research reports

University of Sheffield

2001/2

Director's Introduction:
Professor B.W. Hancock

The Institute for Cancer Studies and Academic Unit of Clinical Oncology are generously endowed by Yorkshire Cancer Research. In addition much other cancer research in Sheffield is supported by Yorkshire Cancer Research programme or project grants. The Clinical Trials Centre at Weston Park Hospital is now well established and is the hub of the new North Trent Cancer Research Network (NTCRN). We are proud that the NTCRN is one of the first networks to be established as part of the strategy within the National Cancer Plan to ensure that more cancer patients across the U.K. have the opportunity to participate in clinical research programmes. On the scientific front the new appointments within the Yorkshire Cancer Research Institute for Cancer Studies (in year 2000) are already starting to bear fruit: innovative research protocols are attracting other outside funding and peer-reviewed publications are starting to flow.

An ethos of collaborative research is very much fostered by the University of Sheffield Medical School. In cancer this is demonstrated by the translational research links between clinicians and scientists, not only in the Institute for Cancer Studies and Academic Unit of Clinical Oncology but also in Biomedical Science, Engineering Materials, Clinical Sciences, Molecular Biology & Biotechnology, and Surgical & Anaesthetic Sciences.


CENTRAL RESEARCH FACILITIES

Flow Cytometry Unit
Dr J. Lawry & Mrs M.O. Smith

The flow cytometry unit continued to provide a useful service to a wide variety of users in 2001, thanks to the generous funding provided by Yorkshire Cancer Research to enable the renewal of the service contracts with Becton Dickenson and Laser Support Services. As well as internal users from the Institute for Cancer Studies, we worked with a number of groups from the University Medical School, and from University Departments at Western Bank. There were also regular users from Sheffield Hallam University and the Jessop Hospital, and occasional users from York, Barnsley and Leeds. The computers and printers were upgraded this year, giving much needed extra data storage capacity and speeding up analysis and presentation.

As well as continuing to analyse transfection efficiency, antibody binding, cell cycle and growth rate, DNA ploidy, apoptosis and calcium uptake, there have been some interesting new additions, such as FRET (fluorescence resonance energy transfer), used to measure molecular interactions in living cells.

One of the major events of 2001 was the departure of Dr John Lawry at the end of the year to take up a position with Becton Dickenson. He will be greatly missed. However as a scientific support specialist with Becton Dickenson he has promised that he will continue to be available to offer his advice and expertise to the unit whenever it is needed.

Clinical Trials Centre
Prof R.E. Coleman & Prof B.W. Hancock

The Academic Unit of Clinical Oncology first established a clinical research facility at Weston Park Hospital in 1992. The principal 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 is able to conduct 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 five years over 2000 patients, with a broad range of different cancers, have been entered into clinical trials. The impressive research output over the past few years in terms of publications and national and international presentations indicates both the excellent quality of research and the potential for future development.

The Academic Unit is now located in new, purpose built facilities. The Cancer Research Centre is a ?2million building, generously funded by the Weston Park Hospital Cancer Appeal with a major donation from the University of Sheffield, that provides facilities for the assessment and treatment of patients, office space for staff with fully networked computer systems, a laboratory, and specialist activities such as bone densitometry. The environment is ideal for both patients and staff and has allowed rapid expansion of the staffing and the range of research that can be performed.

Clinical research relies on collaboration. As a result, strong links have been established with many of the collaborative research groups across the world. These include the National Cancer Research Institute (NCRI), the Medical Research Council (MRC), and the European Organisation for Research and Treatment in Cancer (EORTC) among many others. Additionally, research links with laboratory based research teams both in Sheffield and beyond have flourished and provided opportunities for translational research in bone oncology, melanoma and lymphoma.

The Cancer Research Centre now has a national profile in many areas and international recognition in several key research areas. Over 80 peer-reviewed manuscripts have been published over the past 5 years. Senior members of staff hold important positions on the committees of the NCRI and EORTC tumour specific groups. Additionally, staff are invited on a regular basis to lecture at major international congresses and symposia.

In recognition of our early achievements, we were successful in 1996 in securing a 5 year programme grant from Yorkshire Cancer Research (YCR) to fund key posts. This investment undoubtedly catalysed the successful expansion of the Cancer Research Centre.

In 1998, National Health Service Research and Development (NHS R&D) funds (termed "Culyer" funding) allowed further expansion of the department. This major source of funding was awarded to Weston Park Hospital (on behalf of the North Trent Cancer Network) to increase clinical trials participation in breast cancer and lymphoma across the North Trent network of hospitals. Subsequently, this funding (now called Support for Science) has been extended and enhanced by NHS R&D to continue the work in breast cancer and lymphoma and also expand the clinical trials service into the colorectal and lung cancer clinics across North Trent. This network model of cancer research has been adopted as a national strategy. As a result, we have recently been established as one of the first wave of cancer research networks. Additional funds have been made available through the National Cancer Research Network (NCRN) as part of the NHS Cancer Plan for us to enhance and develop the NTCRN further.


DIVISION OF GENOMIC MEDICINE
Director: Prof G.W. Duff
Deputy Director (Clinical): Prof B.W. Hancock

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

Institute for Cancer Studies
Head: Prof M. Meuth

Genetic Instability and Cancer:
Prof M. Meuth

Hereditary nonpolyposis colon cancer patients inherit a mutant mismatch repair (MMR) gene and inactivate the remaining allele as an early step in tumour development. Most tumour cells deficient in MMR develop a distinctive mutator phenotype characterized by an increased rate of mutation and altered sensitivity to DNA damaging agents. Work in the last year has focussed on a novel step we discovered in the development of this subset of tumours. We found that tumours deficient in MMR also acquire a deficiency of the homologous recombination repair (HRR) pathway. Mutations in genes required for HRR have been found in most MMR deficient tumour cell lines. The loss of the HRR causes these tumour cell lines to become sensitive to several DNA damaging agents including thymidine and mitomycin C. We are currently investigating the possibility that these agents or related ones may provide novel therapies directed specifically against this subset of tumours. The second major project (led by Angie Cox) has been to determine whether common polymorphisms of HRR genes affect the functions of these genes and constitute risk factors for breast cancer. In collaboration with John Thacker we have shown that a common polymorphism of the HRR gene XRCC2 (R188H) can significantly affect sensitivity of cells to DNA damage. Furthermore, carriage of this allele was associated with breast cancer overall (odds ratio 1.3 (95% CI 1.0, 1.8)), and when younger onset cases with a positive family history were compared with older controls with no family history (odds ratio 1.9 (95% CI: 1.0, 3.8)). These results support the hypothesis that subtle variation in DNA repair capacity may influence cancer susceptibility in the population.

Genetics of non-Hodgkin's lymphoma
Dr D.W. Hammond, Ms M. Baird, Ms C. Wardle and Ms K. Rees

The work of the group continues to be focused on the identification and analysis of genetic change in non-Hodgkin's lymphoma by a combination of cytogenetic, molecular genetic and fluorescent in situ hybridisation approaches.

Using the technique of comparative genomic hybridisation (CGH), we have identified a region of DNA copy number gain on the long arm of chromosome 12, which is associated with the transformation of lymphomas from low grade to high grade aggressive diseases.

We have now detected amplifications of a number of genes from within this region. We are now investigating the status of suitable candidate genes in different lymphoma sub groups, in order to identify potential targets for prognostic tests and ultimately for therapeutic intervention.

The role and action of ceramide in cytokine-induced apoptosis
Dr P. Dobson and Prof B. Brown

Apoptosis (programmed cell death) is a normal, genetically regulated process involved in development and tissue homeostasis. In several cancers, cells fail to undergo apoptosis at a normal rate, resulting in DNA-damaged cells being allowed to persist and divide. As lipids are involved in many of the intracellular signalling pathways controlling apoptosis or survival, we investigated initially the role of ceramide. Although direct addition of ceramide analogues initiated apoptosis, stimulators of apoptosis failed to induce ceramide production until after apoptosis was initiated. This suggested that production of ceramide in certain cancer cells is not involved in the initiation of apoptosis; indeed, ceramide production appears to be a consequence of, rather than a cause of apoptosis. Ceramide may have an important secondary role, however, in the amplification of apoptosis.

In terms of anti-apoptosis, we have observed biochemical differences in the cellular events leading to survival in different cancer cell types. Focusing on the lipid kinase, PI3-kinase, and the subsequent downstream events, we have shown that one of the enzymes in this pathway, Akt, is activated and translocated in the cell in response to survival signals.? In particular, Akt is activated by the cytokine, prolactin, in prostate cancer cells. Also, inhibition of the PI3-kinase pathway can enhance the ability of classical anti-cancer drugs to kill cancer cells. The inter-relationships between the biochemical pathways involved in the opposing signals of pro- and anti-apoptosis and the potential of combination therapies of anti-signalling drugs and known treatment regimens are the major focus of our ongoing work.

Response of tumour cells to hypoxia: role of p53 and NFkB
Dr J. Royds, Prof C.E. Lewis and Dr C. Parker (Pathology)

Human tumours contain areas of low oxygen tension (hypoxia) formed when either tumour cell growth outstrips the development of blood vessels and/or newly formed blood vessels collapse. When tumour cells experience hypoxia they release such pro-angiogenic factors as vascular endothelial growth factor (VEGF), which then stimulate vascularisation and re-oxygenation of cells. This means that tumour cells usually experience re-oxygenation after hypoxia in tumours. The transcription factors, p53 and NFkB, are important regulators of cell survival in response to multiple stress signals, including hypoxia. In this project we: (i) studied the movement inside cells of p53 and p65 under hypoxia and re-oxygenation, and (ii) identified the possible interactions between these two factors during hypoxia and re-oxygenation (using anti-sense oligonucleotides to block expression of these proteins).

Hypoxia slowed tumour cell growth and increased VEGF release and cell death. While re-oxygenation also caused cells to apoptose, a proportion started to cycle again and release high levels of VEGF. P53 protein moved to the nucleus during hypoxia (where it regulates expression of other genes). Inhibition of p53 expression had no impact on cell growth or VEGF release, but reduced the number of cells dying in hypoxia and re-oxygenation. P65 was also seen in the nucleus in hypoxia and re-oxygenation, and blockade of this increased cell death and lowered VEGF release in hypoxia and re-oxygenation. In the absence of wild type (wt) p53, p65 exhibited prolonged nuclear localisation in hypoxia and re-oxygenation. Taken together, these studies indicate that loss or mutation of wt p53 may promote tumour progression in hypoxia by prolonging activation of NF B, which in turn enhances tumour angiogenesis by stimulating VEGF expression.


YCR ACADEMIC UNIT OF CLINICAL ONCOLOGY
Head: Prof R.E. Coleman

The Cancer Research Centre is involved in research studies ranging from dose-finding Phase I studies through to post-marketing Phase IV trials. The key areas of research have continued to flourish over recent years and several new interests have developed. The major areas of research interest include:-

• Clinical Trials and Drug Development
• Bone oncology
• Radiotherapy
• Trophoblastic tumours
• Melanoma

Clinical Trials
Team Leader: Prof. R.E. Coleman

Developments in cancer care rely on the evidence generated from clinical trial participation, typically the randomised controlled trial. Trial participation has many benefits for cancer patients. The opportunity to receive new potentially more effective treatments is an obvious advantage, but equally important is the detailed special monitoring and close medical and nursing supervision that comes with participation in a trial. These latter aspects have been shown in themselves to improve the outcome and survival of cancer patients over and above those not treated within a clinical trial. The importance of clinical trials has been appreciated by the cancer charities for many years. Now the NHS is also making efforts to improve clinical trial participation and establish inclusion within a cancer trial as the standard of care. The NHS Cancer Plan has identified specific funds to support research infrastructure across cancer networks with the aim of doubling the numbers of patient recruited to clinical trials considered to be of national importance. Over the last 5-10 years many thousands of our patients have taken part in large multicentre phase III trials.

Recently completed studies have included:

Breast cancer

• Contribution of tamoxifen to chemotherapy in the adjuvant treatment of breast cancer - (EORTC)
• Evaluation of high dose adjuvant chemotherapy in poor prognosis breast cancer - (Anglo-Celtic Group)
• Evaluation of adjuvant docetaxel in node positive breast cancer - (Breast Cancer International Research group [BCIRG] - Canada)
• Contribution of the aromatase inhibitor anastrozole to adjuvant endocrine therapy in early breast cancer - (Cancer Research Campaign [CRC])
• Role of paclitaxel in palliative chemotherapy for advanced breast cancer - (EORTC)

Ovarian cancer

• Evaluation of paclitaxel in the chemotherapeutic management of advanced ovarian cancer - (Medical Research Council [MRC])
• Comparison of topotecan and paclitaxel in salvage treatment of advanced ovarian cancer - (Industry)

Melanoma

• Evaluation of adjuvant low dose interferon in the management of high risk melanoma - (United Kingdom Coordinating Committee for Cancer Research [UKCCCR])
• Comparison of combination chemotherapy with single agent chemotherapy in metastatic malignant melanoma - (Christie Hospital, Manchester)
• Comparison of combination immunotherapy with single agent chemotherapy in metastatic malignant melanoma - (Industry)

Lymphoma

• Role of high dose chemotherapy in Hodgkin's Disease and high grade non-Hodgkin's lymphoma (NHL) - (British National Lymphoma Investigation - [BNLI])
• Evaluation of mitoxantrone in chemotherapy for elderly patients with high grade NHL - (BNLI)
• Comparison of alternating multi-agent chemotherapy with conventional chemotherapy for advanced Hodgkin's Disease - (BNLI)
• Evaluation of the role of growth factor dose in stem cell mobilisation in non-Hodgkin's lymphoma and Hodgkin's Disease. (BNLI)

Sarcoma

• Comparison of growth factor supported chemotherapy with standard combination chemotherapy for advanced soft tissue sarcoma (EORTC)

Lung cancer

• Comparison of growth factor supported chemotherapy with standard combination chemotherapy for small cell lung cancer (MRC)
• Single agent carboplatin versus combination chemotherapy for small cell lung cancer - (Christie)
• Evaluation of the role of the metalloprotease inhibitor marimastat in patients with small cell lung cancer who have had a good response to chemotherapy - (Industry)
• Evaluation of the role of the metalloprotease inhibitor marimastat in patients with non-small cell lung cancer who have had a good response to radical treatment - (Industry)

Drug Development
Team Leaders: Dr P. Lorigan/Dr M. Marples

Improvements in the chemotherapy of advanced cancer continue to be sought through the evaluation of new cytotoxic agents. This is a very important part of our clinical research programme and we maintain a portfolio of studies across many tumour types assessing the activity and suitability of new agents. In patients with breast and ovarian cancer, melanoma, lymphoma, lung cancer and sarcoma, patients are whenever possible entered into multi-centre studies to evaluate new treatment strategies. The majority of studies are at the phase II and phase III level of development but through collaboration with industry a number of phase I studies have recently started or are planned.

Recently completed studies have included:

Breast cancer

• Role of corticosteroids in the prevention of docetaxel induced toxicity
• Phase II study of docetaxel in liver metastases
• Phase II study of multi-target antifolate LY23155
• Phase I study of liposomal doxorubicin (Caelyx)
• Phase II study of antisense oligonucleotide ISIS 2503
• Phase II study of gemcitabine

Ovarian cancer

• Phase II study of altretamine
• Phase II studies of docetaxel plus cisplatin or carboplatin
• Phase I and II studies of the cytotoxic immunoconjugate CMB-401 (hCTM01-calicheamicin)

Melanoma

• Randomised Phase II study of temozolomide +/- interferon +/- thalidomide in advanced malignant melanoma

Lymphoma

• Phase II study of docetaxel in relapsed NHL
• Phase II study of Rituximab in low and high grade NHL
• Phase II study of the stem cell inhibitor MIP-1a with combination chemotherapy in high grade NHL

Sarcoma

• Phase II study of temozolamide
• Phase II study of gemcitabine
• Phase II study of ET743
• Phase II study of the tyrosine kinase inhibitor STI 571

Lung cancer

• Phase I study of the cytotoxic immunoconjugate CMB-401 (hCTM01-calicheamicin)
• Phase II study of liposomal cisplatin in non small cell lung cancr(NSCLC)
• Phase II trial of ZD9331 as second line chemotherapy in NSCLC.

Colorectal

• Phase II studies of various oral fluoropyrimidines

Bone Oncology
Team leader: Prof. R.E. Coleman

The Department has an international reputation in the management of bone metastases especially in the context of advanced breast cancer. We continue to perform a variety of treatment studies with bisphosphonates that have incorporated careful evaluation of pain relief and quality of life along with both biochemical and radiological response to treatment to try and define the type, dose and schedule of bisphosphonate for clinical use. Recently completed studies have included:

• Evaluation of bone resorption inhibitors in monitoring bisphosphonates and response to therapy (EORTC)
• Phase I evaluation of oral ibandronate
• Evaluation of the metalloproteinase inhibitor, marimastat on bone metabolism
• Phase II and III clinical development of the new highly potent bisphosphonate zoledronate
• Assessment of dose-intense and high dose schedules of pamidronate.
• A comparison of clodronate and pamidronate using the dose and schedules currently recommended to determine differences in biochemical or
  symptomatic response.
• Phase I evaluation of recombinant osteoprotogerin (OPG)

Trophoblastic Tumours
Team leader: Prof. B.W. Hancock

Trophoblastic tumours are considered a model for cancer. They present early, are monitored precisely by a unique marker (human chorionic gondatrophin), are curable with chemotherapy and the patients live normal lives thereafter.

Sheffield is one of two UK Centres treating trophoblastic tumours. Therefore, there is a wealth of clinical, biochemical and histopathological data available for research. Recently completed research projects include:

• serosal problems during methotrexate treatment
• post-partum choriocarcinomas
• the role of central nervous system prophylaxis
• an analysis of placental site trophoblastic tumours
• a retrospective comparison of staging and scoring systems for trophoblastic tumours
• an analysis of the risk of second moles
• a report on the Sheffield chemotherapy regimen for high risk and chemoresistant tumours

Radiotherapy Research
Team leader: Dr M. Robinson

Over the last five years our research has focused on the introduction of conformal radiotherapy techniques in head and neck and prostate cancer. This has been achieved via the following completed studies:

• Detailed comparison of the benefit of 3D planning in terms of dose conformity in the head and neck.
• Cost effectiveness assessment of CT planned conformal radiotherapy in head and neck cancer.
• Entry of patients into the RT01 study comparing two dose levels in the treatment of early prostate cancer using conformal radiotherapy
• Detailed assessment of the impact of tissue compensators in the quality of treatment plans used in head and neck cancer
• Improving target volume contouring in radiotherapy for head and neck and brain tumours. This study assessed inter- and intra-observer errors and the value of intravenous contrast use.
• Detailed analysis of the impact of implementing CT simulated conformal radiotherapy in the treatment planning of head and neck cancer.
• Virtual simulation study of palliative radiotherapy for lung cancer
• Development of an automated image registration system.

Melanoma Research
Team leader: Dr P. Lorigan

There is good evidence for an effect of gender and sex steroids on the biological behaviour of melanoma, with females demonstrating a significantly better prognosis than males. Over the last 3 years, we have developed a laboratory model of invasion and metastasis to study this difference in biological behaviour. We initially used a simple reductionist model of invasion and metastasis, characterising the effect of female sex steroids on melanoma cell lines. We then developed a unique reconstructed skin model that is more akin to normal skin. Using this, we have been able to identify that there is a complex interplay of malignant cells, normal skin constituents, growth factors and TIMPS. We are now at a stage where we plan to characterise these interactions in detail.


Academic Unit of Pathology
Head: Prof J.C.E. Underwood

Role of early growth response gene-1 in macrophage responses to hypoxia
Dr. B. Burke, Professor M. Wells and Professor C.E. Lewis

Tumours contain regions of low oxygen (hypoxia) in which white blood cells called macrophages accumulate and respond to the hypoxia present by releasing protein messengers called cytokines, which encourage the formation of new blood vessels. This, in turn, helps the tumour to grow and spread. Despite the important role played by macrophages in tumour progression, surprisingly little is known about how they respond to hypoxia. In this project we investigated the effect of hypoxia on the expression of nuclear proteins called transcription factors (TFs). These are known to control gene expression in cells, including macrophages. We exposed human macrophages to the levels of hypoxia present in tumours, and looked to see whether a range of important TFs were upregulated. We also looked to see whether macrophages in hypoxic areas of human tumours expressed the same panel of TFs using a technique called immunohistochemistry. An earlier paper suggested precursor cells for macrophages (monocytes) might express a pluripotent TF called Early Growth Response gene-1 (Egr-1) when exposed to hypoxia. Although we found no evidence of this in hypoxic human macrophages (in vitro or inside human tumours), we were able to show that these cells produce other important hypoxia-regulated transcription factors such as Hypoxia-Inducible Factors 1 and 2 (HIFs 1 and 2). These proteins then stimulate a number of genes with pro-tumour functions to be expressed in macrophages. This suggests that HIFs play an important part in driving the pro-tumour functions of macrophages in hypoxic areas of tumours, and highlights these as new molecular targets for anti-cancer therapies.


DIVISION OF CLINICAL SCIENCES (N)
Head: Prof R. Eastell

Melanocortins and melanoma invasion
Prof S. Mac Neil & Dr P. Eves, Dr J.W. Haycock (Department of Engineering Materials) and Dr P.C. Lorigan (Academic Department of Clinical Oncology)

Melanoma invasion from the primary tumour through the basement membrane is believed to be the first step in melanoma metastasis. Metastatic melanoma remains one of the most difficult tumours to treat. There is an increasing amount of evidence to support a causative role for the local production of melanocortins in melanocyte transformation and melanoma metastasis. We have previously demonstrated that MSH peptides can confer anti-inflammatory, immunomodulatory and antioxidant activity on melanocytes and melanoma cells and thus propose that endogenous production of such peptides may enable melanoma cells to withstand exogenous stress such as immune cell generated cytokines, oxidative stress, UV radiation and chemotoxic drugs. Hence MSH may play an important role in melanoma invasion and metastasis.

We have investigated the effect of α-melanocyte stimulating hormone (a tridecapeptide derived from proopiomelanocortin (POMC)) on the invasion of three genetically different human melanoma cell lines (HBL, A375SM, C8161) all possessing the MC1 receptor. Using a simple invasion system (consisting of Transwell inserts coated with human fibronectin) we have shown that α-MSH could potently inhibit HBL invasion, partially inhibit A375SM invasion (at very low concentrations of α-MSH), but was ineffective in inhibiting C8161 invasion. This peptide was also markedly effective at inhibiting HBL invasion in a more complex reconstructed skin model. Finally, the ability of α-MSH to influence the response of cells to inflammation (examined using the proinflammatory cytokine TNF-α) was determined. Responses to α-MSH paralleled those seen for invasion through fibronectin in that?α-MSH was particularly effective at opposing TNF-α induced activation of the transcription factor NF-kB in HBL cells, it gave a partial response in A375SM cells but was ineffective in C8161 cells. So far these data support both an anti-invasive and an immunomodulatory role for α-MSH in some human metastatic melanoma cells.


DEPARTMENT OF BIOMEDICAL SCIENCE
Head: Prof P.W. Andrews

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

Embryonal carcinoma (EC) cells are the malignant stem cells of teratocarcinomas which commonly occur as testicular cancers and contain a wide range of cell differentiation. However, many cases of embryonal carcinoma appear to have lost the ability to differentiate. Since differentiation is accompanied by loss of malignancy, those EC cells that do not differentiate presumably arise because of genetic mutations that interfere with differentiation, and so are likely to have a selective advantage in tumour progression. We have shown that hybrids of a pluripotent human EC cell NTERA2 and a nullipotent EC cell, 2102Ep, retain an ability to differentiate. Thus the loss of an ability to differentiate in the 2102Ep EC cells appears to be due to recessive mutations. We are seeking to identify the nature of these mutations, and determine whether there are specific genes involved in permitting cells to differentiate, which may be common targets for such mutations. We are expanding the range of hybrids in which to study this phenomenon, and have produced a selectable subline of the nullipotent EC cell TERA1, which is both HAT sensitive and neomycin resistant. We are currently producing hybrids between these cells and the 2102Ep nullipotent EC line. If different mutations in these cells leads to loss of pluripotency, the resulting hybrids are likely to demonstrate common mutation and regain the capacity for differentiation. On the other hand, if a very limited range of genes are affected by such putative mutations, complementation is unlikely and the expected hybrids will most likely not differentiate. We are further testing the role of various candidate genes p16, p21 and p27 inhibitors of cyclin dependent kinase that may play a role in the switch between undifferentiated and differentiated state.


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

The role of the conserved Cut5/Rad4 protein in initiating the DNA structure checkpoints
Dr C. Price

All eukaryotic cells monitor genome integrity via conserved mechanisms that have come to be termed DNA structure checkpoint control. These control mechanisms act to block cell division in response to either DNA damage or the inhibition of DNA replication. This enables the cell to escape the consequences of these events, which would otherwise lead to mutation. Loss of checkpoint function is a major step in tumour development and has important consequences for cancer therapy. In fission yeast, a widely used model organism for studying eukaryotic cell biology, there are two distinct pathways distinguished by the signals to which they respond; either DNA damage or the inhibition of DNA replication. The Rad4/Cut5 protein acts in the checkpoint response pathways to ?-irradiation and S-phase checkpoint pathways. We have shown that the activity of the conserved downstream effector kinases, Chk1 and Cds1, is dependent upon Rad4p function whilst post-translational modification of the Rad9, Hus1 and Rad26 proteins is not. Genetic experiments have demonstrated that Rad4 acts as a suppressor of mutations in three other conserved checkpoint proteins. This work is now complete and the results have led us to propose a new model for the function of Rad4, which has relevance for human DNA damage responses and which has recently been submitted for publication.

Characterisation of the fission yeast Drc1p homologue
Dr C. Price

The proposal is designed to characterize the role of the fission yeast Drc1 homologue in DNA replication and DNA structure checkpoint control, particularly with respect to its possible interaction with Rad4. It is vital to the understanding of the precise role of Rad4 in the checkpoint response that proteins with which it interacts are identified. On the basis of our previous YCR funded work we have extended this project to include a general screen for proteins, which interact with Rad4 in response to DNA damage. Early results indicate that this search has been successful although much remains to be done. We intend to examine the possibility of physical interaction between Rad4 and the proteins identified in the screen and follow their behaviour when Rad4 function is compromised.