancer.
Mr S.C. Sak, Prof. D.T. Bishop, Dr A.E. Kiltie

One of the major bladder cancer risk factors, namely cigarette smoking, produces chemical carcinogens and reactive oxygen species which damage DNA. Hence DNA repair capacity is a likely factor in bladder cancer predisposition. Mutations in DNA repair genes are found in normal individuals at polymorphic frequency. We are studying single nucleotide polymorphisms (SNPs) in the DNA repair genes XRCC1 (involved in base excision repair) and XPC (involved in nucleotide excision repair) in DNA from bladder cancer patients presenting for surgery at St James's University Hospital, Leeds, and suitable controls. We expect DNA repair mutations to be more frequent in cases, and to exert their influence by their interaction with cigarette smoking. Over 500 patients and 500 controls have been recruited to the study. Questionnaires detailing smoking history, occupational exposure and family history have been administered, a blood sample obtained and DNA extracted from this for each subject. DNA is to be sent to the CR-UK genotyping service in Oxford in April 2004. Twenty two XPC SNPs and fourteen XRCC1 SNPs are to be studied. Those which cannot be genotyped using Taqman will be genotyped using other techniques in Leeds. The data will then be analysed and correlated with the clinical information obtained.

Investigation of the role of TOGp in colorectal cancer.
Dr E. E. Morrison, Dr J. M. Askham and Dr M. Adams

TOGp is a microtubule-associated protein. Its function is controversial, with conflicting evidence suggesting roles in promoting either microtubule growth or shrinkage. By imaging TOGp fragments fused to GFP in living cells we have found that the centrosomal localisation of this protein can be mediated by at least two separate C-terminal regions. An interaction between the TOGp C-terminus and the TACC domain present in the TACC family of proteins has been suggested to target TOGp to centrosomes. We have found that an isolated TACC domain fused to GFP specifically localises to both centrosomes and growing microtubule tips in living cells. This recapitulates the TOGp distribution previously defined by immunostaining, suggesting that the TACC-GFP fusion protein achieves its distribution by directly binding to TOGp already localised at these sites and therefore arguing against a role for TACC proteins in mediating the TOGp centrosomal localisation. This data also supports the hypothesis that TOGp promotes microtubule growth rather than shrinkage. A TOGp C-terminal fragment expressed and purified as a GST fusion protein precipitated gamma-tubulin from cell extracts, providing an alternative mechanism for the TOGp centrosomal localisation. Overexpression of the TACC-GFP fusion protein caused severe defects in microtubule anchorage at centrosomes, consistent with a function for the TOGp-TACC protein interaction in this process. Furthermore, overexpression of a large TOGp N-terminal fragment severely compromised microtubule organisation in transfected cells. Our data suggest that TOGp plays an important role in organising microtubules and promoting microtubule growth in mammalian cells.

Academic Unit of Pathology
Head of Academic Unit: Prof. P. Quirke

Selection of patients for adjuvant therapy in colorectal cancer ¢®V getting it right with molecular profiling.
Prof. P. Quirke, Dr M. Seymour

Many of the projects in this study have started to mature. Quasar 1 data on 630 patients have been analysed. Initial results confirm the strong value of pathology in predicting prognosis in Dukes' B cancers. The low event rate in Dukes' Bs at 5 years precludes identification of predictive markers and we are currently doubling the size of the pathology component. In collaboration with Genentech we have run 70 Dukes' B cancers with follow-up on whole genome Agilent arrays and are currently analysing this data for response to therapy. We hope to test prognostic and predictive genes on the tissue microarrays generated for Quasar 1.

In the MRC FOCUS study we have analysed 17 markers on 800 patients. This confirms thymidylate synthase to be a very interesting marker, with other markers contributing little to the response to 5FU. This has a platform presentation at ASCO. Analysis of response to oxaliplatin and irinotecan are underway. We also have new data on DNA repair pathways that show major abnormalities of double strand break repair. We will apply these to Quasar 1 and FOCUS to identify the clinical relevance of these important proteins.

FACULTY OF BIOLOGICAL SCIENCES
Chairman of Faculty: Professor K.T. Holland

School of Biochemistry and Microbiology
Head of the School: Professor A.J. Turner

Investigation into the role of Bcr-Abl-induced abnormalities in glucose transport regulation in the pathogenesis of chronic myeloid leukaemia.
Dr. K. Barnes, Ms. E. McIntosh, Ms. D. Itchayanan, Dr. J. Bentley, Prof. S.A. Baldwin

An important function of haemopoietic growth factors such as IL-3 appears to be the maintenance of a sufficient rate of cellular glucose uptake to suppress apoptosis. We have demonstrated, using haemopoietic cell lines as models, that maintenance of glucose uptake is dependent on the PI 3-kinase pathway and involves actin- and tubulin-mediated translocation of the GLUT1 glucose transporter from the cell interior to the surface. A key step in the development of chronic myeloid leukaemia (CML) is the inappropriate survival of haemopoietic stem cells in the absence of IL-3, associated with the expression of the chimaeric tyrosine kinase, Bcr-Abl. To investigate the possibility that Bcr-Abl suppresses apoptosis in a fashion similar to that of IL-3 we have exploited the IL-3-dependent haemopoietic cell line TonB210, in which expression of Bcr-Abl is tetracycline-inducible. In these cells, Bcr-Abl expression was found to mimic IL-3 both in signalling (i.e. activation of the kinase Akt) and in its effect on glucose transport: maintenance of a high Vmax for glucose uptake in induced, cytokine-deprived cells was associated with retention of the majority of cellular GLUT1 in the plasma membrane. Treatment of induced cells with the specific Bcr-Abl inhibitor STI571 (Glivec) led to a profound decrease in glucose uptake associated with relocation of the majority of the GLUT1 molecules into the cell interior. Inhibition of glucose transport is therefore likely to play an important role in the therapeutic action of this drug, and the signal transduction pathways involved may offer novel therapeutic targets for CML.

Studies on the expression and intracellular localisation of viral and cellular oncoproteins.
Dr G.E. Blair, Dr S.C. Wright, Dr A. Whitehouse and Dr E.H. Hewitt

This project aims to provide cell biology equipment for the study of viral and cellular oncoproteins in mammalian cells. We have purchased an Amaxa nucleofection system for the high-efficiency transfer of plasmid DNAs into primary human cells and optimised transfer of marker plasmids into human dendritic cells. We are currently tendering for the purchase of a digital microscopy system to visualise protein expression in mammalian cells.

High risk human papilloma virus oncogenes: studies on antigen processing and presentation in transformed cells, organotypic cultures and cell lines.
Dr G.E. Blair, Dr G. Bottley and Ms J. Jarvis

Over the past year we have utilised two cell systems to analyse specific effects of high risk Human Papillomavirus 16 (HPV16) E7 on the expression of MHC class I molecules. The first system uses an inducible cell line, transfected with the HPV16 E7 gene under the control of a tetracycline-repressible promoter. This cell line will only express E7 when cultured in the absence of tetracycline (tet). Thus it has been possible to examine the phenotypic consequences of E7 expression in a non-virally transformed cell line. Removal of tet induced expression of HPV 16 E7 in this cell line and decreased surface MHC class I expression by around 30% after 72 hours, compared to cells cultured in the presence of tetracycline. A parental cell line stably transfected with a control tet-repressor construct showed no significant difference in surface MHC class I levels. The presence of E7-specific transcripts was detected by RT-PCR. The presence of functional E7 protein was demonstrated by determining cell cycle profiles and proliferation, since E7 has previously been reported to stimulate the cell cycle.

The second, complementary, system uses a technique known as RNA interference. Small interfering RNA sequences (siRNAs) specific for the HPV16 E7 gene have been chemically synthesised and transfected into either HPV16-transformed or control cell lines. These RNA sequences will target E7 mRNA for degradation by cellular enzymes for several days following transfection and we have shown that they knock down E7 expression by around 90%. HPV16-transformed human cells (Caski and SiHa) transfected with E7-specific siRNA displayed a small but significant increase in cell surface MHC class I expression. No differences in surface MHC class 1 expression was found in siRNA-treated, non-virally-transformed keratinocyte cell lines.

These results, taken in combination, strongly suggest that high risk HPV E7 actively down-regulates surface MHC class I molecules in transformed cells, thus facilitating the escape of virally-transformed cells from the cellular immune system. These cell systems are now being used to investigate how E7 subverts cellular mechanisms that result in these changes in MHC class I expression.

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

Although survival rates have improved for colorectal cancer, novel approaches are needed. In this project our aim is to investigate the use of immunotherapy as an approach to target colon carcinoma cells. Initially we consulted micro array databases to select targets (tumour associated antigens) which were uniquely expressed or over expressed on colon carcinoma cell lines and tissue. The first target of interest we selected was a protein called CDCP1, a Cub Domain Containing Protein 1. This protein has recently been described to be over-expressed in lung and colon cancer, and has also been associated with later stages of carcinogenesis. Its function is not clearly understood although it is thought to be involved in cellular signalling and cell interactions. At least two transcripts of this protein have been described, transcript 1 (the full length mRNA) which comprises nine exons and transcript 2 which contains only the first four exons.

We are currently investigating the expression of CDCP1 in a panel of 13 colon carcinoma cell lines and an immortalised colon cell line. Three primary cell types (HUVEC, fibroblasts and peripheral blood leukocytes) are being used as negative controls. To investigate the specificity of CDCP1 as a marker of colon cancer, a panel of other carcinoma cell lines has been included. We have designed primers to measure the expression of spliced variants of CDCP1 mRNA. Protein expression of CDCP1 is also being measured using flow cytometry to look at surface CDCP1 and Western Blotting is being used to look at total protein expression.

We have found that CDCP1 is present on most, but not all colon carcinoma cell lines. We are now intending to look at tissue samples of different stages of colorectal cancer. Future work will include the use antigen-presenting cells to stimulate a cellular immune response against CDCP1. We have also identified a number of other protein targets using micro-array data and we will analyse these targets using a similar approach to that adopted for CDCP1.

Functional Genomics of a proteinase gene family (M13; NEP) in human prostate cancer.
Dr. B.A. Usmani and Prof. A.J. Turner

Prostate Cancer (PC) cells which survive androgen withdrawal therapy often switch allegiance to alternative growth pathways, fuelled by mitogenic peptides, which are normally inactivated by the Neprilysin (NEP, M13) family of enzymes. In human PC, NEP loss contributes to metastatic androgen-independent PC progression by allowing mitogenic peptides (e.g. endothelin-1) to stimulate cell proliferation. ECE-1, a close homologue of NEP, generates active endothelin-1. Recent analysis of archive patient PC tissue microarrays revealed 54% high grade and 19% low grade patient tumours expressed ECE, whereas 15% high grade and 25% low grade tumours expressed NEP. ECE-1 exists as four isoforms a,b,c,d. ECE-1c appears to be the common isoform present on cell membranes both in epithellia and stroma of highly malignant tumours. Interestingly, TMA cores representing benign prostatic hyperplasia (BPH) revealed mainly NEP expression (81%).

Co-culture matrigel invasion data show that stromal-epithelial interactions can influence the invasive ability of PC cells as a consequence of their altered NEP and ECE-1 activities (in press). Transient expression of ECE-1c isoform only, in PC cells increased invasion. Primary malignant stroma increased PC cell invasion more than primary benign stroma. Immunohistochemical analysis showed that ECE-1 expression is up-regulated in stroma adjacent to tumour epithelia.

Recently we have recognised the ECE-1a isoform unexpectedly in the nucleus of malignant cells and the significance of this is currently being explored. Also, we are currently investigating a newly recognised suppression of ECE-1 by dihydrotestosterone (DHT) and the results of these studies may be highly relevant in terms of PC therapy.

Kaposi's sarcoma: investigating the human herpesvirus-8 latent-lytic switch.
Dr A. Whitehouse and Dr D. Goodwin

Kaposi's sarcoma-associated herpesvirus or human herpesvirus 8 (HHV-8) is a lymphotrophic g-2 herpesvirus associated with Kaposi's sarcoma (KS), a major neoplasm particularly in AIDs patients. KS is an endothelial neoplasm. However, the primary reservoir of latent virus is established in B lymphocytes. Reactivation to lytic HHV-8 infection from the latently infected lymphoid reservoir is essential for the spread of infectious virus to the endothelium. Therefore, reactivation from the latent state into the lytic cycle, allowing active HHV-8 gene expression and replication is a necessary antecedent step in KS development. However, little is known about the nature of the latent-lytic switch. We and others have demonstrated that the ORF 50 gene products in g-2 herpesviruses are essential transcriptional regulators, activating the lytic replication cycle. Moreover, forced expression of this protein under the control of an heterologous promoter, has the ability to trigger reactivation of the lytic replication cycle from an established latent infection. This suggests that regulation of the ORF 50 promoter itself is a key component of the switch from latency to a lytic replication cycle and is an important determinant of the natural history of the viral infection. Analysis suggests we have identified a specific cellular transcriptional protein, HMG-1, which interacts with the KSHV ORF 50 promoter and synergistically activates it in conjunction with the ORF 50 protein itself. At present further work is being performed to determine whether this cellular factor, and others tentatively identified, play central roles in regulating the latent-lytic switch in gamma-2 herpesviruses.

Identification of genes that are transcriptionally repressed by Mxil.
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 and is overexpressed in many human cancers. The activity of Myc is antagonized by the Mad family (Mad1, Mxi1, Mad3 and Mad4) proteins 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 deregulation 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 Mad family member, Mxi1.

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

The c-Myc proto-oncogene product normally functions to regulate cell proliferation and differentiation; the overexpression of Myc in many human tumours is associated with both an increased rate of cell cycle progression and a block in cellular differentiation. Myc may act as either a transcriptional activator or a repressor to regulate the expression of target genes; transcriptional repression by Myc is thought to be important in cell transformation and tumourigenesis. We have shown that Myc represses transcription of the differentiation-associated Mad family member, Mad4, in proliferating cells; repression of Mad4 by Myc may contribute to the perturbation of cellular differentiation in tumours associated with Myc overexpression. Myc mediates the transcriptional repression of Mad4 via its association with the zinc-finger protein, Miz-1. We have identified other proteins involved in transcriptional regulation by Miz-1 and are currently analyzing the detailed mechanism of Miz-1 activity; this will be relevant for the design of therapeutic strategies aimed at tumours associated with aberrant Myc expression.

YORKSHIRE CANCER RESEARCH PHOTODYNAMIC THERAPY GROUP
Centre for Photobiology and Photodynamic Therapy

A multidisciplinary approach to the development of photodynamic therapy for cancer treatment.

(A multidisciplinary group of staff from the School of Biochemistry and Molecular Biology, the School of Biomedical Sciences, the Department of Colour and Polymer Chemistry, the School of Medicine, Cookridge Hospital, Leeds General Infirmary, St James's Hospital and Goole & District Hospital.)

S B Brown (Principal Investigator) et al

Yorkshire Cancer Research has provided major support for the development of photodynamic therapy (PDT) by its funding of the Leeds Centre for Photobiology and Photodynamic Therapy for more than 15 years. This Centre which is the largest of its kind in Europe has around 30 scientists and clinicians in the Yorkshire region and many collaborations both in the UK and internationally.

As outlined in previous years, PDT is a developing approach which uses a combination of light and a photosensitising drug (a sensitiser) to selectively destroy unwanted tissue such as tumours. The approach is able to achieve a high degree of targeting partly because of the ability of photosensitisers to concentrate preferentially in tumours relative to normal tissue, but also because of our ability to direct light very accurately to the tumour area. Over the years, the YCR Centre has played a major role in the development of PDT globally, has treated many hundreds of patients and has developed a number of new and improved candidate drugs. Photopharmica, the company which has been formed to progress these developments commercially, has made substantial progress in the last year and is planning to lead the later stages of drug development into patients. It is likely that, following preclinical trials, our first PDT drug will be used to treat cancer patients late in 2004. With active involvement of YCR, it is hoped that Photopharmica will provide the vehicle by which the major investment of YCR and the University in PDT can be harnessed to benefit large numbers of patients.

Whilst Photopharmica is leading the later stages of drug development, the Centre is currently focused on developing novel PDT agents and approaches (especially tumour targeting) and also studying the basic mechanisms of action of photosensitising drugs. This report for 2003 concentrates on these aspects of our work and is divided into three sections covering chemical, biochemical and in vivo model studies.

Chemistry
Synthetic work in chemistry has focused on three areas. Firstly, in order to obtain structure-activity relationship data for our phenothiazinium photosensitiser series, we have prepared a wide range of systematically modified derivatives. Useful new compounds with PDT properties equal to our previous best compounds have been found. Secondly, attention has been directed towards a novel photosensitiser system, which is colourless and requires activation by near-infrared light (around 800 nm wavelength). Such compounds have the potential for treating deeper and larger tumours than is feasible with existing photosensitisers, because of the better penetration of infrared light through tissue compared with red light. Compounds have now been made which are very effective at generating singlet oxygen in solution when exposed to 800 nm light, and water soluble compounds, suitable for in vitro testing are in preparation. Finally, we have continued to investigate the concept of covalently linking a photosensitiser moiety to a bioactive molecule, whereby the linkage may be cleaved efficiently on exposure to red light. Suitable linkages have been identified and the preparation of model compounds for demonstrating the effect in vitro, is in progress.

Biochemistry
The biochemistry group has been studying the mechanism of uptake and mode of cell death of two major groups of photosensitisers. We have been able to show that one of the more promising phthalocyanine photosensitisers enters mouse fibosarcoma cells in vitro via a saturable process that is inhibited at low temperatures. These findings suggest that a receptor mediated process is involved although other mechanisms have not yet been ruled out. Identification of such a process may lead to the development of photosensitisers that utilise this mechanism more efficiently.

The initial site of photodynamic action and the subsequent events leading to tumour cell destruction vary dramatically with the structure of the photosensitiser being used. One class of photosensitiser, the phenothiazinium salts, is amenable to extensive chemical modification and therefore enables detailed structure-function studies to be carried out. The phototoxicity of over twenty of these compounds has been studied in vitro together with their physical and photo-physical properties. The studies have identified a number of compounds that are almost three orders of magnitude more phototoxic than methylene blue, due to a combination of increased lipophilicity and more specific mitochondrial targeting. Studies on the mechanism of cell death resulting from PDT with this group of sensitisers have shown that structural determinants also play a role in dictating whether cells die by apoptosis or necrosis. Using flow cytometry we have shown that apoptosis is induced by PDT with some of these photosensitisers, but necrosis is the predominant mechanism with methylene blue. This correlates with fluorescence microscopic analysis which shows that, although the initial sensitiser localisation patterns are similar for all this class of sensitiser, they behave differently upon exposure to light. Methylene blue relocates to the nucleus, whereas the more efficient photosensitisers redistribute to mitochondria and induce apoptosis.

Many photosensitising drugs are initially localised in the lysosomes of the cell. Upon irradiation these lysosomes are damaged releasing their contents into the cytoplasm. This effect can be utilised to deliver cytotoxic agents, which under normal circumstances remain lysosomal and ineffective, but upon release into the cytoplasm relocate to other subcellular targets causing cell damage. We have developed suitable sensitisers for this photochemical internalisation (PCI) technique and have been able to demonstrate efficient cell killing under conditions where PDT or the cytotoxic molecule alone has minimal effect. This novel technique could combine the localised treatment of PDT with the more diffuse treatment with cytotoxic drugs allowing for the treatment of large solid tumours in vivo.

We have continued to develop methods for linking photosensitisers to small peptides and have successfully synthesised and purified a protoporphyrin-peptide conjugate that shows increased binding to RIF-1 fibroblasts.

In vivo studies
Further significant progress has been made in the evaluation of new photosenstisers using in vivo model systems. We have examined the series of methylene blue analogues for their ability to selectively destroy tumour tissue, identifying two molecules which exhibit particularly favourable properties. These agents show greater anti-tumour efficacy and selectivity than the photosensitiser most widely used in clinical practice, without exhibiting the undesirable side-effects associated with many current PDT regimes.

More recently, we have examined a further series of 27 phenothiazinium derivatives and started more detailed characterisation of the most promising of these. Two of these compounds appear to offer further increases in selectivity, relative to the two phenothiazinium derivatives described above.

Advances have also been made in the understanding of how some of the newer photosensitisers can best be formulated for clinical use. By using a blend of solvents as a delivery vehicle and choosing the appropriate photosensitiser salt it has been possible to formulate these materials for intravenous administration without recourse to more complex approaches, such as the use of liposomal preparations. We have demonstrated the importance of drug formulation by showing that this can have a direct effect on important photosensitiser properties, including anti-tumour efficacy and tissue pharmacokinetics.