research reports

University of Sheffield

2004/5

Director's Introduction
Professor B. W. Hancock

Cancer research in Sheffield has well-established programmes in both scientific and clinical research. The main scientific research programme is based in the YCR Institute for Cancer Studies, which occupies 800 sq. metres of recently refurbished space on the top floor of the Medical School. As well as research space for seven investigators, the Institute provides core facilities for cancer researchers throughout Sheffield. These facilities include fluorescent activated cell sorting, DNA sequencing, high throughput DNA sample processing, confocal and time-lapse microscopy, and a radiation source.

The Academic Unit of Clinical Oncology is located in the Cancer Research Centre (CRC), a purpose built clinical trials facility at the Weston Park Hospital. The CRC provides facilities for the assessment and treatment of patients, office space for staff with fully networked computer systems, and specialist facilities including laboratory space and bone densitometry. The North Trent Cancer Research Network is co-ordinated from the CRC.

While these two research components are physically separate, excellent interactions between them have already been established, with numerous collaborations based in cancer genetics, genetic epidemiology and new therapies. Other important cancer research teams are based in the Medical School with specialised facilities within the Division of Genomic Medicine (Academic Units of Pathology, Infection & Immunity and Respiratory Medicine), the Division of Clinical Sciences (S)(Academic Units of Ophthalmology & Orthoptics, Palliative Medicine, Surgical Oncology [the Microcirculation Research Group] and Urology) and in the University Departments of Biomedical Science and Molecular Biology and Biotechnology. There are also researchers now established in the School of Clinical Dentistry (Department of Oral Pathology).


DIVISION OF GENOMIC MEDICINE

Director: Professor G.W. Duff
Deputy Director (Clinical): Professor B.W. Hancock

SECTION OF ONCOLOGY & PATHOLOGY
Section Head: Professor C.E. Lewis

YCR Institute for Cancer Studies
Head:Professor M. Meuth

Genetic Instability and Cancer
Professor M. Meuth

The Ataxia telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks while the related ATR is important in maintaining the integrity of the DNA replication apparatus. Work in our laboratory over the last year has shown that thymidine, which slows the progression of DNA replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated activation of the Chk2 and NBS1/MRE11 pathways and ATR-mediated phosphorylation of Chk1. AT cells or cells expressing a tumour associated mutant form of MRE11 exposed to thymidine showed decreased viability, failed to activate ATM or its signalling through the MRE11 complex, and failed to induce homologous recombination repair (HRR). Taken together, our results implicate both ATM and MRE11 in HRR-mediated rescue of replication forks stressed by thymidine treatment with a specific role for MRE11 in the upstream activation of ATM. This pathway is highly relevant to cancer as our data suggest that it is defective in up to 20% of colon tumours. Furthermore many widely used chemotherapeutic agents cause DNA replication fork stress. In particular, we show that the mutant MRE11 causes sensitivity to an agent (camptothecin) widely used to treat colon cancers. Our findings may improve our ability to identify tumours that respond favourably to this group of agents and lead to the development of new therapies directed against these tumours.

A zebrafish model for HNPCC: isolation of MMR deficient zebrafish
Professor M. Meuth, Professor P.W. Ingham FRS and Dr C.E. Allen (Centre for Developmental Genetics, Department of Biomedical Science)

Zebrafish are a proven and powerful vertebrate model for studying many aspects of human biology and disease. The work undertaken during this funding involvedresearch into isolating mis-match repair (MMR) deficient zebrafish and determining whether enhanced tumour development was evident. An automated reverse genetics programme in the zebrafish (Danio rerio), has been completed - a process more generally termed as TILLING (Targeting Induced Local Lesions in Genomes). This technique facilitates the identification of zebrafish with known mutations in specific, desirable genes of choice. Mutations in many genes, such as cancer causing genes, cannot be revealed by observing associated phenotypes in development, thus we resorted to reverse genetics. We were primarily TILLING for the MMR cancer genes MLH1 and MSH2, which cause hereditary nonpolyposis colorectal cancer (HNPCC) in humans. From a library of 5000 heterozygous fish, many genomic portions from these MMR genes were analysed by direct DNA sequencing. After the subsequent analysis we retrieved a range of mutations in each of these genes and the founder fish were identified and bred to create new zebrafish lines. The families of fish are currently being bred to obtain pure lines and will subsequently be intercrossed. Once a HNPCC model fish line has been established, searches for genetic modifiers of the MMR genes can begin.

Molecular mechanism for genetic instability caused by inactivation of poly (ADP-ribose) polymerase
Dr T. Helleday

Genetic rearrangements are a common cause for disruption of tumour suppressor genes that will eventually lead to cancer. Poly(ADP-ribose) polymerase (PARP) is an enzyme that controls recombination and may have an important role for development of cancer. Here, we study the mechanism how PARP controls recombination. Here we have seen that PARP does not play a direct role in recombination repair. However, it controls the levels of recombination within the cell and how damage is dealt with at replication forks. We have shown that PARP inhibitors specifically kill cells deficient in homologous recombination. This is important for cancer treatment, since several cancers develop due to a defect in this pathway, e.g., inherited form of breast cancer. In this work we have shown that we can specifically kill BRCA2 deficient tumours in xenograft mice. This finding provides a new concept for cancer treatments and provides hope for a cure of inherited breast cancer. During 2004, we licensed out the patent for treating BRCA2 tumours to KuDOS Pharmaceuticals Ltd., Cambridge. In 2005, we will proceed with a phase I clinical trial.

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

The papillomaviruses are the causative agents of warts but are also associated with certain cancers, principally carcinoma of the cervix. Two independent approaches are being employed towards combating viral infection and disease. The first is immunological or vaccine based, aimed at stimulating the body's own immune response to the virus. The second is chemotherapeutic and involves an understanding of important viral proteins as specific targets for potential anti-viral drugs. We are undertaking a comprehensive structural and functional analysis of two viral proteins, known simply as E1 and E2, that coordinate and control viral DNA replication. We have previously determined the molecular structure of part of the E2 protein that interacts with E1 and controls viral replication. Our structure revealed the existence of a novel interaction between two E2 proteins that we now know can regulate its ability to associate with its partner E1. We have also made great in-roads into understanding how E1 functions in viral replication, and are on target to determine how the protein can process the viral DNA during replication.


Academic Unit of Clinical Oncology
Head: Professor R.E. Coleman

Professor R.E. Coleman, Dr D. Greenfield, Professor B.W. Hancock, Dr I. Holen, Dr M. Marples, Dr M.H. Robinson, Professor P.J. Woll, Dr Z.M. Zhu
In collaboration with Professor S. Ahmedzai (Acadaemic Unit of Palliative Medicine, Division of Clinical Sciences (S)), Professor F.C. Hamdy (Academic Unit of Urology, Division of Clinical Sciences (S)), Professor M.W.R. Reed (Academic Unit of Surgical Oncology, Division of Clinical Sciences (S)) and Professor I.G. Rennie (Academic Unit of Ophthalmology & Orthoptics, Division of Clinical Sciences (S))

The Unit first established a clinical research facility at Weston Park Hospital in 1992. The main aim was to provide a centre for the co-ordination of clinical research within Weston Park Hospital, which, by providing facilities and staff for data management, protocol design and data analysis, would enable all clinical staff at Weston Park Hospital to participate in clinical trials.

We now have an experienced and flourishing multi-disciplinary research team, that conducts high quality research studies, to the exacting standards expected by the scientific community and demanded by the regulatory authorities. We are of course interested in the effects of new treatments on both the quality as well as quantity of our patients' lives. Over the past ten years over 5000 patients, with a broad range of different cancers, have been entered into a variety of clinical trials. The Unit is also a national referral centre for gestational trophoblastic neoplasia.

Academic Unit of Pathology
Head: Professor P.G. Ince

Development of a macrophage-based system to target therapeutic viruses to prostate cancer
Professor C.E. Lewis, Professor N. Maitland (YCR Cancer Research Unit, University of York), Professor F. Hamdy (Academic Unit of Urology, Division of Clinical Sciences (S)) and Professor N.J. Brown (Microcirculation Research Group, Academic Unit of Surgical Oncology, Division of Clinical Sciences (S))

This project exploits the ability of macrophages to home to hypoxic areas of prostate tumours to deliver therapeutic adenoviruses to these regions. The main advantages of this system are the bypassing of the liver, the principal target for intravenously administered viral vectors, and the delivery of large quantities of virus to tumours. To achieve this, an E1A/B gene cassette regulated by a hypoxia responsive promoter sequence (HRE) is co-introduced into human macrophages with an E1A/B-deleted adenovirus containing a therapeutic gene (whose expression is driven by a promoter from a gene overexpressed in prostate). After inoculation, the macrophages home to areas of hypoxia within tumours, where the E1A/B induced should result in production of the co-infected therapeutic virus. The capacity of each macrophage to produce more than 104 viral particles will result in infection of surrounding prostate tumour cells and expression of the therapeutic gene. As proof of principle, a trimerised HRE has been used to drive expression of E1A/B expression in HeLa cells in hypoxia, as detected by Western blotting. Co-infection of such cells with an E1A/B deleted adenovirus engineered to express EGFP from a CMV promoter, resulted in complementation and yields of virus which were at least 100 fold higher under hypoxic than normoxic conditions. Optimal conditions for complementation in, and infection of, human macrophages are now being determined, prior to cell inoculation into nude mice bearing orthotopic prostate (PC3) tumours.

Establishment of a zebrafish model of angiogenesis
Professor C.E. Lewis and Professor P.W. Ingham FRS (Centre for Developmental Genetics, Department of Biomedical Science)

Angiogenesis (the sprouting of new blood vessels) is essential for the growth and spread of malignant tumours. This finding prompted the development of various anti-angiogenic drugs. However, current assays for the angiogenic activity of new drug candidates are slow, expensive to run and often require confirmation in cumbersome in vivo angiogenesis assays in rodents. Recent studies have shown that the zebrafish embryo represents a rapid, alternative assay system for testing the potency of putative anti-angiogenic factors. This aims of this project were to establish the zebrafish model of angiogenesis and then to see whether it could be used to identify new anti-angiogenic peptides/proteins and investigate the role of macrophages in the formation and remodelling of new blood vessels (especially following the conclusion of anti-angiogenic therapies). We have now successfully established the zebrafish model of angiogenesis in our laboratories and shown that it responds well to such established anti-angiogenic agents as VEGF-R antagonists and the new agent developed in CEL's laboratory, Alphastatin. We are now using it to screen for novel anti-antiogenic peptides.

Novel use of hypoxia response promoters in salmonella to target genes to hypoxic areas of breast tumours
Professor C.E. Lewis and Dr J. Green

This project only got underway in January 2005 so there is no report yet.


SECTION OF FUNCTIONAL GENOMICS
Head:Professor S.K. Dower

Academic Unit of Infection & Immunity
Head: Dr J.R. Sayers

Novel, highly immunogenic ganglioside based cancer vaccines
Dr A.W. Heath

Gangliosides are small sugary molecules present on a range of human cells, but present in higher numbers on many tumour cells. Ganglioside based vaccines have potential application for therapy of a variety of tumours , but tend to be very poorly immunogenic, and hence methods are required to induce stronger immune responses to these so-called therapeutic vaccines. We have received funding from YCR for a pilot study to address this issue.

We have shown in other work that conjugates of vaccine antigens with antibodies able to bind an immune cell surface molecule called CD40 (CD40mAb), are very highly immunogenic. The pilot study is aimed at producing conjugates of CD40 mAb and ganglioside antigens, with the end result hopefully being that the resultant conjugates are much more immunogenic. Ganglioside based vaccines have potential applications for therapy of a variety of tumours. Chemical conjugates produced between the gangliosides and the antibody must retain ganglioside antigen recognisable to the immune system, and also CD40 binding activity. We have been assessing various methods for producing and assessing successful antibody-antigen conjugates which will retain CD40 binding using model antigens, and we have decided on one particular conjugation method. We are now ready to attempt conjugation with ganglioside antigens using this method.


Academic Unit of Respiratory Medicine
Head:Professor M.K.B. Whyte

HE4 a novel cancer marker? Expression and function
Dr C. Bingle

This project seeks to generate antibody reagents to allow us to test the hypothesis that the recently identified tumour marker gene HE4 may be a useful diagnostic tool in a variety of cancers. It builds on our finding that the gene undergoes complex alternative splicing to yield multiple protein isoforms (Oncogene. 2002 Apr 18;21(17):2768-73.).

The aim of this project is to generate and characterise a panel of specific monoclonal antibodies to all of the splice variants of the human HE4 gene. Our expectation is that such antibodies will subsequently be used to localise expression of these isoforms in tumours from the lung, breast and ovary, as well as to develop quantitative assays to assess HE4 levels in biological samples. These reagents may prove to be of significant value to a wide body of researchers and will aid our own studies on the biology of this potential tumour marker.

Over the past year we have used our antibody reagents to study the expression of HE4 in normal tissues. The protein is expressed in the ducts of both major and minor salivary glands, in the nasal and respiratory epithelium and in both the male and female reproductive tracts. In collaboration with Dr Simon Cross (University of Sheffield) we have also studied the expression of HE4 in a range of human cancers (prostate, colon, breast and lung) using tissue arrays. These results show that HE4 is significantly over expressed in a number of lung cancers (adenocarcinomas and mucoepidermoid tumours) as well as in some lung cancer metasteses. These results suggest that the HE4 may have utility as a marker not only for ovarian cancer (for which it was originally suggested) but also for lung cancer.


DIVISION OF CLINICAL SCIENCES (S)
Director: Professor F.C. Hamdy

Academic Unit of Urology
Head: Professor F.C. Hamdy

Development of novel model systems to study cellular interactions between prostate cancer and bone marrow stroma
Mr A.A.G. Bryden, Dr A.M. Scutt, Dr C.L. Eaton, Mr B. Thomas and Professor F.C. Hamdy

This 'pump priming' project has itself now been completed although work started by this study is continuing and additional funding obtained. The project resulted in two publications in The Prostate and the Journal of Bone and Mineral Research.

The role of Osteoprotegerin in prostate cancer survival
Dr C. Eaton, Professor F.C. Hamdy and Dr I. Holen (Academic Unit of Clinical Oncology, Division of Genomic Medicine)

Prostate cancer frequently metastasises to the skeleton. Osteoprotegerin (OPG) is a bone-derived protein that may protect prostate cancer cells from TRAIL, a natural immune protein that targets and destroys tumour cells. Tumour cells that express OPG may therefore be resistant to TRAIL. Our experimental prostate cancer model expresses OPG. In this study, we aim to genetically modify these prostate cancer cells to reduce their OPG expression, and assess whether they are more susceptible TRAIL-dependent tumour killing. We have now successfully developed strains of cells with reduced OPG production, and we are currently testing these cells to determine whether they are more susceptible to TRAIL than the unmodified prostate cancer cells. We are also investigating the effect of androgens on OPG production. Treatment for prostate cancer involves the depletion of androgens in the patient, which initially prevents tumour growth, however many tumours become androgen resistant and often metastasise to the skeleton. Our experimental prostate cancer system is androgen resistant, although we now have a strain of cells that responds normally to androgens. The effects of androgens on OPG expression are being assessed in these androgen responsive prostate cancer cells. Finally, we are assessing OPG production in paired primary and metastatic prostate cancers. Over the last year we have been increasing our collection of these paired tumours and will shortly commence analysis on these samples.


DEPARTMENT OF BIOMEDICAL SCIENCE
Chairman:Professor P.W. Andrews
Head of Department: Professor C.G.W. Smythe

A screen of candidate regulatory genes that may play a role in the progression of testicular germ cell tumours
Professor P.W. Andrews

Teratocarcinomas are a significant subset of testicular germ cell tumours (TGCT), the most common cancers of young men. Recent research has highlighted several genes that may play a role in regulating self-renewal and preventing differentiation of human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas. With this pilot study grant we used RNA interference (RNAi) to screen several human EC cell lines and showed that, indeed, the transcription factor Oct4 is generally required for maintenance of human EC cells. When levels of Oct4 were reduced, these cells ceased proliferation and differentiated. Thus, Oct4 could be a potential target in developing new therapies for TGCT. In addition we used RNAi to screen several other candidate genes that might play a role in maintenance of the EC phenotype. From this study we have found preliminary evidence that the a gene, RCP32, that encodes a subunit of RNA polymerase, and another gene, dppa4, that encodes a nuclear protein, are required for ES cell survival. This information provides the basis for further studies to explore the function of these genes in stem cell maintenance. The results of such studies could help provide insights into the regulation of stem cell self renewal and its role in carcinogenesis. In this pilot study we also further developed the RNAi methodology using an inducible system that will help in the further analyses of the role in EC cell biology of these specific factors.

Regulation of Pluripotency in Malignant Stem Cells
Professor P W Andrews

Teratocarcinomas are a subset of germ cell tumours (GCT), comprising both differentiated cell types and undifferentiated embryonal carcinoma (EC) cells. 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 which promote EC self-renewal in preference to differentiation may play an important role in GCT progression. Clues to the genetic changes that contribute to tumour progression have also come from observations of culture adaptation of human embryonic stem cells in which karyotypic changes similar to those occurring in human EC cells are often seen. An understanding of the pathways that regulate stem cell self-renewal and differentiation is important to enhance current knowledge of tumorigenesis and for the development of new therapies.

Comparing the differentiation potential of nullipotent and pluripotent EC cell lines, we previously identified p27KIP1 deregulation as a potential contributory factor in the loss of pluripotency. The p27KIP1 protein is a cyclin dependent kinase inhibitor (CDKI) protein, responsible for the regulation of G1/S phase cell cycle progression. However, p27KIP1 protein levels are frequently up-regulated in differentiating cells, suggesting an additional role in the control of cell differentiation. The expression of p27KIP1 is often reduced in a number of tumour types, correlating with aggressive disease and reduced patient survival. The aim of the current study is to determine the pathways by which p27KIP1 regulates EC cell differentiation in response to retinoic acid exposure. We are investigating whether EC cell differentiation occurs via the p27KIP1-mediated inhibition of cyclin-CDK activity, or whether additional pathways are involved. The expression of key p27KIP1 target proteins, such as the cyclin and CDK proteins, will be manipulated in order to determine their precise role in EC cell differentiation.

Maintenance of Confocal Imaging System
Professor P.W. Ingham FRS (Centre for Developmental & Biomedical Genetics)

The Leica SP confocal microscope provided by this grant has continued to be heavily used by a number of groups within the Centre for Developmental Genetics at the University of Sheffield. Indeed confocal microscopy is assuming an increasingly central role in our research such that we will soon be unable to meet the demand for time on our existing machine. To meet this increased demand, we are preparing an application for two new machines to be submitted to the Wellcome Trust. We would however, be grateful for the opportunity to bid for additional funds from YCR, should it be deemed to be an appropriate use of the charity's resources!

Three of the five papers published in 2004 that report research directly supported by the Leica confocal are concerned with the analysis of the Hedgehog signal transduction pathway, a pathway which when inappropriately activated plays a critical role in the development of many tumours (including lung, pancreatic and prostate) that together account for the majority of cancer mortality worldwide. The other two papers report studies of a novel protein that modulates the activity of the Fat tumour suppressor gene product and of the roles of Histone Deacetylase 1 in the repression of Notch target gene expression, two proteins that have also been implicated in a number of cancers. We analyse the roles of these proteins in normal development using genetically tractable organisms such as the fruit fly Drosophila melanogaster and the zebrafish Danio rerio, both of which allow us to manipulate gene activity in the context of the multicellular assemblies and complex organ systems of a living animal. The small size and optical transparency of the embryos of these organisms makes them especially well suited to the high resolution in vivo imaging afforded by confocal microscopy. Our studies have allowed us, for instance, to observe the intracellular movements of a newly identified protein that controls the response of cells to the Hedgehog signalling factor, a process that is likely to influence the behaviour of tumour cells.


DEPARTMENT OF MOLECULAR BIOLOGY & BIOTECHNOLOGY
Head:Professor D.W. Rice

Clinical potential of human antibodies to a known ovarian tumour marker
Dr L.J. Partridge

Because of laboratory refurbishment, the start of this award was delayed until April 14th 2004. There has also been a break in the grant, when the research worker originally employed left. Consequently the award is now due to finish on July 14th 2005.

Ovarian cancer is the most common gynaecological malignancy with an overall 5-year survival rate of only 30% in the UK. In the early stages the disease may have few or vague symptoms and consequently a diagnosis is made only after the original cancer has spread. Unfortunately, advanced ovarian cancer responds poorly to current treatments. There is clearly a need to develop improved early detection methods and new types of therapy for this disease. One strategy, which has shown success in some types of leukaemia and breast cancer, is to use antibodies as 'magic bullets' to target and destroy the cancer.

We generated a panel of six antibodies that recognise a known marker for ovarian cancers. Antibodies to this protein (placental alkaline phosphatase or PLAP) have shown potential for imaging cancers, and in model systems, for targeted killing of cancer cells. Our antibodies were produced using a technique that generates fully human antibodies. This offers advantages over existing antibodies of animal origin, which are recognised as 'foreign' by human patients. We have been further investigating the characteristics of our panel of antibodies to determine which show the most promise for further development. We have also been optimising our production methods, to generate antibodies in a suitable standardised format for assessing their tumour cell killing potential. This work is on-going.

A genetic screen for mutations in cancerVcausing genes that alter exonuclease activity during repair of DNA double strand breaks, leading to loss of heterozygosity
Dr A.S.H. Goldman

Most work this year has concentrated on investigating the importance of XRS2 and SAE2 in regulating the rate of loss of heterozygosity on our assay system.

Initial studies using xrs2? strains indicated that this gene is required for normal regulation of resection at a DNA double-strand break (DSB). We have now looked at the xrs2-11 allele which is deleted for a region of the protein that contains sites that are phosphorylated by the ATM kinase paralogue Tel1. We find an increased rate of loss of genetic material when recombination is induced in this strain.

This is the first time a phenotype has been obtained for this allele and so provides new insight as to why Xrs2 is phosphorylated. We are now continuing investigations to determine if the reason for loss of DNA is due to increased rates of resection or inability by the MRX complex to recognise homology when it is first uncovered.

We have also made further investigations of Mre11p in particular with regard to separation of function alleles and their influence on resection. We find that there are differential effects across the population in these mutants implying a regulatory role for Mre11p.

Seeking the conserved function of the MAGE homology domain present in melanoma antigen
Professor P.W. Piper (Krebs Institute)

MAGE, for melanoma antigen, was a protein first identified by virtue of its tumour-specific cell surface expression. Its true function has remained a mystery, though humans have several MAGE genes. To try to find a function for MAGE, we have begun to investigate the single, essential MAGE protein of yeast (YDR288W; recently renamed Nse3). Nse3 shows homology to the human MAGE proteins, in particular hMAGE B1.

At the commencement of this project we had shown direct binding of the yeast Nse3 and Nse4 proteins (components of a complex which binds the SMC5/SMC6 involved in DNA damage response). So far in this study, we have shown that hMAGE B1 interacts strongly with yeast Nse4, indicating human hMAGE B1 is the human functional equivalent of yeast Nse3. The human Nse4 homolog has been cloned, and work is currently being done to test its interactions with yeast Nse3 and hMAGE B1. Affinity-tagged versions of these proteins are also being produced, to identify further Nse3/Nse4 complex components. A green fluorescent protein-tagged hMAGE has also been expressed in yeast cells, revealing that this is a nuclear protein. Further work will be done to investigate if there is re-localisation of the protein in cells after DNA damage.

In conclusion, yeast Nse3 provides a useful model system in which to study the role of hMAGE proteins, pointing to their possible role in cancer. The necessary tools have been generated to allow for further work on the role of hMAGE B1 and its association with the SMC5/SMC6 complex in the DNA damage response.

SCHOOL OF CLINICAL DENTISTRY

Department of Oral Pathology
Head: Professor P.M. Speight

The role of host defence peptides in epithelial tumour biology
Dr S.A. Whawell, Professor P.M. Speight and Dr D.A. Devine, (Leeds Dental Institute, University of Leeds)

Host defence peptides (HDPs) are secreted by cells including white blood cells and those that line oral tissues. These small molecules have a role in the immune system as well as possessing anti-microbial activity. Previous in vitro studies have indicated that HDPs may be useful in cancer therapy but there is no consensus as to their role in tumour biology. The aim of this study is to examine the effects of HDPs on the growth of normal and cancerous cells from the oral cavity.

Preliminary data using the HDP Cathelicidin (LL37) have shown that both normal and cancer cells are killed by this peptide when used at concentrations greater than 50µg/ml. Cells exposed to the peptide in the absence of serum were more sensitive. Future work will complete these studies and examine the effect of other HDPs which are currently being synthesised. The effect of HDPs on other properties of these cells relevant to tumour biology (migration and invasion) will also be examined.


Academic Unit Of Ophthalmology and Orthoptics
Head of Department: Professor I G Rennie

An in vitro study of posterior uveal melanoma invasion and the potential regulatory influences of HGF and TGF-b
Dr. K. Sisley, Prof. I. G. Rennie

HGF and TGF-b have been shown to be important regulators of tumour growth and invasion. In uveal melanoma, they appear to be important in controlling invasion and successful secondary colonization. Whilst HGF may be a promoter of these processes TGF-b appears to inhibit invasion. We are investigating the interactions of invasive, and non-invasive, uveal melanomas with the extracellular matrix (ECM), hepatic and dermal endothelial cells, and the role of HGF and TGF-b in these processes. Hepatic endothelium, but not dermal endothelium can positively enhance adherence by uveal melanoma cells, and TGF-b appears to be able to upregulate adhesion to endothelium by uveal melanoma cells. This interaction may also be facilitated by the tumour a4 integrin to its ligand, VCAM-1, on the endothelial cells. TGF-b also appears to have more wide ranging effects, and was also observed to strongly stimulate MMP secretion. The response of uveal melanomas to TGF-b was however found to vary depending on whether they were an invasive or relatively non-invasive melanoma. Uveal melanoma cells with a low invasive potential were inhibited by TGF-b whilst in contrast invasive cells tended to be promoted, leading to the possibility, as has been observed in cutaneous melanoma, that there may be a biphasic response to TGF-b by uveal melanomas.