paterson institute for cancer research · of a new pay and grading system for all institute staff....

Paterson Institutefor Cancer ResearchSS CCIIEENNTT IIFF IICC RR EEPPOO RRTT 22000066

Illustration CreditsMany illustrations in this report were taken by Jenny Varley and Paul Cliff

Leukapheresis blood product is depleted of CD25+ lymphocytes prior to infusion back intothe patient. CD4+CD25+ regulatory T-cells are thought to be instrumental in allowing agrowing cancer to evade immunological attack and the rationale behind this clinical trial isthat their removal may unmask an anti-tumour immune response in patients with cancer(See Biological Immune and Gene Therapy Group report on page 36).

Paterson Institutefor Cancer Research

SCIENTIFIC REPORT 2006

CCaanncceerr RReesseeaarrcchh UUKK

Cancer Research UK Paterson Institute for Cancer Research Scientific Report 2006

Edited by Professor Jenny Varley, Assistant Director (Research)Design & Layout by Mark Wadsworth (Web & Graphic Designer)Paterson Institute for Cancer ResearchWilmslow Road, Manchester M20 4BXTel: 0161 446 3156

http://www.paterson.man.ac.uk

ISSN 1740-4525

Copyright © 2006 Cancer Research UK

Printed by Paramount Print

Cancer Research UKRegistered Charity No. 1089464Registered as a company limited by guarantee in England and Wales No. 4325234Registered address 61 Lincoln’s Inn Fields, London WC2A 3PX

Tel +44 (0) 20 7242 0200

http://www.cancerresearchuk.org

P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6

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Contents

DDiirreeccttoorr’’ss IInnttrroodduuccttiioonn .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 66

TThhee MMaanncchheesstteerr CCaanncceerr RReesseeaarrcchh CCeennttrree.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 88

TThhee PPaatteerrssoonn IInnssttiittuuttee ffoorr CCaanncceerr RReesseeaarrcchh

Bioinformatics Group........................................................................ Crispin Miller . . . . . . . . . . . . . . . . . . . . . . . . 10

Carcinogenesis Group .................................................................... Geoff Margison . . . . . . . . . . . . . . . . . . . . . . 12

Cell Cycle Group................................................................................. Karim Labib . . . . . . . . . . . . . . . . . . . . . . . . . 14

Cell Division Group ........................................................................... Iain Hagan. . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Cell Regulation Group...................................................................... Nic Jones . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Cell Signalling Group ......................................................................... Angeliki Malliri . . . . . . . . . . . . . . . . . . . . . . . 20

Clinical and Experimental Pharmacology Group.............. Caroline Dive and Malcolm Ranson . . . . . 22

Immunology Group............................................................................ Peter L Stern . . . . . . . . . . . . . . . . . . . . . . . . 24

Radiochemical Targeting and Imaging Group ...................... Jamal Zweit . . . . . . . . . . . . . . . . . . . . . . . . . 26

Stem Cell Biology Group................................................................ Georges Lacaud. . . . . . . . . . . . . . . . . . . . . . 28

Stem Cell and Haematopoiesis Group .................................. Valerie Kouskoff . . . . . . . . . . . . . . . . . . . . . . 30

Structural Cell Biology Group...................................................... Terence D Allen. . . . . . . . . . . . . . . . . . . . . . 32

TThhee UUnniivveerrssiittyy ooff MMaanncchheesstteerr DDiivviissiioonn ooff CCaanncceerr SSttuuddiieess

Academic Radiation Oncology

Translational Radiobiology Group.............................................. Catharine West . . . . . . . . . . . . . . . . . . . . . 34

Biological, Immune and Gene Therapy Group ................... Robert Hawkins and Peter L Stern. . . . . . 36

Children’s Cancer Group................................................................ Vaskar Saha . . . . . . . . . . . . . . . . . . . . . . . . . 38

Medical Oncology: Breast Biology Group ............................. Rob Clarke . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Medical Oncology: Cell and Gene Therapy Group......... Robert Hawkins. . . . . . . . . . . . . . . . . . . . . . 42

Medical Oncology: Translational Angiogenesis Group.... Gordon Jayson . . . . . . . . . . . . . . . . . . . . . . . 44

Medical Oncology: Proteoglycan Group................................ John T Gallagher . . . . . . . . . . . . . . . . . . . . . 46

Targeted Therapy Group................................................................. Tim Illidge . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

RReesseeaarrcchh SSeerrvviicceess .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 5500

PPuubblliiccaattiioonnss.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 5566

SSeemmiinnaarrss .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 7700

PPoossttggrraadduuaattee EEdduuccaattiioonn .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 7722

OOppeerraattiioonnss SSeerrvviicceess .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 7744

AAcckknnoowwlleeddggeemmeenntt ffoorr ffuunnddiinngg ooff tthhee PPaatteerrssoonn IInnssttiittuuttee .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 7788

CCaarreeeerr OOppppoorrttuunniittiieess .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 8800

HHooww ttoo FFiinndd uuss .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. iinnssiiddee bbaacckk ccoovveerr

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In 1991, the Paterson Institute was incorporatedinto the Christie NHS Trust, a relationship thatexisted until the beginning of 2006. Over theseyears the Institute was the major driver of theTrust’s overall research efforts and close workingrelationships developed between the scientists inthe Institute and the clinical researchers within the

Hospital. The Trust provided significant financial support to the Institute as well as lab-oratory facilities to house our research efforts. However, on the 1st January of this yearthe Institute transferred to become an Institute of The University of Manchester retain-ing its current level of operational autonomy. The major reason for such a move was toinitiate the development of The Manchester Cancer Research Centre (MCRC) which inte-grates all the cancer research efforts of the University and to provide a comprehensiveapproach to cancer research in very close partnership with the Christie NHS Trust andCancer Research UK. The goals and aspirations of the Centre are described in the nextsection and are focused on ensuring that advances in the understanding of the molecularand cellular basis of cancer is translated into new approaches to therapy and meaningful

diagnostics. Through the development of this Centre the Paterson can not only continue to support basicresearch but also facilitate translational and clinical research and thereby achieve its full potential as aCancer Research UK core funded Institute. In addition, not only are our interactions with the rest of theUniversity research activities enhanced, but our interaction within the Christie NHS Trust is also strength-ened through the MCRC-Christie partnership and the increased investment in research that this partner-ship will bring.

A major ambition of the MCRC is to significantly increase the volume of cancer research on thePaterson/Christie site which will necessitate major investment in new laboratory facilities. A new researchbuilding is being planned but in the meantime it is important that existing facilities are renovated and devel-oped to maximise their use for supporting our research efforts. This year saw the completion of a signif-icant phase in such development with the opening of the TRF1 (Translational Research Facilities 1) labo-ratories. These laboratories provide first class facilitiesfor many of our translational research activities – somethat exist already and some for new activities such asdevelopments in molecular pathology. One floor ofthe new facility is devoted to activities in pharmacolo-gy supporting the major phase 1 trial activities in theChristie NHS Trust. This includes the Clinical andExperimental Pharmacology Group headed byCaroline Dive and Malcolm Ranson (see pages 22-23)and the Translational Angiogenesis Group headed byGordon Jayson (pages 44-45). The refurbishment ofthis space has allowed us to optimise the use of thesefacilities, such as the provision of a Good ClinicalLaboratory Practice (GCLP) laboratory that facilitatesour ability to assess clinical samples from early phasetrial patients under the very strict regulations that arenow required. The provision of the GCLP laboratorywas aided by a very generous donation from the PACCAR Foundation. We are very grateful for this sup-port and the support we receive from numerous other private donations (see pages 78-79).

In addition to the academic interactions discussed above, it is vital that we have strong interaction withindustry especially in the translational and clinical research areas. This year has seen the development offruitful interactions with AstraZeneca one of the biggest pharmaceutical companies in the world. An

Director’s Introduction

2006 has been a very important year in the history of thePaterson Institute. It became an Institute of The University ofManchester and at the heart of the newly developedManchester Cancer Research Centre.

Nic Jones, Director ofthe Paterson Institute


The newly refurbished basement labs within TRF1


7

alliance between the MCRC and AstraZeneca has resulted in joint initiatives in biomarker research and intraining which is of considerable benefit to the translational research of the Institute. A novel training pro-gramme for clinical pharmacologists has been initiated which we hope will help to alleviate the shortage ofqualified and experienced researchers in this important area.

This year has also seen the completion and implementationof a new Pay and Grading system for all Institute staff.This has been developed with Cancer Research UK and willoperate across all their core funded Institutes. Although itinvolved considerable effort, overhauling our previous sys-tem was much needed and has resulted in a fair and trans-parent system that allows us to reward achievements andsuccess and hopefully will prove motivating to all Institutestaff.

Our career structure for Group Leaders has two main categories: Junior Group Leaders who are at an earlystage of their independent career development and Senior Group Leaders who have well established, inter-nationally competitive research programmes. It is vitally important that the balance between Junior andSenior Group Leaders reflects the opportunities the Institute has in developing scientific careers but equal-ly important that we ensure that only Junior Group Leaders with outstanding records and a high reputationin their respective fields are promoted to a senior position. This was certainly the conclusion that an inter-national appointments panel reached when they considered the promotion of Karim Labib who was madea Senior Group Leader in August. Karim has made outstanding contributions to our understanding of theprocess of DNA replication, a process that is fundamentally important for the stable inheritance of thegenome and is known to be defective in cancer. It is a very conserved process and Karim’s studies usingbudding yeast as a convenient model system have provided direct insights into this process in mammaliancells. Not surprisingly given the importance of replication and its correct timing in the cell cycle, it is aprocess that is very highly regulated. Karim’s research has led to the identification of a new and essentialcomplex called GINS which interacts with additional proteins to form a ‘Replication Progression Complex’(see pages 14-15). This work is at the forefront of his field and his promotion and consequent expansionof his research group is richly deserved.

The coming year will provide many new and exciting challenges and opportunities. Up to three additionalGroup Leaders will be recruited to enhance our research activities and strengthen our research focus intumour cell biology, tumour microenvironment and stem cell biology. We will initiate new programmes inthe development of advanced mouse models of cancer for pre-clinical research and therapeutic assessmentand in the development of drug discovery. This latter activity is a major new initiative undertaken withCancer Research UK aimed at the development of small molecule drugs against new potential targets someof which will emerge from our basic science programmes. This initiative will strongly complement theresearch that we currently do and plan for the future. The first step in this initiative will be to recruit aSenior Drug Discovery Scientist to lead the development. We will invest further in our research servicesby enhancing our capabilities in mass spectrometry based proteomics and advanced imaging. Finally we willcontinue to play a central role in the development of the MCRC to ensure that the promise and potentialof the Centre will be realised.


8

� Supporting fundamental scientific research to better understand the processes that drive the initiation,progression and maintenance of cancer in different tissues and through which new targets for therapeutic intervention are identified and validated.

� Support translational and clinical research to develop and test new promising therapies as well as advances in diagnosis and prognosis.

� Support extensive clinical and non-clinical cancer research training and development.

In order to achieve these aims the Centre has developed a strategic plan for future development and forthe focus of its cancer research efforts, is coordinating the recruitment of world-class basic and clinicalresearchers, is developing laboratory-based and clinically-based research infrastructure and developing amatrix of basic and clinical research programmes which will provide a framework for matching strengthsin basic research with interests and strengths in translational and clinical research. The Joint ResearchStrategy Group of the MCRC and the Christie Trust is where the main objectives are developed and stepsfor delivery identified.

The opportunities and strengths of the MCRC come from the expertise and ambitions of the partnersinvolved. The University of Manchester is the biggest single-site University in the country with the statedambition to be among the top 25 Universities in the world by 2015. It has significant existing activities incancer research within the Faculties of Medical and Human Sciences and Life Sciences augmented by therecent incorporation of the Paterson Institute. The Paterson Institute is a leading cancer research institutecore-funded by Cancer Research UK, supporting programmes in basic and translational research. TheChristie Hospital NHS Trust is a specialist tertiary centre for cancer treatment and one of the major cen-

tres in the UK for clinical trials and radiotherapy research involv-ing cancer patients. It is the largest single-site cancer treatmentcentre in Europe treating approximately 12,000 new patients peryear and supporting a network population of 3.2 million acrossGreater Manchester and Cheshire. Cancer Research UK is theworld’s leading cancer charity supporting the research activities ofover 3000 scientists, doctors and nurses working across the UK.

Over the next 5-10 years significant investment in people, technol-ogy and infrastructure across the whole basic to clinical researchspectrum will take place. Further investment in basic research willbe made to understand in more depth the events critical for the ini-tiation, progression and maintenance of tumours, the interactionsbetween the tumour cells and their immediate cellular environmentand the mechanistic basis of drug resistance. This will beenhanced by continued provision of state-of-the-art technologies

and new laboratory facilities. At the heart of translational research in the MCRC is experimental therapeu-tics and biomarker discovery. Early phase clinical trial activity is already a significant strength at the Christie

Manchester Cancer Research Centre

The Manchester Cancer Research Centre (MCRC) has been established tointegrate the cancer research efforts within the University of Manchester inpartnership with the Christie Hospital NHS Trust and Cancer Research UK.Its goal is to become a world-leading centre for basic, translational and clini-cal cancer research by 2015 with the major aims of:


Hospital site with dedicated patient facilities and expertise associated with clinical and experimental phar-macology. The MCRC will significantly increase first-into-man trials of mechanism based therapies partic-ularly focussing on targets that match its biological strengths and interests. In addition it will expand cell-based therapies that aim to harness the immune system to recognise and kill tumour cells. This drug devel-opment and biomarker discovery focus is being augmented by further development of molecular imagingand clinical proteomics as well as the development of a new programme in drug discovery. To fully capi-talise on the large patient population associated with the Christie and the associated cancer network, theCentre will increase and strengthen its activities in clinical and population-based research by facilitatingincreased recruitment into clinical trials, developing pharmacogenomic research to identify and validate bio-markers with diagnostic and prognostic potential using major patient cohort studies and sample collectionsand by developing tumour-specific research programmes.

The MCRC is still in its infancy and many challenges lie ahead. However, the opportunities to develop aworld-class comprehensive centre are great. In its first year significant progress has already been made.Most notable is its success in attracting over £3M to expand and develop the phase I clinical trial unit whichwill double its current capacity and in so doing, will become the biggest phase 1 unit in the world. It isanticipated that this expansion will take place over the next year. Recruitment of basic and clinical scien-tists and development of the plans for a new laboratory build will be the focus of the coming year.

M A N C H E S T E R C A N C E R R E S E A R C H C E N T R E

9

Abnormal growth: proliferation, apoptosis, genome stability, signalling

Tumour microenvironment: angiogenesis, hypoxia, adhesion, immunology

Stem cell biology

BasicDiscovery

Labs

Diagnostics:Imaging, Molecular

Pathology, Proteomics,Genomics, Bioinformatics

ExperimentalTherapeutics

Clinical TrialsPharmacogenomics

Abnormal Growth: proliferation, apoptosis, genome stability, signalling

Tumour Microenvironment: angiogenesis, hypoxia, adhesion, immunology

Stem Cell Biology

FundamentalResearch

Translational & ClinicalResearch

Tumour Specific Programmes

Tumour-Specific Programmes: breast, haematology (plus two others)


The majority of the work in the Bioinformatics Groupinvolves interpreting high-throughput gene expression data,most of it arising from the Institute’s Affymetrix system. Thegroup is strongly interdisciplinary by nature (the team’s back-grounds bring together a mix of computer science, mathe-matics, and biology), and the aim is to maintain a friendly andcollaborative environment that brings the informatics andbiology together as much as possible.

Microarray gene expression dataOver the last year, a number of biological collabo-rations have begun to come to fruition; ClaireWilson (a previous postdoc in the group) spent asignificant amount of time working on a microarrayproject investigating the effects of oestrogen ongene expression in epithelium and stroma of nor-mal human breast tissue in collaboration with AndySims and the Breast Biology group, headed by RobClarke. Claire has also worked with Tony Whetton’sStem Cell and Leukemia Proteomics Laboratory(SCALPL) at the University of Manchester, onwork that revealed post-translational control as aregulatory factor in primary hematopoietic stemcells. Her contribution was towards the underlyingbioinformatics necessary to bring the quantitativeproteomics data alongside Affymetrix gene expres-sion data. Other projects, including one looking atFormalin Fixed Paraffin Embedded Tissue(FFPET) in collaboration with Stuart Pepper andthe Molecular Biology Core Facility (MBCF; page54), Kim Linton, Tony Freemont at the Universityof Manchester and John Radford at the ChristieHospital are also generating promising results, andwe have continued to work extensively with other

groups in the Institute, most notably throughGraeme Smethurst and Laura Edwards, who havejoint PhD projects with Peter Stern (page 24) andGeorges Lacaud (page 28).

Detailed annotation of microarray dataThe more computational side of our research isalso yielding exciting data. We have continued ourwork with BioConductor and R, and have con-tributed the package plier to the BioConductorproject, which provides access to Affymetrix’ plieralgorithm for expression summarization.Affymetrix microarrays use 25-mer oligonucleotideprobes to target individual transcripts of interest.Typically, 11 probes are used to target each tran-script, and these are grouped in software to form aprobeset, and their combined data summarized togenerate an estimated concentration for each tran-script. Michal Okoniewski has been interested inthe specificity of the probeset to transcript relation-ship, and, along with Tim Yates, built a database ofprobe-probeset-transcript mappings by searching,in silico, the target probe sequences against a data-base of well annotated mRNA sequences. Michalwas able to use this database to identify ‘multiply-targeted’ probes that were able to bind to multipletranscripts, possibly from different genes, and thento show that this was a significant effect that couldlead to apparent, but spurious, relationshipsbetween genes. This work is also interestingbecause these events are most likely to happenwhen sequences are similar. Since sequence similar-ity is often used to infer functional relationships, wemight expect the effect to be most frequentlyobserved between functionally related genes. Thisis important to consider, when, for example, usingcorrelation based techniques to predict functionalassociations by clustering gene expression data.

Bioinformatics Grouphttp://www.paterson.man.ac.uk/groups/bioinf.jsp

GROUP LEADERCrispin Miller

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSMichal OkoniewskiCarla Möller Levet(with Catharine West)Páll Jónsson

RREESSEEAARRCCHHAAPPPPLLIICCAATTIIOONNSSPPRROOGGRRAAMMMMEERRTim Yates

SSYYSSTTEEMM AADDMMIINNIISSTTRRAATTOORR// SSCCIIEENNTTIIFFIICCPPRROOGGRRAAMMMMEERRZhi Cheng Wang

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSGraeme Smethurst (with Peter Stern)Laura Edwards (with GeorgesLacaud)Danny Bitton

10


Analysis of Exon arraysRecently, Affymetrix released a new generation ofmicroarrays that aim to interrogate every knownand predicted exon in the human genome. Theability to investigate transcription at such fine levelsof detail is clearly exciting, but makes some signifi-cant demands on data analysis, not least, becausetheir interpretation requires access to a comprehen-sive description of the fine-grained structure ofevery gene of interest. Michal and Tim have beenapplying their expertise in databases and theirunderstanding of probeset-transcript-gene relation-ships to these arrays for some time now, and havedeveloped a set of software tools to support theiranalysis. Tim’s database, X:MAP, can be foundonline at http://xmap.picr.man.ac.uk. We are nowstarting to use their software to explore microarraysat the exon level, and have a number of collabora-tive exon array projects underway.

Underpinning this work was a validation study donein collaboration with the MBCF and CancerResearch UK Affymetrix service (page 51), to con-sider how well these new arrays perform in compar-ison to the previous generation of expression arraysfrom Affymetrix. We found that with the right dataanalysis high-correspondence could be found, and

this has given us enough confidence to conductsome new projects to these arrays.

Development of novel statistics for gene expres-sion analysisMeanwhile, Carla Möller Levet has been workingwith Catharine West from the University ofManchester’s Academic Department of RadiationOncology (ADRO), looking at clinical microarraydata. Carla has also been developing novel analysistechniques, and this has led to the development ofa promising new method for finding correlationswithin gene expression, important because correla-tion based techniques are a fundamental set of toolsfor gene expression analysis.

Finally, in the last few months, Danny Bitton hasjoined us as a PhD student investigating novel tran-scriptional and translational events in microarrayand proteomics data, and Páll Jónsson has justarrived as a postdoc to bring knowledge of proteininteraction networks. Over the next year we aim tobegin to bring all of these different strands togeth-er, and to continue to apply them to our datasets.

B I O I N F O R M AT I C S

Publications listedon page 56

11

X:MAP, an annotation database representing the relationship between Affymetrix Exon 1.0STarray probesets and gene structure.


Many types of cancer treatment exploit the cell killing effectsof therapeutic agents that can generate a variety of types ofdamage in DNA. But the cell killing effects can be preventedby a variety of cellular DNA repair pathways that operate toremove this potentially lethal damage from DNA.Understanding how DNA damage leads to cell death, andhow these repair systems process the damage, may provideopportunities to improve the effectiveness of existing cancertherapies, and develop new agents. Our main focus is onDNA damage and the ensuing DNA repair processes thatfollow exposure to certain types of alkylating agents, oneexample of which is the CR-UK drug, Temozolomide. Otherprojects include studies of the relationship between singlenucleotide polymorphisms in the O6-methylguanine-DNAmethyltransferase (MGMT) gene and cancer risk, and thecharacterisation, in fission yeast, of a close relative of MGMT,which appears to be part of a novel DNA alkylation damagerepair pathway.

BBaacckkggrroouunnddChemotherapeutic alkylating agents generate vary-ing amounts of a dozen different types of lesions inDNA and there is increased understanding of themechanism/s by which some of these lesions resultin cell killing. Thus, methylating agents such asDacarbazine and Temozolomide produce O6-methylguanine in DNA and this kills cells via theaction of the post replication mismatch repair(MMR) system. These agents also generate 3-methyladenine, which kills cells by blocking DNAreplication.

Repair pathways that probably evolved to deal withlow levels of endogenously produced damage, andwhich may be important in preventing the carcino-genic effects of such damage, reduce both the ther-

apeutic efficiency and also the toxic side effects ofalkylating agents. Thus there is increasing interestin attenuating the expression of such pathways intumours, and enhancing them in normal tissues, inorder to increase the differential killing effects andthus to improve clinical outcome. These repairprocesses, especially that repairing damage at theO6-position of guanine, i.e. MGMT, and morerecently alkylpurine-DNA glycosylase (APG) whichinstigates the base excision repair system thatprocesses 3-methyladenine, have therefore becometargets for modulation. MGMT removes alkylgroups from the O6-position of guanine by stoi-chiometric transfer to a cysteine residue in its activesite, a process that results in its irreversible inactiva-tion. Because of this, inactivation of MGMT resultsin increased cellular sensitivity until more protein issynthesised.

DDeevveellooppmmeenntt ooff tthhee MMGGMMTT iinnaaccttiivvaattiinngg ddrruugg,,LLoommeegguuaattrriibbOver the last decade, in collaboration with ProfBrian McMurry and the late Dr Stanley McElhinney(and their group at the Chemistry Department,Trinity College, Dublin), and with the support ofCR-UK Drug Development and Formulation Unitsand also Cancer Research Technology, we havedeveloped a drug that is a “pseudosubstrate” forMGMT. This drug is PaTrin-2, officially calledLomeguatrib (Figure), and it effectively inactivatesMGMT and sensitises human tumour xenografts tothe killing effect of Temozolomide. The first-time-into-man Phase I clinical trial of this drug startedhere at Christie Hospital in 2000, and established adose combination of Lomeguatrib andTemozolomide for use in Phase II trials. These tri-als, carried out under the auspices of KuDOSPharamaceuticals (who were recently purchased byAstra-Zeneca), to whom Lomeguatrib is licensed,are ongoing in several centres in the UK, USA andAustralia. The dose and schedule of the two drugsis currently being optimised. In addition, another

Carcinogenesis Grouphttp://www.paterson.man.ac.uk/groups/carcino.jsp

GROUP LEADERGeoff Margison

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSAmna ButtBarbara VerbeekStephen Wharton

CCLLIINNIICCAALL FFEELLLLOOWWPhil Crosbie

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRSSGail McGown Mary ThorncroftMandy Watson

GGRRAADDUUAATTEE SSTTUUDDEENNTTAndrew Marriott

UUNNDDEERRGGRRAADDUUAATTEESSTTUUDDEENNTTSSGhazala BegumCatia CaetanoJill HunterJeff HollinsNatalia Rukazenkova

12


trial, sponsored by CR-UK is addressing the effec-tiveness of Lomeguatrib in inactivating MGMT in anumber of tumour types in order to establish thedoses required for complete inactivation. These tri-als have required us to develop and validate toGood Clinical Laboratory Practice standards, quan-titative assays for both functionally active and totalMGMT protein. Brain, prostate, breast and col-orectal tumours are being examined in dose escala-tion studies. Once the effective dose is established,Phase II trials will be undertaken. We are also inves-tigating the role of MGMT promoter methylationin regulating MGMT expression and influencingresponse.

Aspects of these clinical trials and also of our can-cer chemotherapy molecular epidemiology studieshave involved the determination of the major DNAmethylation product, 7-methylguanine in DNA bymeans of a sensitive immunoslot-blot assay. Thelevels of this product in lymphocyte DNA oftemozolomide-treated patients varies very consider-ably and recent results suggest that this may be aconsequence of the activity of APG (see above).

MMGGMMTT ppoollyymmoorrpphhiissmm ssttuuddiieessOur previous single nucleotide polymorphisms(SNP) studies that demonstrated allelic expressionimbalance (AEI) of the two MGMT alleles in sub-jects that are heterozygous for an expressed poly-morphism have been confirmed and extended. Ahuman cell line that contains an expressed polymor-phism has been identified and this may allow us toestablish a possible mechanism of MGMT AEIthat could then be explored in human samples.

PPrreecclliinniiccaall ssttuuddiieess wwiitthh MMGGMMTT iinnaaccttiivvaattiinngg aaggeennttssWe are also involved in preclinical studies aimed atestablishing clinical strategies that will reduce thedose-limiting bone marrow toxicity of agents likeTemozolomide, work that we had been undertakingin collaboration with the late, and greatly missed,Lez Fairbairn and his group. We have shown thatthe P(140)K mutant of MGMT is highly resistant toinactivation by Lomeguatrib and that a humanhaemopoietic cell line (K562) transduced with aretroviral vector encoding MGMT(P140K) is high-ly resistant to the cytotoxic effects ofTemozolomide in combination with Lomeguatrib.This is therefore a suitable combination for chemo-protective gene therapy. We have also shown in col-laborations with Prof Chris Baum (Hamburg andCincinnati) and Dr Samy Chinnasamy (Milwaukee)that self-inactivating gammaretroviral and lentiviralvectors with comparable internal expression cas-settes had similar MGMT expression properties.These vectors are of interest because of the safetyconcerns associated with the use of the standard

retroviral vectors.

It has been reported previously that over-expres-sion of MGMT might be disadvantageous tohaematopoietic cells. To allow regulated functional-ity of MGMT, a 4-hydroxytamoxifen (4-OH-T)inducible MGMT(P140K) was constructed by fus-ing a mutated version of the mouse oestrogenreceptor (ERT) to the C-terminus ofMGMT(P140K). Following transduction with aretrovirus containing the MGMT(P140K)-ERTfusion, the human cell line K562, which does notexpress endogenous MGMT, expressed high levelsof MGMT activity. Growth analysis showed thatthe negative effect of MGMT(P140K) overexpres-sion in the absence of drug selection was reducedby the ERT-fusion. Further evaluation is in hand.

Alkyltransferase-like (ATL) proteins: a novelDNA repair pathwayThe alkyltransferase-like (ATL) proteins containprimary sequence motifs resembling those found inMGMT but in the putative active site of some ATLproteins a tryptophan residue replaces the cysteinefound in the alkyltransferases. In collaboration withDavid Williams (Sheffield) we have shown using gelshift assays that the ATL from E.coli binds to shortsingle- or double-stranded oligonucleotides con-taining a number of O6-alkyl-substituted guaninesincluding Benzyl, hydroxyethyl and 4-bromothenyl(i.e. Lomeguatrib embedded into an oligonu-cleotide, which is the most potent MGMT inactivat-ing agent so far described). Inactivation of theATL gene (that we have christened Atl1) in S. pombesensitises them to the toxic effects of a wide rangeof O6-alkylating agents, demonstrating that, in wild-type cells, the Atl1 protein confers resistance tosuch agents. The mechanism of this repair pathwayis currently being investigated.

C A R C I N O G E N E S I S


13

S

Br

N N

NNNH H2

O

Chemical structure of the MGMT inactivating drug,Lomeguatrib (O6-(4-bromothenyl)guanine; previously calledPaTrin-2, and having the trade mark, Patrin™)


Our group studies chromosome replication and cytokinesisin eukaryotic cells. During 2006 we identified a key role forthe four-protein GINS complex at DNA replication forks.We showed that GINS allows the MCM helicase to interactwith the Cdc45 protein that is essential for the progressionof DNA replication forks. In contrast, we found that theSld3 protein is required to establish but not maintain MCM-Cdc45-GINS complexes. Sld3 is recruited to origins beforeGINS but is subsequently displaced during the initiationprocess, and does not normally travel with DNA replicationforks. In parallel with this work, we identified a novel factorthat we call “Ingressin” or Inn1, which is required for cytoki-nesis. We found that Inn1 associates with the actomyosinring at the end of mitosis and is required for contraction ofthe ring to be coupled to ingression of the plasma mem-brane.

Regulation of the establishment and progressionof DNA replication forksIn a systematic study of the essential budding yeastproteins of previously unknown function we iden-tified four factors required for chromosome replica-tion (Kanemaki, Sanchez-Diaz et al, (2003), Nature423, 720-724). Together with Hiro Araki’s group inJapan we showed that these proteins interact toform a complex called GINS. By purifying GINSfrom yeast cell extracts, Aga Gambus found thatGINS associates with the MCM helicase at nascentDNA replication forks and allows large “ReplisomeProgression Complexes” (RPCs) to form aroundMCM (Gambus et al, 2006). It appears that RPCsexist uniquely at DNA replication forks as they areonly found during S phase, on chromatin, requireprior recruitment of the MCM helicase to originsduring G1 phase, and are preserved if the progres-sion of replication forks is blocked artificially.GINS is required for the normal progression ofDNA replication forks and Aga identified one

important reason for this, namely that GINS main-tains the interaction of MCM with Cdc45 that isalso essential for fork progression.

Hiro Araki’s lab previously identified Sld3 as aDNA replication protein that appeared to behave ina very similar manner to Cdc45. Both proteins arerecruited to early origins during G1 phase in bud-ding yeast, in a mutually dependent manner, butSld3 and Cdc45 are only recruited to late originsduring S phase around the time that these originsare activated. Sld3 and Cdc45 can apparently inter-act with each other, and Sld3 is required to establishthe interaction between Cdc45 and MCM duringinitiation. More recently, Araki’s lab showed thatSld3 could also interact with GINS in 2-hybridscreens, and Sld3 is required for loading of GINS atorigins during the establishment of DNA replica-tion forks.

We were struck, therefore, by the fact that Sld3 isnot a component of Replisome ProgressionComplexes. This observation led us to suspect thatSld3 may only be required during initiation to medi-ate events such as the recruitment of Cdc45 andGINS, but might then be excluded from the nas-cent replisome, in contrast to Cdc45 and GINS thatare required together with MCM for progression offorks. To address this possibility, Masato Kanemakiused a sensitive and quantitative chromatinimmunoprecipitation (ChIP) assay based on real-time PCR to follow the association of replicationproteins with DNA replication forks (Kanemakiand Labib, 2006). He found that Sld3 is indeed dis-placed from origins during initiation withoutbecoming incorporated into the replisome, in con-trast to Cdc45 and GINS that move away withDNA replications forks, presumably as part ofRPCs. He then used degron technology to gener-ate a novel and extremely tight new allele of SLD3.This required the addition of the degron cassette toa version of SLD3 that also contained a mutationthat contributed to the resultant temperature-sensi-tive phenotype. Masato used this new allele, called

Cell Cycle Grouphttp://www.paterson.man.ac.uk/groups/cellcycle.jsp

GROUP LEADERKarim Labib

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSBen HodgsonMasato KanemakiAlberto Sanchez-Diaz

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRFrederick van Deursen

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSAgnieszka GambusVanessa MarchesiHiroko NishikawaMagdalena Przywara

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sld3-7td, to show that Sld3 is not required for thecompletion of chromosome replication after theestablishment of DNA replication forks, in agree-ment with our finding that Sld3 is not part of RPCsand does not move with DNA replication forks(Kanemaki and Labib, 2006).

Ingressin links contraction of the actomyosinring to ingression of the plasma membrane during cytokinesisIn our systematic screen for new cell cycle proteinsAlberto Sanchez-Diaz identified a protein that wecall “Ingressin” or Inn1, which is essential for celldivision but dispensable for the continuation of thenuclear cycle of replication and mitosis. In collab-oration with Terry Allen’s group we have used elec-tron microscopy to show that cells lacking Inn1 aretruly defective in cytokinesis.

Alberto fused Inn1 to Green Fluorescent Protein(GFP) and found that it associates at the end ofmitosis with the actomyosin ring that marks the sitewhere cell division will subsequently occur.Contraction of the actomyosin ring is coupled byan unknown mechanism to ingression and subse-

quent abscission of the plasma membrane, thusproducing two cytoplasms from one. Alberto hasshown that the localisation of Inn1 requires otheressential components of the ring, and Inn1 remainsassociated with the ring during subsequent contrac-tion.

He then used the degron strain to show that Inn1 isspecifically required for ingression of the plasmamembrane. The Inn1 protein contains a predictedlipid-binding domain, and a purified recombinantform of Inn1 is able to bind to inositol phospho-lipids in vitro. These data suggest that Inn1 may linkthe actomyosin ring to the plasma membrane dur-ing cytokinesis.

C E L L C Y C L E


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Figure 1A model for the role of Sld3 during the initiation of chromosome repli-cation. See text for details.

Figure 2Inn1-GFP associates with the contractile actomyosin ring atthe end of mitosis. The picture shows cells that express Inn1fused to Green Fluorescent Protein, as well as a fusion of RedFluorescent Protein to a Spindle Pole Body component.Cells were fixed and stained with a DNA binding dye.


Microtubule behaviour underpins many aspects of cancerbiology. Alteration in the function of these filaments withinthe mitotic spindle can affect chromosome transmission toalter the balance of tumour suppressor and tumour promot-er genes and so precipitate carcinogenic changes in genomecomposition. Microtubules also play an integral part in themigration of these cancer cells during their invasion of sec-ondary sites to form metastases and again in the generationof the vasculature to provide the blood supply that is essen-tial for the survival of these secondary tumours. Thus, drugsthat disrupt microtubule function, such as taxol, have provedhighly effective chemotherapeutic agents. Further, under-standing microtubule biology will extend therapeutic optionsby identifying novel ways to target the microtubulecytoskeleton and define better ways to exploit existingdrugs. As the structure, composition and function of themicrotubule cytoskeleton is highly conserved, we study thesimple and highly malleable, unicellular yeasts to learn lessonsthat can then be applied to tumour biology.

Microtubule growthMicrotubules form by the polymerisation of tubu-lin heterodimers. They are polar structures.Subunits add onto one end much faster than theother. Consequently this end is known as the plusend and the opposing end the minus end. Freetubulin subunits only associate with microtubuleswhen it has bound GTP. The incorporation ofGTP tubulin into the polymeric microtubule latticethen stimulates the hydrolysis of the GTP to GDP.This hydrolysis induces a conformational switch inthe tubulin molecule. Thus, the microtubule poly-merises as a flat sheet of GTP tubulin that curls upupon GTP hydrolysis to form a tube of GDP tubu-lin. If there are no GTP associated molecules at theend of the microtubule, the structural change

induced by GTP hydrolysis within the latticeinduces the subunits to peel away from the micro-tubule lattice leading to the rapid depolymerizationof the microtubule. Thus, if at any point in the lifeof a microtubule, the hydrolysis rate exceeds thepolymerisation rate, the microtubule will loose itsprotective cap of GTP tubulin and depolymerise.As the loss of a GTP cap is a random event, micro-tubules are constantly switching from growth toshrinkage independently of one another. The resultis a dynamic microtubule cytoskeleton that is con-stantly probing the 3-dimensional space. Thisenables it to rapidly establish contacts that are crit-ical in cell migration or chromosome segregation.

The +TIP class of microtubule associated pro-teinsWhile the inherent GTPase activity of tubulin gen-erates a dynamic cytoskeleton, it is the activity of anumber of molecules that associate with micro-tubules that modulate microtubule behaviour and itis the control of these microtubule associated pro-teins (MAPs) that underpins the activity of themicrotubule cytoskeleton. There are several typesof MAP: motor proteins that migrate along micro-tubule lattice, structural MAPs that associate alongthe length of the microtubule and the +TIPs thatassociate specifically with the +ends of micro-tubules. Because it is the dynamic +end of themicrotubule that mediates many microtubule func-tions, there is considerable interest in understand-ing how these +TIPs function. The +TIP CLASPwas identified by virtue of its association with thefounder +TIP, CLIP170, leading to the proposalthat the association with CLIP170 enables CLASPto modulate microtubule dynamics. Subsequentanalyses demonstrated that a number of other+TIPs also associate with one another, and the pro-posal that CLASP influences microtubule functionthrough its association with a second +TIP, EB1.

Cell Division Grouphttp://www.paterson.man.ac.uk/groups/celldiv.jsp

GROUP LEADERIain Hagan

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSAgnes GrallertNimesh JosephTamara KrsmanovicNajma RachidiAlasdair RobertsonVictor Tallada

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRSSDeepti Wilks

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSDorota FeretDaphne GarcinKate Sloan

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Fission yeast CLASPWe have been studying fission yeast CLASP. Likeits higher eukaryotic counterparts, fission yeastCLASP regulates microtubule behaviour at the cellcortex. When microtubules arrive at the ends ofthese highly polarised cells they establish an end-onassociation with the cortex, continue to polymerisefor a while at this attached end before finallydepolymerising so that their end ultimately shrinksaway from the cell tip. The polymerisation rate ofmicrotubules that have established end-on associa-tions at cell tips is slower than before contact ismade. We found that this reduction in polymerisa-tion rate depended upon CLASP function. Otheraspects of cortical function were also altered byablating CLASP function and many microtubulesfailed to make an end-on attachment but continuedelongating to curl around cell tips. Unexpectedlywe failed to find strong evidence for any interplaybetween fission yeast CLASP and its CLIP170 orEB1 homologues. Instead, we found a strong linkbetween CLASP and the multi-subunit motor pro-tein dynein. Because dynein moves towards theminus end of microtubules it either transports car-

goes to minus ends or pulls the plus ends towards astructure to which the dynein is anchored. Wefound striking similarities in the consequences oflosing the function of CLASP and dynein.

Lessons from yeastThe ability to manipulate genes at will in a simpleorganism whose primary purpose is to divide isenabling us to explore the finer points of micro-tubule biology. This information informs studies inhigher systems that, in turn, raise models that canbe most readily tested in yeast. This re-iterativecycle of comparative studies ensures that greatstrides are being made in understanding micro-tubule biology.

C E L L D I V I S I O N


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Left: Consecutive images of a wild type and a clasp mutant cell. Arrowsindicate the ends of dark microtubules that elongate to cell tips wherethey pause before de-polymerising. Right: Placing mutations in a back-ground that produces highly elongated cells shows that clasp plays no rolein morphogenesis.


Cells commonly respond to extracellular signals by modulat-ing the activity of specific transcription factors and subse-quently the expression of many target genes. We are partic-ularly interested in the response to cytotoxic and genotoxicstress which results in the mobilisation of a battery of pro-tective and repair mechanisms or the induction of apoptosis.Failure to respond appropriately can result in cellular damageand thereby drive tumourigenesis.

The AP-1 transcription factor plays a key role in theresponse of cells to extracellular signals. In mam-malian cells it is regulated by a plethora of physio-logical and pathological stimuli including mitogens,hormones, genotoxic agents, stress signals, viralinfections and cytokines. Not surprisingly there-fore, it has been linked to many cellular eventsincluding cell proliferation, differentiation as well asapoptosis. AP-1 plays important roles in tissuestress responses such as inflammation and ischemiaand is implicated in the onset and progression oftumours. The factor and its regulation is complexsince it is not a single entity but rather a mixture ofdimeric complexes composed of members of theJun, Fos, ATF and MAF protein families. Differentdimeric combinations can recognise slightly differ-ent sequence elements and be regulated by distinctsignalling pathways. A well characterised signallingcascade involves the activation of the mitogen-acti-vated protein (MAP) kinases ERK and stress-induced MAP kinases JNK and p38 which directlyphosphorylate and modulate the activity of variousmembers of the AP-1 complex. Over the last fewyears considerable progress has been made in eluci-dating the function of individual AP-1 proteinsthrough the characterisation of genetically modifiedmice and cells that derive from them.

Homologues of AP-1 family proteins are found inall eukaryotic organisms and their involvement in

stress responses is highly conserved. In fissionyeast the major transcriptional responses to stressconditions are coordinated by the transcription fac-tors Atf1 and Pap1, which are related to mammalianATF and Jun proteins respectively. In addition theactivity of Atf1 is regulated by the Sty1 kinase, ahomologue of the mammalian p38 kinase. Thusfission yeast serves as a useful model for under-standing the role and regulation of AP-1 proteins inmediating stress responses.

Functional Characterisation of ATF2ATF2 is a member of the AP-1 family and can bindto DNA either as a homodimer or as a heterodimerwith other AP-1 family members, most prominent-ly c-Jun. ATF2 is activated by the p38 or JNKkinases through phosphorylation of two N-termi-nal threonine residues T69 and T71. Many reports,mostly using in vitro systems, have implicated ATF2in numerous growth and developmental programsand in response pathways after stimulation withgeno- and cytotoxic stresses.

To better understand the biological importance ofATF2 we have generated a number of geneticallymodified mice where ATF2 activity is compro-mised. The germline deletion of the DNA bindingdomain in ATF2 leads to postnatal lethality due todeficiencies in the control of breathing. A similardefect is uncovered by specifically deleting ATF2 inneuronal cells by Cre/loxP mediated conditionalgene inactivation. Therefore, we could show thatATF2 has essential roles in the central nervous sys-tem, and at least one essential function is the estab-lishment or correct placement of respiratory cen-tres in the hindbrain.

The essential role of ATF2 in embryonic develop-ment is uncovered by simultaneously deleting ATF2and its closest homologue ATF7 in the germline.The resulting double homozygous embryos display

Cell Regulation Grouphttp://www.paterson.man.ac.uk/groups/cellreg.jsp

GROUP LEADERNic Jones

AASSSSOOCCIIAATTEE SSCCIIEENNTTIISSTTCaroline Wilkinson

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSJulien AckermannWolfgang BreitwieserYujun Di Dominic JamesClare LawrenceWolfgang Reiter

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSGemma Thornton Orestis Mavroudis-Chocholis

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRSSKeren DawsonSteve Lyons

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severe deficiencies in the developing liver due toseverely enhanced apoptosis. During large periodsof embryogenesis, the embryonic liver consists ofdeveloping hepatocytes as well as haematopoieticprecursor cells and in the absence of functionalATF2 and ATF7, both cell types showed increasedapoptosis. This increase is driven by high levels ofactivated p38 kinase and is reversed by the additionof a specific p38 inhibitor. The accumulation ofactive p38 is due to a decrease in a number of dualspecificity phosphatases, including DUSPI/MKPI,which serve to negatively regulate the activity ofMAP kinases. ATF2 binds directly to the promot-er regions of these phosphatases and regulates theirtranscription, thereby establishing a negative feed-back loop. We also generated mice containing anallele of ATF2 where the phospho-acceptor threo-nine residues (T69 and T71) are altered to alanines(ATF2-AA) and thus rendering ATF2 inactive forresponse to upstream MAP kinases. On an ATF7mutant background, these mice demonstrate thesame embryonic phenotypes as the deletion in theDNA binding domain. Furthermore, since theATF2/7 mutations phenocopy the germline muta-tion of JNK and p38 activating kinases MKK4, andMKK7, it is likely that during liver developmentATF2/7 are essential effectors of MAP kinase sig-naling.

Cells derived from ATF2/7 mutant embryos havebeen established in culture. Compared to wild-typecontrol cells ATF2/7 mutant cells are characterisedby saturation at a higher cell density suggesting adefect in contact inhibition of growth. The cellsare also sensitive to specific pro-apoptopic signalssuch as TNFα. When immortalised and expressingoncogenic Ras, ATF2 deficient cells generate signif-icantly larger tumours following injection intoimmunodeficient mice suggesting a negative rolefor ATF2 in tumour growth. In this context, theimportance of regulation of MAP kinase activitythrough the transcriptional activity on MAP kinasephosphatase genes is currently being established.

ATF2/7 knockout cells, as well as other tissue spe-cific knockout models, are being used to furtheraddress the role of ATF2 in tumourigenesis or theresponse of tumour cells to therapeutic approach-es.

Stress Response in Fission YeastWe use fission yeast as a model system for studyingstress responses since there appears to be remark-able conservation involving similar signalling path-ways and the mobilisation of closely related tran-scription factors. All cells sense and react tochanges in their environment. Single-celled organ-

isms, in particular, must contend with fluctuationsin nutrients, pH, temperature and external osmolar-ity, as well as exposure to UV irradiation and a rangeof potentially toxic environmental compounds.Appropriate responses to these environmentalstresses must be induced for cell survival and pro-liferation.

Both the Sty1 kinase and the Atf1 transcription fac-tor are crucial for fission yeast to respond normallyto a range of different stress conditions by orches-trating the expression of a common set of environ-mental stress response genes encoding numerousdefence and repair proteins. We are investigatinghow Sty1 modulates Atf1 activity. Atf1 is directlyphosphorylated by Sty1 and we have shown thatthis regulates Aft1 stability. Our results suggest thatAtf1 phosphorylation by Sty1 is not required foractivation of gene expression per se. However, Atf1-dependent transcription absolutely requires Sty1which suggests that the MAPK plays some otherrole in the activation of gene expression. We arecurrently investigating the mechanism whereby Sty1activates expression and find that the kinase getsrecruited to Atf1 dependent promoters upon stress.

We have gone on to identify an E3 ubiquitin ligaseinvolved in Atf1 turnover and are currently charac-terising the mechanisms by which phosphorylationinterferes with this turnover. Furthermore, this lig-ase itself seems to be subject to stress dependentregulation. In addition, we have used mass-spec-trometry approaches to identify a number of novelprotein interactions with Atf1. The functionalimportance of these interactions is being charac-terised and should lead to a more detailed under-standing of how this factor is regulated.

C E L L R E G U L AT I O N


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Chemical inhibition of p38 kinase blocks growth arrest in ATF2/7 mutantembryonic hepatocytes.


Tumour initiation and progression result from inappropriateactivation of intracellular signalling cascades. Rho-likeGTPases are molecular switches in signalling pathways thatregulate cytoskeletal and junctional organisation, as well asgene transcription. In this way, Rho proteins influence cellmorphology, adhesion, motility, as well as cell cycle progres-sion and cell survival. Rho proteins are essential for Ras-mediated in vitro transformation. Recently, data has emergedto directly implicate Rho proteins in tumour initiation andprogression in vivo. Our group’s focus is on identifying sig-nalling events downstream of Rho proteins that modulatetumour susceptibility and disease progression.

Similarly to Ras, Rho proteins such as Rac1, RhoAand Cdc42 are guanine nucleotide binding proteinsthat cycle between an inactive GDP-bound stateand an active GTP-bound state. In the active state,Rho proteins bind and stimulate effector moleculesthat in turn govern cell morphology, adhesion,motility, as well as cell cycle progression and cellsurvival. Of relevance to cancer, Rho proteins aretransforming in in vitro assays, particularly whenexpressed in combination with Ras effectors, andthey are required for Ras-induced transformation.The activity of Rho proteins is controlled by gua-nine nucleotide exchange factors (GEFs) andGTPase-activating proteins (GAPs). GEFs activatesmall GTPases by promoting the exchange of GDPfor GTP, whereas GAPs enhance the intrinsic rateof hydrolysis of bound GTP for GDP, leading toinactivation. Tiam1 (for T-lymphoma invasion andmetastasis protein) belongs to the GEF family ofproteins and selectively activates Rac in response togrowth factors and cell-substrate interactions.Interestingly, Tiam1 preferentially associates withactivated GTP-bound Ras through a Ras-bindingdomain (RBD). Activated Ras and Tiam1 synergizeto induce formation of Rac-GTP (Lambert et al.,Nature Cell Biol 2002; 4: 621). Further, Tiam1-deficient cells are resistant to Ras-induced cellular

transformation (Malliri et al., Nature 2002; 417:867).

Tiam1/Rac signalling and tumorigenesis in vivoMice deficient for Tiam1 are resistant to the forma-tion of skin tumours induced by application of atwo-stage chemical carcinogenesis protocol (Malliriet al., Nature 2002; 417: 867). This protocol entailstumour initiation in epidermal keratinocytes bytreatment with the carcinogen 7,12-dimethyl-ben-zanthracene, which induces oncogenic activation ofthe c-Ha-Ras gene. Subsequent repeated treatmentswith the tumour promoter 12-O-tetradecanoylphor-bol 13-acetate (TPA) result in the outgrowth andprogression of initiated cells. Tiam1-deficienttumours were not only fewer but also smaller thanwild-type tumours and this correlated withincreased apoptosis and reduced proliferation incarcinogen-exposed skin of Tiam1-deficient mice.

Tiam1 is also a potent modifier of intestinaltumourigenesis (Malliri et al., J Biol Chem 2006;281: 543). The majority of intestinal tumours arecaused by mutations in the canonical Wnt signalingpathway, leading to its activation. However, fewgenes targeted by this pathway have been demon-strated to affect tumour development in vivo. Tiam1is a Wnt-responsive gene. It is expressed in the pro-liferative compartments (crypts) of the adult mam-malian intestine where the Wnt pathway is normal-ly active. It is also up-regulated in adenomas frompatients with either sporadic colorectal polyps orfamilial adenomatous polyposis (FAP), as well as inadenomatous polyps in Min (multiple intestinalneoplasia) mice. In each instance, the Wnt pathwayis hyperactivated due to a mutation in the apctumour suppressor gene. Further, by comparingtumour development in Min mice expressing orlacking Tiam1, it was found that Tiam1 deficiencysignificantly reduces the formation as well asgrowth of polyps in vivo (Malliri et al., J Biol Chem2006; 281: 543).

These two studies on tumourigenesis in vivo demon-

Cell Signalling Grouphttp://www.paterson.man.ac.uk/groups/cellsig.jsp

GROUP LEADERAngeliki Malliri

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSEduardo CastanedaSaucedoSonia Castillo-Lluva(funded by anEMBO Long-Termfellowship) Simon Woodcock

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRGavin White

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSLucy DaltonNatalie ReevesClaire Rooney

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strate that two independent oncogenic signallingpathways of major clinical significance (Ras andWnt) recruit the Tiam1-Rac signalling pathway byspecific, albeit distinct mechanisms. In the contextof oncogenesis, activation of this signaling modulepromotes tumour initiation and growth. Moreover,this role is specific to Tiam1 since its loss cannot becompensated for by other Rho GEFs.

Tiam1/Rac signalling and tumour invasionThe skin carcinogenesis model revealed an addi-tional role for Tiam1 in tumourigenesis. The fewskin tumours arising in Tiam1-deficient mice pro-gressed more frequently to malignancy than thosein wild-type mice, suggesting that Tiam1 deficiencypromotes malignant conversion (Malliri et al.,Nature 2002; 417: 867). Analysis of Tiam1 expres-sion in skin tumours of wild-type mice revealed thatbenign papillomas maintained high levels of Tiam1expression, whereas expression was reduced insquamous cell carcinomas and was completely lostin highly invasive spindle cell carcinomas.Paradoxically, the increased Ras signalling associat-ed with advanced skin malignancies (resulting fromamplification of the mutated Ras allele) seems to beresponsible for the reduction or loss of Tiam1expression in the later stages of tumour progres-sion, as demonstrated in vitro for Ras-transformedMDCK cells (Zondag et al., J Cell Biol 2000; 149:775). Thus, while Tiam1/Rac co-operate with Rasin establishing tumours, they antagonize Ras duringtumour invasion. Similarly in intestinal tumours,lack of Tiam1 increased the invasiveness of malig-nant cells (Malliri et al., J Biol Chem 2006; 281: 543).

One probable mechanism by which Tiam1/Racantagonizes malignant progression is through theirability to stimulate cell-cell adhesion. In vitro studieshave shown that over-expression of activated Racor Tiam1 can promote the formation of adherensjunctions and the accompanying induction of anepithelioid phenotype in a number of cell lines(Malliri & Collard, Curr Opin Cell Biol 2003; 15:583). Moreover, using both RNA interference andcells derived from Tiam1-deficient mice, it has beenshown that endogenous Tiam1 is required for boththe formation as well as the maintenance of cad-herin-based adhesions (Malliri et al., J Biol Chem2004; 279: 30092). Intriguingly, Asef, another Racspecific exchange factor, promotes intestinaltumour cell migration and invasiveness in in vitromodels through down-regulating cadherin-mediat-ed adhesion (Kawasaki et al., Nature Cell Biol 2003;5: 211).

The diverse and even opposing roles of Rho GEFsin certain processes clearly indicate that Rho GEFs

do more than simply activate Rho molecules, andseveral studies now point to their role in influencingthe choice of biological response elicited by a givenRho protein. GEFs have been shown to bind toeffectors directly or to scaffold proteins that com-plex with components of effector pathways. Tiam1contributes to the signalling specificity downstreamof Rac via associating with IB2/JIP2, a scaffoldthat promotes Rac activation of p38 kinase cascadeover JNK MAP kinase cascade (Buchsbaum et al.,Mol Cell Biol 2002; 22: 4073). Tiam1 can also influ-ence Rac signalling specificity through its interac-tion with spinophilin, a scaffold that binds to p70S6K, another kinase regulated by Rac. Spinophilinbinding supresses the ability of Tiam1 to activatePak1, a different Rac effector (Buchsbaum et al., JBiol Chem 2003; 278: 18833). In our lab, we areusing biochemical approaches to identify Rac andRac GEF interacting proteins involved in differentaspects of transformation including malignant pro-gression (acquisition of invasiveness). We are alsoinvestigating the potential role of the Tiam1 homo-logue, Stef, in tumourigenesis and the impact ofdown-regulating more than one GEF simultaneous-ly in different aspects of the transformed pheno-type.

C E L L S I G N A L L I N G


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The Rho GTPase cycle. Rho-like GTPases cyclebetween an active GTP-bound and an inactive GDP-bound form. This is regulated by guanine nucleotideexchange factors (GEFs) and GTPase-activating pro-teins (GAPs). Guanine nucleotide dissociationinhibitors (GDIs) inhibit nucleotide dissociation andcontrol cycling of Rho GTPases between membraneand cytosol. Signals like growth factors, extracelullarmatrix (ECM) or lysophosphatidic acid (LPA) are ableto activate Rho-like GTPases. Active GTPases interactwith effector molecules to elicit various cellularresponses. Additionally GEFs could work as scaffoldproteins by either binding directly to Rac effectors orother scaffold proteins that bind to effectors.


CEP incorporates teams in pre-clinical drug target validationand biomarker discovery. Within the recently refurbished,specialised PACCAR Good Clinical Laboratory Practice(GCLP) laboratory CEP also develop, validate and imple-ment pharmacokinetic (PK) and pharmacodynamic (PD)assays for Phase I trials at the Christie Hospital’s DerekCrowther Unit (DCU). Our research focuses on novelagents targeted to apoptosis pathway components (e.g. IAPand Bcl-2 inhibitors) and, in collaboration with GordonJayson, we are validating a panel of biomarkers for trials ofanti-angiogenic drugs (e.g. GSAO). Technology developmentinitiatives in CEP include generation of preclinical modelswith inducible expression of wild type or mutant drug tar-gets, optimisation of serum proteomics in a new clinical pro-teomics facility, the isolation of circulating tumour cells, theuse of Q Dots to enhance immuno-histochemical (IHC)analyses and implementation of multiplexed bead arrays.

Clinical Trial Facilities at the Christie Hospital’sDCU Our translational research is associated directly withclinical trials in DCU. During 2005/2006, DCUhad 118 trials involving 635 patients. The ChristieHospital NHS Trust, became an ExperimentalCancer Medicines Centre (ECMC) led by MalcolmRanson in 2006 and recent award of a ClinicalResearch Infrastructure bid will fund a doubling ofDCU capacity making it one of the largest units ofits kind worldwide. The CR-UK Phase I trial ofAegera Therapeutics’ AEG35156 (anti-senseXIAP) showed that it is well tolerated up to125mg/m2 x 7 days every 3 weeks with dose limit-ing toxicity of asymptomatic, reversible increase inliver enzymes. There was preliminary evidence ofsingle agent anti-tumour activity in refractory breastcancer and in refractory non-Hodgkin’s lymphoma.The Phase I Trial of Allos Therapeutics’ bio-reduc-tive alkylating agent RH-1 concluded this year.Clinical PK/PD data are now being integrated andmodelled in collaboration with Prof Leon Aarons,UoM.

Clinical and ExperimentalPharmacology Grouphttp://www.paterson.man.ac.uk/groups/cep.jsp

GROUP LEADERSCaroline DiveMalcolm Ranson (Director of the DerekCrowther Unit)

SSEENNIIOORR LLEECCTTUURREERR //PPAAEEDDIIAATTRRIICC OONNCCOOLLOOGGIISSTTGuy Makin

STAFF SCIENTISTSTim WardJeff Cummings

POSTDOCTORAL FELLOWSClare DempseySarah HoltJian Mei HouDeema HusseinDavid MooreFlavia Moreira LeiteChristopher MorrowDarren RobertsStephen St George SmithKathryn TaylorArek Welman

CLINICAL FELLOWSRuth BoardEmma DeanAlastair GreystokeGavin Wilson

SCIENTIFIC OFFICERSJuliana BalesKaren Brookes Fouziah ButtMartin DawsonOlive Denenny Martin GreavesCassandra HodgkinsonAlison HoggAmanda LomasLourdes Ponce PerezNigel SmithElizabeth Sweeney

GRADUATE STUDENTSJane BarracloughMartin BrandenburgChristina FernandezDimitra MichaIsabel Pires

UNDERGRADUATE STUDENTEmma Judson

KEY COLLABORATORSGordon Jayson (Medical Oncologist, gynaecological tumours)Fiona Blackhall (Medical Oncologist, lung cancer)Mark Saunders (GI Clinical Oncologist)Noel Clarke (GU Surgeon)Richard Byers (Pathologist)Tony Whetton (ClinicalProteomics)Crispin Miller (Bioinformatics)Andrew Hughes (Translational Oncology)

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The Critical Importance of Biomarkers in EarlyClinical Trials In an era of mechanism-based therapies there isclear imperative to determine the right dose andschedule for the right patient and thus increasedrequirement for pharmacodynamic (PD) biomark-ers. These biomarkers allow proof of conceptdemonstrations of target modulation in a patient;they can facilitate rational choice of correct doseand schedule, and, if implemented to the appropri-ate quality standard they can contribute to clinicaldecision making. Ultimately biomarkers can helpexplain or predict clinical outcomes and decreasecosts of drug development. CEP has therefore putincreased emphasis on development and validationof PD biomarkers and on biomarker discoverystrategies.

Pharmacodynamic Assay Development CEP has focussed on IHC, flow cytometric andELISA-based PD biomarkers. With a Bcl-2 smallmolecule inhibitor phase I trial due to be initiated inDCU in 2007, we worked up a series of IHC meth-ods to detect Bcl-2 family proteins and apoptosis.We have progressed and validated markers oftumour vasculature and vascular density assessmentwithin biopsies. In collaboration with AstraZenecaand Richard Byers (UoM), CEP is also evaluatingthe utility of Q-dot technology for multi-analytedetection in IHC. Multiple serological ELISA-basedbiomarkers have been evaluated for clinical useincluding three surrogate markers of cell death anda panel of biomarkers of angiogenesis. CEP is cur-rently developing methods to utilise cytometricbead arrays to measure multiple analytes in smallvolume clinical samples. Protocol development isongoing to implement 4 colour flow cytometricanalysis of apoptosis in serial samples from lym-phoma patients (see Figure).

Development of a GCLP Quality System To use biomarkers for clinical decision making,studies must be performed to GCLP standard.With expansion of the QA team and relocation tothe TRF, the QA system now covers facilities com-pliance, equipment qualification, resource logs andarchiving. CEP continues to innovate in QA/labmanagement with introduction of pro forma labbooks and fast track validation, establishing thelab/clinic (CEP/DCU) QA interface and develop-ment of custom designed electronic resources.

Pre-clinical validation of novel agents againstpaediatric tumours. RH-1 and a novel HDAC inhibitor demonstratedeffectiveness against paediatric tumour cell lines invitro; RH-1 also showed efficacy in vivo. We validat-ed two agents that target XIAP which is expressed

across our paediatric cell line panel. Repression ofXIAP by shRNAi or antisense oligonucleotide cansensitise to cytotoxic agents. A novel XIAP smallmolecule inhibitor is effective against these cell linesand synergizes with conventional cytotoxic agentsin a number of them.

Biomarker DiscoveryIn a joint venture with Tony Whetton’s group(UoM) and in collaboration with Beyond GenomicsMedicine Inc, we are optimising a workflow forlarge-scale proteomic screening of human plasma.With a pilot clinical study and process validationunderway, and recruitment of a bioinformatician(in collaboration with Crispin Miller, PICR), weanticipate our first clinical proteomics data setsearly in 2007.

Biomarkers Club and the AstraZenecaSerological Biomarker AllianceAZMU Biomarkers Club, a forum to discuss bio-markers of efficacy and toxicity with colleagues atAstraZeneca met on the topics of apoptosis, inva-sion and hypoxia. The recruitment of three staffand purchase of an ELISA Robotic station in 2006activated the AZ/CEP Serological BiomarkerAlliance to investigate surrogate markers of tumourcell death in AstraZeneca-driven clinical trials.

C L I N I C A L A N D E X P E R I M E N TA L P H A R M AC O L O G Y


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Four-colour Flow Cytometric Apoptosis Assay

Cell death in lymphoma cells from patients treated withAEG 35156 XIAP antisense was assessed using CD20 pos-itive staining to discriminate lymphoma cells, Annexin V forearly apoptosis and cleaved caspase 3,7 for late apoptosiswith membrane permeable (necrotic) cells staining positive-ly for 7-AAD.


A consistent theme of the Immunology Group’s research hasbeen the investigation of shared properties of developmentaltissues and tumours or cancer cells with a view to identifyingnew targets/markers of value in diagnosis, prognosis or ther-apy. One particular success has been the 5T4 oncotro-phoblast molecule where several different types ofimmunotherapies have progressed through preclinical andearly clinical studies and are now entering phase III studies.Our clinical studies of 5T4 and HPV related therapies arenow reported in the Biological Immune and Gene Therapysection. In the past year we have investigated the early dif-ferentiation of embryonic stem cells of both mouse andhuman to further study the cellular role of 5T4 molecules.In addition we have exploited the early upregulation of sur-face 5T4 expression of embryonic stem (ES) cells committedto differentiation rather than self renewal, to identify addi-tional gene expression changes which may be important inboth development and cancer.

5T4 expression in ES cellsThe 5T4 oncofoetal antigen is a cell surface glyco-protein that is transiently expressed during mouseES cell differentiation and correlates with decreasedpluripotency of such cells. We have now shownthat the 5T4 antigen is a transient marker of humanES cell differentiation and that 5T4 phenotype,colony seeding density and culture conditions sig-nificantly influence the maintenance of pluripotenthES cells and their differentiation to neural lineag-es (Exp Cell Res. 2006; 312: 1713-26). In collabo-ration with Chris Ward, we have shown that mouseand human ES cell differentiation is also associatedwith loss of cell surface E-cadherin and up-regula-

tion of N-cadherin proteins. The E- to N-cadherinswitch during ES cell differentiation correlates withup-regulation of Snail and Slug proteins, both ofwhich show nuclear localisation in OCT-4 negativecells. ES cell differentiation is also associated withup-regulation of transcripts encoding matrix metal-loproteinases (MMP)-2 and -9 and increased gelati-nase activity. Although E-cadherin is down-regulat-ed during ES cell differentiation it is not requiredfor maintenance of undifferentiated ES cells.Furthermore, E-cadherin, N-cadherin and 5T4 pro-teins are independently regulated during ES cell dif-ferentiation and are not required for induction ofepithelial-mesenchymal transition (EMT)-associat-ed transcript expression. Abrogation of E-cadherinmediated cell-cell contact in undifferentiated EScells resulted in a reversible mesenchymal pheno-type in the absence of EMT-associated transcriptexpression. The loss of cell surface E-cadherin inundifferentiated ES cells results in translocation ofthe 5T4 antigen from the cytoplasm to the cell sur-face in an energy-dependent manner. We concludethat E-cadherin protein is likely to function in EScells to stabilise cortical actin cyoskeletal arrange-ment and this can prevent cell surface localisationof the 5T4 antigen, a newly identified componentof the EMT process. Interestingly, a study (withRenehan, O’Dwyer et al. Dept. Surgery) ofpseudomyxoma peritonei (PMP), a rare neoplasmof mainly appendiceal origin, demonstrated a spe-cific pattern of adhesion-related protein expres-sions of increased N-cadherin, reduced E-cadherin,and increased vimentin (P=0.004), a phenotypesuggesting a possible epithelial–mesenchymal tran-sition state. The similarities between PMP and col-orectal adenocarcinoma, also reveal a specific cad-herin phenotype that may characterise the distinctnon-metastasising behaviour of PMP (Brit. J.Cancer 2006; 95: 1258–1264)

Immunology Grouphttp://www.paterson.man.ac.uk/groups/immuno.jsp

GROUP LEADERPeter L Stern

PPOOSSTTDDOOCCTTOORRAALL FFEELLLLOOWWSSEyad ElkordHui-Rong JiangDavid ShawTom Southgate

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRSSDebbie BurtKate Mulryan

CCLLIINNIICCAALL RREESSEEAARRCCHH FFEELLLLOOWWSSLikhith AlakandySai Daayana (with HC Kitchener)Christy Ralph (with Medical Oncology)Ursula Winters (with HC Kitchener)

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSGraeme Smethurst (withCrispin Miller)Helen Spencer (with ChrisWard)

ROTATION STUDENTSEster MartinRebecca Roberts

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5T4 has been used as a marker to define and sepa-rate populations of pluripotent mouse ES cells(5T4 negative) and early differentiating cells (5T4positive) for comparison by microarray analysis.Many established transcriptional changes that occurduring differentiation were identified, including thedown-regulation of the transcription factorsKrüppel like factor 4 and oestrogen receptor relat-ed ß, very recently suggested to have roles in ES cellself-renewal. In addition, a number of previouslyunreported transcriptional changes were identified.One of these was the significant down-regulationof CD26 during differentiation. CD26 is a multi-functional cell surface molecule which inhibitsSDF-1 chemokine induced cell migration. In vari-ous cancers loss of CD26 expression is associatedwith increased metastatic potential and SDF-1responsiveness (Figure). The inverse correlationbetween 5T4 and CD26 expression during mouseES cell differentiation, and the known roles ofthese molecules in cell migration/motility, suggeststhat molecular events are common to both ES celldifferentiation and tumour metastasis.Understanding the co-ordination of these eventsmay provide new strategies for cancer treatment.

In our ongoing studies into the 5T4 oncofoetalantigen we have backcrossed 5T4 KO heterozygotemice against the 129 and C57BL/6 backgroundsallowing us to assess the consequences of the KOphenotype. The null 5T4 C57Bl/6 animals areviable but adult animals show some structural disor-ganisation within the brain and exhibit a high fre-quency of hydrocephaly. Importantly, the 5T4 nullmice have been used to generate useful monoclon-al antibodies and cytotoxic T cell responses againstthe mouse 5T4 target. This will allow the detailedanalysis of the consequences of generation ofautologous antigen specific immunity in animals asa result of vaccination (Cancer ImmunolImmunother. 2006; 56 : 165-180), adoptive transferof natural or targeted T cells or their combinationuse (e.g. J Immunology 2006; 177: 4288-98), all ofwhich are potential goals for clinical trials in man.

5T4 immunotherapyWe have shown that human CD8-T cell repertoireversus 5T4 antigen exists and can be detected in theabsence of CD4 T cells (either helper or regulatory)and HLA-A2 epitopes have been identified (Int JCancer. 2006; 119: 1638-47). Significant 5T4responses of CD4 T cell enriched populations aremost apparent with depletion of the CD25+ T cellsand some MHC Class II restricted epitopes havebeen identified. These observations imply that theavailability of the 5T4 repertoire of both CD4 andCD8 T cells may be optimally accessed in theabsence of T regulatory activity. In many differenttypes of cancer there are significantly increased

numbers of CD4+CD25high regulatory cells whichexpress the FOXP3 marker. We are examining themodulation of repertoire of recognition of 5T4 inpatients in trials aimed at reducing T regulatoryactivity in CD25+ depletion in renal cell carcinomaand in upper gastrointestinal cancer patients receiv-ing anti-CTLA-4 therapy.

HPV studies High risk HPV infection is a necessary cause of cer-vical cancer. An ideal vaccine would have both pro-phylactic and therapeutic effects. Such a vaccinewould have immediate benefit in contrast to theinevitable delay before the clinical impact of pro-phylactic vaccines. A fusion protein comprisingHPV16 L2, E6 and E7 is a candidate combinationpreventive and therapeutic HPV vaccine. In collab-oration with Richard Roden at Johns HopkinsUniversity we have analysed sera from previousclinical trials of the TA-CIN vaccine for L1 and L2-specific and neutralising serum antibody titers. Inparticular vaccination three times at monthly inter-vals in a phase I randomised double-blind placebocontrolled dose escalation trial in 40 healthy volun-teers, and a phase II trial of TA-CIN at the maxi-mum dose in 29 women with high grade anogenitalintraepithelial neoplasia (AGIN). Vaccination ofhealthy volunteers induced L2-specific serum anti-bodies that were detected one month after the finalvaccination and neutralized both HPV16 andHPV18 in vitro. There was also an antigen-specificproliferative response of the vaccinated healthy vol-unteers that showed a significant trend with increas-ing vaccine dose and correlated with the L2-specif-ic antibody response. However, the AGIN patientsresponded less effectively to vaccination at thehighest dose than the healthy patients with respectto the induction of HPV16 L2-specific and neutral-ising antibodies and proliferative responses. We areexploring the means to increase such cross neutral-ising antibody titres with different adjuvants.

I M M U N O L O G Y

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Research activity over the last year focussed on i) developingmulti-modality imaging with radio-quantum dots, allowingboth radionuclide and fluorescent signals to be detectedusing the same probe and ii) investigation of 18F-Fluorothymidine ([18F]-FLT) as a PET probe for cell prolifera-tion. In the quantum dot project, a 109CdSe(ZnS) probecapable of detecting a combined fluorescence and radionu-clide signal was developed and evaluated in in vitro and invivo studies. Radio-quantum probes were synthesized at atracer level, hence overcoming a major toxicity issue associ-ated with conventional fluorescence only quantum dots. Thebio-distribution and autoradiography profile of intravenouslyadministered probe showed accumulation in reticulo-endothelial system (RES) tissues, particularly the liver andspleen, followed by metabolism and significant clearance over24 hours. To demonstrate biological targeting, the radio-quantum dot was conjugated to the monoclonal antibodyretuximab and found to bind at the surface of lymphomacells expressing the anti-CD20 protein target. In the secondproject we have investigated the uptake kinetics of [18F]-FLTand enzyme activity in tumour cell lines and related thenucleoside kinase activity to the concentration of radiotracerin tumour samples. Nucleoside kinase activity (pmoles perµg protein) of tissues from H460 tumour-bearing mice werehigher for 3H-thymidine and [18F]-FLT than for radioiodinatedIUdR and FIAU. The higher in vivo stability of [18F]-FLT com-pared to the iodinated analogues enabled better cell incor-poration and its improved overall kinetics showed betterreflection of S-phase enzyme activity than in the case of theiodinated nucleosides.

Radiotracer quantum dotsWe have established a model system, based on109CdSe, for the development and validation of the

radio-quantum dot approach to multi-modalityimaging. We have synthesised radiotracer quantumdots which emit both gamma-ray and fluorescencesignal. Such probes were found to be non-toxic invarious cell lines and were significantly (40-60% in30 minutes) incorporated in lung and colorectaltumour cells. Whole body autoradiography of109CdSe/ZnS quantum dots (630nm) showed accu-mulation mainly in the RES system followed bymetabolism and clearance through the liver and kid-neys (figure 1). In tumour-bearing mice, the uptakeof 109CdSe/ZnS quantum dots at 24 hours was 10-fold higher than at 1 hour. Uptake is mainly drivenby endocytosis with greater retention in tumourcells than in normal organs including non-RES tis-sues. To demonstrate non-toxicity, stability andbiological compatibility, the radio-quantum dotswere conjugated to the anti-CD20 monoclonal anti-body rituximab and the binding of the probe wasdemonstrated in lymphoma xenografts expressingthe CD20 protein (figure 2).

Nucleoside-based PET probesThe development of [18F]-FLT as a probe to meas-ure cell proliferation has continued over the lastyear. In terms of thymidine kinase activity, in bothnormal and tumour bearing animals, FLT showedhigher level of the kinase in proliferative tissues(bone marrow, spleen, intestine) as well as inNSCLC xenografts. Despite high thymidine phos-phorylase activity of [124I]-IUdR, measured as thenumber of cleaved iodouracil molecules, the higherstability of [18F]-FLT resulted in higher and morespecific uptake of the PET signal in both tumoursand the proliferative cells of the bone marrow,spleen and intestine. These studies, along with ourprevious observation, of the [124I]-IUdR kineticprofiles in animal and human studies indicate thatdespite the higher DNA incorporation of [124I]-IUdR, it has lower specific signal sensitivity com-pared to [18F]-FLT due to the extensive metabolismof the radioiodinated pyrimidine analogue. Thisobservation was substantiated in our pilot clinicalstudy which showed a biodistribution pattern main-

Radiochemical Targeting and ImagingGroup http://www.paterson.man.ac.uk/groups/rti.jsp

GROUP LEADERJamal Zweit

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSJames GilliesChristian PrenantDavid Shaw (with Immunology)

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRSSJohn BaileyLynn DisleyNigel Smith

GGRRAADDUUAATTEE SSTTUUDDEENNTTSadia Ashraf

26


ly of radiolabelled metabolites in the case of the[124I]-IUdR compared to [18F]-FLT.

Imaging pharmacokinetics of superantigen tar-geted therapy Clinical trials of superantigen targeted therapydirected against the tumour associated antigen 5T4have shown clinical efficacy in lung cancer (unpub-lished) and in renal cancer (Shaw et al in press). Inthe latter study, a sub-set of patients was assessedfor clinical effect using FDG-PET, which gave areliable, early indication of disease control whichcorrelated with long term survival. In order to

study the pharmacokinetics of such therapy, wehave, in collaboration with Immunology andMedical Oncology, designed, optimised and validat-ed a clinical labelling strategy to label the drugANYARA with the positron emitting isotope 124I.Proof of principle of drug targeting was demon-strated in renal cancer patients and the radiolabelleddrug uptake was found to be antigen-specific as val-idated by immuno-histochemistry on biopsy sam-ples.

R A D I O C H E M I C A L TA R G E T I N G A N D I M A G I N G


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Figure 1. Whole body autoradiographs of 109CdSe/ZnS radio-quantum dots following intravenous administration of the probesinto mice.

Figure 2. Conjugation of 109CdSe/ZnS radio-quantum dots to the anti-CD20 monoclonal antibody rituximab (top) and binding ofthe conjugated antibody to the surface of lymphoma cells expressing the CD20 protein (bottom).


The AML1/Runx1 transcription factor is a frequent target ofgene rearrangements and mutations in human acute myel-ogenous leukemia (AML) and acute lymphoblastic leukemia(ALL). Consistent with its initial implication in leukemias,Runx1 has been shown to be critical for normalhaematopoietic development. The MOZ gene is involved inthree independent myeloid chromosomal translocations fus-ing MOZ to the partner genes CBP, P300 or TIF2.

Using the in vitro differentiation system based onmouse embryonic stem (ES) cells and mouse mod-els, our goals are to further define the role of Runx1and MOZ in early haematopoietic development andhow alterations of their function leads to leukemo-genesis.

Early haematopoietic development and haemangioblastThe earliest site of blood cells development in themouse embryo is the yolk sac where blood islands,derived from mesodermal cells, develop at approxi-mately day 7.5 of gestation. The yolk sac bloodislands consist of two lineages, a population ofprimitive erythroid cells surrounded by a layer ofangioblasts that eventually form the developing vas-culature. The parallel development of these lineag-es in close association provided the basis for thehypothesis that they arise from a common precur-sor, a cell called the haemangioblast.

The differentiation of embryonic stem (ES) cells inculture offers a powerful alternative approach tostudy the development of lineages that are estab-lished very early in embryonic life. Using thismodel system, a precursor was identified that gen-erates blast colonies containing precursors ofendothelial and haematopoietic lineages. The blastcolony-forming cells (BL-CFC) that generate thesecolonies represent a transient population thatappears in the embryoid bodies (EBs) prior to theemergence of any other haematopoietic lineageprecursors. The characteristics of the BL-CFC sug-gest that it represents the in vitro equivalent of thehaemangioblast and as such the earliest stage ofhaematopoietic development described to date.

The existence of the haemangioblast has beendemonstrated recently in vivo in early embryos.

Development of blast colonies and characteriza-tion of the onset of blood cell development.We have initiated a series of studies to determinethe series of cellular events leading to the genera-tion of a blast colony from a BL-CFC. For this, wehave followed the maturation of the precursor intime lapse experiments and identified differentdevelopmental stages. In collaboration with TerryAllen (Structural Cell Biology Group) we are cur-rently further investigating by electron microscopythese early events of generation of blood cells.

A critical function of Runx1 in Haemangioblastdevelopment.To investigate the role of Runx1 at the earliest stageof haematopoietic commitment, we have analysedits expression pattern and function during ES/EBdifferentiation and in early yolk sac development.Expression analyses indicated that Runx1 isexpressed in yolk sac mesodermal cells prior to theestablishment of the blood islands and within theBL-CFC in EBs. Analysis of early EBs revealed aprofound defect in the potential of the Runx1-/-ES cells to generate blast colonies. Altogether theseresults provide evidence that Runx1 does function

Stem Cell Biology Grouphttp://www.paterson.man.ac.uk/groups/scbio.jsp

GROUP LEADERGeorges Lacaud

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSMark HollandChristophe LancrinFlor Perez-Campo

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRCatherine Gavin

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSKelly Chiang (with V. Kouskoff)Laura Edwards (with C. Miller)Patrycja SroczynskaMalgorzarta Gozdecka

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Figure1: Electron microscopy of early haematopoietic cells generated fromES cells.


at the haemangioblast stage of development andposition Runx1 as a master regulator of the onset ofblood development.

Downstream transcriptional targets of Runx1 atthe onset of blood developmentOur results have indicated that Runx1 is requiredfor definitive haematopoietic development at thelevel of the BL-CFC and is therefore likely to regu-late a specific set of genes at this time of develop-ment. To identify these genes, we have comparedthe patterns of gene expression of haemangioblast-enriched cell populations or haemangioblast-derived cell populations from either Runx1 deficientor Runx1 competent ES cells. We have then validat-ed the differential expression of candidates on sam-ples generated from the ES/EB system. We havefurther documented the regulation by Runx1 of thetranscription of several of these genes by promot-er assays or chromatin immunoprecipitation. Weare currently evaluating the specific function ofthese genes at the onset of haematopoietic develop-ment.

Runx1 DNA binding partnersWe have created Runx1 variants containing a N- orC- tag which can be biotinylated in vivo by a biotinligase enzyme. We have validated that both con-structs are still able to rescue the haematopoieticdefects in Runx1 deficient cells. We have expressedboth constructs in human Jurkat T cells and puri-fied proteins associated with Runx1 using strepta-vidin beads. Peptide sequencing and mass spec-trometry indicated the association of Runx1 withseveral components of the nucleosome remodelingand histone deacetylase (NuRD) complex, one ofthe major transcriptional co-repressor complexes inmammalian cells. Future experiments will investi-gate using this experimental approach the nature ofthe proteins associated with Runx1 at the onset ofhaematopoietic development.

Runx1 isoformsPrevious studies have shown that Runx1 isexpressed as multiple naturally occurring splicedisoforms that generate proteins with distinct activi-ties on target promoters. One intriguing hypothesisis that the different isoforms of this transcriptionfactor could fulfill distinct functions at the differentstages of the establishment of the haematopoieticsystem. We have determined that expressions ofsome of these isoforms are differentially regulatedduring early haematopoietic development. We havegenerated ES cells containing reporter genes knock-in in the different isoforms or knock-out altering

the specific expression of these isoforms. We arecurrently investigating the respective expression ofthese isoforms, the biological potential of the cellsexpressing them and the respective function ofeach isoform.

A critical function of MOZ in haematopoieticdevelopment.MOZ is found translocated in cases of AML witheither CREB binding protein (CBP), the nuclearreceptor TIF2 or the P300 transcriptional co-activa-tor. All these genes encode enzymes containing ahistone acetyl transferase domain (HAT) suggestingthat aberrant modification of histones or other fac-tors could provide the first step in the route tooncogenicity. To evaluate the role of the HATactivity of MOZ, we have created a mouse modelwith a point mutation inactivating the HAT activityof MOZ. Analysis of these mice has revealed aprofound defect in haematopoiesis. The numbersof haematopoietic foetal liver precursors is dramat-ically affected by the mutation. Haematopoieticstem cells carrying the mutation present a strongdefect in competitive repopulation assays. These invivo results were substantiated with ES cells mutat-ed for the HAT activity of MOZ. Much less primi-tive and definitive haematopoietic precursors weregenerated with these mutated cells. These studieswith embryonic stem cells allowed us to further pin-point the defect in absence of the HAT activity ofMOZ to the balance between normal proliferationand differentiation of haematopoietic precursors.We are currently trying to identify the critical geneswhose expression is epigenetically regulated by theHAT activity of MOZ.

S T E M C E L L B I O L O G Y


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colonies generated by HAT+/+, HAT+/- and HAT-/-.


The differentiation of embryonic stem (ES) cells offers apowerful approach to study mechanisms implicated in cellfate decision. A major hurdle however is to promote thedirected and efficient differentiation of ES cells toward a spe-cific lineage. To overcome that challenge, we have estab-lished a serum-free culture system allowing the selective andefficient differentiation of ES cells toward haematopoieticprogenitors. We have defined in serum-free media the mini-mal growth factors that are required to control each step ofthe differentiation process. BMP4 (Bone morphogeneticprotein 4) promotes the very efficient formation of meso-derm, FGF (Fibroblast growth factor) and Activin A inducethe differentiation of these mesodermal precursors to thehaemangioblast fate, and VEGF (Vascular endothelial growthfactor) is required for the production of fully committedhaematopoietic progenitors. This step-wise control of differ-entiation is extremely efficient and provides a perfect systemfor understanding the molecular machineries involved inblood progenitor commitment.

In vitro differentiation of ES cells as a model sys-tem to study lineage specification:Upon differentiation, mouse ES cells can give riseto primitive and definitive haematopoietic precur-sors, an in vitro process that was shown to accurate-ly recapitulate the in vivo development of yolk sachaematopoiesis. This progressive differentiationcan be monitored by measurement of gene expres-sion and quantitative analysis of biological poten-tial. As they lose their self-renewal and pluripotentcharacteristics, ES cells form epiblast-like cellswhich differentiate further to give rise to mesoder-mal precursors. The first blood precursor, the hae-mangioblast, derives from the mesoderm and givesrise to primitive and definitive haematopoiesis,smooth muscle and endothelium. This carefullyorchestrated process can be reproduced in vitroupon the differentiation of ES cells in well-defined

conditions, fetal calf serum been one of the keycomponents of the culture conditions. Althoughthe inherent characteristics of ES cell lines are like-ly to be important for an efficient differentiation,the batch of serum used appears critical in obtain-ing the most effective differentiation. This factillustrates the complexity of serum compositionand the many parameters that may modulate posi-tively or negatively the differentiation process. Todissect and understand the molecular mechanismsof cell fate specification, refined culture conditionsare needed to allow the specific and efficient differ-entiate of ES cells toward haematopoiesis.

Selective and efficient differentiation of ES cellstoward the formation of blood progenitors:To define the minimal requirement for the genera-tion of blood progenitors from mouse ES cells inabsence of serum, we assessed several serum-freemedia and soluble factors previously shown toinduce some degree of mesoderm orhaematopoiesis specification. To monitor mesoder-mal differentiation, we used an ES line (GFP-Bry)carrying the cDNA for green fluorescence proteintargeted to the Brachyury locus. We have previous-ly shown using this ES cell line that GFP expressionrecapitulated effectively Brachyury expression andcould be used to track mesoderm formation in vitroand in vivo. Using the in vitro differentiation ofGFP-Bry ES line, we have studied the requirementfor each step of the differentiation (figure 1): EScells to epiblast-like cells (step 1), epiblast-like cellsto mesodermal precursors (step 2), mesodermalprecursors to haemangioblast (step 3) and haeman-gioblast to committed blood precursors (step 4).

Altogether, our data demonstrate that commitmentto the haematopoietic lineage can be driven effi-ciently by only four factors in the absence of serumand that each step of this differentiation can becontrolled by only one or two factors (Figure 1).The removal of leukemia inhibitor factor (LIF) andfeeder cells which together keep ES cell undifferen-tiated, is sufficient to trigger the progression from

Stem Cell and Haematopoiesis Grouphttp://www.paterson.man.ac.uk/groups/sch.jsp

GROUP LEADERValerie Kouskoff

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSArnaud GandilletAlicia Gonzalez-SerranoAndrew Williamson

SSCCIIEENNTTIIFFIICC OOFFFFIICCEERRStella Pearson

GGRRAADDUUAATTEE SSTTUUDDEENNTTSSRebecca Baldwin (50%, with Cathy Merry)Katalin BorosKelly Chiang (with Georges Lacaud)Sarah Lewis

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ES cell to epiblast-like cell stage (step 1). Thesedata suggest that no added factors are necessary forthe transition from ES cells to epiblast-like cells.The transition from epiblast-like cells to mesoder-mal precursors (step 2) requires the addition ofBMP4 in a dose dependent manner. The specifica-tion of haemangioblast from the mesodermal pre-cursors (step 3) is induced very efficiently and rap-idly upon stimulation with FGF and Activin A. Thelast step of differentiation (step 4), allowing the for-mation of committed blood precursors from hae-mangioblast is triggered upon the addition ofVEGF to the culture already stimulated by thesequential addition of BMP4, Activin A and FGF.The very high fraction of haematopoietic cellswithin the EBs underscores the efficiency withwhich this combination of factors specifically pro-motes haematopoietic commitment at the expenseof other lineages.

Molecular analysis of haemangioblast precursorspecification:A robust increase in the number of haemangioblastprecursors can be detected within a few hours ofstimulation with Activin A and FGF (Figure 2).This very rapid response suggest that Activin A/FGF might promote the specification of mesoder-mal precursors to the haematopoietic programrather that inducing the proliferation of a few hae-mangioblast already present in the EBs. To dissectthe molecular mechanisms implicated in haeman-gioblast commitment, we performed a screening forgenes implicated in this process via Affymetrixmicroarray analysis. Three independent samples foreach time point were harvested: (T0) prior to stim-ulation with Activin A and FGF and 6 hours later

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either without (T6) or with the addition of ActivinA/FGF (T6+). Secondary replating assay was per-formed to assess the number of haemangioblastprecursors in each population (Figure 2A). Analysisof the microarray data revealed that 33 genes weredifferentially regulated with a fold change greaterthan 2 and with a p value lower than 0.001 (Figure2B). We are now in the process of investigating therole of these selected genes in the specification ofhaemangioblast precursors.

S T E M C E L L A N D H A E M ATO P O I E S I S


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Figure 1: Schematic representation of ES cell differentiationto the haematopoietic fate.

Figure 2: (A) Kinetic of blast colony formation upon stimulation with Activin Aand FGF. (B) Heat map representation of transcripts found to be differentiallyexpressed upon Activin A and FGF induction.


Higher eukaryotes are characterised by the separation ofnucleus and cytoplasm with a nuclear membrane, whichundergoes two major dynamic events during the cell cycle.At mitosis the entire structure is dismantled and reformed,and during interphase the nuclear surface area and porecomplex populations increase by a factor of two. Ourexperimental endpoint is direct, in situ, 3D macromolecularvisualisation of specific proteins and multiprotein complexes.These studies incorporate GFP labelling for live cell imagingcoupled with immunogold localization at the EM level. Weare also investigating the roles of condensin and cohesin inthe processes of chromatin condensation and chromosomeseparation. Collaborations within the Institute currentlyinvolve projects on apoptosis, yeast mitosis and haematopoi-etic stem cell interactions.

Nucleoporin interactions during nuclear poreformation.New nuclear pore complexes (NPC) are inserteddirectly into the nuclear envelope (NE) throughoutinterphase. At mitotic NE reformation however,the nucleoporin (nup)107/160 complex associatesdirectly with the chromatin surface before NEmembrane reformation. This complex locates tokinetochores during prophase, but also over thechromosome arms from metaphase onwards.Labelling for other structural nucleoporins by mAb414 is absent from the chromosomal surface untilanaphase, and appears to be concentrated aroundthe spindle poles. Thus although NPC formation isinitiated by binding of the nup107/160 complex tochromosome surfaces at late metaphase, it may notbe completed until the chromosomes have migrat-ed towards the spindle poles for recruitment of theremaining NPC structural elements.

The structural visualisation of cohesin and con-densin in complex with condensing chromatin. Cohesin is a multisubunit complex that mediatessister-chromatid cohesion. Cohesin complexes linkreplicated DNA duplexes by forming huge ringstructures which ‘embrace’ both duplexes untilcleavage is brought about during anaphase by thecysteine protease separase. We are studying cohesinloading onto DNA duplexes in S-phase, investigat-ing both the possible de novo assembly of cohesin onchromatin and the transient opening of the cohesinring to allow entry of DNA molecules. Cohesinmay also mediate the repair of double-strand DNAbreaks. We are using immunogold and fluoro-nanogold labeling by light microscopy (LM) andField Emission In Lens Scanning ElectronMicroscopy (FEISEM) to investigate the predictedring structure of cohesion.

Condensins are structurally related to cohesin, pos-sessing two SMC (Structural Maintenance ofChromosomes) core subunits, but differ in action toproduce condensation of replicated chromatin.Condensins I and II consist of the hSMC2 andhSMC4 core subunits in combination with threenon-SMC regulatory subunits, forming distinctcomplexes which interact with chromatin at specif-ic stages during condensation. Our data support amodel in which the condensins function as molecu-lar hinges, contacting two points on a chromatinfibre in their open conformations and bringingthem into closer proximity upon its closure.

Maintenance and regulation of structural organization at the nuclear surfaceOur studies into the function of the human ortho-logue of the integral membrane nucleoporin gp210have involved down-regulation of protein expres-sion using the pSUPER vector system. FEISEMstudies have revealed that a reduction in gp210expression results in a loss of spatial organisationwhere individual NPCs are no longer maintained at

Structural Cell Biology Grouphttp://www.paterson.man.ac.uk/groups/scb.jsp

GROUP LEADERTerence D Allen

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSSheona DrummondFiona Gardiner

SSCCIIEENNTTIIFFIICCOOFFFFIICCEERRSS Sandra RutherfordStephen Murray

VVIISSIITTIINNGG FFEELLLLOOWWSSElena Kiseleva

32


a uniform distance from each other. Ultra-structur-al visualisation indicates that the nuclear laminainteracts with the “spoke-ring” component ofNPCs. This is a large annular structure, thought tobe at least partly composed of gp210 oligomers,that encircles the channel through which moleculesare transported and is embedded within the lumenof the NE. Therefore it is likely that this structurecontributes to the positional regulation of NPCswithin the plane of the NE. FACS analysis ofHeLa cells transfected with our pSUPER gp210knock-down constructs shows that a significantproportion of these cells contain activated caspase-3. This strongly suggests that gp210 is an essentialprotein without which cells initiate apoptosis.

Ultrastructural aspects of cell deathAccidental (necrosis) versus Programmed (apopto-sis) cell death is now too limited an option todescribe the spectrum of cellular demise. Necrosisis best used for features that appear after the cellhas died, and Oncosis, (Greek –swelling), describesaccidental death. Apoptosis is the mechanism forreducing cell numbers in tissue, characterized bynuclear alteration of chromatin condensation andfragmentation, blebbed membranes, loss of endo-plasmic reticulum and mitochondrial integrity, fol-lowed by phagocytosis by adjacent cells. Of thevarious pathways leading to an apoptotic endpoint,‘anoikis’ (Greek - state of homelessness) is trig-gered by ‘loss of cell anchorage’ The ability toovercome detachment-induced apoptosis is a cru-cial step in the oncogenic transformation pathway.In a recent collaborative study involving an induced

increase in c-SRC expression levels in a colorectalcancer cell line (Welman et al Neoplasia 2006, 8,905), ultrastructural observations confirmed theinduction of a cell death pathway in detached cellscharacterised by nuclear alteration and intense cyto-plasmic vacuolization, which was associated withthe accumulation of cells in the G2 phase (Figure1).

In collaboration with the Stem Cell Biology group,we have initiated investigations into the cellularinteractions during the early stages of blast colonyforming cell differentiation (see page 28). Figure 2shows images visualizing cell interactions inhaematopoietic differentiation in vitro. Cultureshave been observed by LM time lapse (Fig 2:2),then fixed in situ for both SEM and TEM with spe-cific areas relocated for direct comparison by verti-cal or ‘en face’ sectioning by TEM, (Fig 2:3) orwhole cell interactions by SEM (Fig 2:1).

S T R U C T U R A L C E L L B I O L O G Y


33

Fig 1 Increased c-SRC expression in HCT 116 colorectalcancer cells results in intense cytoplasmic vacuolation andnuclear disorganization of detached cells, which undergo celldeath in a characteristic manner, but without classic apoptot-ic morphology. (top micrographs from attached cells, lowermicrographs from detached cells).

Fig 2 Haematopoietic cell association in culture, visualized by SEM (1),LM (2), and sections cut parallel to the growing surface for TEM (3).

3


The Translational Radiobiology Group, part of AcademicRadiation Oncology (ADRO) in the Christie Hospital (headProf Pat Price) has laboratory space within the PatersonInstitute. The group explores and develops methods for theindividualisation of radiotherapy. With the advent of high-throughput techniques, the group is interested in characteris-ing molecular profiles that reflect relevant biological pheno-types and predict tumour and normal tissue response toradiation.

Development of a hypoxia transcriptome in headand neck cancer The group has a long interest in evaluating methodsfor assessing tumour hypoxia and a prospectivestudy was established in patients with head andneck cancer to investigate the potential of RNAmicroarrays (Priy Silva). This work involves collab-oration with Prof Adrian Harris at the WeatherallInstitute of Molecular Medicine in Oxford, DrFrancesca Buffa at the Gray Cancer Institute, DrCrispin Miller (Bioinformatics Group), Dr NickSlevin (Clinical Oncology), Mr Jarrod Homer(Surgery) and Prof Phil Sloan (Pathology,Manchester Royal Infirmary). Tumour (n=59) andnormal (n=11) tissue samples were arrayed and asignature representing 99 up-regulated genes wasderived by clustering around the tumour expressionof 10 well-validated hypoxia-associated genes (egVEGF, CA9). The median RNA expression of all99 genes was used to investigate the prognostic abil-ity of the signature (HS-up). In multivariate analy-sis in an independent head and neck cancer datasetincluding an intrinsic classifier and established clin-ico-pathological prognostic variables, HS-up was asignificant and independent prognostic factor out-performing the intrinsic classifier. HS-up also hadprognostic information in a published breast cancer

dataset, again independent of established variablesand a trained intrinsic signature. During the pastyear work was established to validate the signaturein terms of investigating the hypoxia induction ofnovel genes identified in the work (Sara Bhana).

Development of a homogeneous database ofhead and neck patientsValidation work for the hypoxia signature willinclude examining the prognostic significance ofnovel genes. A homogeneous database of head andneck cancer cases was established, therefore, com-prising patients with oropharyngeal cancers (ton-sil/tongue base) who received radiotherapy for theirprimary tumour; 133 patients were identified andtumour blocks obtained from 79 (Priy Silva, HelenValentine). Features associated with poor locore-gional control were low Hb level (p=0.035) andadvancing disease stage (p=0.007). HIF-1α expres-sion was a more significant adverse prognostic fac-tor in tonsil (HR=23.1, 95% CI 3.04-176.7) thantongue base (HR=2.86, 95% CI 1.14-7.19) tumours(p=0.03, interaction test). High tumour HIF-1αexpression associated with low Hb levels (p=0.03).In multivariate analysis, the only independent prog-nostic factors for locoregional control were increas-ing Hb level (HR=0.84, 95% CI 0.72-0.98, p=0.03),tongue base cancer (HR=2.03, 95% CI 1.04-3.96,p=0.04) and high tumour HIF-1α expression(HR=6.60, 95% CI 2.92-14.86, p<0.001). Thework showed differences in radiotherapy outcomewithin a homogeneous subsite of head and neckcancers related to molecular marker expression.

Assessment of hypoxia-associated markers inoesophagogastric cancerEvidence for the presence and prognostic signifi-cance of hypoxia is lacking in oesophagogastriccancer. The latter, along with the need to develop

Cancer Studies Academic Radiation Oncology: Translational Radiobiology Group

GROUP LEADERCatharine M.L. West

POSTDOCTORALFELLOWSSara BhanaCarla Möller Levet (joint with Bioinformatics)Ruth Swann (joint with GenitourinaryGroup)

CLINICAL RESEARCHFELLOWSEwen GriffithsAshirwad Merve Karim Sillah

SCIENTIFIC OFFICERSHelen ValentineJoely Irlam-Jones

GRADUATE STUDENTStephanie Donaldson (jointwith Imaging Science andBiomedical Engineering)

UNDERGRADUATESTUDENTSGemma Foley

SCIENTIFICADMINISTRATORSJo Cresswell (30%)Rebecca Elliott

RESEARCH NURSETeresa Bailey

34


approaches for selecting patients likely to benefitfrom adjuvant radiotherapy, led to the initiation ofa project looking at the role of hypoxia in the dis-ease. This work (Ewen Griffiths, Helen Valentine)involves collaboration with Mr Ian Welch and DrSue Pritchard (Wythenshaw Hospital). Hypoxia-associated markers were studied in oesophageal andgastric carcinogenesis sequences, and 177 surgical-ly-treated oesophagogastric cancers. Marker expres-sion increased with progression along the carcino-genesis sequences. HIF-2α was expressed late inthe Barrett’s sequence, and was only seen in dyspla-sia and cancer samples. HIF-1α and HIF-2α wereexpressed in 53% (2% >30% staining) and 63%(44% >30% staining) of tumours, respectively.HIF-1α expression at the invasive tumour edge wasassociated with a median survival of 18 vs 33months for negative tumours (p=0.019). HighHIF-2α was an adverse prognostic factor(p=0.015). Neither protein provided independentprognostic information in multivariate analysis.The late expression of HIF-2α in the carcinogene-sis models and its high expression in tumours(Figure) suggest it is worth further study as a mark-er of disease progression in patients with Barrett’sdysplasia and as a cancer therapeutic target.

Radiogenomics: assessment of polymorphismsfor predicting the effects of radiotherapy (RAP-PER)Individual radiosensitivity is considered to be aninherited complex trait dependent on interactionsbetween multiple genes/gene products. RAPPERis designed to explore associations between singlenucleotide polymorphisms (SNPs) in candidategenes and radiotherapy toxicity. It is a multi-centrestudy with a planned recruitment of 2200 patientsand is powered to detect common alleles conferringa moderate risk of late morbidity and rarer oneswith larger effects. Breast, prostate or gynaecolog-ical cancer patients are being recruited to identifypolymorphisms affecting radioresponse acrosstumour types. The project involves collaborationwith Drs Neil Burnet and Alison Dunning(Cambridge), Prof Soeren Bentzen (Wisconsin) andnumerous clinical oncologists locally and nationally.The day-to-day administration of RAPPER is car-ried out by Rebecca Elliott. The work began in July2005 and sample accrual is on target with blood col-lected from 1075 patients. Radiation toxicity isbeing scored using LENT-SOMA. Rapid develop-ments in high-throughput genotyping may allowstudy of 120 genes using SNP-tags to cover allcommon variation in each gene. Selection of thecandidate list will be finalised in ~2 years but willfocus on cell cycle checkpoint control, DNA dam-age response and cytokine pathways.

VORTEX-BIOBANKFunding was obtained from the CR-UK for VOR-TEX-BIOBANK. VORTEX is a national ran-domised trial currently in set-up. Patients with softtissue sarcoma of the extremity who undergo post-operative radiotherapy: 66 Gy in 33 fractions oncedaily for 5 days a week over 6.5 weeks will be ran-domised to one of two different radiation volumesand the primary outcome measures are limb func-tionality and time to local recurrence. The targetaccrual is 400 patients based on non-inferiority oflocal control but improved limb function in patientswith reduced irradiation volumes. Joely Irlam-Joneswill manage the prospective sample collection forthe trial. The translational hypothesis underlyingthe work is that a pre-treatment tumour molecularprofile will define patients with a high risk of treat-ment failure following surgery and post-operativeradiotherapy, and that the profile could be used in asubsequent study to identify patients who mightbenefit from adjuvant systemic therapy. The sec-ondary hypothesis is that there is an associationbetween SNPs in relevant candidate genes and indi-vidual patient variability in normal tissue radiationtoxicity. It is envisaged that samples for microarrayanalysis will be collected at surgery from at least halfof the patients. Paraffin blocks for tissue microar-rays and blood samples for future genotypingshould be collected from the majority of patients.

CANCER STUDIES ACADEMIC RADIATION ONCOLOGY: TRANSLATIONAL RADIOBIOLOGY GROUP


35

Box and whisker plotof HIF-2α expressionin the Barrett’s (15columnar-lined meta-plasia, 20 intestinalmetaplasia, 17 dyspla-sia, 20 adenocarcino-ma) carcinogenesissequence (a). TumourHIF-2α expression vsoverall survival in 177oesophagogastric can-cer patients (b).Tumour sectionshowing high HIF-2αexpression (c).

a

b

c


In order to translate laboratory discoveries into effectiveclinical therapies, partnerships involving scientific and clinicalresearchers as well as the commercial sector are key. It isfrom this platform that the Biological, Immune and GeneTherapy Group has been established. The focus of thegroup is on development of novel immunotherapies with aparticular interest in cellular based therapies, genetic vaccinesand antibodies. This report details the group’s current clinicaltrial activity and highlights the translational interface thatexists from target discovery through to later stage Phase IIand III trials. In recent years the group has spearheaded thedevelopment of two novel agents from pre-clinical develop-ment through Phase I and on to Phase III trials. The group isalso involved with other trials and is the largest recruiter totrials in the Christie Clinical Trials Unit (Derek CrowtherUnit).

Clinical trials of TroVax – 5T4 vaccineWith Oxford Biomedica we have been involved inthe development of 5T4 vaccines from pre-clinicaland through all phases of clinical trials. The mainPhase I trial (Clin Can Res. 2006; 12: 3416-24) ofTroVax in colorectal cancer showed safety andimmunological efficacy as well as encouraging cor-relates of survival with immune responses.Subsequent Phase II studies combining TroVaxwith chemotherapy in colorectal cancer and withinterferon in renal cancer have been successfullycompleted and have lead to the development ofPhase III studies in each of these diseases. Aninternational Phase III study of TroVax in combi-nation with standard therapy for renal cancer hasbeen initiated by Oxford Biomedica withManchester as the lead UK site. For colorectal can-cer we are developing a national study with the

NCRN colorectal group combining TroVax withchemotherapy and Bevacizumab.

A mechanistic study sponsored by CR-UK investi-gated the effect of TroVax in patients undergoingresection of colorectal cancer liver metastases. Theobjectives in this trial included assessment of sys-temic immune responses before and after surgery aswell as local tumour immunity and clinical out-comes. Twenty patients undergoing resection ofliver metastases by David Sherlock at NorthManchester General Hospital received 2 vaccina-tions pre-operatively, 2 post-operatively and 2 fur-ther boosting vaccinations if immune responseswere detected. Immunological assays indicate that15 of the 20 patients have T-cell or T-cell plus anti-body responses to 5T4 or subunit peptide within 14weeks. Of the 16 protocol-compliant patients 13have demonstrated responses. Currently we areinvestigating the levels of regulatory T-cells (Tregs) inperipheral blood and local immune factors byimmunohistochemistry and isolated tumour infil-trating lymphocytes.

5T4 superantigen therapy Targeting of superantigen to a tumour-associatedantigen can direct T-cell mediated cytotoxicity.Building on previous successful Phase II studieswith a first generation agent (Shaw et al in press) wehave now undertaken a Phase I trial (with ActiveBiotech) with a re-engineered molecule. Thisshows improved tolerability and encouraging clini-cal results. A PET mechanistic study has also beencompleted (with Peter Julyan / David Hastings /Jamal Zweit) and this shows tumour localization ofthe fusion protein in all patients tested. AnInternational Phase II/III study in renal cancer isdue to commence shortly with Manchester as thelead centre.

Cancer Studies: Biological, Immuneand Gene Therapy Group

GROUP LEADERSRobert E HawkinsPeter L Stern

SENIOR CLINICALRESEARCH FELLOWFiona Thistlethwaite

POSTDOCTORAL SCIENTISTSEyad Elkord(Immunology)David Gilham (Cell andGene Therapy)Ryan Guest (at NBS)Dominic RothwellZihni Yazici

SCEINTIFIC OFFICERSHayley BarthaDebbie BurtNatallia Kirillova (at NBS)

RESEARCH NURSESJackie FenemoreCarmel GarnerCaroline HamerJackie O’Dwyer

ADMINISTRATORSDebbie MaskellCatriona ParkerJane Rogan

CLINICAL RESEARCHFELLOWSAdam DangoorRichard GriffithsChristy RalphAlaaedin ShablakUrsula Winters (with HCKitchener)

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Adoptive transfer of Treg depleted autologous

T-cellsWith the Urology Group (Noel Clarke/VijayRamani) we have demonstrated thatCD4+CD25high Tregs are present in increased num-bers in the peripheral blood of patients withadvanced cancer compared to normal controls. Tregs

suppress proliferation and cytokine release fromeffector cells and, in animal models, depletion ofCD25+ T-cells can induce tumour responses. Wetherefore initiated a study to examine the effects ofTreg depletion on renal cancer. Six patients withadvanced renal cancer were recruited and under-went leukapheresis followed by conditioningchemotherapy with cyclophosphamide and fludara-bine. The autologous leukapheresis product wasdepleted of CD25+ cells by magnetic selectionusing the CliniMACS® system then reinfused intothe patient. Treg depletion of the leukapheresisproduct was efficient and there was no toxicity ofthe infused cellular product. Transient reductionsin Tregs in the peripheral blood of patients werenoted with evidence of increased T-cell responsesto the tumour-associated antigen 5T4 in at least onepatient. This was associated with mixed/minorresponses in two patients. We are now looking tocombine this treatment with vaccines or with engi-neered T-cells.

Anti-CTLA-4 monoclonal in advanced oesopha-go-gastric cancerCTLA4 is a transmembrane protein which is a cru-cial inhibitor of T-cell activation. Anti-CTLA4antibody blockade has been shown to have thera-peutic effects in melanoma. We are thereforeexploring the effects of an anti-CTLA4 monoclon-al antibody (Ticilimumab, Pfizer) in a Phase II studyin advanced oesophago-gastric cancer. As well asclinical efficacy we are examining pharmacodynam-ic effects on lymphocyte phenotype (eg. CD25,CTLA4, and FoxP3), and for the development ofactive immune responses against 5T4. The trialopened in September, and has recruited elevenpatients to date. Toxicity has been acceptable, andthe first patients are due formal re-staging of theirdisease by the New Year.

Sequential therapy of imiquimod and photody-namic therapy for vulval intraepithelial neoplasia(VIN)The aim of this Phase II study (with HenryKitchener) was to demonstrate the tolerability ofImiquimod and photodynamic therapy (PDT) usedsequentially and to assess lesion and immunologicalresponse in women with VIN. The hypothesis was

that VIN lesions pre-treated with an immuneresponse modifier would respond more favorablyto PDT. Treatment consisted of Imiquimod andPDT using methylated ALA as a photosensitiser.Follow up at 12 months is available for 10 womenand response rates are maintained at this time pointwith 3 complete responses and 4 partial responses.Pharmacodynamic analysis showed that Imiquimodsignificantly increased CD8 T-cells within VINlesions and that non-response to Imiquimod wasassociated with an increase in Tregs within VIN

lesions.

Trials with engineered T-cellsTrials of engineered T-cells (see Cell and GeneTherapy Group) are awaiting final approval by theMHRA following recent changes in the regulationsgoverning first into man trials of immunotherapy.For our CR-UK sponsored trial targeting CEA theGMP virus has been produced and production runsof gene modified T-cells carried out to GMP (withEric Austin at Manchester Blood Centre). Likewiseassay validation for immunological and molecularmonitoring of the trial has been completed and eth-ical approvals are in place. The trial targeting CD19is at a similar point although delays in production ofGMP virus mean the trial could not start beforeMarch 2007.

Other large scale trialsWe are also involved in trials of agents targetingother biological pathways in renal and oesphago-gastric cancer. In renal cancer a number of majortrials have been completed and these are changingthe way we treat renal cancer. Building on success-ful recruitment to trials of anti-angiogenic drugs weare investigating how these interact with theimmune system as a means of rationally developingcombination trials. Following our presentation atESMO of interesting results from a large Phase IIItrial with Lapatinib (Glaxo) in renal cancers over-expressing EGFR we are also planning to look atcombination studies.

SummaryThe group, through interactions with laboratorygroups in Manchester and elsewhere and with thepharmaceutical industry, has a vibrant trials portfo-lio focusing on a variety of ways to manipulate theimmune system.

CANCER STUDIES: BIOLOGICAL, IMMUNE AND GENE THERAPY GROUP


37


The Children’s Cancer Group (CCG), formed in 2000, waspreviously located within the laboratories of the Institute ofCancer, Queen Mary University of London Charterhousecampus. In October of 2006, the group relocated to thePaterson Institute for Cancer Research at the University ofManchester. The focus of our research lies in improving theoutcome of children with acute lymphoblastic leukaemia(ALL). On the clinical front, this involves designing and run-ning of novel international clinical trials for those with highrisk disease. In the laboratory we are exploring biologicalexplanations for the variations in response to therapy.

Clinical Trials:

ALL R3This trial is for children with ALL who relapse afterinitial therapy. The trial recruits patients from all 21paediatric centres in UK and Ireland as well as fromcentres in Netherlands, Australia and New Zealand.An indigenously designed web-based remote entrydatabase with decision support tools has been madeto run the trial. Minimal residual disease estimationafter 5 weeks of therapy is used to risk stratify chil-dren. Those who have high levels of disease at thisstage are offered allogeneic stem cell transplanta-tion. The trial opened in the UK in 2003 and todate has recruited 178 patients. The overall survivalof patients is 70% at 3-years. A number of clinicaland cytogenetic markers of poor prognosis havebeen identified. An interim analysis is being per-formed to better inform us of the progress of thetrial and help us design the next trial when this onecloses in 2010.

EsPhALLThis trial is randomising the use of imatinib in chil-dren with Philadelphia positive (Ph+) ALL. Allchildren with this disease in the UK are eligible for

allogeneic transplantation after completing 5 blocksof treatment. We have obtained funding from theLeukaemia Research Fund to monitor the speed ofresponse to therapy, identify key mutations andunique gene expression patterns in these patients.Our preliminary studies suggest that mutations inthe ABL kinase domain are present in some chil-dren with Ph+ ALL, but this does not necessarilypredict for a poor response to therapy. On theother hand mutations in cell cycle kinase genesappear to do so. This trial will continue to 2010.

BIOV-111This is a phase II trial of the drug Clofarabine inresistant and relapsed ALL. It is currently recruit-ing in 10 countries throughout Europe and willclose early next year. At the moment the responserate is 28% and some refractory patients have beentransplanted in remission. This is highly promisingfor a single agent in this population. Both pharma-cokinetics and molecular pharmacology studies areincorporated into this trial.

Laboratory Investigations:

Targets of ETV6 The ETS-related transcription factor ETV6 is fre-quently found disrupted in many human malignan-cies, in particularly in childhood ALL, where 25%of patients have ETV6-RUNX1, t(12;21) transloca-tion. ETV6 binds to a specific DNA sequence con-sisting of GGAA/T core motif within the promot-ers of its target gene and as a result, repress its tran-scription. To date, there are few known targetgenes of ETV6, namely BCLXL, MMP3 and Fli1.

We have used a Gene-ACE technique to convertETV6 into an activator and used microarray analy-sis to identify putative direct targets of ETV6.Promoter and chromatin immunoprecipitationassays identify two new target genes of ETV6,namely the cytokines CCL3 and CSF2.

Children’s Cancer Group

GROUP LEADERVaskar Saha

NON-CLINICAL LECTURERLouise Jones

POSTDOCTORAL FELLOWOlga Yiannikouris

SCIENTIFIC OFFICERSSeema AlexanderNaina Patel

CLINICAL FELLOWSShekhar Krishnan Doaa Khater Frederik van Delft

GRADUATE STUDENTShai Senderovich

CLINICAL TRIALS MANAGERCarly Leighton

DEPARTMENT ADMINISTRATORMarita Marshall

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Chromosome 21 and childhood ALL We have identified and characterised a new cytoge-netic sub-type of childhood ALL. This is charac-terised by a intrachromosomal amplification ofchromosome 21 (iAMP21). A common region ofamplification (CRA) has been identified between33.192 and 39.796Mb and a common region ofdeletion (CRD) between 43.7 and 47Mb in 100%and 70% of iAMP21 patients, respectively.Supervised gene expression analysis showed a dis-tinct signature for eight patients with iAMP21, with10% of overexpressed genes located within theCRA. The mean expression of these genes was sig-nificantly higher in iAMP21 when compared toother ALL samples. Although genomic copy num-ber correlated with overall gene expression levelswithin areas of loss or gain, there was considerableindividual variation. A unique subset of differen-tially expressed genes, outside the CRA and CRD,were identified when gene expression signatures ofiAMP21 were compared to ALL samples withETV6-RUNX1 fusion or high hyperdiploidy withadditional chromosomes 21. From this analysis,AEP was shown to be overexpressed in patientswith iAMP21. Genomic and expression data hasfurther characterized this ALL subtype, demon-strating high levels of 21q instability in thesepatients.

AEP over expression and high risk ALLWe have now further demonstrated that AEP isoverexpressed in high risk ALL and those withextramedullary relapse. Western analysis identifieshigh levels of active AEP in blast cells obtainedfrom these patients. Leukaemic cell lines over-expressing AEP show increased migration and inva-siveness in vitro assays and produce extramedullary

tumours in NOD-SCID mice. This behaviour isinhibited by AEP specific inhibitors. AEP alsocleaves the drug Asparaginase and we believe thisnot only activates the drug but may increase itsMHC Class II processing resulting in the produc-tion of inactivating antibodies.

Plans for 2007 The group is in the process of reorganisation andwill add a new senior post doctoral fellow and clin-ical senior lecturer in 2007. The clinical trials willmove towards further integration with the laborato-ry with the development of a national cell bank inManchester for relapsed and refractory ALL. Weplan to use the new chromosome 21 tiling arrays tofurther investigate the differences in the transcrip-tome of chromosome 21 in childhood ALL. TheETV6 targets will be further examined using a ChIPon chip approach and we will investigate the possi-bility of collaboration between ETV6 and RUNX1on the CSF2 and CCL3 promoter. The role ofAEP in resistant disease is being investigated usinga number of different tools.

C H I L D R E N ’ S C A N C E R G R O U P


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HE section of glioblastoma showing cells highly positive for AEPexpression (brown) at the edge of the tumour.

The AEP expressing cells are tumour associated macrophages.Blue = DAPI; Red = antiCD68; Green = AEP


Normal tissues are maintained by the self-renewal capacityof a rare population of stem cells, which divide asymmetrical-ly both to replace themselves and to generate progenitors.After limited cell division, progenitor cells produce the non-dividing differentiated cells specific for each tissue. Anemerging concept is that in leukemia as well as in neural andepithelial cancers, including breast cancer, only a minority ofcells, i.e. the “cancer stem cells”, have the capacity to initiatetumours; the others are committed to differentiation path-ways and senescence. Thus, characterising the cancer stemcell and understanding the molecular basis for dysregulatedself-renewal will be crucial for identification of targets foreffective therapeutic intervention.

Stem cell self-renewal pathwaysIdentification of stem cell self-renewal pathways isemerging as important for cancer prevention, andfor the future therapy of treatment-resistant cancerstem cells that have the capacity to initiate tumoursand recurrence. Although some cell-specific mark-ers and signal transduction pathways are similar innormal and cancer stem cells, the tight regulation ofself-renewal that is operative in the normal stem cellmay well be disrupted in cancer. Thus, understand-ing the regulation of self-renewal will be crucial forimproving therapeutic intervention by targeting thecancer stem cell that is predicted to be inherentlychemo- and endocrine resistant and to initiatetumour recurrence.

Our current aim is to exploit culture and in vivotechniques to address the regulation of normal andcancer stem cells in the breast. We have alreadyestablished methods to isolate human mammaryepithelial stem cells from normal tissue, using

Hoechst dye-efflux to obtain the mammary cell‘side population’. More recently, we have exploitedthe expression of ALDH1 in stem cells by using thecommercial substrate Aldefluor to enrich for stemcell populations. Both these methods can be cou-pled with the use of mammary stem cell surfacemarkers. To identify the factors that regulate breaststem cell phenotype, we are collecting material fromconsenting patients undergoing surgery for eitherbreast reduction, removal of a benign or primarytumour, ductal carcinoma in situ or pleural effusions(from advanced breast cancer patients).

One current focus is the signalling pathways regu-lating stem cell self-renewal of which Notch, Wnt,ErbB, Prl and ovarian hormones are of particularinterest. We have shown the aberrant activation ofthe Notch receptor signaling pathway in breast can-cer (see Stylianou et al., 2006). We have extendedthese studies of Notch signaling and have datademonstrating its importance in regulation ofbreast cancer stem-like cells, using methods fornon-adherent mammosphere suspension culture(see figure), analogous to neurosphere culture thatenriches for brain stem cells. Our recent resultsindicate that in this culture system self-renewingstem cells can be enriched and passaged, and thecontribution of the above signalling pathways canbe assessed. Their importance in an in vivo modelof tumourigenesis (the gold standard stem cellassay) is also being actively investigated.

Gene expression and functional genomicsGene expression arrays and functional genomicsmethods are being employed to identify novel path-ways that participate in stem cell regulation. For thefunctional genomics studies, we are collaboratingwith Rene Bernards’ group at the NKI, Amsterdamto use a retroviral short hairpin (sh) RNA librarythat targets approximately 8,000 genes (Nature,

Medical Oncology:Breast Biology Group

GROUP LEADERRob Clarke

POSTDOCTORALFELLOWSGillian FarnieHelen Kalirai Rebecca Lamb Andy Sims

CLINICAL FELLOWSKai Ren OngRachael Johnson

SCIENTIFIC OFFICERSKath SpenceRognald Blance Pam Flint

GRADUATE STUDENTSNgoc-Sa Nguyen-HuuHannah Harrison

ROTATION STUDENTSStephanie JoblingClair Thomas

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2004, 428: 431-7). We use the library to screen forgenes that function in stem cell self-renewal byusing several rounds of mammosphere culture toenrich for integrated shRNA sequences thatincrease production of non-adherent spherecolonies. DNA must be collected at each passageand ‘bar-coded’ by amplification, labelling andhybridisation to custom arrays of the shRNAsequences. The enriched sequences light up on thearray and the most important ones will be increasedby each passage of mammospheres. We are cur-rently testing the candidate shRNA sequences inour normal and cancer stem cell assays.

Gene expression and risk of breast cancerAnother area of focus is gene expression and riskof breast cancer where we compare tissues fromwomen at high or normal risk of breast cancer, forexample, parous versus nulliparous women or thosewith a family history of breast cancer versus popu-lation controls. To inform these investigations, wehave developed an in vivo model where nulliparoushuman breast tissue is exposed to pregnancy levelsof ovarian hormones and we can observe signifi-cant changes in genes previously identified in othermodel species. If these genes can be modulated in

women, then it may be possible to reduce theincreasing incidence of breast cancer. We thereforecollaborate with Professors Howell and Evans atthe South Manchester Family History Clinic toexamine the effects of preventative strategies to seewhether relevant gene expression is altered follow-ing a clinical intervention such as a calorie restrict-ed diet.

It is hoped that the results of these investigationsshould lead to an increased understanding of thebiology of the normal human breast which, in turn,could lead to the development of new strategies ornew targets for breast cancer prevention andtherapy.

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The photomicrograph shows a bright field image of mammospheres grown from dissociated normal human breastepithelial cells obtained from a reduction mammoplasty specimen. The mammospheres are colonies grown in sus-pension from single stem-like cells. They are analogous to neurospheres that have been demonstrated to enrich forbrain stem cells. Magnification x400


The primary focus of the Cell and Gene Therapy group isupon the development of engineered T cells to eradicatecancer. Pre-clinical studies generated over the past five yearshave formed the basis of two clinical trials which arepresently at the final regulatory hurdle (see Biological,Immune and Gene Therapy Group report). Consequently,the focus of our work has moved towards the use of modelsystems to investigate the mechanisms of activity of engi-neered T cell therapies with the aim of improving the overalltherapeutic approach. A key recent development is theATTACK programme which is a Framework 6 EU fundedproject (Co-ordinator Professor Hawkins) which bringstogether 14 laboratories to work on the pre-clinical devel-opment of engineered T cell strategies. Importantly, localcollaborative work with various groups within the PICR(including Immunology and Carcinogenesis) is exploitingthese model systems in order to discover how combinationsof therapeutic approaches may synergise thereby producingmore potent therapies.

Engineered T cellsThe core activity of the research group is the genet-ic engineering of T cells. In essence, this involvesthe stable introduction of genes encoding chimericimmune receptors (CIR) into primary T cells. CIRsconsist of scFv (derived from antibodies) fused tothe signalling domain of important T cell receptors(e.g. CD3ζ). The group has spent some consider-able time optimising methods for the gene modifi-cation of primary human and mouse T cells withthe result that we have protocols which use retrovi-ral vectors to reliably and efficiently introduce the

CIR gene into T cells. T cells engrafted the CIRthen target tumour cells through the scFv part ofthe CIR resulting in the specific destruction of thetumour cell. There are two principal targets used inthe group – carcino-embryonic antigen (CEA –gastro-intestinal cancers and others) and CD19 (Bcell lymphoma in collaboration with Prof. J.Radford, Medical Oncology) and these are the tar-gets of the two clinical trials currently held up inregulation (see clinical trial section). Human T cellsengrafted with anti-CEA.CD3ζ or anti-CD19.CD3ζ receptors kill their respective antigen-expressing tumour cell lines in vitro and also caneffectively challenge the growth of these tumourlines in immuno-compromised animal models.

While immuno-compromised animal models pro-vide a setting to investigate the functionality ofhuman T cells, these models lack the power to fullyexplore the implications of adoptive cell therapy inthe context of a normal, functioning host immunesystem. To this end, we have developed immuno-competent models for both CEA and CD19 andmouse T cells harbouring the respective receptorsare indeed active in challenging the growth oftumours. However, in order to achieve high levelsof T cell engraftment, lymphodepletingchemotherapy is required which transiently reducesthe number of white cells in the circulation therebyallowing the therapeutic T cells to survive longenough to carry out their anti-tumour function.Optimising the combination of chemotherapy andengineered T cell infusions in these model systemsis the key to the future translation of the therapy.However, one safety concern is the possibility ofauto-immunity driven by the engineered T cells tar-geting tissues expressing normal or physiologicallevels of target antigen. To investigate this, we areusing mice transgenic for CEA (CEATg) which

Medical Oncology:Cell and Gene Therapy Group

GROUP LEADERRobert Hawkins

POSTDOCTORALFELLOWSDavid Gilham Eleanor CheadleJennifer LocontoSimon DovediVivien WatsonMichael Lie-a-Ling

CLINICAL RESEARCHFELLOWSWasat MansoorKallingal Riyad

SCIENTIFIC OFFICERSHayley BathaAlison O’NeillAmanda Russell

ADMINISTRATORIsabelle Wartelle

GRADUATE STUDENTSJohn BridgemanAlex SmithGrazyna Lipowska-Bhalla

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express CEA protein to levels similar to thatobserved in man. Initial experiments have provenencouraging where CEATg animals givenchemotherapy and CEA specific T cells remainedtumour-free with no evidence of auto-immunitywhile cohorts treated with the same regime but withnon-specific control T cells developed tumour.

The concept of combining therapies is beingexplored in a broader context. Together with theImmunology group, we have recently demonstratedthat mouse T cells engineered with specificity forthe 5T4 tumour-associated antigen synergise with5T4 vaccination and dendritic cell therapy to signif-icantly improve therapy against established tumours(Jiang et al., 2006). We are currently seeking toimprove the 5T4 specific chimeric receptor in orderto investigate the mechanisms underlying thisobservation. In a similar vein, in a further collabo-ration with the Immunology and CarcinogenesisGroups, we are exploring whether DNA damagingchemotherapy may potentiate immune therapies inour model systems. Furthermore, improving thepower of the receptor itself is being undertakenwith more powerful signalling domains being incor-porated which may further boost the activity of theengineered T cell.

Chimeric immune receptors in other immunecell types.T cells are potent immune effector cells. However,many other immune cells can play an importantrole in eradicating tumour and exploiting these cellsthrough chimeric receptor targeting has been oneavenue of research. Our group was the first to suc-cessfully show that monocyte/macrophages can betransduced to express a chimeric receptor and toeffectively combat the growth of tumour cells invivo (Biglari et al. 2006). Current studies are work-ing upon the expression of chimeric receptors inNatural Killer cells (collaboration withImmunology) and also seeking to exploit gene-modified haematopoietic stem cells as a source tore-populate recipient animals with multiple lineagesof immune cells harbouring tumour specificchimeric receptors.

Homing of T cells to tumour.As an aside to our studies investigating engineered

T cell function in patients with advanced CEA+

tumours, we have identified the presence of anunusual chemokine within colorectal tumourswhich have metastasised to the liver. Chemokinesplay an important role in controlling the migrationof cells around the body and we have identified thatthe chemokine Eotaxin-2 is present to very highlevels in colorectal metatastases and is also present

in primary colorectal tumours (Cheadle et al., sub-mitted). Eotaxin-2 is more commonly found play-ing a role in allergy and at present it is not clearwhat the biological significance of the presence ofthis chemokine is in colorectal tumours. However,the presence of specific chemokines in tumoursdoes provide a potential tag with which to attemptto target engineered T cells to specifically migrateto tumour and our future work will involveattempts to manipulate T cells to respond to thepresence of these tumour derived chemokines.

SummaryThe translational research carried out by our grouphas formed the basis for two clinical trials which aredue to commence in 2007. Further development togenerate future trial proposals will be dependentupon model systems being currently used in thelaboratory to investigate the potential and also toassess the potential risks of gene and immune ther-apies.

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Photo of ATTACK / CRUK sponsored Symposium inManchester December 2006. The meeting featured theglobal leaders in the field.


We have established a Translational Angiogenesis Group incollaboration with Professor Caroline Dive with the aim ofdefining biomarkers for anti-angiogenic agents. We will runthe translational research programme for two MRC/NCRNinternational randomised trials of anti-angiogenic agents inovarian cancer. The laboratory programme has identifiedcritical FGFs in ovarian cancer and developed heparinoligosaccharide synthesis.

Ovarian Cancer Laboratory ProgrammeThe obligate dependency of the Fibroblast GrowthFactors (FGF) on heparan sulphate (HS) for theirbiological activity led us to examine this axis inovarian cancer. In previous work we had shownthat stromal syndecan 1 was of prognostic signifi-cance in the disease and that syndecan 3, conven-tionally found on neuronal tissue, was aberrantlyexpressed in the tumour endothelium. Using aunique molecular probe we showed that theheparan sulphate on the tumour vascular endotheli-um has the capacity to activate FGF2. While thestroma bound this probe only moderately, thetumour cells were largely negative.

We investigated why the tumour cells do notexpress growth factor activating heparan sulphateand showed, using in situ hybridisation, that thetumour cells strongly express 6-O-sulphotrans-ferase, an enzyme implicated in the synthesis ofbiologically active heparan sulphate. In addition weshowed that the core proteins for proteoglycanswere present on the surface of ovarian cancer cells.These data implied that the tumour cells makeheparan sulphate but that active heparan sulphate isnot present on the cell surface. By adding exoge-

nous FGF2 we were able to probe for non-activat-ing sequences of HS but this was also reduced onthe tumour cell surface suggesting that there is aglobal rather than sequence-specific reduction incell surface HS. Both heparanase and H-sulf1 wereexpressed by the tumour cells but only heparanasecould account for the findings. Thus these datasuggest that heparanase is a key mediator ofheparan sulphate depolymerisation at the tumourcell surface, augmenting its known role in invasionand angiogenesis.

In an extensive study of FGFs in ovarian cancer wehave identified a switch in the receptor type upontransformation. This is associated with theresponse of the tumour cells to the appropriate lig-and FGFs and enhanced chemosensitivity in inhibi-tion studies. Thus our data (manuscript in prepara-tion) suggest that FGF2 is relevant to angiogenesiswhile other FGFs play a role in transformation andthe malignant phenotype, highlighting the potentialof FGF inhibitors in the disease.

Therapeutic ProgrammeWe had previously shown that heparin octasaccha-rides inhibit angiogenesis in vivo and over the lastyear have developed the organic chemistry to makesynthetic heparin octasaccharides. This work isongoing and will provide uniquely pure reagents totest structure-function relationships in the nextyear.

Clinical Trial ProgrammeWe have completed phase I trials of anti-angiogenicagents that include a pure anti-αv integrin antibodyand a pure anti-VEGFR2 antibody. These trialshave yielded unique insights into the possible rolesthat the drugs might have. In the integrin trial weshowed that the antibody was very well tolerated

Medical Oncology:Translational Angiogenesis Group

GROUP LEADERGordon Jayson

PPOOSSTTDDOOCCTTOORRAALLFFEELLLLOOWWSSBader Al Salameh Alison Backen Claire ColeSteen HansenWill Stimpson

CCLLIINNIICCAALL FFEELLLLOOWWSSAndrew ClampSin LauClaire MitchellDaniela Rosa

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and that one patient with angiosarcoma underwentrapid improvement of her disease with single agenttherapy. In the anti-VEGFR2 trial we demonstrat-ed growth retardation using serial imaging strategies(collaborators: Jackson, Parker, Univ. Manchester)but did not see changes in vascular permeability,thereby challenging the dogma that all active VEGFinhibitors reduce vascular permeability as assessedby dynamic contrast enhanced Magnetic ResonanceImaging.

In collaboration with Prof. Sean Kehoe (Oxford)we have gained approval from CTAAC to expandthe preliminary randomised study of neoadjuvantchemotherapy for the treatment of ovarian cancer.The CHORUS trial will now be completed over the

next 2 years and will test the acceptability of defer-ring surgery in ovarian cancer through the use ofpre-operative chemotherapy.

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AA.. HS6ST1 ISH shows RNA to be present in ovary tumour and endothelial cells, but absent instroma and normal ovary (inset). BB.. HS6ST2 ISH shows RNA to be present in ovary tumour cells, but absent in endothelial cells,stroma and normal ovary (inset). Each scale bar represents 400mm.


Heparan sulphate (HS) is a near-universal cell surface co-receptor for many growth factors and cytokines. Its mode ofaction is still unclear, although specific structural motifs in theHS polysaccharide chain are known to be essential for bind-ing growth factors and enabling their efficient engagementwith tyrosine kinase receptors. We are investigating themolecular design of HS in different cell types, includingembryonal stem (ES) cells, and examining how its uniquedomain structure and conformational flexibility drive theassembly of ligand-receptor signalling complexes on the plasma membrane.

Glycosaminoglycan recognition sites in complexmodular proteins

Hepatocyte Growth Factor/Scatter FactorHepatocyte growth factor/scatter factor (HGF/SF)is comprised of an N-terminal domain (N), fourkringle domains (K1 to K4) and a serine proteinasehomology domain (SP). It contains both Metreceptor and glycosaminoglycan (GAG) co-recep-tor-binding sites. The alternatively transcribedNK1 fragment of HGF/SF retains the majorGAG-binding site that recognises two structurally-distinct GAGs, HS/heparin and dermatan sulphate.We have recently investigated the independentGAG-binding properties of the six individual N, Kand SP domains (provided by Dr ErmannoGherardi, MRC Centre, Cambridge), using gelmobility shift and HPLC size exclusion chromato-graphic assays. Interestingly, GAG-binding siteswere revealed in three distinct domains, with rela-tive affinities of N>K1>>SP. All three sites boundheparin but only the N domain bound dermatansulphate with high affinity. NMR titration data (incollaboration with Dušan Uhrín, University ofEdinburgh), also indicate a substantial overlap of

the highest affinity heparin- and dermatan sulphate-binding sites within NK1. Together, these data sug-gest that the N domain provides a single high affin-ity site for both GAGs, but that long GAG chains,especially HS/heparin, may be able to engage inadditional, weaker, cooperative interactions withother HGF/SF modules.

Factor H Complement Regulatory ProteinIn collaboration with Dušan Uhrín, we have inves-tigated the GAG-binding properties of factor H, aninhibitor of complement amplification on self-sur-faces. Factor H is comprised of 20 tandem comple-ment control protein modules. Our gel mobilityshift assay demonstrated binding of a fully-sulphat-ed heparin tetrasaccharide to the C-terminal 19-20module pair. NMR chemical shift mapping allowedthe GAG-binding site to be delineated within thenewly-derived 3D solution structure of this modulepair (Herbert et al. J. Biol. Chem. 2006; 281:16512).Strikingly, missense mutations associated with atyp-ical haemolytic uraemic syndrome congregate inthis GAG-binding site and may thus disrupt theGAG self-recognition properties of factor H.Similarly, we have also shown that the Tyr-to-Hismutation at residue 402 in module 7, that is knownto predispose to acute macular degeneration, caus-es a reduction in affinity of the isolated module 7for heparin oligosaccharides. This is consistentwith the positioning of this residue on the edge ofa separate GAG-binding site.

Studies on mouse embryonic stem cells

Characterisation of ES cells with mutations inHS endosulphatasesThe Sulfs are a family of extracellular enzymeswhich act to modify mature HS chains, removing 6-O-sulphate groups from specific sites. The loss ofthese groups significantly alters the biological func-

Medical Oncology:Proteoglycan Group

GROUP LEADERJohn Gallagher

SENIOR FELLOWMalcolm Lyon

POSTDOCTORALFELLOWSChris RobinsonClaire Johnson

SCIENTIFIC OFFICERSNijole GasiunasGraham RushtonJon Deakin

GRADUATE STUDENTSRebecca Baldwin (jointwith Stem Cell Biology)Annie WatEun –Ang Raber(joint with SalfordUniversity)

PLACEMENT STUDENTNicole Yan

RESEARCH COLLABORATORSValerie Kouskoff and GeorgesLacaudStem Cell Biology Group

Professor Gordon Jayson andColleaguesTranslational AngiogenesisGroup

Dr Catherine Merry, Materials Science, Universityof Manchester

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tion of HS by influencing the ability of the chainsto interact with a variety of growth factors andmorphogens, such as BMP and FGF. Althoughtheir activities are tightly controlled during develop-ment, Sulfs are mis-regulated in various tumoursincluding bladder and breast cancer. Working withcollaborators in Germany (Prof. Dierks, Bielefeld)we analysed the structure and function of HSextracted from mice mutants of one or both mem-bers of the Sulf family. Surprisingly, we found thatthere was an element of co-operativity in the func-tions of these enzymes, a feature not observed inprevious studies. In addition, we demonstrated thatthe pattern of HS sulphation observed in these invivo models was different to that predicted from invitro experiments, with considerably more 6-O-sul-phate groups being targeted by the enzymes thanpreviously thought. We have developed a non-inva-sive assay for detecting Sulf activity, and we hope touse this method to define the extent of Sulfloss/gain in and around tumour sites in vivo.

HS and the regulation of ES cell biologyES cells are a useful model system to study theinter-relationships between HS structure and func-tion. We can manipulate the levels of HS biosyn-thetic enzymes and core proteins using eitherRNAi, or by deriving new ES cell lines from exist-ing mutant mice. As pluripotent ES cells differen-tiate and commit to a defined lineage (e.g. neural,haematopoietic), they change the sulphation pat-terns of their HS chains. The expression of line-age-specific HS may influence the ability of differ-entiating cells to respond to the range of HS-dependent factors that direct ES cell differentiation.ES cells deficient for the iduronate-2-O sulpho-transferase enzyme (Hs2st) appear to be restrictedin their ability to differentiate to specific neural lin-eages. Various members of the FGF family are crit-ical in this pathway and we are investigating howloss of 2-O-sulphation influences their activity.There is growing interest in the role of extracellularmatrix components in directing ES cell behaviour,and we have been able to conduct experimentsdemonstrating that specific HS oligosaccharidescan be used to influence neural and mesodermaldifferentiation pathways. We are also working with

the Manchester Stem Cell Centre on a variety ofprojects.

Observations on the interaction of HS withFGF1 using an enzyme protection-basedapproach The fibroblast growth factors (in particular FGF1and FGF2) are an important area of researchbecause of the roles they play in tumour growthand angiogenesis. At the cell surface, 2:2 complex-es of FGF and FGFR (FGF-receptor) are respon-sible for signal transduction; however, these com-plexes cannot form in the absence of HS or heparina chemical analogue of the sulphated domains (S-domains) of HS. We have demonstrated that spe-cific heparin saccharides drive the assembly of 2:1FGF1: saccharide complexes by a co-operativebinding mechanism that correlates with the capaci-ty of these saccharides to induce FGF1-mediatedcell proliferation. We have now begun to analyseFGF1 interactions with HS using a protection assayin which preformedHS-FGF1 complexes are incu-bated with heparinase enzymes to degrade unpro-tected HS. This showed that FGF1 conferredalmost complete protection on the heparinase Icleavage sites in HS (i.e. the S-domains). This wassurprising since only a minority of S-domains bindFGF1 following their excision from HS. Anothersurprising observation was that FGF1 also protect-ed transition zones (T-zones) of HS from degrada-tion. T-zones are positioned at the interface of theS-domains and the non-sulphated sections of HS.They have an intermediate level of sulphation andwere not expected to interact with FGF1. Ourfindings indicate that FGF1 binding to T-zones andS-domains may be facilitated by co-operative con-formational effects resulting from their close appo-sition in the HS chain. It is unclear whether FGF1is dimerized at any of these binding regions in HS.In collaboration with Professor Tom Blundell andcolleagues (University of Cambridge), we are nowexploring the interaction of FGF-receptors withHS-FGF1 complexes with a view to identifying theactive sites in HS for assembly of signalling com-plexes.

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SSuullff eennzzyymmeess ““eeddiitt””tthhee ssuullpphhaattiioonn ppaatt--tteerrnn ooff HHSS

Sulphation at C6 ofN-sulphated disac-charides in HS isdynamically regulatedby an interplay ofsu lphotr ans fer aseand endosulphatase(Sulf) enzymes. Theextracellular Sulfsspecifically remove 6-O-sulphate groupsand in so doing regu-late HS-mediated cellgrowth.


The research aims of the Targeted Therapy Group are toincrease our understanding of the interactions betweenmonoclonal antibodies (mAb) and irradiation in the treat-ment of cancer and to develop novel clinical trial protocolsusing radionuclide targeted therapies. There are two researchdomains which inter-relate to form a cohesive programme ofpreclinical work under the title of radioimmunotherapy (RIT)of cancer, which include optimisation of RIT and irradiationand immunoregulation. In the first project, the roles of mAbas both vectors to deliver radiotherapy to tumours and indirect cancer cell killing are further explored. The specificaims are to further understand the mechanisms of action ofRIT and define the relative contributions of targeted radia-tion and mAb effector mechanisms to the clearance oftumour. In the second project, mAb are used to augment T-cell responses to tumour by blocking or stimulating co-receptors in the immune system. This project aims to furtherdefine the factors which are important in combining irradia-tion and immunoregulatory mAb.

Preclinical GroupThe last year has been very successful in creating acohesive preclinical laboratory and clinical transla-tional groups. In February 2006 Dr JamieHoneychurch relocated from Southampton andtwo new post-doctoral fellows Dr JamesHainsworth and Dr Lijun Mu were appointed laterin 2006.

Over the last years, we have substantially increasedour understanding of the relative contributions ofantibody effector mechanisms and targeted radia-tion to the eradication of tumour by using welldefined syngeneic animal models of B-cell lym-phoma. We have demonstrated for the first time ina variety of different syngeneic models of lym-phoma that successful RIT leading to long-termtumour protection consists of both targeted irradi-

ation and mAb effector mechanisms. Our resultsconfirm that RIT is much more than targeted radi-ation and provide a scientific rationale to supportthe use of selecting combinations of mAb in RITrather than using a single mAb. We have shownthat a radiation dose response exists for RIT in B-cell lymphoma, when the targeted dose of radiationis augmented in the presence of signalling mAb.We have demonstrated for the first time in vivo thatthis type of combination mAb approach is effectivein RIT and are attempting to translate these preclin-ical findings to clinical studies. Our recent work hasfocused on the importance of the micro-distribu-tion of radiolabelled mAb to the delivery of target-ed radiotherapy to tumour. We have found impor-tant therapeutic differences between targeting dif-ferent tumour antigens and their ability to delivertumouricidal doses of radiation at the microscopiclevel.

Another line of investigation has been the intracel-lular signalling pathways underlying the combinedtreatment of anti-CD20 antibodies and irradiationin non-Hodgkin lymphoma cells. We haveobserved a synergistic cytotoxic effect when theanti-CD20 mAb B1 was combined with irradiation.The additive effect seen with B1 mAb and radiationwas not however observed with another anti-CD20mAb, Rituximab. This synergistic effect wasaccompanied by activation of ERK/MAPK path-way and could be reversed with MEK inhibitorsand siRNA approaches.

Our other preclinical laboratory interests involveexploiting the therapeutic potential of combiningimmuno-modulatory mAb such as anti-CD40 withirradiation or cytotoxic chemotherapy. We havebeen able to demonstrate that irradiation and anti-CD40 mAb can act in concert to eradicate lym-phoma and induce long-term survival under condi-tions whereby either treatment alone is ineffective.When anti-CD40 mAb is given in combination withEBRT a clear radiation dose-dependent increase insurvival is seen with long-term CD8 T-cell depend-ent protection. We are hopeful that this type of

Cancer Studies:Targeted Therapy Group

GROUP LEADERTim Illidge

PRECLINICAL GROUP

POSTDOCTORAL FELLOWSJames HainsworthJamie HoneychurchLijun Mu

GRADUATE STUDENTAndrei Ivanov

CLINICALRADIOIMMUNOTHERAPYGROUP

SENIOR CLINICAL SCIENTIST Maureen Zivanovic

RADIOPHARMACIST Anna McNicholas

SENIOR PHYSICISTMathew Guy

CLINICAL TRIALS ADMINISTRATOR Emma Burke

RESEARCH NURSES Sue NeelsonTracy Gardener

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combination approach using tumour cytoreductionwith standard anti-cancer approaches such as cyto-toxic chemotherapy and irradiation followed byhost immunostimulation may provide an excellenttherapeutic opportunity for future clinical testing.Our current research is focused on improving ourunderstanding of the fate of irradiated tumour cellsin vivo following combination therapy, and deter-mining which type of cell death provides the great-est stimuli to the host immune system and whichare more “immunologically silent”. To addressthese issues we are currently developing a numberof systems for manipulating individual antigen pre-senting cell (APC) populations in vivo, includingtransgenic models, specific depleting agents, andmechanisms for altering the recognition of dyingtumour cells by phagocytes. Using fluorescentlylabelled tumour cells, we have been able to demon-strate that following EBRT lymphoma cells are rap-idly cleared in vivo by macrophages in a radiationdose-dependent manner. These studies are current-ly being extended to assess the role of macrophagesand other APC such as dendritic cells and B-cells inpriming immune responses following combinationtherapy. To increase our understanding of how themode, site and quantity of cell death can affectimmunogenicity we are also characterising andcomparing the efficacy of cells treated ex vivo withchemotherapy or radiotherapy in prophylactic andtherapeutic immunisation protocols.

Clinical Translational research A major focus of the targeted therapy group is towork alongside and to interact with the clinical trialprogramme. The clinical RIT group has madetremendous progress this year in building sufficientinfrastructure to enable the delivery of high qualitynovel clinical research over the coming years. Wewere delighted to welcome Maureen Zivanovic, asenior clinical scientist who relocated fromSouthampton, Anna McNicholas a radiopharmacistand to have secured the services of Mathew Guy tolead the clinical radionuclide dosimetry. A majorproject over the coming year and beyond will be thebuilding of a new radiopharmacy following therecent Christie Trust approval.

One of the major successes from the laboratoryprogramme which we have translated from the clin-ic to the laboratory has been the use of fractionat-ed RIT as well as the development of a novel anti-Idiotype against Rituximab that can be used tomeasure serum Rituximab levels in patients. Wehave completed a Phase I/II dose escalation studyof fractionated 131I Rituximab and are now embark-ing on a multicentre investigator led study usingfractionated RIT. This phase II study (FIZZ) in fol-licular lymphoma will be the first study to be per-formed with fractionated 90Y Ibritumomab tiuxe-

tan (Zevalin) in previously untreated follicular lym-phoma and will be led from the Christie and involvecollaborations with investigators in France and Italy.

The Phase I/II study in diffuse large B cell lym-phoma is the first attempt to integrate RIT into acombination chemotherapy schedule. It is being ledfrom the Christie along with 5 other UK sites.More recently the CR-UK and an industrial partner-ship have funded a further novel trial approachusing abbreviated chemotherapy followed by RIT inrelapsed follicular lymphoma. This study (SCRIFT)will again be led from the Christie but will runthrough the NCRI lymphoma group.

Another novel clinical trial developed inManchester and launched this year is the GemBexstudy in Cutaneous T cell lymphoma. This is firstmulti-institutional national study ever to have beenattempted in this rare cancer and is again funded bythe CR-UK. An important element of this Phase IIclinical trial using Gemcitabine and Bexarotene inaddition to the clinical response rates, will be theskin assessment but a detailed quality of life assess-ment. Finally we plan to develop the anti-Idiotypeagainst Rituximab in clinical trials to measure serumRituximab levels in national clinical trials. This willbe done via a collaboration with the ClinicalExperimental Group in the Paterson Institute, ledby Caroline Dive.

CollaboratorsSue Owens Nuclear Medicine; John RadfordMedical Oncology; Richard Cowan ClinicalOncology (all Christie Hospital) and members ofManchester Lymphoma Group; Richard MyersPathology, MRI; Martin Glennie, Peter Johnson,Mark Cragg; Cancer Sciences Division,Southampton University; Franck Morschausser,Lille, France; Giovanni Martenelli, Milan, Italy

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ERK protein activated in cells undergoing homotypical adhe-sion following treatment with the anti-CD20 antibody B1.Immunofluorescence of active ERK (Alexa fluor 488, green)and DNA (Propidium Iodide, red)


There have been a number of significant developments overthe past year in the Research Services. A review of therequirements of the Institute for Electron Microscopy hasresulted in the formation of a Transmission EM service unitwhich will roll out in January 2007, and which will be run byStephen Murray who currently works for Terry Allen. Theproteomics service component of the Molecular BiologyCore Facility has been boosted tremendously by theappointment of Duncan Smith who is working closely withYvonne Connolly to provide an excellent proteomics serv-ice. We have continued to invest in equipment, includingcomputers and software, to make sure that the servicesremain at the forefront. This year major purchases haveincluded a new Qiagen robot for the MBCF and a new timelapse microscopy system. The facilities work closely togetherto provide a range of integrated activities such as cell sortingor laser capture microdissection for microarray work, andgenotyping transgenic pups.

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Advanced Imaging FacilitySteve Bagleyhttp://www.paterson.man.ac.uk/facilities/advimg.jsp

One of the principal challenges in the study of bio-logical processes is the temporal localisation ofevents hence the facility has concerned itself overthe last five years with the visualisation of some-time fleeting relationships between proteins and astudy of their function. Working in conjunctionwith other facilities within the institute, the analysisof structure, function and relationship assists inbuilding up a snapshot of biological activity.

Over the previous year the institute has purchasedanother time lapse microscope which has beendesigned for the analysis of biological activitywhere the environment (temperature, nutrition,vibration) around the microscope is neutral to the

cell and the very act of imaging the cell has as lowphoto-toxic effect as possible. This microscopesystem enables the researcher to record not only ina manner that does not affect the biological activityof the cell but also allows large numbers of cells tobe examined thus producing statistically viableresults. The requirement for specific processingtools to achieve numerical characterisation of parti-cle and cell movement has increased which has ledto the purchase of another 64-bit workstation andthe building of software which utilises mathemati-cal modelling software.

Currently usage of the microscopes is up to 80%which includes overnight and weekend usage, hencethe amount of useful data being generated increas-es exponentially year on year. As a result of thisamount of data, together with that generated byother facilities and groups in the Institute, ITdepartment is looking at a storage and archival solu-tion due to the terabytes of data being generated.

Challenges over the coming year include highthroughput imaging and three dimensional histo-logical imaging which would allow the automatical-ly imaging and creation of whole tissue visualisa-tion. An important avenue of study would also bethe localisation of specific protein interactions withtissue array data. The requirement for high resolu-tion, low light, and low impact imaging also increas-es as it becomes a technique adopted by severalresearch groups within the institute consequentlythere is a constant drive for the update of the cur-rent equipment and software to allow the systemsto become more photon efficient and hence impactless upon biological activity.

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Biological Resourceshttp://www.paterson.man.ac.uk/facilities/bru.jsp

The Biological Resources unit is a modern trans-genic facility that continues to support the scientif-ic research programmes at the Paterson whilstensuring that the highest quality of welfare and

Research Serviceshttp://www.paterson.man.ac.uk/facilities/scifacs.jsp

HEAD OF RESEARCH SERVICESJenny Varley

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health status of the animals is maintained at alltimes. This year has seen the introduction of addi-tional animal caging as more genetically alteredstrains are developed and maintained.

The use of animals in medical research does stillremain a controversial issue but where ever possiblealternative methods such as tissue culture are usedbut there is still a need for some research involvinganimals to allow the understanding of cancer thatwill lead to the development of better treatmentsfor patients. All animal work at the PatersonInstitute that involves the use of rodents is coveredby both Project and Personal licences that areissued by the Home Office and reviewed by a localethical committee.

Since 2005 we have brought in several new geneti-cally altered strains of mice through our Quarantinesuite. These new models are vital to ensure weremain at the forefront of scientific research. Oncethe animals have been re-derived they are then inte-grated into the BRU where they are housed in indi-vidually ventilated cages (IVCs). These cages pre-vent the spread of potential disease from one cageto another and each cage has an individual Hepa fil-tered air supply that gives approximately 72 airchanges per hour and a fixed exhaust system. Inaddition to protecting the mice from disease thesecages also help to protect the staff from exposureto allergens from the mice and bedding. Exposureof staff to Laboratory Animal Allergens (LAA) is acurrent hot topic with the Health and SafetyExecutive (HSE). Long term exposure to LAA caninduce asthma and skin or eye allergies in staff han-dling the animals. The use of IVC caging dramati-cally reduces this risk.

All the cages are provided with environmentalenrichment, in the form of nesting material, a vari-ety of mouse houses, wooden chew blocks or playtunnels. The addition of these items increasessocialisation and environmental stimulation for themice and reduces aggression amongst some strainsof males.

We undertake routine health screening from ourcolonies to ensure that the mice are free from a listof specific pathogens (SPF) and any new strainsbrought into the unit are health screened beforeintroduction into the facility.

Two thirds of the space in the unit has been givenover to the development and breeding of genetical-ly altered mice, which are important in providing

mouse models of human disease or understandingthe function of a particular gene in the living organ-ism. The transgenic service has continued to devel-op with pronuclear and ES cell injections, and overthe last year we have successfully developed severalnovel strains for groups within the Institute. Theroutine archiving of mouse lines continues throughcryo-preservation and sperm freezing and re-deri-vation of embryos.

The Molecular Biology Core facility provides a fullgenotyping service of the genetically altered miceproduced on site for the users which allows us toefficiently identify the required genotypes.

The remaining third of the unit is dedicated to thecare and housing of experimental mice, and a dedi-cated staff of highly skilled technicians can under-take a range of procedural studies involvingxenograft models and associated therapies. There isclose liaison between the BRU staff and the scien-tific groups within the Institute and the Universityof Manchester.

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Cancer Research UK GeneChip MicroarrayService.Head: Stuart Pepperhttp://bioinf.picr.man.ac.uk/mbcf/index.jsp

The last year has seen numerous developments tothe GeneChip service. Since joining with CR-UKResearch Services and introducing full cross charg-ing we have expanded in size and increased therange of services. Notably this year we have start-ed offering a SNP array service in conjunction withMike Churchman, early in the year we undertooktraining on the Affymetrix 50K SNP array, howev-er such is the pace of change in microarrays thatbefore the end of the year we have already movedon to arrays with 250K SNPs, and early in 2007 wewill be offering half a million SNPs on one array.

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During this year we have also added two extra poststo the team. Firstly a bioinformatician has beenrecruited to add in house support for the service.Having a bioinformatician as part of the team isproving to be very valuable for day to day trou-bleshooting and protocol comparison work, thispost also provides a string link between themicroarray service team and Crispin Miller’sBioinformatics group.

Last year we had started working with Epistem whowere providing access to technology for amplifica-tion of small samples. Although we have stoppedshort of offering single cell profiling as a routineservice, we have now supported several projectsbased on either LCM samples or small numbers ofFACS sorted cells. This work has been helped byimprovements in sensitivity of the AgilentBioanalyzer Picochips, such that we can now per-form QC on less than 1ng of total RNA.

The major development for this year has been theintroduction of new arrays known as Exon arrays.These arrays are significant step forwards in expres-sion profiling as they allow detection of splice vari-ants. Traditional arrays have detect transcripts atone location, however the Exon arrays have a stun-ning 1.4 million probe sets, such that each exon ofa transcript is analysed independently, the arraysalso include a large number of probe sets targetedagainst introns sequences as controls. These arraysuse completely different chemistry to previousAffymetrix expression arrays, and require a very dif-ferent approach to data analysis. Support from thePICR Bioinformatics Group has been particularlyimportant to enable work with these arrays, theyhave put a significant effort into writing novel soft-ware aimed at allowing streamlined analysis of suchlarge data sets. Our two in-house pilot studies havebeen presented at the European Affymetrix CoreLab Directors Meeting and have generated a lot ofinterest; we are now offering these arrays as a stan-dard service and already have four projects at vari-ous stages of processing.

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Central ServicesHead: Martin Chadwickhttp://www.paterson.man.ac.uk/facilities/censerv.jsp

A modern and efficient central services facility pro-vides a vital role in supporting the research carriedout in the Institute. The roles carried out by thegroup include the preparation of media and sterileservices, central stores, porters. The services pro-

vided can often go un-noticed by staff as it runssmoothly and efficiently, which is a sign that thegroup works well.

During the last year we have had three new mem-bers of staff including a Central Services AssistantManager in Maurice Cowell who is learning the jobinside out so that he can cope with any problem inMartin’s absence. We have also had Catherine andAndrew join the team to replace the retirements ofKen, Colin and Gordon. The service has not onlyexpanded in personnel but also the services thegroup provide, in the last year we have taken overthe lunch time cover of reception as well as provid-ing a delivery service on the institute freezer pro-grams. The planned integration of the service iscontinuing with the new employees and some ofthe old able to transfer between departments tocover which ever may be understaffed. This inte-gration is set to continue with a few more ideas toprovide an even more efficient and effective service.

The barcode system is now fully operational andhelping in the department with recharges stockmonitoring. In the last twelve months we have alsointroduced an online intranet ordering system notonly for our stores but also for our six onsitefridge/freezer programs providing the media andenzymes from Bioline, Invitrogen, Promega,Qiagen, Roche, and Sigma.

Stores continues to grow at a rapid rate, there areover 200 different products available in stores forthe end users, and with over 1500 individual prod-ucts being delivered by the porters a month.

The media and plate pouring service is provingmore popular than ever now with four members ofstaff having to work hard to keep up with increas-ing demand in an attempt to provide a next daydelivery service, the service still continues to groweach month. All the lab aides are assigned labswhich they tend to spend a few hours a week inhelping with basic technical duties along side the labstaff. Along side the Media Services the SterileServices provide an important role ensuring the labsare fully stocked with sterile glassware, simplebuffers and boxes of tips. All lab aides are respon-sible for ensuring the smooth running of the labsthey are assigned.

The porters have been running smoothly and stillprovide an essential service and remove all rubbishfrom the Institute as well as moving equipment.They also not only deliver the normal deliveries butalso the goods ordered from the ‘intranet ordering

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system’ for both stores and freezers to the labs dur-ing the day. We have also managed to switch sup-plier of our dry ice contact with no disruption tothe institute and save over £500 a week.

The Porters now recycle as much of the Institute’swaste as possible, existing contracts mean we areable to recycle the plastic inserts from tip trays, aswell as all the cardboard and waste paper that leavesthe Institute.

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Flow Cytometry UnitHead: Mike Hugheshttp://www.paterson.man.ac.uk/facilities/facs.jsp

The Flow Cytometry service unit has two cellsorters, the FACSVantage and the FACSAria thatcan sort up to 22,500 cells a second. They are oper-ated by two full time staff. There are also two multi-user cell analysis machines, the FACScan and theFACSCalibur. All staff wishing to operate thesemachines are given an initial training course and latethis year a more advanced course on compensationand red laser alignment has been offered. The serv-ice is undergoing a review so that we can accommo-date an expected increase in demand. A new data-base has been produced for monitoring usage andperformance of the service.

There have been several new projects this year onthe cytometers. The Targeted Therapy Group hasfocused on the immunogenicity of apoptotic cells.Tumour cells were treated with different chemicaldrugs to induce apoptosis. These cells were used invivo to see whether they can enhance the immuneresponse or not. Doxirubicin, one of the drugsused, is red in colour with a broad spectrum, mak-ing it impossible to use viability dyes that could beexcited with a 488nm laser. DAPI was thereforeused to measure the exact number of apoptoticcells and the FACSVantage with the UV laser usedto excite the DAPI.

The Medical Oncology group has started using theFACSAria as part of the framework 6 ATTACKproject to optimize in vitro culture conditions forchimeric immune receptor expressing T cells. Inorder to identify potentially useful cytokine combi-nations for culture, they want to look for differ-ences in receptor expression patterns betweenpatient groups with different cancers and to followreceptor expression during culture. To do this theyhave developed a 5-colour (FITC, PE, PECy5,PECy7, Pacific Blue) panel of antibodies that canbe run on the FACSAria that enables them to studyexpression of a number of surface molecules(CD3, CD4, CD8, CD25, IL-7Ralpha, IL-15Ralpha,IL-18Ralpha, IL-21Ralpha and FOXP3) in just 4tubes, thus minimizing the number of cells requiredand maximizing the information gained. This panelmay also be extended in the future to make use ofthe Aria’s capacity to run more colours in a singlesample.

During this year we have been working with theMolecular Biology Core Facility to develop proto-cols that allow microarray profiling of small num-bers of FACS analysed cells. By working togetherthe services aim to provide users with a seamlessservice from initial cell sort through to microarraydata. Various options for cell collection have beentested for their compatibility with downstreamRNA extraction and microarray analysis. Althoughthis work is technically challenging it has alreadybeen applied to projects with some success and nowprovides an excellent option for detailed expressionanalysis of small cell populations.

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HistologyHead: Garry Ashtonhttp://www.paterson.man.ac.uk/facilities/histology.jsp

The unit specialises in the histological preparationand analysis of a wide range of samples includingmouse model tissues, whole mouse embryo preps,cell preps, zebra fish and human biopsies. Requestsfrom groups new to histology, as well as increases inworkload from our existing customers, have result-ed in a very busy year for the unit. All our key serv-ices have once again seen heavy demand.

Once again the unit has worked closely with CellRegulation to help characterise the developmentalphenotypes arising from functional deletion of thetranscription factor ATF2. Protocols have beenoptimised to detect proliferation, differentiationand apoptosis markers in the ATF2 embryonic


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knockout mice. In addition, research into the 3Dreconstruction of tissues from optimally preparedsections has been undertaken in collaboration withthe Advanced Imaging Facility.

Immunohistochemistry was used to show thatCEA+ colorectal hepatic metastases produce andsecrete large quantities of the chemokine Eotaxin-2compared to adjacent normal liver. Primary boweltumours were also shown to produce Eotaxin-2that was elevated compared to non-cancerousbowel. These data might be of use in the targetingof T-cell immunotherapies to sites of tumour or inthe development of other targeted therapies.

Adoptive transfer of mouse T-cells genetically engi-neered to express a chimeric receptor consisting ofan anti CD19 scFv linked to the CD3zeta signallingmolecule leads to the regression of established B-cell lymphomas in vivo. Immunohistochemical tech-niques are currently being employed to study the T-cell infiltrate in these tumours before they aredestroyed by the transferred T-cells.

With the Immunology group, laser capturemicrodissection has been used to isolate regions oftumour and normal tissue to study HLA expressionin patients undergoing surgical resection of col-orectal carcinoma metastases to the liver. The iso-lated RNA has been converted to cDNA and usedto assess the transcription from different HLA classI gene loci between the normal and tumour areasusing real time PCR. These data will then be com-pared to immunohistochemical analysis of HLAclass I expression using locus- and allele-specificantibodies. This work will identify evidence andmechanism of HLA down-regulation of primaryimportance to escape from tumour immunity.

Recently and in conjunction with the Cell Signallinggroup, and again using LCM, the expression ofTiam1, STEF and pRex1 homologues in zebra fishis being determined in wild type, benign and malig-nant melanophores.

Tissue microarray construction has been expandedover the last year. Several hundred samples havenow been incorporated into new TMA blocks,allowing for high throughput and standardisation ofmethodology, both of which are essential in trans-lational research. Improvements in template designand post construction processes have resulted inincreased numbers of samples per array.

Finally, as well as a new request form and sampletracking system being introduced, the unit hopes to

automate some of the antibody optimisation andtroubleshooting services by purchasing an automat-ed staining robot over the next few months. Linkedwith this, two new epitope retrieval systems will alsobe introduced, allowing for improvements in stan-dardisation.

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Molecular Biology Core FacilityHead: Stuart Pepper.http://www.paterson.man.ac.uk/facilities/mbcf.jsp

The Paterson Institute Molecular Biology CoreFacility has now been in existence for 6 years. Inthat time it has grown from a team of two peopleoffering DNA sequence and plasmid isolation to ateam of nine people offering a full range of state ofthe art techniques for analyzing DNA, RNA andprotein. This year a total of three extra posts joinedthe team, two of which are supporting the CR-UKwide GeneChip Microarray service which is detailedseparately on these pages.

Duncan Smith has joined the team and brings withhim a strong background in chromatography andMS protocol development and implementation.Over this year phosphorylation mapping hasbecome a standard service and data have now beengenerated for approximately ten projects. These



services have had a significant impact on the workof groups in the Institute, allowing research pro-grams to include phosphorylation mapping as amatter of routine. As the year draws to a close thefacility is developing strategies that will enablequantitative phosphorylation mapping. Techniquesfor analyzing global phosphorylation patterns arealso being evaluated, and we would hope that nextyear both quantitative and global phosphorylationmapping will be offered to research groups withinPICR.

In 2005 we had started using an EpMotion robot toset up real time reactions in 384 well format so thatlarge quantitative PCR projects could be undertak-en more efficiently. Over the last year this systemhas been used to complete several large projectswhich have been written into publications. We arecurrently testing a new qPCR system fromBeckman which may well offer another significantjump in throughput for qPCR; pilot studies shouldbe completed early in 2007.

The core services of DNA sequencing and smallscale plasmid isolation have remained stable thisyear; our new Qiagen 8000 robot has performedreliably since install in May, and the 3100 sequencesystem has run as reliably as ever – the 3100 hasnow been running every day for 6 years and hasdelivered over 80 000 sequences for the Institute.

Looking ahead to next year there will be more chal-lenges – particularly the growing interest in microRNA analysis will require more development with-in the facility.

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Kostoris LibraryHead: Steve Gloverhttp://www.christie.nhs.uk/profinfo/departments/library/default.htm

The Kostoris Library provides a service to staff andstudents at Christie Hospital NHS Trust and thePaterson Institute for Cancer Research. Sited nearClinical Oncology on the 2nd floor of the NathanHouse courtyard, the library holds textbooks, PhDtheses and journals.

Staff of the Paterson Institute can access the onlinejournals of the University of Manchester from theirdesktop PCs within the PICR. The KostorisLibrary staff work in close liaison with the healthsciences and electronic resources teams at the uni-

versity and provide an onsite point of contact foraccess to resources. Access to university resourcesis primarily based on IP address but certainresources require usernames or access via Athens.

In addition to a liaison role with the university theKostoris Library provides an onsite document sup-ply service. Documents not available from theInternet, or through subscriptions or in print copyin the library are sourced from other research insti-tutes, hospitals, or the British Library DocumentSupply Centre. In 2006 the library supplied 101articles to PICR staff.

The library also provides a centralised alerting serv-ice using publishers’ table of contents (etocs). In2006 the library supplied over 20,000 etoc alerts toChristie and PICR staff for over 154 different jour-nal titles. PICR and Christie staff downloaded over53,000 full text PDFs from subscribed journals.The American Association of Cancer Researchjournal Cancer Research was the most heavily down-loaded title with a figure of 4059.

The library provides a database surveillance serviceand will set up monthly queries against PubMed, ISIWeb of Science, EMBASE, or BIOSIS. The resultscan be sent to PICR staff via email on a monthly orweekly basis. The library carried out 730 set queriesfor PICR staff in 2006.

The library aims to support the information needsof research staff and has developed its services togive access to published research as soon as it isavailable. This includes delivering information tothe desktop and alerting users to newly publishedarticles within their fields of interest. Integratingonline services with a responsive enquiry serviceand efficient document supply system enhances thecore oncology and related sciences of the referencecollection available onsite.


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BIOINFORMATICS GROUP (page 10)Crispin Miller

Refereed Research Papers

Okoniewski, M.J. and Miller, C.J. (2006)Hybridization interactions between probesets inshort oligo microarrays lead to spurious correla-tions. BMC Bioinformatics, 7, 276.

Unwin, R.D., Smith, D.L., Blinco, D., Wilson, C.L.,Miller, C.J., Evans, C.A., Jaworska, E., Baldwin, S.A.,Barnes, K., Pierce, A., Spooncer, E. and Whetton,A.D. (2006) Quantitative proteomics reveals post-translational control as a regulatory factor in pri-mary hematopoietic stem cells. Blood, 107, 4687-4694.

Wilson, C.L., Sims, A.H., Howell, A., Miller, C.J. andClarke, R.B. (2006) Effects of oestrogen on geneexpression in epithelium and stroma of normalhuman breast tissue. Endocr Relat Cancer, 13, 617-628.

Other Publications

Pepper, S.D., Hey, Y., Newton, G., Okoniewski, M.and Miller, C. (2006) A core lab case study – exonarray challenges and opportunities. The Core –Affymetrix Core Lab Communication Bulletin.September.(http://www.affymetrix.com/userForum/technolo-gy/arraysContents/CoreLabCaseStudy_Pepper.uf)

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CARCINOGENESIS GROUP (page 12)Geoff Margison


Chinnasamy, D., Milsom, M.D., Shaffer, J.,Neuenfeldt, J., Shaaban, A.F., Margison, G.P.,Fairbairn, L.J. and Chinnasamy, N. (2006)Multicistronic lentiviral vectors containing theFMDV 2A cleavage factor demonstrate robustexpression of encoded genes at limiting MOI. VirolJ, 3, 14.

Harrison, K.L., Crosbie, P.A., Agius, R.M., Barber,P.V., Carus, M., Margison, G.P. and Povey, A.C.(2006) No association between N7-methyldeoxyguanosine and 8-oxodeoxyguanosinelevels in human lymphocyte DNA. Mutat Res, 600,125-130.

Hornillo-Araujo, A.R., Burrell, A.J., Aiertza, M.K.,Shibata, T., Hammond, D.M., Edmont, D., Adams,H., Margison, G.P. and Williams, D.M. (2006) Thesyntheses and properties of tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanineand O6-methylguanine. Org Biomol Chem, 4, 1723-1729.

Horsman, M.R., Bohm, L., Margison, G.P., Milas,L., Rosier, J.F., Safrany, G., Selzer, E., Verheij, M.and Hendry, J.H. (2006) Tumor radiosensitizers—current status of development of various approach-es: report of an International Atomic EnergyAgency meeting. Int J Radiat Oncol Biol Phys, 64,551-561.

Lees, N.P., Harrison, K.L., Hall, C.N., Margison,G.P. and Povey, A.C. (2006) Human colorectalmucosal O6-alkylguanine DNA-alkyltransferaseactivity and DNA-N7-methylguanine levels in col-orectal adenoma cases and matched referents. Gut,epub Aug 4.

Margison, G.P., Povey, A.C. and Santibanez-Koref,M.F. (2006) Genetic variation at the humanMGMT locus and its biological consequences. CurrPharmacogenomics, 4, 133-144.

Pearson, S.J., Wharton, S., Watson, A.J., Begum, G.,Butt, A., Glynn, N., Williams, D.M., Shibata, T.,Santibanez-Koref, M.F. and Margison, G.P. (2006)

Publications

56P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 6


A novel DNA damage recognition protein inSchizosaccharomyces pombe. Nucleic Acids Res, 34,2347-2354.

Povey, A.C., O’Donnell, P., Barber, P., Watson, M.,Margison, G.P. and Santibanez-Koref, M.F. (2006)Smoking is associated with a decrease of O6-alkyl-guanine-DNA alkyltransferase activity in bronchialepithelial cells. Int J Cancer, 119, 463-466.

Ranson, M., Middleton, M.R., Bridgewater, J., Lee,S.M., Dawson, M., Jowle, D., Halbert, G., Waller, S.,McGrath, H., Gumbrell, L., McElhinney, R.S.,Donnelly, D., McMurry, T.B. and Margison, G.P.(2006) Lomeguatrib, a potent inhibitor of O6-alkyl-guanine-DNA-alkyltransferase: phase I safety, phar-macodynamic, and pharmacokinetic trial and evalu-ation in combination with temozolomide in patientswith advanced solid tumors. Clin Cancer Res, 12,1577-1584.

Saad, A.A., O’Connor, P.J., Mostafa, M.H., Metwalli,N.E., Cooper, D.P., Margison, G.P. and Povey, A.C.(2006) Bladder tumor contains higher N7-methyl-guanine levels in DNA than adjacent normal blad-der epithelium. Cancer Epidemiol Biomarkers Prev, 15,740-743.

Schambach, A., Bohne, J., Chandra, S., Will, E.,Margison, G.P., Williams, D.A. and Baum, C. (2006)Equal potency of gammaretroviral and lentiviralSIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells. MolTher, 13, 391-400.

Schambach, A., Schiedlmeier, B., Kuhlcke, K.,Verstegen, M., Margison, G.P., Li, Z., Kamino, K.,Bohne, J., Alexandrov, A., Hermann, F.G., von Laer,D. and Baum, C. (2006) Towards hematopoieticstem cell-mediated protection against infection withhuman immunodeficiency virus. Gene Ther, 13,1037-1047.

Shibata, T., Glynn, N., McMurry, T.B., McElhinney,R.S., Margison, G.P. and Williams, D.M. (2006)Novel synthesis of O6-alkylguanine containingoligodeoxyribonucleotides as substrates for thehuman DNA repair protein, O6-methylguanineDNA methyltransferase (MGMT). Nucleic AcidsRes, 34, 1884-1891.

Southgate, T.D., Garside, E., Margison, G.P. andFairbairn, L.J. (2006) Dual agent chemoprotectionby retroviral co-expression of either MDR1 orMRP1 with the P140K mutant of O6-methylguanine-

DNA-methyl transferase. J Gene Med, 8, 972-979.

White, S.C., Lorigan, P., Margison, G.P., Margison,J.M., Martin, F., Thatcher, N., Anderson, H. andRanson, M. (2006) Phase II study of SPI-77 (ster-ically stabilised liposomal cisplatin) in advancednon-small-cell lung cancer. Br J Cancer, 95, 822-888.

Woolford, L.B., Southgate, T.D., Margison, G.P.,Milsom, M.D. and Fairbairn, L.J. (2006) The P140Kmutant of human O6-methylguanine-DNA-methyl-transferase (MGMT) confers resistance in vitro andin vivo to temozolomide in combination with thenovel MGMT inactivator O6-(4-bromothenyl)gua-nine. J Gene Med, 8, 29-34.

Active Patents

O6-Substituted guanine derivatives , a process fortheir preparation and their use in treating tumourcells. McMurry, T.B.H., McElhinney, R.S.,McCormick, J.E., Elder, R.H., Kelly, J., Margison,G.P., Rafferty, J.A., Watson, A.J. and Willington,M.A. International Patent ApplicationPCT/IE94/0031, published (WO 94/29312, 70pages) through CR Technology Ltd. (Chem. Abs.1995, 122, 239458e).

Pyrimidine derivatives and guanine derivatives, andtheir use in treating tumour cells. McMurry, T.B.H.,McElhinney, R.S., McCormick, J.E., Donnelly, D.J.,Murray, P., Carola, C., Elder, R.H., Kelly, J.,Margison, G.P., Watson, A.J., Rafferty, J.A.,Willington, M.A. and Willington, M.R. InternationalPatent Application PCT/IE96/00084, published(WO97/20843, 129 pages) through CR TechnologyLtd. (Chem. Abs., 1997, 127, 108802t).

Potentiation of temozolomide in human tumourcells. Baer, J., Freeman, A.A., Newlands, E.S.,Watson, A.J., Rafferty, J.A., and Margison, G.P.(USP: 5731304) through CR Technology Ltd.

Ionizing radiation or diathermy-switched gene ther-apy vectors and their use in antitumour therapy.Margison, G.P. Marples B., Scott, S.D. and Hendry,J.H. through CR Technology Ltd.


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CELL CYCLE GROUP (page 14)Karim Labib


Gambus, A., Jones, R.C., Sanchez-Diaz, A.,Kanemaki, M., van Deursen, F., Edmondson, R.D.and Labib, K. (2006) GINS maintains associationof Cdc45 with MCM in replisome progressioncomplexes at eukaryotic DNA replication forks.Nat Cell Biol, 8, 358-366.

Kanemaki, M. and Labib, K. (2006) Distinct rolesfor Sld3 and GINS during establishment and pro-gression of eukaryotic DNA replication forks.EMBO J, 25, 1753-1763.

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CELL DIVISION GROUP (page 16)Iain Hagan


Grallert, A., Beuter, C., Craven, R.A., Bagley, S.,Wilks, D., Fleig, U. and Hagan, I.M. (2006) S.pombe CLASP needs dynein, not EB1 or CLIP170,to induce microtubule instability and slows poly-merization rates at cell tips in a dynein-dependentmanner. Genes Dev, 20, 2421-2436.

Other Publications

Hagan, I.M. and Palazzo, R.E. (2006) Warming upat the poles. EMBO Rep, 7, 364-371.

Hagan, I.M., Grallert, A. and Bagley, S. (2006) Can

you see the light: simple and affordable steps toexploit recent advances in fluorescence imaging.Microbiology Today, 33, 112-116.

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CELL REGULATION GROUP (page 18)Nic Jones


Sinclair, J., Weeks, M., Butt, A., Worthington, J.L.,Akpan, A., Jones, N., Waterfield, M., Allanand, D.and Timms, J.F. (2006) Proteomic response ofSchizosaccharomyces pombe to static and oscillat-ing extremely low-frequency electromagnetic fields.Proteomics, 6, 4755-4764.

Weeks, M.E., Sinclair, J., Butt, A., Chung, Y.L.,Worthington, J.L., Wilkinson, C.R., Griffiths, J.,Jones, N., Waterfield, M.D. and Timms, J.F. (2006)A parallel proteomic and metabolomic analysis ofthe hydrogen peroxide- and Sty1p-dependent stressresponse in Schizosaccharomyces pombe.Proteomics, 6, 2772-2796.

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CELL SIGNALLING GROUP (page 20)Angeliki Malliri


Malliri, A., Rygiel, T.P., van der Kammen, R.A.,Song, J.Y., Engers, R., Hurlstone, A.F., Clevers, H.and Collard, J.G. (2006) The RAC activator Tiam1is a Wnt-responsive gene that modifies intestinaltumour development. J Biol Chem, 281, 543-548.

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CLINICAL AND EXPERIMENTALPHARMACOLOGY GROUP (page 22)Caroline Dive and Malcolm Ranson


Blackhall, F., Ranson, M. and Thatcher, N. (2006)Where next for gefitinib in patients with lung can-cer? Lancet Oncol, 7, 499-507.


Booton, R., Ward, T., Heighway, J., Taylor, P., Power,F., Ashcroft, L., Morris, J. and Thatcher, N. (2006)Xeroderma pigmentosum group D haplotype pre-dicts for response, survival, and toxicity after plat-inum-based chemotherapy in advanced nonsmallcell lung cancer. Cancer, 106, 2421-2427.

Cummings, J. and Dive, C. (2006) A chromato-graphic screening method to identify agents thatreverse intrinsic drug resistance in colon cancercells. LC GC Europe, 16, 363-370.

Cummings, J., Ethell, B.T., Jardine, L. and Burchell,B. (2006) Glucuronidation of SN-38 andNU/ICRF 505 in human colon cancer and adjacentnormal colon. Anticancer Res, 26, 2189-2196.

Cummings, J., Ranson, M., Lacasse, E.,Ganganagari, J.R., St-Jean, M., Jayson, G., Durkin, J.and Dive, C. (2006) Method validation and prelim-inary qualification of pharmacodynamic biomark-ers employed to evaluate the clinical efficacy of anantisense compound (AEG35156) targeted to theX-linked inhibitor of apoptosis protein XIAP. Br JCancer, 95, 42-48.

Hussein, D., Estlin, E.J., Dive, C. and Makin, G.W.(2006) Chronic hypoxia promotes hypoxia-inducible factor-1alpha-dependent resistance toetoposide and vincristine in neuroblastoma cells.Mol Cancer Ther, 5, 2241-2250.

Isa, A.Y., Ward, T.H., West, C.M., Slevin, N.J. andHomer, J.J. (2006) Hypoxia in head and neck can-cer. Br J Radiol, 79, 791-798.

Ranson, M., Middleton, M.R., Bridgewater, J., Lee,S.M., Dawson, M., Jowle, D., Halbert, G., Waller, S.,McGrath, H., Gumbrell, L., McElhinney, R.S.,Donnelly, D., McMurry, T.B. and Margison, G.P.(2006) Lomeguatrib, a potent inhibitor of O6-alkyl-guanine-DNA-alkyltransferase: phase I safety, phar-macodynamic, and pharmacokinetic trial and evalu-ation in combination with temozolomide in patientswith advanced solid tumors. Clin Cancer Res, 12,1577-1584.

Southgate, T.D., Sheard, V., Milsom, M.D., Ward,T.H., Mairs, R.J., Boyd, M. and Fairbairn, L.J. (2006)Radioprotective gene therapy through retroviralexpression of manganese superoxide dismutase. JGene Med, 8, 557-565.

Welman, A., Barraclough, J. and Dive, C. (2006)Generation of cells expressing improved doxycy-cline-regulated reverse transcriptional activatorrtTA2S-M2. Nature Protocols, 1, 803-811.

Welman, A., Cawthorne, C., Ponce-Perez, L.,Barraclough, J., Danson, S., Murray, S., Cummings,J., Allen, T.D. and Dive, C. (2006) Increases in c-Src expression level and activity do not promote thegrowth of human colorectal carcinoma cells in vitroand in vivo. Neoplasia, 8, 905-916.

White, S.C., Lorigan, P., Margison, G.P., Margison,J.M., Martin, F., Thatcher, N., Anderson, H. andRanson, M. (2006) Phase II study of SPI-77 (ster-ically stabilised liposomal cisplatin) in advancednon-small-cell lung cancer. Br J Cancer, 95, 822-888.

Williams, H., Julyan, P., Ranson, M., Zweit, J.,Gillies, J. and Hastings, D. (2006) Does (124)Iodo-deoxyuridine measure cell proliferation in NSCLC?Initial investigations with PET imaging and radio-metabolite analysis. Eur J Nucl Med Mol Imaging,epub Nov 16.

Wilson, G.R., Cramer, A., Welman, A., Knox, F.,Swindell, R., Kawakatsu, H., Clarke, R.B., Dive, C.and Bundred, N.J. (2006) Activated c-SRC in duc-tal carcinoma in situ correlates with high tumourgrade, high proliferation and HER2 positivity. Br JCancer, 95, 1410-1414.

Other Publications

Taylor, K., Micha, D., Ranson, M. and Dive, C.(2006) Recent advances in targeting regulators ofapoptosis in cancer cells for therapeutic gain.Expert Opin Investig Drugs, 15, 669-690.

Ranson, M. (2006) Panitumumab : a viewpoint byMalcolm Ranson. Drugs, 66, 2015-2016.


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IMMUNOLOGY GROUP (page 24)Peter Stern


Ali, S., Mulryan, K., Taher, T. and Stern, P.L. (2006)Immunotherapy success in prophylaxis cannot pre-dict therapy: prime-boost vaccination against the5T4 oncofoetal antigen. Cancer Immunol Immunother,56, 165-180.

Bibi, R., Pranesh, N., Saunders, M.P., Wilson, M.S.,O’Dwyer S, T., Stern, P.L. and Renehan, A.G.(2006) A specific cadherin phenotype may charac-terise the disseminating yet non-metastatic behav-iour of pseudomyxoma peritonei. Br J Cancer, 95,1258-1264.

Biglari, A., Southgate, T.D., Fairbairn, L.J. andGilham, D.E. (2006) Human monocytes express-ing a CEA-specific chimeric CD64 receptor specif-ically target CEA-expressing tumour cells in vitroand in vivo. Gene Ther, 13, 602-610.

Elkord, E. (2006) Role of regulatory T cells inallergy: implications for therapeutic strategy.Inflammation Allergy - Drug Targets, 5, 211-217.

Elkord, E., Hopcraft, L., Burt, D. and Stern, P.L.

(2006) Bead-isolated human CD4+CD25+ T regu-latory cells are anergic and significantly suppress

proliferation of CD4+CD25- T responder cells.Clin Immunol, 120, 232-233.

Elkord, E., Rowbottom, A.W., Kynaston, H. andWilliams, P.E. (2006) Correlation between CD8+T cells specific for prostate-specific antigen andlevel of disease in patients with prostate cancer.Clin Immunol, 120, 91-98.

Gambhira, R., Gravitt, P.E., Bossis, I., Stern, P.L.,Viscidi, R.P. and Roden, R.B. (2006) Vaccination ofhealthy volunteers with human papillomavirus type16 L2E7E6 fusion protein induces serum antibodythat neutralizes across papillomavirus species.Cancer Res, 66, 11120-11124.

Green, K.L., Southgate, T.D., Mulryan, K.,Fairbairn, L.J., Stern, P.L. and Gaston, K. (2006)Diffusible VP22-E2 protein kills bystander cells andoffers a route for cervical cancer gene therapy. Hum

Gene Ther, 17, 147-157.

Jiang, H.R., Gilham, D.E., Mulryan, K., Kirillova,N., Hawkins, R.E. and Stern, P.L. (2006)Combination of vaccination and chimeric receptorexpressing T cells provides improved active therapyof tumors. J Immunol, 177, 4288-4298.

Jiang, H.R., Hwenda, L., Makinen, K., Oetke, C.,Crocker, P.R. and Forrester, J.V. (2006)Sialoadhesin promotes the inflammatory responsein experimental autoimmune uveoretinitis. JImmunol, 177, 2258-2264.

Smyth, L.J., Elkord, E., Taher, T.E., Jiang, H.R.,Burt, D.J., Clayton, A., van Veelen, P.A., de Ru, A.,Ossendorp, F., Melief, C.J., Drijfhout, J.W.,Dermime, S., Hawkins, R.E. and Stern, P.L. (2006)CD8 T-cell recognition of human 5T4 oncofetalantigen. Int J Cancer, 119, 1638-1647.

Southgate, T.D., Garside, E., Margison, G.P. andFairbairn, L.J. (2006) Dual agent chemoprotectionby retroviral co-expression of either MDR1 orMRP1 with the P140K mutant of O6-methylguanine-DNA-methyl transferase. J Gene Med, 8, 972-979.

Southgate, T.D., Sheard, V., Milsom, M.D., Ward,T.H., Mairs, R.J., Boyd, M. and Fairbairn, L.J. (2006)Radioprotective gene therapy through retroviralexpression of manganese superoxide dismutase. JGene Med, 8, 557-565.

Ward, C.M., Eastham, A.M. and Stern, P.L. (2006)Cell surface 5T4 antigen is transiently upregulatedduring early human embryonic stem cell differenti-ation: effect of 5T4 phenotype on neural lineageformation. Exp Cell Res, 312, 1713-1726.

Winters, U., Roden, R., Kitchener, H.C. and Stern,P.L. (2006) Progress in the development of cervi-cal cancer vaccine. Therapeutics and Clinical RiskManagement, 2, 259-269.


Woolford, L.B., Southgate, T.D., Margison, G.P.,Milsom, M.D. and Fairbairn, L.J. (2006) The P140Kmutant of human O6-methylguanine-DNA-methyl-transferase (MGMT) confers resistance in vitro andin vivo to temozolomide in combination with thenovel MGMT inactivator O6-(4-bromothenyl)gua-nine. J Gene Med, 8, 29-34.

Active Patents

Stern, P.L. and Hole, N. (1989). Improvementrelating to antigens. Publication informationEP0336562.

Stern PL and Hole N (1999). 5T4 antigen fromhuman trophoblasts Publication informationUS5869053

Ward C.M and Stern (2005) 5T4 antigen expressionas an indicator of stem cell differentiation.Publication information. US2005260591

Melief C, Ossendorp F, Drijfhout JW, Stern, PL.Epitopes of 5T4 antigen for treating preventing anddiagnosing cancer Filed October 2005

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RADIOCHEMICAL TARGETING ANDIMAGING (page 26)

Jamal Zweit


Gillies, J.M., Najim, N. and Zweit, J. (2006)Analysis of metal radioisotope impurities generatedin [(18)O]H(2)O during the cyclotron production offluorine-18. Appl Radiat Isot, 64, 431-434.

Gillies, J.M., Prenant, C., Chimon, G.N., Smethurst,G.J., Dekker, B.A. and Zweit, J. (2006) Microfluidictechnology for PET radiochemistry. Appl RadiatIsot, 64, 333-336.

Gillies, J.M., Prenant, C., Chimon, G.N., Smethurst,G.J., Perrie, W., Hamblett, I., Dekker, B. and Zweit,J. (2006) Microfluidic reactor for the radiosynthe-sis of PET radiotracers. Appl Radiat Isot, 64, 325-332.

Williams, H., Julyan, P., Ranson, M., Zweit, J.,Gillies, J. and Hastings, D. (2006) Does (124)Iodo-deoxyuridine measure cell proliferation in NSCLC?

Initial investigations with PET imaging and radio-metabolite analysis. Eur J Nucl Med Mol Imaging,epub Nov 16.

Workman, P., Aboagye, E.O., Chung, Y.L., Griffiths,J.R., Hart, R., Leach, M.O., Maxwell, R.J., McSheehy,P.M., Price, P.M. and Zweit, J. (2006) Minimallyinvasive pharmacokinetic and pharmacodynamictechnologies in hypothesis-testing clinical trials ofinnovative therapies. J Natl Cancer Inst, 98, 580-598.

Other Publications

Flower, M., Zweit, J. and Atthey, M. (2006)Unsealed radionuclide targeted therapy. In:Handbook of Radiotherapy Physics: Theory andPractice. Eds Mayles, P., Nahum, A. andRosenwald, J.C. Institute of Physics

Robson, L., McKenna, P., McCanny, T., Ledingham,K.W.D., Gillies, J.M. and Zweit, J. (2006) High-power laser production of PET isotopes. In: Lecturenotes in physics. Lasers and nuclei. Vol 694 pp 191-203.Springer Berlin/Heidelberg

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STEM CELL BIOLOGY GROUP (page 28)Georges Lacaud


Chan, W.Y., Follows, G.A., Lacaud, G., Pimanda,J.E., Landry, J.R., Kinston, S., Knezevic, K., Piltz, S.,Donaldson, I.J., Gambardella, L., Sablitzky, F.,Green, A.R., Kouskoff, V. and Gottgens, B. (2006)The paralogous haemopoietic regulators Lyl1 andSCL are co-regulated by Ets and GATA factors yetLyl1 cannot rescue the early SCL-/- phenotype.Blood, epub Oct 19.


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STEM CELL AND HAEMATOPOIESISGROUP (page 30)Valerie Kouskoff


Chan, W.Y., Follows, G.A., Lacaud, G., Pimanda,J.E., Landry, J.R., Kinston, S., Knezevic, K., Piltz, S.,Donaldson, I.J., Gambardella, L., Sablitzky, F.,Green, A.R., Kouskoff, V. and Gottgens, B. (2006)The paralogous haemopoietic regulators Lyl1 andSCL are co-regulated by Ets and GATA factors yetLyl1 cannot rescue the early SCL-/- phenotype.Blood, epub Oct 19.

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STRUCTURAL CELL BIOLOGYGROUP (page 32)Terry Allen


Drummond, S.P., Rutherford, S.A., Sanderson, H.S.and Allen, T.D. (2006) High resolution analysis ofmammalian nuclear structure throughout the cellcycle: implications for nuclear pore complex assem-bly during interphase and mitosis. Can J PhysiolPharmacol, 84, 423-430.

Welman, A., Cawthorne, C., Ponce-Perez, L.,Barraclough, J., Danson, S., Murray, S., Cummings,J., Allen, T.D. and Dive, C. (2006) Increases in c-Src expression level and activity do not promote thegrowth of human colorectal carcinoma cells in vitroand in vivo. Neoplasia, 8, 905-916.

Other Publications

Allen, T.D., Drummond, S.P., Murray, S. andRutherford, S.A. (2006) Visualisation of thedynamics of nuclear envelope reformation in mam-malian cells. Scanning, 28, 96-97.

Allen, T.D., Rutherford, S., Murray, S., Gardiner, F.,Drummond, S. and Kiseleva, E. (2006)Visualisation and analysis of nuclear and chromo-some structures in situ by Field Emission ScanningMicroscopy. In Proceedings 16th InternationalMicroscopy Congress. Vol 1 Biol and Med Sci, p 1184.Pub Japan Mic Soc.

Drummond, S.P. and Allen, T.D. (2006)Nucleoporin relocation to kinetochores duringmitosis: analysis of nup37 and nup43. Scanning, 28,98-99.

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CANCER STUDIES – ACADEMICRADIATION ONCOLOGY: TRANSLATIONAL RADIOBIOLOGYGROUP (page 34) Catharine West


Charnley, N., West, C.M., Barnett, C.M., Brock, C.,Bydder, G.M., Glaser, M., Newlands, E.S., Swindell,R., Matthews, J. and Price, P. (2006) Early changein glucose metabolic rate measured using FDG-PET in patients with high-grade glioma predictsresponse to temozolomide but not temozolomideplus radiotherapy. Int J Radiat Oncol Biol Phys, 66,331-338.

Griffiths, E.A., Pritchard, S.A., Valentine, H.R.,Whitchelo, N., Bishop, P.W., Ebert, M.P., Price,P.M., Welch, I.M. and West, C.M.L. (2006)Hypoxia-inducible factor 1α expression in the gas-tric carcinogenesis sequence and its prognostic rolein gastric and gastro-oesophageal adenocarcinomas.Br J Cancer, epub Dec 19, printed 2007, 96, 95-103

Kabuubi, P., Loncaster, J.A., Davidson, S.E.,Hunter, R.D., Kobylecki, C., Stratford, I.J. and West,C.M. (2006) No relationship between thymidinephosphorylase (TP, PD-ECGF) expression andhypoxia in carcinoma of the cervix. Br J Cancer, 94,115-120.


Nordsmark, M., Loncaster, J., Aquino-Parsons, C.,Chou, S.C., Gebski, V., West, C., Lindegaard, J.C.,Havsteen, H., Davidson, S.E., Hunter, R., Raleigh,J.A. and Overgaard, J. (2006) The prognostic valueof pimonidazole and tumour pO2 in human cervixcarcinomas after radiation therapy: a prospectiveinternational multi-center study. Radiother Oncol, 80,123-131.

Other Publications

Burnet, N.G., Elliott, R.M., Dunning, A. and West,C.M. (2006) Radiosensitivity, radiogenomics andRAPPER. Clin Oncol (R Coll Radiol), 18, 525-528.

Isa, A.Y., Ward, T.H., West, C.M., Slevin, N.J. andHomer, J.J. (2006) Hypoxia in head and neck can-cer. Br J Radiol, 79, 791-798.

Laking, G.R., West, C., Buckley, D.L., Matthews, J.and Price, P.M. (2006) Imaging vascular physiolo-gy to monitor cancer treatment. Crit Rev OncolHematol, 58, 95-113.

Silva, P., Slevin, N., Sloan, P., Price, P., West, C.,Homer, J., Hampson, I. and Hampson, L. (2006)The role of lung resistance protein in radiationtreated head and neck carcinoma. Clin Otolaryngol,31, 247.

West, C. and McKeown, S. (2006) Translationalresearch in radiotherapy trials. Br J Radiol, 79, 716-718.

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CANCER STUDIES: BIOLOGICAL,IMMUNE AND GENE THERAPYGROUP (page 36)Robert Hawkins and Peter Stern


Board, R.E., Thistlethwaite, F.C. and Hawkins, R.E.(2006) Anti-angiogenic therapy in the treatment ofadvanced renal cell cancer. Cancer Treat Rev, epubOct 26.

Chong, G., Bhatnagar, A., Cunningham, D.,Cosgriff, T.M., Harper, P.G., Steward, W.,Bridgewater, J., Moore, M., Cassidy, J., Coleman, R.,Coxon, F., Redfern, C.H., Jones, J.J., Hawkins, R.,Northfelt, D., Sreedharan, S., Valone, F. andCarmichael, J. (2006) Phase III trial of 5-fluo-

rouracil and leucovorin plus either 3H1 anti-idio-type monoclonal antibody or placebo in patientswith advanced colorectal cancer. Ann Oncol, 17,437-442.

Clayton, A.J., Mansoor, A.W., Jones, E.T., Hawkins,R.E., Saunders, M.P., Swindell, R. and Valle, J.W.(2006) A phase II study of weekly cisplatin andgemcitabine in patients with advanced pancreaticcancer: is this a strategy still worth pursuing?Pancreas, 32, 51-57.

Dangoor, A., Thistlethwaite, F. and Hawkins, R.(2006) Cancer vaccines: a clinical perspective. Br JHosp Med (Lond), 67, 365-369.

Elkord, E., Hopcraft, L., Burt, D. and Stern, P.L.

(2006) Bead-isolated human CD4+CD25+ T regu-latory cells are anergic and significantly suppress

proliferation of CD4+CD25- T responder cells.Clin Immunol, 120, 232-233.

Friess, H., Langrehr, J.M., Oettle, H., Raedle, J.,Niedergethmann, M., Dittrich, C., Hossfeld, D.K.,Stoger, H., Neyns, B., Herzog, P., Piedbois, P.,Dobrowolski, F., Scheithauer, W., Hawkins, R.,Katz, F., Balcke, P., Vermorken, J., van Belle, S.,Davidson, N., Esteve, A.A., Castellano, D., Kleeff,J., Tempia-Caliera, A.A., Kovar, A. and Nippgen, J.(2006) A randomized multi-center phase II trial ofthe angiogenesis inhibitor Cilengitide (EMD121974) and gemcitabine compared with gemc-itabine alone in advanced unresectable pancreaticcancer. BMC Cancer, 6, 285, epub Dec 11.

Gambhira, R., Gravitt, P.E., Bossis, I., Stern, P.L.,Viscidi, R.P. and Roden, R.B. (2006) Vaccination ofhealthy volunteers with human papillomavirus type16 L2E7E6 fusion protein induces serum antibodythat neutralizes across papillomavirus species.Cancer Res, 66, 11120-11124.

Harrop, R., Connolly, N., Redchenko, I., Valle, J.,Saunders, M., Ryan, M.G., Myers, K.A., Drury, N.,Kingsman, S.M., Hawkins, R.E. and Carroll, M.W.(2006) Vaccination of colorectal cancer patientswith modified vaccinia Ankara delivering the tumorantigen 5T4 (TroVax) induces immune responseswhich correlate with disease control: a phase I/IItrial. Clin Cancer Res, 12, 3416-3424.

Jiang, H.R., Gilham, D.E., Mulryan, K., Kirillova,N., Hawkins, R.E. and Stern, P.L. (2006)Combination of vaccination and chimeric receptor


expressing T cells provides improved active therapyof tumors. J Immunol, 177, 4288-4298.

Redchenko, I., Harrop, R., Ryan, M.G., Hawkins,R.E. and Carroll, M.W. (2006) Identification of amajor histocompatibility complex class I-restrictedT-cell epitope in the tumour-associated antigen,5T4. Immunology, 118, 50-57.


Trumper, M., Ross, P.J., Cunningham, D., Norman,A.R., Hawkins, R., Seymour, M., Harper, P., Iveson,T., Nicolson, M. and Hickish, T. (2006) Efficacyand tolerability of chemotherapy in elderly patientswith advanced oesophago-gastric cancer: A pooledanalysis of three clinical trials. Eur J Cancer, 42,827-834.

Whitworth, M.K., Sheen, A., Rosa, D.D., Duff, S.E.,Ryder, D., Burumdayal, A., Wiener, K., Hawkins,R.E., Saunders, M., Valle, J.W., Sherlock, D. andJayson, G.C. (2006) Impact of laparotomy andliver resection on the peritoneal concentrations offibroblast growth factor 2, vascular endothelialgrowth factor and hepatocyte growth factor. JCancer Res Clin Oncol, 132, 41-44.

Winters, U., Roden, R., Kitchener, H.C. and Stern,P.L. (2006) Progress in the development of cervi-cal cancer vaccine. Therapeutics and Clinical RiskManagement, 2, 259-269.

______________________________

CANCER STUDIES – CHILDREN’SCANCER GROUP (page 38) Vaskar Saha


Strefford, J.C., van Delft, F.W., Robinson, H.M.,Worley, H., Yiannikouris, O., Selzer, R., Richmond,T., Hann, I., Bellotti, T., Raghavan, M., Young, B.D.,Saha, V. and Harrison, C.J. (2006) Complexgenomic alterations and gene expression in acutelymphoblastic leukemia with intrachromosomalamplification of chromosome 21. Proc Natl Acad SciU S A, 103, 8167-8172.

Other Publications

Shankar, A. and Saha, V. (2006) Non Hodgkin’slymphomas of childhood. In: Lymphomas 2nd Edition.Eds Canellos, G.P., Lister, T.A. and Young, B.D.W.B. Saunders

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CANCER STUDIES – MEDICALONCOLOGY: BREAST BIOLOGYGROUP (page 40)Rob Clarke


Bramley, M., Clarke, R.B., Howell, A., Evans, D.G.,Armer, T., Baildam, A.D. and Anderson, E. (2006)Effects of oestrogens and anti-oestrogens on nor-mal breast tissue from women bearing BRCA1 andBRCA2 mutations. Br J Cancer, 94, 1021-1028.

Flasza, M., Nguyen Huu, N.S., Mazaleyrat, S.,Clemence, S., Villemant, C., Clarke, R. and Baron,M. (2006) Regulation of the nuclear localization ofthe human Nedd4-related WWP1 protein by Notch.Mol Membr Biol, 23, 269-276.

Stylianou, S., Clarke, R.B. and Brennan, K. (2006)Aberrant activation of notch signaling in humanbreast cancer. Cancer Res, 66, 1517-1525.

Warnberg, F., White, D., Anderson, E., Knox, F.,Clarke, R.B., Morris, J. and Bundred, N.J. (2006)Effect of a farnesyl transferase inhibitor (R115777)on ductal carcinoma in situ of the breast in a human


xenograft model and on breast and ovarian cancercell growth in vitro and in vivo. Breast Cancer Res, 8,R21.

Wilson, C.L., Sims, A.H., Howell, A., Miller, C.J. andClarke, R.B. (2006) Effects of oestrogen on geneexpression in epithelium and stroma of normalhuman breast tissue. Endocr Relat Cancer, 13, 617-628.

Wilson, G.R., Cramer, A., Welman, A., Knox, F.,Swindell, R., Kawakatsu, H., Clarke, R.B., Dive, C.and Bundred, N.J. (2006) Activated c-SRC in duc-tal carcinoma in situ correlates with high tumourgrade, high proliferation and HER2 positivity. Br JCancer, 95, 1410-1414.

Other Publications

Clarke, R.B. (2006) Ovarian steroids and thehuman breast: regulation of stem cells and cell pro-liferation. Maturitas, 54, 327-334.

Clarke, R.B. and Smith, G.H. (2006) Mammarystem cells. In: Tissue Stem Cells. Eds Potten, C.S.,Clarke, R.B., Wilson, J. and Renehan, A.G. pp 71-88. Taylor and Francis New York

Kalirai, H. and Clarke, R.B. (2006) TGFbeta inhi-bition of ERalpha-positive cell proliferation. BreastCancer Online, 9, e25.

Kalirai, H. and Clarke, R.B. (2006) Human breastepithelial stem cells and their regulation. J Pathol,208, 7-16.

Sims, A.H., Ong, K.R., Clarke, R.B. and Howell, A.(2006) High-throughput genomic technology inresearch and clinical management of breast cancer.Exploiting the potential of gene expression profil-ing: is it ready for the clinic? Breast Cancer Res, 8,214.

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CANCER STUDIES – MEDICALONCOLOGY: CELL AND GENE THERAPY GROUP (page 42)Robert Hawkins


Biglari, A., Southgate, T.D., Fairbairn, L.J. andGilham, D.E. (2006) Human monocytes express-ing a CEA-specific chimeric CD64 receptor specif-ically target CEA-expressing tumour cells in vitroand in vivo. Gene Ther, 13, 602-610.

Board, R.E., Thistlethwaite, F.C. and Hawkins, R.E.(2006) Anti-angiogenic therapy in the treatment ofadvanced renal cell cancer. Cancer Treat Rev, epubOct 26.

Cheadle, E.J. (2006) MT-103Micromet/MedImmune. Curr Opin Mol Ther, 8,62-68.

Chong, G., Bhatnagar, A., Cunningham, D.,Cosgriff, T.M., Harper, P.G., Steward, W.,Bridgewater, J., Moore, M., Cassidy, J., Coleman, R.,Coxon, F., Redfern, C.H., Jones, J.J., Hawkins, R.,Northfelt, D., Sreedharan, S., Valone, F. andCarmichael, J. (2006) Phase III trial of 5-fluo-rouracil and leucovorin plus either 3H1 anti-idio-type monoclonal antibody or placebo in patientswith advanced colorectal cancer. Ann Oncol, 17,437-442.

Clayton, A.J., Mansoor, A.W., Jones, E.T., Hawkins,R.E., Saunders, M.P., Swindell, R. and Valle, J.W.(2006) A phase II study of weekly cisplatin andgemcitabine in patients with advanced pancreaticcancer: is this a strategy still worth pursuing?Pancreas, 32, 51-57.

Dangoor, A., Thistlethwaite, F. and Hawkins, R.(2006) Cancer vaccines: a clinical perspective. Br JHosp Med (Lond), 67, 365-369.

Friess, H., Langrehr, J.M., Oettle, H., Raedle, J.,Niedergethmann, M., Dittrich, C., Hossfeld, D.K.,Stoger, H., Neyns, B., Herzog, P., Piedbois, P.,Dobrowolski, F., Scheithauer, W., Hawkins, R.,Katz, F., Balcke, P., Vermorken, J., van Belle, S.,Davidson, N., Esteve, A.A., Castellano, D., Kleeff,J., Tempia-Caliera, A.A., Kovar, A. and Nippgen, J.(2006) A randomized multi-center phase II trial ofthe angiogenesis inhibitor Cilengitide (EMD


121974) and gemcitabine compared with gemc-itabine alone in advanced unresectable pancreaticcancer. BMC Cancer, 6, 285, epub Dec 11.

Harrop, R., Connolly, N., Redchenko, I., Valle, J.,Saunders, M., Ryan, M.G., Myers, K.A., Drury, N.,Kingsman, S.M., Hawkins, R.E. and Carroll, M.W.(2006) Vaccination of colorectal cancer patientswith modified vaccinia Ankara delivering the tumorantigen 5T4 (TroVax) induces immune responseswhich correlate with disease control: a phase I/IItrial. Clin Cancer Res, 12, 3416-3424.

Jiang, H.R., Gilham, D.E., Mulryan, K., Kirillova,N., Hawkins, R.E. and Stern, P.L. (2006)Combination of vaccination and chimeric receptorexpressing T cells provides improved active therapyof tumors. J Immunol, 177, 4288-4298.

Redchenko, I., Harrop, R., Ryan, M.G., Hawkins,R.E. and Carroll, M.W. (2006) Identification of amajor histocompatibility complex class I-restrictedT-cell epitope in the tumour-associated antigen,5T4. Immunology, 118, 50-57.


Trumper, M., Ross, P.J., Cunningham, D., Norman,A.R., Hawkins, R., Seymour, M., Harper, P., Iveson,T., Nicolson, M. and Hickish, T. (2006) Efficacyand tolerability of chemotherapy in elderly patientswith advanced oesophago-gastric cancer: A pooledanalysis of three clinical trials. Eur J Cancer, 42,827-834.


______________________________

CANCER STUDIES – MEDICALONCOLOGY: TRANSLATIONALANGIOGENESIS GROUP (page 46)Gordon Jayson


Board, R.E., Bruijns, C.T., Pronk, A.E., Ryder, W.D.,Wilkinson, P.M., Welch, R., Shanks, J.H., Connolly,G., Slade, R.J., Reynolds, K., Kitchener, H.C. andJayson, G.C. (2006) Stage- and CA125-related sur-vival in patients with epithelial ovarian cancer treat-ed at a cancer center. Int J Gynecol Cancer, 16 Suppl1, 18-24.

Buonaccorsi, G.A., Roberts, C., Cheung, S., Watson,Y., O’Connor, J.P., Davies, K., Jackson, A., Jayson,G.C. and Parker, G.J. (2006) Comparison of theperformance of tracer kinetic model-driven regis-tration for dynamic contrast enhanced MRI usingdifferent models of contrast enhancement. AcadRadiol, 13, 1112-1123.

Clamp, A., Blackhall, F.H., Henrioud, A., Jayson,G.C., Javaherian, K., Esko, J., Gallagher, J.T. andMerry, C.L. (2006) The morphogenic properties ofoligomeric endostatin are dependent on cell surfaceheparan sulfate. J Biol Chem, 281, 14813-14822.

Clamp, A.R., Maenpaa, J., Cruickshank, D.,Ledermann, J., Wilkinson, P.M., Welch, R., Chan, S.,Vasey, P., Sorbe, B., Hindley, A. and Jayson, G.C.(2006) SCOTROC 2B: feasibility of carboplatinfollowed by docetaxel or docetaxel-irinotecan asfirst-line therapy for ovarian cancer. Br J Cancer, 94,55-61.


Cummings, J., Ranson, M., Lacasse, E.,Ganganagari, J.R., St-Jean, M., Jayson, G., Durkin, J.and Dive, C. (2006) Method validation and prelim-inary qualification of pharmacodynamic biomark-ers employed to evaluate the clinical efficacy of anantisense compound (AEG35156) targeted to theX-linked inhibitor of apoptosis protein XIAP. Br JCancer, 95, 42-48.

Duff, S.E., Jeziorska, M., Rosa, D.D., Kumar, S.,Haboubi, N., Sherlock, D., O’Dwyer, S.T. andJayson, G.C. (2006) Vascular endothelial growthfactors and receptors in colorectal cancer: implica-tions for anti-angiogenic therapy. Eur J Cancer, 42,112-117.

Jayson, G. and Harris, J. (2006) How participantsin cancer trials are chosen: ethics and conflictinginterests. Nat Rev Cancer, 6, 330-336.

Parker, G.J., Roberts, C., Macdonald, A.,Buonaccorsi, G.A., Cheung, S., Buckley, D.L.,Jackson, A., Watson, Y., Davies, K. and Jayson, G.C.(2006) Experimentally-derived functional form fora population-averaged high-temporal-resolutionarterial input function for dynamic contrast-enhanced MRI. Magn Reson Med, 56, 993-1000.

Rosa, D.D., Clamp, A., Mullamitha, S., Ton, N.C.,Lau, S., Byrd, L., Clayton, R., Slade, R.J., Kitchener,H.C., Shanks, J.H., Wilson, G., McVey, R., Hasan, J.,Swindell, R. and Jayson, G.C. (2006) The intervalfrom surgery to chemotherapy in the treatment ofadvanced epithelial ovarian carcinoma. Eur J SurgOncol, 32, 588-591.

Rosa, D.D., Harris, J. and Jayson, G.C. (2006) Thebest guess approach to phase I trial design. J ClinOncol, 24, 206-208.


Other Publications

Baka, S., Clamp, A.R. and Jayson, G.C. (2006) Areview of the latest clinical compounds to inhibitVEGF in pathological angiogenesis. Expert OpinTher Targets, 10, 867-876.

Thistlethwaite, F. and Jayson, G.J. (2006) Anti-angiogenic therapy in colorectal cancer: an update.Gastrointestinal Cancer Abstracts, 17, 2-6.

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CANCER STUDIES – MEDICALONCOLOGY: PROTEOGLYCANGROUP (page 46)John Gallagher


Clamp, A., Blackhall, F.H., Henrioud, A., Jayson,G.C., Javaherian, K., Esko, J., Gallagher, J.T. andMerry, C.L. (2006) The morphogenic properties ofoligomeric endostatin are dependent on cell surfaceheparan sulfate. J Biol Chem, 281, 14813-14822.

Harmer, N.J., Robinson, C.J., Adam, L.E., Ilag, L.L.,Robinson, C.V., Gallagher, J.T. and Blundell, T.L.(2006) Multimers of the fibroblast growth factor(FGF)-FGF receptor-saccharide complex areformed on long oligomers of heparin. Biochem J,393, 741-748.

Herbert, A.P., Uhrin, D., Lyon, M., Pangburn, M.K.and Barlow, P.N. (2006) Disease-associatedsequence variations congregate in a polyanionrecognition patch on human factor H revealed inthree-dimensional structure. J Biol Chem, 281,16512-16520.

Lamanna, W.C., Baldwin, R.J., Padva, M., Kalus, I.,Ten Dam, G., van Kuppevelt, T.H., Gallagher, J.T.,von Figura, K., Dierks, T. and Merry, C.L. (2006)Heparan Sulphate 6-O-endosulphatases - Discretein vivo activities and functional cooperativity.Biochem J, 400, 63-73.


Mahalingam, Y., Gallagher, J.T. and Couchman, J.R.(2006) Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin requireheparan sulfate with specific properties. J Biol Chem,epub Nov 27.

Ozensoy, O., Kockar, F., Arslan, O., Isik, S.,Supuran, C.T. and Lyon, M. (2006) An evaluationof cytosolic erythrocyte carbonic anhydrase andcatalase in carcinoma patients: an elevation of car-bonic anhydrase activity. Clin Biochem, 39, 804-809.

Robinson, C.J., Mulloy, B., Gallagher, J.T. andStringer, S.E. (2006) VEGF165 binding sites with-in heparan sulfate encompass two highly sulfateddomains and can be liberated by K5 lyase. J BiolChem, 281, 1731-1740.

Theoleyre, S., Kwan Tat, S., Vusio, P., Blanchard, F.,Gallagher, J., Ricard-Blum, S., Fortun, Y., Padrines,M., Redini, F. and Heymann, D. (2006)Characterization of osteoprotegerin binding to gly-cosaminoglycans by surface plasmon resonance:role in the interactions with receptor activator ofnuclear factor kappaB ligand (RANKL) andRANK. Biochem Biophys Res Commun, 347, 460-467.

Uchimura, K., Morimoto-Tomita, M., Bistrup, A.,Li, J., Lyon, M., Gallagher, J., Werb, Z. and Rosen,S.D. (2006) HSulf-2, an extracellular endoglu-cosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects onVEGF, FGF-1, and SDF-1. BMC Biochem, 7, 2.

Other Publications

Gallagher, J.T. (2006) Multiprotein signalling com-plexes: regional assembly on heparan sulphate.Biochem Soc Trans, 34, 438-441.

Lyon, M. (2006) The co-factor role of gly-cosaminoglycans in the activity of hepatocytegrowth factor/scatter factor and the possibilities fortherapeutic inhibition. Chapter 15 In: NewDevelopments in Therapeutic Glycomics. Eds Delehedde,M. and Lortat-Jacob, H. Research Signpost,Trivandrum India

Sarrazin, S., Adam, E., Lyon, M., Depontieu, F.,Motte, V., Landolfi, C., Lortat-Jacob, H., Bechard,D., Lassalle, P. and Delehedde, M. (2006) Endocanor endothelial cell specific molecule-1 (ESM-1): Apotential novel endothelial cell marker and a newtarget for cancer therapy. Biochim Biophys Acta,1765, 25-37.


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CANCER STUDIES – TARGETEDTHERAPY GROUP (page 48)Tim Illidge


Kalejs, M., Ivanov, A., Plakhins, G., Cragg, M.S.,Emzinsh, D., Illidge, T.M. and Erenpreisa, J. (2006)Upregulation of meiosis-specific genes in lym-phoma cell lines following genotoxic insult andinduction of mitotic catastrophe. BMC Cancer, 6,6.

Weigert, O., Illidge, T., Hiddemann, W. andDreyling, M. (2006) Recommendations for the useof Yttrium-90 ibritumomab tiuxetan in malignantlymphoma. Cancer, 107, 686-695.

ADDITIONAL PUBLICATIONS


Araki, Y., Lau, C.K., Maekawa, H., Jaspersen, S.L.,Giddings, T.H., Jr., Schiebel, E. and Winey, M.(2006) The Saccharomyces cerevisiae spindle polebody (SPB) component Nbp1p is required for SPBmembrane insertion and interacts with the integralmembrane proteins Ndc1p and Mps2p. Mol BiolCell, 17, 1959-1970.

Brown, M.D., Hart, C.A., Gazi, E., Bagley, S. andClarke, N.W. (2006) Promotion of prostaticmetastatic migration towards human bone marrowstoma by Omega 6 and its inhibition by Omega 3PUFAs. Br J Cancer, 94, 842-853.

Lalloo, F., Varley, J., Moran, A., Ellis, D., O’Dair, L.,Pharoah, P., Antoniou, A., Hartley, R., Shenton, A.,Seal, S., Bulman, B., Howell, A. and Evans, D.G.(2006) BRCA1, BRCA2 and TP53 mutations invery early-onset breast cancer with associated risksto relatives. Eur J Cancer, 42, 1143-1150.

Olivier, M., Langerod, A., Carrieri, P., Bergh, J.,Klaar, S., Eyfjord, J., Theillet, C., Rodriguez, C.,Lidereau, R., Bieche, I., Varley, J., Bignon, Y.,Uhrhammer, N., Winqvist, R., Jukkola-Vuorinen,A., Niederacher, D., Kato, S., Ishioka, C., Hainaut, P.and Borresen-Dale, A.L. (2006) The clinical value

of somatic TP53 gene mutations in 1,794 patientswith breast cancer. Clin Cancer Res, 12, 1157-1167.


2005 saw a lot of disruption to the seminar programme ofthe Institute due to refurbishment of the lecture theatre.This was all completed by 2006, so we could return to fullstrength and hosted some excellent speakers. In addition tothe external speaker programme, there are regular GeneTherapy seminars and also seminars in the Christie Hospital.Last, but definitely not least, the postdocs continue to run aninternal seminar series which is extremely well attended bystaff in the Institute.

Peter AngelGerman Cancer Research Centre, Heidelberg,Germany.

Frances BalkwillCentre for Translational Oncology, Institute ofCancer, London.

Stephen BellMRC Cancer Cell Unit, MRC Research Centre,Cambridge.

Dominique BonnetCancer Research UK London Research Institute.

Allan BradleyThe Wellcome Trust Sanger Institute, WellcomeTrust Genome Campus, Cambridge.

John CouchmanDivision of Biomedical Sciences, Imperial CollegeLondon.

John DiffleyCancer Research UK, Clare Hall.

Amanda FisherMRC Clinical Sciences Centre, Imperial CollegeFaculty of Medicine, London.

Holger GerhardtCancer Research UK London Research Institute.

Ermanno GherardiMRC Research Centre, Cambridge.

Eyal GottliebCancer Research UK Beatson Institute for CancerResearch, Glasgow, Scotland.

Paul HarkinQueen’s University, Belfast, Northern Ireland.

Christine HarrisonLeukaemia Research Fund Cytogenetics Group,University of Southampton.

Ron HayCentre for Interdisciplinary Research, University ofDundee, Scotland.

Marja JäätteläApoptosis Department, Institute of CancerBiology, Copenhagen, Denmark.

Stephen KeyseStress Response Laboratory, Biomedical ResearchCentre, Dundee.

Seminar Series 2006

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Michael KnopEMBL, Heidelberg, Germany.

Walter KolchCancer Research UK, Beatson Institute for CancerResearch, Glasgow, Scotland.

Willy KrekInstitute of Cell Biology, Swiss Federal Insititute ofTechnology, Zurich, Switzerland.

Johan KreugerUppsala University, Sweden.

Dieter MarméTumor Biology Center, Freiburg, Germany.

Eric MiskaThe Wellcome Trust/Cancer Research UK GurdonInstitute, University of Cambridge, Cambridge.

Inke NäthkeCell & Developmental Biology, University ofDundee, Scotland.

Daniel PeeperDivision of Molecular Genetics, NetherlandsCancer Institute, Amsterdam, The Netherlands.

Francesc PosasUniversitat Pompeu, Fabra, Barcelona, Spain.

Kai RothkammMedical Sciences Division, University of Oxford,Oxford.

Erik SahaiCancer Research UK, London Research Institute.

Owen SansomCancer Research UK, Beatson Institute for CancerResearch, Glasgow, Scotland.

Viesturs SimanisCell Cycle Control Laboratory, ISREC,Switzerland.

Helle UlrichCancer Research UK, Clare Hall Laboratories.

Maarten Van LohuizenThe Netherlands Cancer Institute, Amsterdam,The Netherlands.

Laura van’t VeerThe Netherlands Cancer Institute, Amsterdam,The Netherlands.

Fiona WattCancer Research UK, London Research Institute.

Michael WhiteUniversity of Liverpool, Liverpool.

Gareth WilliamsDepartment of Pathology, University CollegeLondon

Wolfgang ZachariaeMax-Planck Institute of Molecular Cell Biology andGenetics, Dresden, Germany.

Pascale ZimmermannMolecular Genetics Section, K.U. Leuven, Belgium.

Publications listedon page

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Postgraduate Educationhttp://www.paterson.man.ac.uk/education/

POSTGRADUATETUTORGraham J Cowling

EDUCATIONCOORDINATORJulie Edwards

EDUCATION COMMITTEE 2006Iain Hagan – Chairman Fiona BlackhallNoel ClarkeGraham CowlingCaroline DiveDave GilhamTim IllidgeGordon JaysonValerie KouskoffKarim Labib Catherine Merry Crispin MillerMark Saunders Jenny Varley

STUDENT REPRESENTATIVES 2006Hiroko Morohashi Helen Spencer Claire Rooney Katalin Boros Martin Brandenberg

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The Paterson PhD ProgrammeIn 2006 we welcomed eight new Paterson four-yearPhD students from the around the world, who wereselected from over 30 interviewed candidates whowere chosen from 250 applications received by theInstitute during the year. The School of Medicinealso awarded two strategic PhD studentships inMedical Oncology and a further four new clinicalfellows who started work in the Institute. Two ofthese were the prestigious AstraZeneca ClinicalFellowships who will spend half their degree work-ing in Professor Caroline Dive’s laboratory and therest working at AstraZeneca leading the clinical tri-als of new cancer drugs. The contributions of allour postgraduates to the scientific work of theInstitute are described elsewhere in this report. Thefour-year PhD students from previous years contin-

ue to master a remarkable range of skills and areachieving a strong basis for their final thesisresearch. Several have already published parts oftheir work. We say goodbye to Graeme Smethurst atthe end of 2006 who is our first four-year PhD stu-dent and wish him all success as he begins a newcareer in investment with a city bank. Our pro-gramme is still evolving and, in 2007, we are offer-ing more of these flexible PhD training pro-grammes. Successful UK, European andInternational candidates can opt to work on onemain project or three 10 week options in their firstyear.

Research Degree StructurePostgraduate students entering the Institute tostudy for the degree of PhD are under the directguidance of an appointed Supervisor(s) and are alsoallocated an Advisor, with whom they meet regular-ly to review and record progress, set new targets andidentify any assistance required. All postgraduatesare also required to participate in regular researchmeetings within their research group and to attendan organised series of seminars by national andinternational speakers, which runs throughout theyear in the Paterson Institute, Christie Hospital andManchester Universities. To further enhance ourtraining programme, the monthly series of cancerbiology master-classes has been continued, allowingLead Researchers in the Paterson and ChristieHospital to explain the “what”, the “how” and the“why” of their area of research. These relaxedevening sessions are proving extremely popular withall of our postgraduate students and many of thepostdoctoral fellows take part in the scientific dis-cussions. Students also participate in a structuredFaculty graduate training programme of short

Developing first rate cancer research and treatment in futuredecades depends on today’s quality training of our clinicaland science graduates. The Paterson Institute continues toact as the centre for postgraduate study in many aspects ofmolecular, cellular and translational cancer research by pro-viding the major elements required of a training programmefor postgraduate research students and clinical research fel-lows studying for MPhil, MD and PhD degrees by research.As the major centre for research and teaching in theDivision of Cancer Studies, School of Medicine, University ofManchester, our active research environment serves all ourstudents equally and they can draw on the wide expertise ofour scientists and clinicians as well as gaining benefit fromour modern laboratories and first-rate service units.


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courses and workshops depending on their researchneeds. This includes sessions on statistics and datahandling (project dependent), bioinformatics(optional), pharmacology (optional), safety (com-pulsory), innovation (compulsory), written and oralpresentations (compulsory), careers, animal usage(compulsory if using animal models) and ethics(compulsory if using human tissue). The FacultyTraining sessions has continued to allow students todevelop generic research and personal skills.

In the Division of Cancer Studies all new postgrad-uate research projects are assessed for quality.Based on Research Council principles, theEducation Committee, with help from internal andexternal assessors ensure that the work will formthe basis of a stimulating and intellectually challeng-ing postgraduate programme. This process oftentakes place before the candidate is chosen. TheEducation Committee, a body made up of seniorscientists, postdoctoral fellows and student repre-sentatives, continue to assess student progressthroughout their 3-4 years of study and ensure,along with their Supervisors and Advisors, that thestudent achieves their degree goal. Assessment isthrough written reports at 3 months (literaturereview), 1 year and 2 years and short talks to theInstitute at similar times (6, 12, 24 months and as

part of the final viva voce). All students have theopportunity to give written feedback on any aspectof their degree programme. Students can askadvice of any member of the Education Committeeor use their student representatives. All Studentsare automatically members of the Cancer StudiesPostgraduate Association, an independent studentbody at the Institute and Christie Hospital.

When our students are not working, our studentrepresentatives have been busy organising a pro-gramme of activities outside of the laboratory.This begins in September when all our students jointhe Institute for the annual Colloquium. Thesethree days of intensive science also includes twonights of relaxation that allow new students to findtheir feet for hard work and play. All students areinvited to annual student socials to meet other newpostgraduates and postdoctoral fellows and to fin-ish the year, the Simon students social takes theform of ice skating followed by a meal.

Paterson students 2006


Operations provide the foundations upon which thePaterson Institute runs. This encompasses Administrationand Reception, the Director’s office, Estates, Finance andPurchasing, Health & Safety, HR, IT and the MCRC team.

2006 has been a very interesting and busy year for theOperations teams. On 1 January 2006 we legally transferredthe Paterson Institute from the Christie Hospital NHS Trustto The University of Manchester. This meant that all the HR,Payroll and Finance systems changed and so the staff had toadapt very quickly to these new systems. I am delighted toreport that the changeover was so smooth that the majorityof Paterson staff were unaware of it. I would like to thank allthe operations staff for their perseverance and patience dur-ing the transfer.

As well as managing the Paterson transfer, Operations staffwere also responsible for implementing the new CR-UKcontribution-based national Pay & Grading scheme.Following a consultation period with the union and staff, thiswas agreed and implemented in September. Ricky VanDeursen (Cell Cycle) suggested that staff from within thePaterson should design the performance review forms thatwill be used with the new system. To this end a small cross-section of staff have been meeting and the new forms willbe ready for distribution early in the New Year. TowersPerrin, the consultants working on the project, will be run-ning training sessions for all managers, staff and the perform-ance review panel in January and February 2007 ready forthe first cycle of reviews, scheduled for March/April nextyear.

The MCRC team were recruited within the year to providesupport in establishing the MCRC.

____________________________________

Admin & Reception ServicesManager: Julie HallettSharon Barnes (temp), Trevor Haughton, ShirleyLeonard, Carl Oluwole

There were a number of staffing changes in theAdministration Department during 2006. JulieHallett returned from maternity leave and SharonBarnes, who had been providing additional cover,left. Sharon made a valuable contribution to theteam, assisting Shirley Leonard, who was ‘acting up’into the Manager’s position. Shirley adapted to thisrole extremely well and enjoyed the challenge thatthis opportunity provided.

Trevor Haughton, Front of HouseSecurity/Receptionist, also decided it was time tomove on to pastures new. Carl Oluwole took overthe reins and settled in well.

Despite these changes the team continued to pro-vide a comprehensive secretarial / administration /reception service to the Institute. The needs of thebusiness continue to evolve and the diverse natureof the work being given is proving to be a very chal-lenging experience for the team.

____________________________________

Director’s OfficeDirector’s PA: Elaine Mercer

Throughout 2006, the Director’s office has contin-ued to organise the Paterson Seminar series with afull and exciting programme of eminent speakersvisiting the Institute. This has served to foster col-laboration and encourage positive interaction with-in the wider scientific community, covering adiverse range of fields. The 2007 series has nowbeen planned and guest speakers invited, with a fullprogramme for the start of the year.

Following the merger with The University and thedevelopment of the MCRC, this office has under-

Operations Serviceshttp://www.paterson.man.ac.uk/facilities/adminfacs.jsp

Director of OperationsPippa McNichol

DDEEPPUUTTYYMargaret Lowe

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taken a great deal of work with regard to the plan-ning and organisation of meetings involving bothinternal and external personnel. Another majorfocus has been on the recruitment of potential newGroup Leaders with emphasis on the developmentof research programmes.

The newsletter has continued to be a great successwith regular meetings of the Newsletter EditorialBoard taking place. Copies have been sent out to awider audience over the past year, with much posi-tive feedback from the recipients. Members of thepublic who have made regular donations to theInstitute and those that have shown an interest infund-raising, have all appreciated being able to readabout the work and the staff of the Institute.

This office has continued to support the Directorand his management team, in an administrativecapacity, through an extremely busy and eventfulyear.

____________________________________

EstatesManager: Steve AlcockGraham Hooley, John Lord, Dennis O’Shea, TonyWoollam,

The Estates function has a new look to it this year,since John Lord left and was replaced by GrahamHooley and Tony Woollam. The additional postwas created in recognition of the expanding work-load and the opening of TRF1. Graham and Tonyhave made quite an impact since joining in June,with response times to outstanding work and faultreports improving considerably.

Several capital schemes have been undertakenthroughout the year including laboratory refurbish-ments and a replacement lift for the central stair-well. Dennis O’Shea managed the TRF schemewhich was handed over in summer 2006, so manythanks to Dennis for all his hard work.

An Estates user group was established this year togive users an opportunity to comment upon theservices they receive. The team has been pro-activethroughout the year which has improved the overallservice to the Institute.

____________________________________

Finance & PurchasingManager: Margaret LoweYana Anderton, Catherine Bentley, Liz Fletcher,David Jenkins, Denise Owen, Debbie Suthern

Once the Paterson Institute transferred to TheUniversity of Manchester, life within the FinanceDepartment became a series of challenges.Contacts within the various departments withinThe University were established and obstacles wereovercome. The University introduced a newFinance System (Oracle) that went live on 1st June;this proved to be an additional challenge for theteam as they had just become acclimatised to theprevious system. As a result of its transfer to TheUniversity, the Paterson now has two differentfinancial years that it needs to work within – Aprilto March for CR-UK (and the Christie HospitalNHS Trust) and August-July for The University.

The team would like to think that the majority ofthe Institute have been unaware of the problemsthey have faced. They have all given immense sup-port to provide an efficient service to keep thegroups and service units functioning.

During the year the Paterson instigated a Europeantender process for the new IT storage and archivingsystem (overseen by The University’s ProcurementManager) which will be implemented during 2007.

O P E R AT I O N S S E R V I C E S

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CR-UK have introduced a new budget planningprocess and so the team have been working toensure that the Paterson complies with this.

In 2007 it is planned to introduce InternetProcurement throughout the Institute. Several keymembers of staff within the groups and serviceunits have already received training and courses areon-going for any further staff wishing to attend.

____________________________________

Health & SafetyManager: Colin Gleeson

Wide-ranging health and safety advice and guidanceis provided to staff at all levels within the Institute.The main issues for staff are around biological andchemical hazards. To address these concerns anumber of initiatives have been developed andimplemented in the last twelve months.

Firstly, an Institute wide Bio-safety survey wasdesigned and implemented which surveyed theInstitute’s laboratories. This focused on laboratoryenvironment and estate-related issues and on labo-ratory waste disposal procedures. As a result of thissurvey, estates-related and waste disposal procedur-al deficiencies have subsequently been rectified orare programmed to be so.

Secondly, an Institute-wide review of genetic mod-ification (GM) risk assessments was undertaken toascertain their sufficiency and suitability in light ofthe regulations. Some improvements in the level ofdetail of these risk assessments have been made, tobetter reflect the hazard and risk potential posed bythe work.

It is pleasing to note that the Paterson Institute’sGM risk assessment format and its administrativeprocedures for GM risk assessment review and itsGM committee’s operations are looked onfavourably by the University; and are held up as amodel of good practice for the main Universitycampus and other campuses.

Thirdly, the training presentations for working withbiological agents and hazardous chemicals havebeen further developed to address frequently occur-ring themes and concerns posed by staff, over thelast year. Adapting these and other presentations toaddress concerns raised by staff will be continuednext year.

More generally, departmental health and safety

inspections are run on a rolling annual programme.This helps to spread any necessary minor remedialworks more evenly throughout the year. Statutoryperformance-testing of the Institute’s hundredfume cupboards and microbiological safety cabi-nets, (including those in the Kinnaird Road labs),have been carried out. Finally our relationship withThe University’s Health & Safety Services teamcontinues to develop, especially via contributions tothe Biological and GM Hazard Advisory Group.

____________________________________

HRManager: Anna PearsonLaura Humes

The HR department has continued to provide pro-fessional advice to managers and staff on allemployment-related matters such as policy guid-ance, legislation and best practice.

Throughout 2006, the team has undertaken severallarge tasks including the co-ordination of HR issuesrelating to the merger with The University ofManchester, the administration of the new CR-UKPay and Grading system, the evaluation andimprovement of the induction process and thetransfer of all staff data onto The University’s pay-roll software, Resourcelink.

A Joint Negotiation Committee with three manage-ment representatives and three union members hasbeen created to ensure effective partnership work-ing in the development of HR policies and otherchange processes.

The HR Manager is currently developing the HRstrategy based on high standards of employmentpractice to ensure it is aligned with the needs of theInstitute. This will almost certainly produce anexciting and varied workload for the team.

____________________________________

ITManager: Malik PervezBrian Poole, Steve Royle, Zhi Cheng Wang, MattYoung

The IT department has continued to maintain highlevels of support to users in terms of responsetimes to support calls and extremely low downtimesensuring that research and development is support-ed at the highest level.

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The key challenge facing the Institute is the everincreasing demand for higher levels of storagecapacity. In response to this plans are being put inplace to develop a new storage solution that willquadruple the available storage capacity. The newstorage solution is scheduled for implementation inearly 2007 and can be easily added to, which shouldprovide adequate storage for subsequent years. Tomeet legal and legislative requirements an archivesolution is also being developed in conjunction withthe storage strategy. The new solution will initiallyprovide 50 Terabytes (Tb) of storage and archiving,with the potential to increase this to 200 Tb in thefuture.

____________________________________

Web & Graphic DesignMark Wadsworth

To enhance the image of the Institute there areplans to redevelop and modernise the Institute'swebsite. A small committee has been working onthis and the revamped site will go live in Spring2007. The internal intranet will also be redesignedand this will be ready by Summer 2007.

The design of the Paterson newsletter, annualreport, stationery and other promotional materialhas now been brought in-house, giving greater flex-ibility and allowing us to increase our profilethrough a variety of different media and events.


77

Operations team


The major source of funding (77%) of the Paterson Institute is through a core grant from Cancer ResearchUK (CR-UK). This is divided between the various scientific groups and service units within the Instituteto enable them to carry out their research. In addition to this a further 3% of funding has been receivedfrom CR-UK for Project Grant Work and Studentships.

The infrastructure of the Institute is funded by the Christie Hospital Endowment Fund and together withspecific project grants accounts for just over 12% of the total income.

The final 8% of the Institute’s funding is received from a number of additional sources. The research car-ried out through these additional projects enhances and supports the research undertaken by the core fund-ing.

These sources are as follows:• National Translational Cancer Research Network (NTRAC)• European Commission• Medical Research Council• Astra Zeneca• Wellcome Trust• Active Biotech• Trillium Therapeutics

We are immensely grateful to all our sponsors.

Donations to the Institute in 2006

Legacies• In memory of Mrs Catherine Ord• In memory of Mrs Mary Hyde

Acknowledgement for Funding of the Paterson Institute

78

77%

8%

12%

3%

CRUK Core Grant

CRUK Project Grants

Christie Hospital Endowment Fund

Other Sources

PATERSON INSTITUTE FUNDING 2006


Donations

• The PACCAR foundation (through CR-UK)• Mr Neil Raghib• Oldham SNU Church• Mrs E Barton• In memory of Jeremy Daintree: Mr & Mrs S Rubin; Mr & Mrs B L Singer; Mr & Mrs A Miller;

Mr & Mrs V Harris; Mr & Mrs M Moss; Mr & Mrs M Collins; Mr & Mrs H I Silverberg; Mr & Mrs P Showman; Mr & Mrs B Rothmer; Mr & Mrs Goldenfield; Ms M Milton & Mr M Press; Mrs G Daintree; Mrs B Kay; Mrs A Nicholson; Mrs A Halton; Mrs J Reuben.

• Mr G T Mercer in memory of Mr H Roberts• Mrs P Johnson• In memory of Rhoda Luscott-Evans (formerly Rhoda Langmead-Smith): Mr & Mrs R Evans; Mrs E

Haworth; Mrs B Teale and Friends at Kidderminster Health Centre; Mrs A Henderson; Mr M Luscott-Evans; Jane Johns and Friends.

• ServiceMaster Contract Services• In memory of Audrey Beardmore: Mr J Beardmore, Mrw H Roberts; Mrs M K Sutcliffe and members

of the Alkrington Townwomen’s Guild; Mrs Neta Smart.• Ms Eileen Hamilton• Mrs H M Emerson in memory of Mr G Emerson• Mrs M Collier in memory of Mr Bill Collier• The Ladies Committee of Prosperity Lodge No 5206, Accrington• Mr M Gannon• Mr V Timson in memory of Mrs Eileen Bottom• Women’s Trust Fund

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Stuart Heys (left) formally opened the new PACCAR laboratory in October. The opening was attended by senior members of the University, the Christie Hospital NHS trust and Paterson Institute. Also shown (leftto right) are Nic Jones, David Newbigging (Chairman of CR-UK), Caroline Dive and Alex Markham (ChiefExecutive of CR-UK)


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Career Opportunities

Opportunities exist at a number of levels in theInstitute. We have a well-established programme ofdegrees by research which is described in the sec-tion on Postgraduate Education (page 72). Weencourage applications from suitable qualified grad-uates to apply to join either the PhD or MD pro-grammes. Graduates with a first or 2.1 honoursdegree in a biological science can apply each year totrain for a four-year PhD in one of our research lab-oratories. First year students will compliment theirlaboratory skills by attending a small number ofspecialised postgraduate taught and training coursesallowing them to gain a sound knowledge base ofthe latest developments in cancer treatment andresearch. The Institute also has a well developedprocess for ensuring suitable pastoral care and men-toring for all students.

Postdoctoral applicants of high calibre are regularlysought. Although post docs will be encouraged toapply for their own fellowships, funded positionsare available for outstanding candidates. Interestedapplicants should contact the Group Leaders direct-ly, with details of their area of interest and recentexperience. Links to sources of potential fundingfor fellowships are provided on http://www.pater-son.man.ac.uk/djs?jid=222.

In addition to postgraduate and postdoctoralopportunities, the Institute is still seeking to recruitoutstanding candidates (clinical and non-clinical) tothe positions of Junior and Senior Group Leaders.The packages provided are extremely attractive andcommensurate with the experience of the applicant,with significant funding for personnel, recurrentexpenditure and equipment. Junior Group Leadersare appointed for an initial six-year period, withSenior Group Leaders appointed to non-time limit-ed positions.

Specific vacancies can be found on our web pages(http://www.paterson.man.ac.uk/vacancies), butsuitably qualified and enthusiastic individualsshould contact the Institute at any time to enquireabout career possibilities.

The Paterson Institute is located alongside the ChristieHospital, and has a strong programme of basic and transla-tional research. There are very close links with clinical andtranslational research groups throughout the ChristieHospital site. Recently the Manchester Cancer ResearchCentre has been created with partners including thePaterson Institute, the Christie Hospital NHS Trust, TheUniversity of Manchester and Cancer Research UK(http://www.mcrc.manchester.ac.uk/). This is an extremelyexciting development which will enhance all aspects of can-cer research, education and treatment. The Institute offersexcellent laboratory facilities and outstanding core facilities,including molecular services, a microarray platform, pro-teomics, the production of knock-in/knock-out animal mod-els, real-time PCR and advanced imaging. Details of allgroups and facilities are given throughout this report, andcan guide interested parties to the appropriate contacts.

The Paterson is wellplaced for both nationaland international travel,with Manchester Airportonly around a 30 minutedrive away. The region isvery well-served by themotorway network andthe West Coast mainlinerail service

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