research reports

University of Sheffield

2002/3

Director's Introduction
Professor B. W. Hancock

Over the past few years Sheffield has built up well-established programmes in both basic and clinical research, based on excellent cross-University collaborations and strong links with the NHS. On the laboratory side Professor Mark Meuth's strategy is to create a vertically integrated approach to cancer research highlighting, through the Institute for Cancer Studies, work being done in cancer genetics and genetic epidemiology. From the clinical point of view, Professor Robert Coleman leads our clinical trials and bone oncology programme, and there have been major funding initiatives for prostate cancer (by Professor Freddie Hamdy). Add to this other research strengths, (for example angiogenesis and tissue hypoxia, radiotherapy and imaging, health services and ocular oncology) and links with basic research being undertaken by the Departments of Biomedical Science and of Molecular Biology and Biotechnology, it is not surprising that cancer has been accorded priority status in both University and NHS research strategy.


CENTRAL RESEARCH FACILITIES

Clinical Trials Centre
Professor R. E. Coleman and Professor B. W. Hancock

The Yorkshire Cancer Research Programme Grant to support infrastructure within the Cancer Research Centre formally ended in December 2001. However, we are grateful for a 6 month extension which enabled our diverse programme of research to continue pending successful funding from other sources.

The Cancer Research Centre (CRC) in Sheffield now employs over 60 staff devoted to improving the treatment of cancer and increasing our understanding of some of the underlying mechanisms and biological processes involved in cancer. The North Trent Cancer Research Network is co-ordinated from the CRC and provides a research infrastructure to the Cancer Units in North Trent as well as the Cancer Centre in Sheffield. This has allowed increased participation in clinical trials and stimulated links with laboratory scientists.

Our aims are to promote participation in clinical research protocols as the standard of care for cancer patients and ensure that the research portfolio is of relevance to our patients and conducted to standards of the highest quality.


DIVISION OF GENOMIC MEDICINE

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

SECTION OF ONCOLOGY & PATHOLOGY
Section Head: Professor M. Meuth

YCR Institute for Cancer Studies
Head:Professor M. Meuth

Mechanisms of genetic instability in tumour cells.
Mr A. Mohindra, Ms J. Lee, Dr S. Cross (Academic Unit of Pathology), Professor M. Meuth

Our major research goal has been to understand the molecular mechanisms underlying the genomic instability apparent in tumor cells. Loss of mismatch repair (MMR) leads to a complex mutator phenotype that drives the development of a subset of colon cancers. We have shown that MMR deficient tumour cell lines are highly sensitive to the toxic effects of thymidine. This sensitivity was not a direct consequence of MMR deficiency or alterations of DNA precursor metabolism. Instead, MMR defective tumour cell lines were also defective in homologous recombination repair (HRR) induced by DNA double-strand breaks. Two mutations were found in MMR defective tumour cells that could account for this sensitivity. The first (found in greater than 90% of these cancers) was a frameshift in an intronic (T)11 tract of MRE11 that leads to altered splicing, exon loss, and reduced expression of this key gene involved in DSB repair and S-phase checkpoints. The second was a frameshift of the human RAD51 paralog XRCC2 found in a MMR deficient uterine tumour cell line. When these mutant genes were introduced into MMR proficient cells they conferred thymidine sensitivity and defects in some forms of homologous recombination. These data suggest that the thymidine sensitivity of MMR deficient tumour cell lines is a consequence of defects in the HRR pathway and may be a common event in the development of these tumours. The increased thymidine sensitivity and the loss of this important pathway for the repair of DNA double-strand breaks create new opportunities for therapies directed specifically against this subset of tumours.

A cytokine involvement in the pathogenesis and tumour progression of pituitary adenomas
Dr J.A. Royds, Dr. T.H. Jones (Academic Unit of Endocrinology, Section of Functional Genomics)

The aim of this study was to examine the role of the cytokines, IL1 and IL6 on the growth and invasion of human pituitary tumours using the novel human pituitary tumour cell line HP75.
We have shown that that IL6 at low dose stimulates growth, however at higher doses IL6 was inhibitory. This diverse phenomenon for IL6 has been shown in other cell types and caused by receptor downregulation. IL6 secretion by HP75 cells is high but using appropriate doses of an IL6 blocking antibody, 76% of basal growth was inhibited over six days. This finding demonstrates that the growth of the pituitary tumour cells is IL6 dependent. IL1 inhibited growth after 3 days.

Vascular Endothelial Growth Factor (VEGF) is important for new blood vessel formation in tumours and essential for tumour growth and invasion. We have found a strong positive correlation between the amount of VEGF and IL6 secreted by human pituitary tumours in culture. We demonstrated that HP75 cells secrete VEGF and its release stimulated by IL1 but not by IL6. Significantly, we have demonstrated that IL6 secretion has a strong correlation with tumour size.

We did not find any effect of either cytokine on MMP protease activity. In addition, no effect on specific inhibitors of MMP 2,3 and 13 on protease activity.
Invasion studies performed over 24 hours demonstrated that invasion could be strongly blocked in the presence of IL6 neutralising antibody and to a lesser extent by IL1 neutralising antibody. Furthermore, an inhibitor of MMP13 also significantly blocked invasion.

In summary, we have provided evidence that IL6, which is produced by the majority of human pituitary tumours, plays an important role in the promotion of both tumour cell growth and invasion in the HP75 cell model. If we can now proceed to show that it has a similar role in primary human pituitary tumours then therapies could be devised to block IL6 production. It is known that IL6 production can be inhibited by corticosteroids that may be a potential treatment.

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. PARP inhibitors have been shown to be very useful in treatments of cancer and the outcome of this work will help understanding the mechanisms of action.


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

Support for Cancer Research Centre infrastructure
Professor R. Coleman, Professor B. Hancock. Dr M. Robinson, Dr M.Marples, Dr P. Lorigan, Sister F. Armitage, Mrs L. Bruce, Mrs C. Radstone, Dr J. Horsman, Miss E. Hodgkinson, Mrs K. Gibbins.

Clinical Trials and Drug Development

• More than 10 years experience in cancer trials
• Established team of specialist oncology research nurses and dedicated data management support
• Translational research links to renowned laboratory expertise in genetic epidemiology, angiogenesis research, tissue hypoxia, cell signalling and biomarker development
• Well established clinical and research network providing cancer care for a catchment population of 1.7 million.
• Weston Park Hospital receives over 5000 cancer referrals each year, and more than 600 patients are entered into clinical trials annually
• Dedicated research support to multidisciplinary teams across the clinical network in place for most common cancer sites
• Experience in both the development and co-ordination of national clinical trials
• Long standing major commitment to breast cancer and lymphoma trials
• National and international links to collaborative site specific groups

Bone oncology

• Internationally recognised centre for research into cancer induced bone disease
• Development of bone specific drugs and the use of biochemical bone markers to tailor therapy
• Studies into the effects of cancer treatments on skeletal health and bone metabolism
• Laboratory programme of research evaluating the bone microenvironment
• Ongoing major grants with industrial partners

Technical Advances in Radiotherapy

Ongoing evaluations of state of the art radiotherapy technology including

• intensity modulated radiotherapy treatment (IMRT)
• virtual simulation
• image registration facilities
• dynamic contrast magnetic resonance imaging in treatment planning

Trophoblastic tumours

Clinically based research projects continue aimed broadly at:

• improvement in our understanding of the aetiology of this range of diseases associated with pregnancy
• assessment of prognosis when chemotherapy is required
• evaluation of long-term physical and psychological morbidity.

Melanoma

• Research into the role of sex steroids, cytokines and tissue inhibitors of metalloproteases (TIMPS) in invasion and metastasis of malignant melanoma

Role of intensity-modulated radiotherapy in the treatment of parotid tumors.
Mr C.M. Bragg, Mr J. Conway (Department of Radiotherapy Physics, Weston Park Hospital) and Dr M.H. Robinson (YCR Academic Unit of Clinical Oncology)

This year has seen the completion of the first phase of the work studying IMRT in the treatment of tumours of the parotid gland. A class solution, on which all subsequent treatment plans for these tumours will be based, has been produced and will be implemented clinically within the next three months. This should result in improved quality of life for patients treated with radiotherapy for this condition by reducing the severity of the dry mouth from which they suffer. A comparison showed that IMRT resulted in a lower dose to normal tissues close to the tumour than with conformal radiotherapy, the technique currently used. This will reduce the side-effects of the radiotherapy treatment. New techniques for comparing the quality of treatment plans have been implemented.

A similar study was performed for sarcomas. While some potential benefits from the use of IMRT were seen with sarcomas in the pelvic and trunk regions, sarcomas are considerably more varied than parotid tumours and the development of class solutions was therefore more difficult.

Progress has been made in developing class solutions for the treatment of tonsil and base of tongue tumours, again comparing IMRT plans produced with conformal radiotherapy. The results of this work will be clinically implemented within the next six months.


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, York), Professor F. Hamdy (Academic Unit of Urology, Section of Surgical & Anaesthetic Sciences) and Dr N.J. Brown (Academic Unit of Surgery, Section of Surgical & Anaesthetic Sciences)(Division of Clinical Sciences (S))

Areas of low oxygen tension (hypoxia) are common in prostate tumours and immune cells called macrophages accumulate in these sites. These findings led to Professor Lewis's group to propose that macrophages could be used as cellular vectors to carry hypoxically-activated therapeutic genes into hypoxic sites in tumours [Griffiths et al. (2000). Gene Therapy 7:255-62], which are the most drug resistant areas of tumours. In our current project, we are extending this gene therapy to target the production of prostate-specific, therapeutic adenoviruses to prostate tumours as well as their skeletal metastases. We are generating human macrophages that transiently express the adenoviral genes, E1A/B, under the control of a strong hypoxia-inducible DNA element (HRE), as hosts for E1A/B-deleted therapeutic human adenovirus vectors. Following injection, these macrophages will home to hypoxic tumour areas, where expression of the E1A/B would be induced, resulting in a productive infection and release of replication defective therapeutic viruses. These would then transfer the therapeutic gene to surrounding tumour cells where the anti-tumour gene would be expressed.

Initially, we have characterised a suitable HRE for use in human macrophages - the HRE from the MMP-7 gene - which shows high inducibility in hypoxic macrophages. We have established PC3 tumour spheroids as an in vitro model of tumour hypoxia. We are also testing the E1A/B adenoviral complementation system by transiently transfecting the macrophage cell line, U937, with a plasmid carrying E1A/B under a strong constitutive promoter, CMV, and have demonstrated high E1A/B protein expression in these cells using Western blotting. We are currently testing the ability of these E1 proteins to activate expression of a replication deficient adenovirus in U937 cells. The CMV promoter will then be replaced by the MMP-7 HRE to drive E1A/B, and transfected macrophages injected into mice bearing prostate tumours to see if they migrate into (and release adenovirus in) hypoxic areas of these tissues.


SECTION OF FUNCTIONAL GENOMICS
Head:Professor S. Dower

Academic Unit of Respiratory Medicine
Head:Professor M. Whyte

Regulation of splicing of the cellular survival gene MCL-1
Dr C. Bingle, Professor M. Whyte, Dr J.T. Reilly (Academic Unit of Haematology, Section of Genetics & Informatics)

Apoptosis, is the stereotypic program of cell suicide by which organisms eliminate redundant, damaged, or infected cells. Apoptosis is highly regulated since defects in the pathway leads to extended cell survival and may contribute to development of cancer. Induction of apoptosis has been proposed as treatment for a wide range of cancers. Mcl-1 has been implicated in the inappropriate cell survival found in haematological malignancies. We identified a novel variant of Mcl-1, that induces cell death. Our hypothesis was that expression of these two Mcl-1 proteins regulates cell death in human leukaemias and the current study seeks to investigate this hypothesis.

We have so far recruited 23 patients with a variety of leukaemias for the study. Using these patient samples and others from established leukaemic cell lines we have expanded our analysis of regulation of the Mcl-1 gene. It is clear from these studies that the levels of the two Mcl-1 protein isoforms are highly variable between the different patient samples. So far no clear association with disease type has been seen. We are continuing to recruit patients for these studies.

The most striking development in this work has been the discovery of 3 further Mcl-1 protein isoforms. Much of our efforts have been applied to understanding the biology of these. One of the new isoforms is also a potent cell killer whereas the other two proteins function in the same anti-apoptotic manner as Mcl-1. We are presently attempting to correlate the expression of these proteins in the different type of leukaemic samples that we are studying.

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

At present we are using a combination of methods to generate recombinant HE4 isoforms.

1. We are generating HE4 proteins as His tagged proteins in E coli
2. We are generating stable cell lines expressing HE4 isoforms as His and V5 epitope tagged soluble proteins.
3. With the help of Dr Guy Tremblay, University of Laval, Quebec, Canada, we are also making recombinant HE4 using yeast.

We expect the yeast and mammalian expression systems to generate sufficient protein for antibody production within the next few months.


DIVISION OF CLINICAL SCIENCES (N)
Head: Professor P.G. Hellewell

Academic Department of Human Metabolism
Head: Professor R.J.M. Ross

Obesity and breast cancer
Dr J. Newell-Price, Dr K.A. Al-Sakkaf (Institute for Cancer Studies, Section of Oncology & Pathology, Division of Genomic Medicine), Professor M. Reed (Academic Unit of Surgical Oncology, Division of Clinical Sciences (S)), Professor B.L. Brown (Academic Unit of Endocrinology, Section of Functional Genomics, Division of Genomic Medicine), Dr P.R.M. Dobson (Institute for Cancer Studies, Section of Oncology & Pathology, Division of Genomic Medicine), Professor R.J.M. Ross

The aim of this project is to investigate whether the increased mortality from breast cancer in obese patients is the result of an interaction between the obesity hormone, leptin, and oestrogen. We know that obesity is associated with a poorer prognosis in patients with breast cancer and that the heaviest women have the highest mortality. Leptin is a hormone produced in the fat cells and circulates in the bloodstream. We know that the receptors on the surface of cells for this hormone are present in breast cancer tissue. Because of this we are studying the effects of leptin on breast cancer cells, and also whether there is an interaction between leptin and the female hormone oestrogen. It is well known that oestrogen plays an important role in breast cancer. These studies may allow us to find novel interactions between these hormones and have, in the future, potential to lead to new therapies.


DIVISION OF CLINICAL SCIENCES (N)
Head: Professor P.G. Hellewell

Academic Department of Human Metabolism
Head: Professor R.J.M. Ross
and
DEPARTMENT OF ENGINEERING MATERIALS
Head: Professor A.R. West

Melanocortins and Melanoma Invasion
Professor S. Mac Neil (Division of Clinical Sciences (N) & Department of Engineering Materials), Dr. J.W. Haycock (Department of Engineering Materials), Dr. P. Lorigan (Academic Unit of Clinical Oncology, Section of Oncology & Pathology, Division of Genomic Medicine)

a-melanocyte stimulating hormone (a-MSH) is a peptide hormone derived from proopiomelanocortin, which regulates several aspects of melanocyte (and melanoma) function and some, but not all, epidemiological studies suggest that non-functional variants of the melanocortin receptor (MC-1) are associated with increased melanoma metastases. We have identified using immunofluorescent microscopy and western blotting techniques that both human cutaneous (HBL, A375SM and C8161) and uveal (196b, VUP and 177w7B7) melanoma cell lines express the MC-1 receptor. However, in collaboration with Prof. G. Ghanem (Belgium) and Prof. J.C. Garcia-Borrn (Spain) we have shown that whilst the HBL melanoma has a high number of MSH receptor binding sites and a wild type receptor (from receptor sequencing data), the other two cutaneous melanomas have low receptor binding sites and MC-1 receptor polymorphisms (A375SM, homozygous Cys 151; C8161, Arg (wt) 151 / Cys 151). These findings corroborate our functional data showing thata-MSH can potently inhibit the invasion of HBL melanoma cells, has only a partial effect on A375SM cells and is ineffective at inhibiting C8161 invasion. While the receptor status of the three uveal melanomas has not yet been fully established,a-MSH at a concentration of 10-9 M significantly inhibited the invasion of all three cell lines. Furthermore, we have recently demonstrated thata-MSH can attenuate pro-inflammatory cytokine (TNF-a)-induced increases in invasion for one of the cutaneous (HBL) cell lines and for all three uveal melanomas. Interestingly,a-MSH had no effect on TNF-a induced increases in C8161 invasion. The data so far indicates that for cutaneous melanoma cells, MC-1 receptor polymorphisms may contribute to melanoma invasion and that MSH may also be relevant to the metastatic potential of uveal melanomas.


DIVISION OF CLINICAL SCIENCES
Director: Professor H. F. Woods

SECTION OF SURGICAL & ANAESTHETIC SCIENCES

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

Regulating effects of HGF and TGF-b on uveal melanoma invasion
Dr K. Sisley, Professor I.G. Rennie

Uveal melanomas are highly aggressive tumours that selectively target the liver. Regulation of their invasive ability is controlled by a number of factors, and recent evidence suggests that HGF and TGF-b may play a part respectively in the positive and negative control of uveal melanoma invasion. The mechanism by which they control invasion is unknown. In this current investigation, using adhesion assays, we are studying 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. Our initial findings have shown that HGF, TGF-b (1 and 2), and endothelial cells themselves, can regulate expression of adhesion molecule by uveal melanomas. Invasive uveal melanomas were found to be more adherent to endothelium than non- invasive cells, and differences were also observed in the effect produced by hepatic, but not dermal endothelial cells. Our findings would suggest that hepatic endothelium may assist in the targeting of uveal melanoma cells to the liver, and that the process is in part regulated by binding of tumoura4 integrin to its ligand, VCAM-1, on the endothelial cells.


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, Professor F.C. Hamdy

The aim of this project was to isolate and grow prostate cancer cells and associated bone cells from bone marrow biopsies taken from patients with metastatic prostate cancer growing in their skeletons.

Twelve bone marrow cell lines from prostate cancer patients have been isolated and studied in detail. We have also maintained prostate cancer cells from patients for up to 4 weeks in primary culture. We have been able to use the bone marrow cells we have isolated in combination with prostate cancer cell lines already available to us to study factors produced by bone cells that could affect tumour cell growth.

We have shown that the bone marrow cells we have isolated produce a protein named osteoprotegerin or OPG. This protein inactivates a mechanism by which a patient's immune system kills prostate cancer cells. That these cells do this is very important as it means that, for the first time, we have clear evidence to show that cells in the bone marrow can help neighbouring tumour cells to survive in the skeleton. The presence of OPG in the bone marrow biopsies themselves has been confirmed in tissue sections using antibodies raised against OPG. We collected 30 matched primary prostate cancers and metastatic tumours from the same patients for histological analyses. This is a unique and valuable resource. In addition to these studies we have been able to show that the level of OPG in the circulation of patients with metastatic, aggressive disease was significantly higher than those with disease confined to the prostate or non-cancer patients. This again suggests that OPG is produced in and around metastatic tumours and is released into the circulation where it could be useful as a marker of disease progression.


DEPARTMENT OF BIOMEDICAL SCIENCE
Chairman:Professor P.W. Andrews

Genetic determination of the ability of malignant stem cells to differentiate
Professor P.W. Andrews

Embryonal carcinoma (EC cells), the malignant stem cells of teratocarcinomas, a subset of testicular germ cell tumours (TGCT), are pluripotent, i.e. capable of differentiating into a wide range of cell types. Since differentiation is accompanied by a loss of malignancy these cells are subject to strong selection for mutations that limit their capacity for differentiation, i.e. they are nullipotent. EC cells provide a paradigm for the concept that cancer arise from genetic changes that affect the behaviour of stem cells in a wide range of tissues, and particularly the mechanisms that regulate the balance between their self-renewal and differentiation. Our genetic studies of a nullipotent EC line, 2102Ep, and a pluripotent EC line, NTERA2, suggest that nullipotency is due, at least in part to recessive, loss of function mutations. Whereas 2102Ep cells activate retinoic acid (RA) responsive genes after exposure to RA, in contrast to NTERA2 cells they do not differentiate in response to this inducer. Therefore the putative mutations are downstream of the RA response mechanism. We have now focused on the potential role of p27, a key regulator at the G1:S boundary of the cell cycle as one possible target for such mutations. We have found that p27 is up-regulated in NTERA2 but not 2102Ep cells, in response to RA, suggesting that p27 up-regulation is a primary response to RA and that this protein is a central element for mediation between RA and cell differentiation. However, if p27 is over-expressed in 2102Ep cells by transfection, there is a marked reduction in growth rate but without significant differentiation, even after exposure to RA, although neural differentiation is enhanced in p27-transfected NTERA2 cells. Our results suggest that regulation of p27 is necessary but not sufficient for differentiation to proceed and that genetic changes in 2102Ep cells have affected further downstream components of the differentiation pathway as well as regulation of p27 itself.

Maintenance of Leica imaging system
Professor P.W. Ingham FRS (Centre for Developmental Genetics)

The confocal microscope is key piece of equipment used by cell and developmental biologists to visualize specific proteins in individual cells. The information obtained about the distribution of proteins gives us important clues about their function in normal development and in cancer. One of our major focuses is the family of Frizzled proteins that act as extracellular docking proteins - or receptors - for secreted signalling proteins of the Wnt family. Defects in Wnt/Frizzled signalling have been implicated in a wide range of human diseases including cancers. Our studies of the subcellular localisation of Frizzled have revealed that it accumulates asymmetrically within the cell in a complex with other conserved proteins. We have demonstrated a role for these complexes in modulating the activity of another receptor protein called Notch that is also implicated in cancers (Strutt et al 2002). Using similar methodologies, we have also been able to demonstrate that Frizzled has two separable functions that are both involved in polarizing the cell (Strutt and Strutt, 2002), a process likely to be of importance for cell migration and hence for tumour mestastasis. Another major focus of our group is the family of Hedgehog proteins. These are deployed extensively during embryonic development in humans; importantly, inappropriate Hedgehog signalling activity has been shown to cause several cancers including basal cell carcinoma, medulloblastoma and rhabdomyosarcoma. We have discovered that Hedgehog signalling plays an important role in the development of the inner ear, specifically distinguishing the back of the ear from the front (Hammond et al 2003). Understanding how the signal can give cells specific identities in this way will give us new insights into the response of cells to aberrant activity of the signal and hence into the oncogenic process.


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

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

This work began with engineering the DNA of our model organism, Saccharomyces cerevisiae, to be able to answer the molecular questions being asked. This period of work required complex genetic engineering in bacteria and transfer of the new DNA sequence to yeast cells. This step was successful and was followed by testing the engineered DNA sequence to check that it can report the mode of DNA repair in the way it was designed. Genetic and molecular experiments have been used to induce damage into the engineered DNA and confirm that way it gets repaired is according to expectations.

The next step was to make our model system mutant for the genes whose function we wish to test in DNA repair. Three genes have been deleted or mutated creating 5 new strains ?mre11, mre11-58, mre11-125N, ?xrs2, ?rad50. Each of these mutants is now being tested to check the effect of loosing gene function on how the engineered DNA is repaired. Results so far indicate that the genes are required to be present to initiate repair in some cells. In other cells repair does take place but in a less regulated way that leads to more deletions of DNA, increasing the rate of loss of heterozygosity.

We are now preparing to make many mutant forms of the three gene MRE11, XRS2 and RAD50 to determine find out exactly how their proteins work to control repair of damaged DNA. Phase of the work is just starting and is on schedule to meet to demands of the proposed project.