research reports

University of Sheffield

2003/4

Director's Introduction
Professor B. W. Hancock

Cancer research in Sheffield has well-established programmes in both basic and clinical research. The main scientific research programme is based in the YCR Institute for Cancer Studies, which occupies 800 sq. meters of newly 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 specialized facilities within Surgical and Anaesthetic Sciences, Pathology, Genomic Medicine and Tissue Engineering, and in the University Departments of Biomedical Science and of Molecular Biology and Biotechnology.


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

A tumour derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks
Dr A. Mohindra, Ms E. Bolderson, Dr J. Stone (Department of Histopathology, Royal Hallamshire Hospital, Sheffield Teaching Hospitals NHS Trust), Professor M. Wells (Academic Unit of Pathology, Division of Genomic Medicine, University of Sheffield), Dr T. Helleday, Professor M. Meuth

Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in a HRR defective tumour cell line, we introduced 342delT into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA

The ATM-NBS1-MRE11 pathway is essential for the homologous recombination repair mediated rescue of DNA replication forks impaired by thymidine
Dr J. Lee, Ms E. Bolderson, Dr T. Helleday , Dr C.G.W. Smythe (Department of Biomedical Science, University of Sheffield), Professor M. Meuth

In interphase cells, genetically distinct checkpoints, activated as a consequence of either replicational arrest or ionising radiation-induced DNA damage, integrate the appropriate DNA repair responses into the cell cycle programme. The ATM protein kinase blocks cell cycle progression in response to DNA double strand breaks while the related ATR kinase is important in maintaining the integrity of the DNA replication apparatus in response to lesions that arrest replication fork progression. We have found that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, is dependent on both ATM and ATR. We have found a functional link between ATM, the MRE11/NBS1/Rad50 DNA repair complex and homologous recombination in the cellular response to thymidine-mediated replicational slowing. Thymidine treatment induces ATM-mediated activation of the Chk2 and NBS1/MRE11 pathways as well as ATR-mediated phosphorylation of Chk1. Furthermore, on exposure to thymidine, both AT cells and cells expressing a tumour associated mutant form of MRE11 show decreased viability, fail to activate the NBS1/MRE11 complex, and fail to induce homologous recombination repair. Taken together, our results implicate both ATM and MRE11 in homologous recombination repair-mediated rescue of replication forks impaired by thymidine treatment. Given that this damage response pathway is disrupted in greater than 90% of MSI+ tumors, our finding have important implications for treatment strategies directed against this subset of tumors.

Distinct patterns of microsatellite instability are seen in tumours of the urinary tract
Dr J.W. Catto (Academic Unit of Urology, Clinical Sciences Centre (S), University of Sheffield), Dr A.R. Azzouzi (Academic Unit of Urology, Clinical Sciences Centre (S), University of Sheffield), Dr N. Amira (Dpartement dUrologie, Facult de Mdecine, Paris, France), Dr I. Rehman (Academic Unit of Urology, Clinical Sciences Centre (S), University of Sheffield), Dr K.M. Feeley (Department of Pathology, Royal Hallamshire Hospital, Sheffield), Dr S.S. Cross (Academic Unit of Pathology, Division of Genomic Medicine, University of Sheffield), Dr G. Fromont (Service dAnatomo-Pathologie, Institut Mutualiste de Montsouris, Paris, France), Dr M. Sibony (Service dAnatomo-Pathologie, Hpital Tenon, Paris, France), Professor F.C. Hamdy (Academic Unit of Urology, Clinical Sciences Centre (S), University of Sheffield), Dr O. Cussenot (Dpartement dUrologie, Facult de Mdecine, Paris, France), Professor M. Meuth

Two forms of microsatellite instability (MSI) have been described in human cancer. MSI typical of hereditary nonpolyposis colon cancer (HNPCC), is due to deficient DNA mismatch repair (MMR) and is defined with mono- and dinucleotide repeat microsatellites. A second variety of instability is seen at selective tetranucleotide repeats (EMAST; elevated microsatellite alterations at select tetranucleotides). While MSI occurs infrequently in bladder cancers, EMAST is common. Sporadic tumours with the largest proportion showing MSI are those found most frequently in HNPCC kindreds. While bladder cancer is not frequently seen in HNPCC, upper urinary tract tumours (UTTs) are. Having previously found a low frequency of MSI in bladder cancer, we sought to determine the relative levels of MSI and EMAST in transitional cell carcinoma (TCC) of the upper and lower urinary tracts. Microsatellite analysis was performed at 10 mono- and dinucleotide and eight tetranucleotide loci, in 89 bladder and 71 UTT TCC. Contrasting patterns of instability were seen in urinary tumours. In bladder cancer, MSI was rare and EMAST was common. The presence of EMAST was not related to tumour grade, stage, subsequent outcome or immunohistochemical expression of the MMR proteins. In UTT, while MSI occurred frequently, EMAST was seen less frequently than in bladder cancer. When TCC of the upper and lower urinary tracts are compared, MSI-H is more frequent in UTT and EMAST more frequent in bladder cancer. Our findings show that the pattern of MSI varies with location in the urinary tract. In addition, we have confirmed that MSI and EMAST are discrete forms of MSI, and that the presence of EMAST does not affect tumour phenotype.

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 recently discovered 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 breast cancer cells with a defect in BRCA2, while other somatic cells are unaffected following the same treatment.

A structural investigation of the papillomavirus E1 ATPase/helicase protein
Dr C.M. Sanders, Dr F. Anston (University of York Structural Biology Laboratory)

Replication of papillomaviruses, including the high-risk oncogenic human types, is controlled by the viral replication initiator protein E1. E1 hydrolyses ATP and uses the energy thus released in several ways. First, an ATP-dependent switch governs the conversion of a specific pre-replication complex to a series of replication active E1 initiator complexes. As E1 then progressively multimerises on the origin DNA, ATP is required for complex stability, origin DNA melting, and finally to drive helicase activity that unwinds the DNA. The mechanisms that underlie these ATP-dependent processes are not understood. To learn more, we have begun a structural investigation of E1 and the viral transcription factor E2, its binding partner in the pre-replication complex. Protein fragments from E1 and E2 that interact, the E1 helicase domain and the E2 transactivation domain, have been over produced in E. coli and purified to homogeneity. In collaboration with Dr Fred Anston, these fragments have been crystallised for structural analysis. The structure of the E2 fragment has been solved to 3 Angstroms. The crystallisation parameters for E1 are under refinement, as we also explore other E1 related proteins for expression, purification and crystallisation. Our ultimate aims are to solve molecular structures for E1 in complex with E2, and an E1 fragment with ATPase and helicase activities. As potential therapeutic targets, these structures may prove invaluable in drug design or validation.

Fluorescence Imaging System
Dr D.W. Hammond, Professor M. Meuth

This equipment, sited in the Institute for Cancer Studies, is primarily used for analysing large scale changes in tumour genomes. It can additionally facilitate the physical mapping of DNA clones.


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

Professor R.E. Coleman, Dr D.W. Hammond, Professor B.W. Hancock, 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 S MacNeil (Division of Clinical Sciences (N) & Department of Engineering Materials), Professor M.W.R. Reed (Academic Unit of Surgical Oncology, Division of Clinical Sciences (S)), Professor I.G. Rennie (Academic Unit of Ophthalmology & Orthoptics, Division of Clinical Sciences (S))

With the establishment of a cancer research theme across University and NHS the team has been able to capitalise on excellent existing collaborations (academic/ NHS clinicians, clinicians/translational scientists) and use to the full specialist clinical and laboratory facilities. The network serves a population of almost 2M giving an excellent resource of clinical material for which Sheffield provides the specialist oncology facilities (chemotherapy, radiotherapy, laboratory studies). Sheffield is also a national referral centre for gestational trophoblastic neoplasia and ocular melanoma.

Key research areas supported in part by YCR funding

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, Section of Surgical & Anaesthetic Sciences) and Dr N.J. Brown (Microcirculation Research Unit, Academic Unit of Surgery, Section of Surgical & Anaesthetic Sciences)(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, Professor P.W. Ingham (FRS Centre for Developmental Genetics, University of Sheffield)

This project has not yet commenced; grant awarded in October 2003.


SECTION OF FUNCTIONAL GENOMICS
Head:Professor S. Dower

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

Regulation of splicing of the cellular survival gene MCL-1
Dr C. Bingle, Professor M.K.B. Whyte, Dr J.T. Reilly (Academic Unit of Haematology, Division of Genomic Medicine)

Mcl-1, an antiapoptotic member of the BCL-2 family, 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 this study sought to investigate this hypothesis.

We recruited 30 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. 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. These isoforms have different distribution within cells with at least one of the proteins being localised to the nucleus. We have studied the expression of these protein isoforms in the leukaemic samples as well as in normal neutrophil precursors isolated from bone marrow. We have also investigated the induction of expression of these different isoforms using a variety of in vitro and in vivo models and have been able to show clear regulation of protein levels. These results suggest that the Mcl-1 gene undergoes complex processing to generate functionally distinct isoforms, which may play a role in mediating cell lifespan in the myeloid lineage. Modulation of expression of these protein isoforms may form the basis of a novel therapy for leukaemia.

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.

We have generated and isolated human and mouse HE4 expressing CHO cell lines from which we are able to isolate recombinant HE4 for immunisation and for use as positive controls.

We have also obtained some monoclonal antibodies for human HE4, which we are presently in the process of characterising by western blotting (using HE4 expressing lung cancer cell lines) and immunohistochemistry (using tissue arrays of normal tissues and a variety of cancers). Initial indications are that these antibodies recognise different epitopes of human HE4 and may be useful for identifying the individual protein isoforms.

These reagents will now be used to support additional funding applications.


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

ACADEMIC SECTION 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 (Section of Oncology & Pathology, Division of Genomic Medicine), Professor R.J.M. Ross

Obesity is associated with a poorer prognosis in patients with breast cancer. The heaviest women have the highest mortality. Leptin is a hormone produced in the fat cells and circulates in the bloodstream at higher levels in heavier women. We know that the receptors on the surface of cells for this hormone are present in some breast cancer tissue. We hypothesised that leptin may cause proliferation of the breast cancer cells and that this could explain the relationship between obesity and breast cancer, and that antagonists of leptin could provide a potential therapy.

We have, therefore, investigated the effects of leptin on breast cancer cells. In contrast to other investigators we have found that only low levels of the active form of the leptin receptor is present on the breast cancer cells that we have studied. Moreover, these cells do not respond to leptin. These data suggest that it is unlikely that the proliferative effects of leptin explain the link between breast cancer and obesity, and that it is unlikely that antagonists of leptin would be of benefit in the treatment of breast cancer.


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

SECTION OF SURGICAL & ANAESTHETIC SCIENCES

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

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

HGF and TGF-ß have been shown to be important regulators of tumour growth and invasion. Recent evidence has indicated they may have a significant role in controlling uveal melanoma invasion, particularly in relation to their selective targeting of the liver. In this investigation we have studied 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 results have shown that hepatic endothelium, but not dermal endothelium can positively enhance adherence by uveal melanoma cells. This interaction appears to be facilitated by the interactions of tumour a4 integrin to its ligand, VCAM-1, on the endothelial cells. We have been able to confirm that HGF, TGF-ß (1 and 2), and endothelial cells themselves, can regulate expression of adhesion molecule by uveal melanomas. More specifically TGF-ß appeared to have more wide ranging effects, and was also observed to strongly stimulate MMP secretion. The response of uveal melanomas to TGF-ß was however found to vary depending on whether they were an invasive or relatively non-invasive melanoma, leading to the possibility, as has been observed in cutaneous melanoma, that there may be a biphasic response to TGF-ß by uveal melanomas.


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.  

This 'pump priming' project has itself now been completed although work started by this study is continuing and additional funding obtained.      


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 testicular germ cell tumours (TGCT), are 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.  EC cells provide a paradigm for the concept that cancers arise from stem cells in a wide range of tissues, and involve dysfunction of the mechanisms that regulate self-renewal and differentiation.  A possible target for such mutations is p27, a key regulator at the G1:S boundary of the cell cycle.  Our results suggest that regulation of p27 is necessary but not sufficient for differentiation to proceed.  Separately, we have also observed that human embryonic stem (ES) cells, of which EC cells are the malignant equivalent, acquire chromosomal changes in culture mirroring the characteristic chromosomal changes of EC cells in TGCT.  This suggests similar selection pressures, affecting the control of self renewal, differentiation and apoptosis, apply in cultured ES cells as in TGCT, and provide a new paradigm for investigating the way in which genetic change may promote the progression of stem cell based cancers.  

Regulation of Pluripotency in Malignant Stem Cells
Professor P W Andrews  

The control of self-renewal and differentiation of stem cells is the key to understanding tumorigenesis.  We will study how dysfunctional control of p27, a Cyclin Dependant Kinase inhibitor, associated with G1 cell cycle arrest and with differentiation, contributes to the loss of pluripotency of Embryonal Carcinoma cells and the progression of germ cell tumours.  We will determine how p27 controls differentiation in EC cells, identifying early response gene targets, mutations in which contribute to the development of nullipotent EC lines with enhanced tumorigenicity.  We will also determine whether dysfunctional control of p27 in nullipotent EC lines may be due to failure to modulate its degradation by a proteosome mechanism.      


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  

Due to refurbishment of the Department of Molecular Biology and Biotechnology, University of Sheffield , Dr Partridge's research group faced two laboratory moves at the end of 2003.  Because of the attendant disruption to research, it was decided to delay starting work on this proposal until the group had moved into new premises in 2004.  

Ovarian cancer is the most common gynaecological malignancy with an overall 5-year survival rate of only 30% in the UK. Despite improvements in surgical techniques and chemotherapy, advanced ovarian cancer responds poorly to current treatments and there is clearly a need to develop new types of therapy. One strategy, which has recently shown success in treating some types of leukaemia and breast cancer, is to use antibodies as "magic bullets" to target and destroy the cancer. We have generated a panel of five antibodies that recognise a known marker for ovarian cancers. These antibodies were produced using a technique that generates fully human antibodies, offering advantages over existing antibodies that are of animal origin, limiting their use in therapy. The aim of the current study is to assess the clinical potential of these human antibodies through detailed characterisation of their properties.  

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.H. 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.