research report

University of Sheffield

2007/08

Faculty of Medicine
School of Medicine and Biomedical Sciences  

SECTION OF ONCOLOGY  
YCR Institute for Cancer Studies  


The effect of single nucleotide polymorphisms on clinical outcome and survival in the FOCUS clinical trial of metastatic colorectal cancer  
Dr. A. Cox and Profs. P. Quirke and M. Seymour, (University of Leeds)  

The aim of this project, which was funded in December 2005, is to identify genetic variants that are associated with outcome and survival from metastatic colorectal cancer. The mainstay for many years for first line chemotherapy in colorectal cancer has been 5-fluorouracil, but the FOCUS clinical trial has helped determine the best way to use newer agents such as oxaliplatin and irinotecan. However, many patients do not respond well to these therapies and it would be of benefit to be able to identify which patients are likely to respond. We have previously shown that inherited variation in the genes involved in the cellular response to DNA damage can be related to patient survival. DNA samples are available from around 1200 patients enrolled in FOCUS, and we have obtained approval from the Local Research Ethics Committee for use of these samples to determine whether DNA damage response genes are associated with treatment outcome in the FOCUS patients. We have developed a panel of single nucleotide polymorphisms (SNPs) in 8 genes involved in the DNA damage response, including XRCC2, ATM, XRCC3, RAD51, BRCA2, MRE11, RAD50 and NBS1. The SNPs have been selected to capture the common variation present in the population for each of these genes. We are currently genotyping these SNPs and will analyse the data in relation to clinical outcome and survival data, to determine whether any of these genes can predict treatment response or survival.    

Variation in apoptosis genes and susceptibility to common cancers  
Dr A Cox, Professor F Hamdy, Professor M Reed, Professor J Donovan and Professor D Neal  

Identification of the inherited component of cancer allows us to identify the pathways involved and to design better treatments. The inherited component of most common cancers will comprise a spectrum of gene variants from rare highly-penetrant mutations (e.g. those in BRCA1 and BRCA2), to common low penetrance variants. The latter are just beginning to be identified in large collaborative studies. The Breast Cancer Association Consortium, has recently shown that a common variant of the apoptosis (programmed cell death) gene caspase-8 is associated with breast cancer (Cox et al, Nature Genetics 2007). The aim of this project is to identify the causative variant(s) in the caspase-8 gene and determine the extent to which apoptosis pathway genes are involved in breast, prostate and colon cancer, in collaboration with partners in the UK and USA.  

The project began in August 2007 and Mr. Graeme Elliott has been employed as technician on the project. Over 10,000 DNA samples have been assembled in Sheffield in preparation for this study, and genotyping is in progress. Thus far, genotyping of variants in the caspase-8 gene has been carried out on over 3000 samples from colon cancer case and control subjects from Utah, Sheffield and Leeds, and these data are currently being analysed. We are also in the process of identifying further variants (in other apoptosis genes) that we plan to genotype. This study should give us a clear picture of the role of the apoptosis pathway in susceptibility to breast, prostate and colon cancer.    

Liquid handling robot for cancer genetic epidemiology  
Dr A Cox  

One approach to the identification of cancer pathways is to examine inherited variants, which can affect a person's susceptibility to cancer or their response to treatment. Genetic studies in cancer require large sample sizes to achieve good statistical power., and this necessitates the use of high throughput systems in order to carry out the large number of genetic assays that are needed.  

This equipment award supports other Yorkshire Cancer Research funded research projects in Sheffield by providing a liquid handling robot for such studies. In particular, project S288 investigates the effect of genetic variation in DNA damage response genes on survival from metastatic colon cancer, and project S295 examines the role of apoptosis genes in susceptibility to common cancers. The robotic equipment (Biomek NXp, Beckman Coulter Ltd) was purchased in January 2008 following a tendering procedure involving tender bids from 3 companies. It has now been installed, and staff training has taken place. The first task of the robot is currently underway; this involves processing of DNA samples from women with advanced breast cancer as part of another YCR-funded project. We anticipate that this equipment will make a valuable contribution to cancer genetics research in Sheffield in the coming years.    

The role of Poly(ADP-ribosyl) polymerase in replication  
Prof T. Helleday and Dr H. Bryant  

If replication forks stall, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. In this project we set out to investigate whether poly(ADP-ribosyl) polymerase (PARP) has a role to play at stalled replication forks. So far we have seen that PARP1 binds to stalled replication fork structures in vitro and that these structures induce recombinant PARP activity. Supporting these results in vivo, we see activation of PARP in cells treated with the replication inhibitor hydroxyurea. We have also shown that PARP1 is required for restart of hydroxyurea stalled replication forks.  Homologous recombination is required for restart of stalled forks and we have gone on to show that both PARP1 and PARP2 are required for recombination repair induced at stalled forks. In addition we have demonstrated that PARP interacts with both Mre11 and NBS1 (DNA repair proteins) following replication stress, an interaction that is disrupted by PARP inhibition. This provides a potential mechanism for the way in which PARP promotes homologous recombination following replication fork stalling and will the subject of our future studies. During this study we have observed that PARP activity is elevated in BRCA2 defective cells, there are today no amenable biomarkers for recombination defective cancer cells responding to PARP inhibitors. The increased PARP activity in cells with high levels of replication stress such as HR-defective cells, is easily identified by the large number of poly(ADP-ribose) polymers present in those cells and may potentially serve as a good biomarker for recombination defective cells as well as responsiveness to PARP inhibitors in the clinic. We hope to investigate this further.  

The role for RPA in maintaining genomic stability  
Prof. T. Helleday and Dr. K. Sleeth  

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. In this project we set out to look at the role of RPA in maintaining genomic stability following stalling of replication forks in human cells. We have depleted cells of the RPA protein using siRNA and seen that this inhibits the formation of RAD51 nuclear foci (a marker of homologous recombination) after hydroxyurea-induced replication fork stalling. Depletion of RPA also led to persistent unrepaired DNA double-strand breaks (DSBs), these data together suggest that RPA is required for repair of HU induced replication stalling by promoting homologous recombination. We have confirmed a role for RPA in the promotion of recombination following replication arrest with HU using a cell line containing a recombination reporter construct. When ssDNA regions are generated the DNA damage signalling checkpoint kinase 1 (Chk1) is activated, we have seen that RPA is dispensable for (Chk1) activation suggesting that its role in promoting recombination is downstream of Chk1. We then looked to see if RPA interacted with any recombination proteins and saw that it binds RAD52 upon replication stress, suggesting a mechanism by which RPA promotes recombination repair. In addition we have shown that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.    

Genetic instability and cancer  
Prof M. Meuth  

Cells respond to DNA damage by triggering cell cycle arrest, DNA repair, or death.  DNA damage response pathways are frequently disrupted during tumour development leading to genetic instability, loss of cell cycle checkpoints, defects in the induction of apoptosis, and altered responses to widely used chemotherapeutic agents.  The objectives of our work are to elucidate the interactions of pathways controlling the response to DNA replication fork stress, to determine how these pathways are altered in tumour cells, and to determine how they may be exploited to improve therapy. One of our major lines of work is aimed at understanding the role of one of the primary regulators of the DNA damage response (a protein called ATM) in the cellular response to disruption of DNA replication.  We have discovered that a subset of human colon cancer cell lines are defective in ATM activation following replication fork disruption and identified a mutant allele of another regulator (called MRE11) that suppresses ATM activation and ATM-mediated rescue of disrupted DNA replication forks. Our results have important implications for the regulatory pathways and suggest a novel and therapy specific for tumour cells defective in this response.  A second line of work is aimed at understanding how cells "decide" whether to repair DNA and restart DNA replication or die following disruption of replication.  We have shown that the efficacy of a clinically relevant agent that disrupts DNA replication (camptothecin) can be enhanced by manipulation of S-phase signalling pathways. DNA replication inhibitors that do not normally induce cell death can be converted into potent inducers of apoptosis by depletion of the Chk1 checkpoint kinase.  Thus combinations of agents that disrupt S-phase checkpoints with those that inhibit DNA replication may be particularly effective in the treatment of some types of tumors.  

A structural investigation of the papillomavirus replication initiation complex  
Dr C.M. Sanders and Dr A. Anston (Structural Biology Laboratory, University of York)  

The papillomaviruses are important disease organisms in man and are the causative agents of certain cancers principally carcinoma of the cervix.  The viral replication apparatus is an attractive target for anti-virals that have the potential to eradicate a communicable form of cancer.  We set out to understand the function of the viral E1 protein that drives replication.  E1 belongs to a class of proteins known as helicases that unwind double stranded DNA in preparation for replication.  Our approach was to determine the three-dimensional structure of the protein by X-ray crystallography from which we could build and test models for DNA unwinding.  We solved the structure of E1 protein to reveal a six-member protein ring.  A central channel formed by multimerisation can accommodate single stranded DNA, and specific DNA structures in each subunit could be identified.  Active sites for ATP binding and hydrolysis form at the subunit interfaces and their configurations reveal insights into the mechanism of DNA unwinding.  Aspects of these mechanisms have been tested by site directed mutagenesis and our understanding of the unwinding mechanism of E1 is now better understood.  This information will be essential in designing or validating any ant-viral drugs that target the E1 protein.        


Academic Unit of Surgical Oncology  

Influence of Tumour Cell Death Mechanisms and Immunoregulatory T Cells on Tumour Growth and Immunity  
Professor N.J. Brown, Professor A.G. Pockley, Professor M.W.R. Reed and Dr S.L. Hankin  

Functionally suppressive, naturally-occurring CD4⁺CD25⁺ regulatory T (T_reg) cells are elevated in the blood and tumours of cancer patients. Tumour cell death might influence the induction of protective anti-tumour immunity, as necrosis is an inflammatory stimulus, whereas apoptosis can have anti-inflammatory consequences, at least some of which appear to be mediated via the induction of T_reg cells. The influence of tumour cell death on the development of protective anti-tumour immunity is being evaluated by administering viable (Annexin^-/PI^- cells sorted on a BD FACSAria^™ flow cytometer), apoptotic (24 hr incubation in PBS containing 2% v/v BSA; Annexin⁺/PI^-) and necrotic (freeze-thaw treatment; Annexin⁺/PI⁺) EMT6 cells to Balb/c mice bearing a subcutaneous EMT6 mammary carcinoma. The EMT6 populations being used are >90% pure. The number of splenic CD4⁺CD25^highFoxp3⁺ T_reg cells in mice bearing EMT6 mammary tumours was 3-fold higher than that in naïve mice.  The intravenous administration of viable EMT6 cells to tumour bearing mice induces haemorrhage, a concomitant peripheral leukocytic infiltrate and tumour growth (n=6). Ongoing studies are evaluating the influence of apoptotic and necrotic EMT6 cells. Current studies are also evaluating the influence of the different tumour cell populations on the recruitment, expansion and function of CD4⁺CD25⁺ regulatory T cell populations. These findings will provide further insight into immunoregulatory mechanisms controlling anti-tumour immunity and promote the development of more effective immunotherapeutic approaches.    

The relationship between vascular endothelial growth factor and tissue factor in breast cancer  
Dr C.A. Staton, Prof M.W.R. Reed and Prof N.J. Brown  

Many haemostatic proteins modulate tumour angiogenesis resulting in either stimulation or inhibition, thus understanding the relationships between these processes has implications for cancer therapy. Tissue Factor (TF), a key haemostatic molecule, up-regulates vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor. Thus far our studies show a significant increase in VEGF expression between normal and hyperplastic/pre-invasive breast cancer tissue, and between pre-invasive and invasive carcinomas. In contrast TF was not expressed in normal hyperplastic breast epithelial or pre-invasive cancer cells, but was expressed in approximately 45% of invasive cancer specimens.  VEGF was expressed in the endothelium of normal breast, whilst TF was not.  However, both VEGF and TF were expressed in endothelial cells in hyperplastic breast and in pre-invasive and invasive carcinomas.  Our data indicate that angiogenesis is initiated at the earliest signs of dysplasia and increases rapidly between pre-invasive and invasive cancer.  VEGF and TF expression patterns suggest these factors play a significant role in this process. In vitro assays of angiogenesis (endothelial cell proliferation, migration and tubule formation) are being used to assess the relative contribution of each of these factors to angiogenesis using recombinant proteins.  Moreover, a breast cancer cell line known to express high levels of TF and VEGF is currently being grown in the mammary fat pad and knockdown experiments are underway, following a visit to Dr Mohammed Amarzguioui at Oslo University, to genetically alter the levels of VEGF and TF expression in these cells to establish their direct effects on tumour growth and angiogenesis.    


Academic Unit of Ophthalmology and Orthoptics  

Identifying novel regulators of uveal melanoma invasion within the aqueous and vitreous humours  
Dr. K. Sisley and Prof. I. Rennie  

Uveal melanomas (choroid and ciliary body) are highly aggressive eye cancers readily metastasising to the liver.  Iris melanomas however are relatively benign.  We have recently shown that the behaviour of uveal melanomas may reflect interaction between the tumour and its local environment; specifically exposure of melanomas to the vitreous humor induced a significant increase in tumour invasion.  In addition, sections of posterior tumours demonstrated a change of cell type and cell cycle regulators following invasion into the anterior chamber.  In this current investigation we have explored how exposure to the aqueous or vitreous affects the regulation of uveal melanomas.  Using immunocytochemistry we are looking for the changes in the expression of a number of cell cycle regulators including p21, p27 and cyclin D1 following exposure to the aqueous and vitreous.  A series of primary uveal melanomas have been studied. Our initial data suggests that p21 expression is not affected by the aqueous or vitreous.  Primary uveal melanoma explants are comprised of a mixture of spindle (less aggressive) and epithelioid cells (aggressive).  Preliminary data indicates that the differentiation marker p27, as expected, is mainly expressed by spindle cells.  In less aggressive tumours (determined by Fluorescence In Situ Hybridization) p27 was however up-regulated in response to exposure to the aqueous. These initial findings confirm that the aqueous and the vitreous are capable of regulating uveal melanoma behaviour.  We are currently continuing to study Cyclin D1 expression and are using a proteomic approach to analyze the regulators within the aqueous and vitreous that may regulate uveal melanoma behaviour.    


Section of Infection, Inflammation and Immunity  
Academic Unit of Pathology

Novel use of hypoxia response promoters in Salmonella to target genes to hypoxic
Profs C. Lewis and J. Green, Drs J. Harmey and R. Ryan.

Targeting genes is central to gene therapy-based cancer treatment but remains challenging because many anti-cancer gene delivery vectors are unable to penetrate the poorly vascularised, hypoxic regions of tumours, leaving these sites untreated. We therefore developed a new approach for targeting gene therapy to these hypoxic sites in tumors, using a bespoke highly hypoxia-inducible bacterial promoter (FF+20*) and demonstrated its specificity using a novel bacterial, cytotoxic protein, haemolysin E, targeted to murine mammary tumours. We engineered an attenuated strain of the bacterium Salmonella typhimurium, to contain a hypoxia-inducible reporter gene construct, FF+20*-lacZ, and showed that the bacteria infiltrated into tumour spheroids in vitro or orthotopic tumours in vivo, and expressed high levels of ß-galactosidase only in hypoxic/necrotic sites. We then adapted the system to deliver a gene encoding the novel cytotoxin, haemolysin E, to hypoxic regions of murine mammary tumours, resulting in widespread tumour necrosis and markedly reduced tumour growth. This new system represents a promising new strategy for delivering cytotoxic gene therapy to hypoxic regions of solid tumours.  

Macrophage responses to hypoxia: relative contribution to tumour angiogenesis and progression.
Prof C. Lewis, Drs C. Murdoch and R. Johnson.  

Recent studies have shown tumour-associated macrophages (TAM) play a crucial part in driving tumour angiogenesis and metastasis. A key microenvironmental factor that promotes angiogenesis in tumours is hypoxia (the low oxygen tension found at multiple sites in tumours). We showed recently that TAM gather in such hypoxic tumour sites where they are stimulated by hypoxia to express a pro-angiogenic phenotype. This is thought to be mediated by their upregulation of the hypoxia-inducible factors (HIFs) 1 and 2, which in turn trigger the expression of a wide array of pro-angiogenic and pro-metastatic genes.  However, the exact role of each form of HIF in this pattern of hypoxic gene expression has yet to be outlined.  We have successfully devised a way to block the expression of HIF-1 and HIF-2 mRNA in primary human macrophages (using transient transfection with siRNA), and have used Affymetrix cDNA arrays to examine the effect of this on hypoxic gene expression in these cells.  This has identified a subset of genes that are either independent of HIFs, or regulated by HIF-1, HIF-2 or both HIF under hypoxia.  We have also discovered that hypoxic macrophages express elevated levels of Tie2, the cell surface receptor for angiopoietins.  This receptor was hitherto thought to be restricted to endothelial cells.  Moreover, we then found that the pro-angiogenic cytokine, angiopoietin-2 (which is expressed by hypoxic tumour cells) exerts profound inhibitory effects on hypoxic macrophages and essentially switches off their ability to perform anti-angiogenic functions (eg to release the potent anti-angiogenic cytokine, IL-12). Together these studies show how tumour hypoxia stimulates macrophages to perform various pro-tumour functions.    

Integrative modelling of the molecular pathogenesis of Barrett's metaplasia  
Dr N Green, Dr J Bury, Professor S MacNeil, Dr B Corfe and Dr G Battaglia  

Cancer of the oesophagus is increasing in incidence in the western world.  It is a disease with a high mortality, largely because it tends to be advanced at presentation. Our group's aim is improve out understanding of a pre-malignant condition called Barrett's metaplasia (BM) in which the normal oesophageal squamous epithelium is replaced by columnar epithelium. This is a key risk factor for the subsequent development of oesophageal adenocarcinoma.  We are investigating the hypothesis that exposure of the normal squamous epithelium to bile salts and acid in gastrooesophageal reflux acts through NF-kappa-B signalling to upregulate the Cdx2 homeobox gene, which then induces inappropriate differentiation to the columnar phenotype.  To date almost all research in this field has relied upon simple cell culture systems, often using cells that are already malignant.  We think this is unrealistic, so to support our work we have developed a novel three-dimensional tissue-engineered cell culture system.  This incorporates oesophageal fibroblasts as well as squamous cells and a connective tissue matrix, which we believe provides a more realistic model for studying the pathogenesis of Barrett's metaplasia.  Our initial experiments have focussed on establishing the specific sensitivity to the oesophageal squamous cells and the supporting fibroblasts to acid exposure.  We have established that fibroblasts are far more sensitive to acid exposure that the squamous cells they support.  We suspect that any response of the squamous cells to noxious agents may be enhanced by intercellular signals from the fibroblasts nearby, a phenomenon that Professor MacNeil's team have observed in analogous experiments in the skin.  We are now exploring this hypothesis, as well as moving on to characterise the impact of bile salts on oesophageal squamous cells and fibroblasts.    


Academic Unit of Infectious Diseases  

Tumour vaccines with enhanced Immunogenicity  
Dr A.W. Heath and Dr J. Carlring-Wright  

Idiotypic antibody from the surface of B lymphoma cells represents the best most tumour specific target antigen known. Patient specific therapeutic vaccines are currently produced by conjugation of the purified antibody with a large carrier molecule, keyhole limpet hemocyanin (KLH). Vaccines produced in this way are effective only in a proportion of patients due to poor immunogenicity.  The project involves the production of what we hope will be a highly immunogenic vaccine based on a bivalent antibody which is half lymphoma surface immunoglobulin (idiotype) and half anti-CD40 antibody. We have successfully produced CD40/lymphoma bivalent antibody and have used it for immunisation of mice. In a preliminary experiment enhanced responses to the idiotypic protein were seen, and the system is now being optimised in preparation for comparisons to be made between this system and the currently used KLH conjugation method.     


Department of Biomedical Sciences  

The role of Crumbs2 in epithelial to mesenchymal transition and tumour progression  
Dr P. Rashbass, Dr. S.S. Cross (Department of Pathology), Mr J. Catto (Academic Unit of Urology),  

As cancer progresses to an invasive, metastatic state, the tumour loses its normal tissue appearance.  The cancer cells themselves change their shape (polarity) and behaviour so that they no longer attach properly to their neighbours and instead can move away (migrate) from their original location.  This is known as epithelial to mesenchymal transition (EMT). During this project we have found that levels of Crumbs2 (a protein associated with cell polarity) are altered in a range of tumours including colorectal, breast and prostate cancers.  Our work over the last year has extended these observations to determine what happens when we alter the levels of Crumbs2 in tumour cell lines.  Our results show that modulating the levels of Crumbs2 in a neuroblastoma cell line has a dramatic effect on a variety of different cell behaviours including proliferation, ability to polarise and rate of migration.  Combined, the work from this project will determine whether Crumbs2 can be used as a new screening marker for tumour progression. In the longer-term, we aim to establish whether modulating Crumbs2 levels could be used a therapeutic tool to inhibit metastatic progression.    

Regulation of Pluripotency in Malignant Stem Cells  
Prof P. W. Andrews  

Teratocarcinomas are a subset of germ cell tumours, comprising a mixture of differentiated cell types and undifferentiated embryonal carcinoma (EC) cells.  Undifferentiated EC cells constitute the malignant stem cell component of the tumour, whilst their differentiated derivatives display a limited proliferative capacity and are non-tumorigenic.  Aberrations that promote EC self-renewal in preference to differentiation may play an important role in germ cell tumour progression.  A further understanding of the pathways involved in stem cell self-renewal and differentiation is important in order to enhance our current knowledge of tumorigenesis and aid the development of novel therapies.  The cyclin dependent kinase inhibitor (CDKI) protein p27^KIP1 is a negative regulator of cell cycle progression.  Its expression is frequently lost in a number of tumour types. Differentiating cells often contain elevated p27^KIP1 protein levels, suggesting an additional role in the control of cellular differentiation.  In this study we have investigated the mechanisms by which p27^KIP1 mediates differentiation in EC cells.  In addition we have also found that, on prolonged passage, human embryonic stem (ES) cells may acquire chromosomal abnormalities that mirror those found in EC cells from GCT.  It seems likely that these chromosomal abnormalities arise because of a common selection pressure for variant cells that undergo self renewal at the expense of differentiation.  They therefore offer a novel approach to identifying the key genes that may influence the progression of GCT and we extended our study to identify these.  The results provide the basis for a new project to examine the mechanisms that lead to genetic instability and the generation of variant cells that possess enhanced malignancy because of dysregulation of the mechanisms that control the decision of stem cells between self renewal, differentiation and death.  

Genetic changes in cultured hES cells as a model for germ cell tumour development
Professors P.W. Andrews and M. Meuth  

Teratocarcinomas contain malignant stem cells (EC cells), providing a paradigm for the stem cell concept of cancer. Human embryonic stem (hES) cells, resembling EC cells, undergo culture adaptation on prolonged passage, marked by amplification of chromosomes typically amplified in ECs, suggesting common underlying causes for genetic instability. Thus, elucidation of mechanisms of genetic instability in cultured hES cells can provide a model for exploring mechanisms of progression of stem cell based tumours. As preliminary data showed reduced activation of Checkpoint kinase 1 (Chk1) in hES cells following treatment with DNA replication inhibitors, we hypothesize that defects in the response to replication fork stress may underlie observed chromosomal instabilities that occur during adaptation and germ cell tumour development. To test this hypothesis we will examine the integrity of Chk1 function in hES and EC cells and determine the cellular and genetic consequences of dysfunction of this DNA damage response pathway.    


SCHOOL OF CLINICAL DENTISTRY  
Department of Oral Pathology  

Regulation of malignant epithelial cell behaviour by integrin-ADAM interactions
Dr S. Whawell, Prof. P.M. Speight (Sheffield) and Prof A.J. Turner (Leeds)  

Our project, which we have just commenced, aims to investigate the mechanism by which oral cancers cells migrate and invade tissue locally. We believe that ADAM (A Disintegrin And Metalloproteinase domain) proteins play a role in this process through their interaction with extracellular matrix receptors (integrins) on the cell surface. The first objective has already been completed, that is to create an oral cancer cell line over-expressing the α9ß1 integrin which interacts with the largest number of ADAMs. Full length human α9 DNA was transfected into the H357 oral epithelial cell line by retroviral transfection methods. Null cells containing the vector alone were also created. Expression of α9 integrin was confirmed by flow cytometry in H357 wild type, null cells and transfected cells and the function of α9 expressing cells was studied using migration assays. 99% of the transfected cell population showed expression of α9 integrin. The α9 expression pattern of H357 null vector cells was similar to the H357 wild type cells. The migration of transfected cells towards Collagen I and Tenascin-C was significantly higher that that of null and wild type cells and this increased migration could be significantly reduced by anti-α9 antibody. Migration towards fibronectin was not significantly different between the cell lines but showed a trend towards higher migration in α9 expressing cells. We can now utilise these cells to investigate which ADAMs are interacting with this integrin and the functional significance of integrin-ADAM interactions in malignant epithelial cell behaviour.