research reports

University of York

2004/5


YCR Cancer Research Unit
Director: Professor N. J. Maitland

Targeting of baculoviral vectors to the prostate for gene therapy
Mr Vincent Dussupt, Ms Katrin Tiemann, Ms Stephanie Swift and Prof Norman Maitland
Collaborators: EU FP5 funded personnel: Dr Lindsay Stanbridge, Ms Regina Nugent, Ms Lucy Hudson, YCR Cancer Research Unit, University of York;
Dr Robert Kraaij and Dr Chris Bangma, Erasmus MC University, Rotterdam.

The baculovirus (Bv) is an insect virus with a rod-shaped nucleocapsid, surrounded by a membrane with a high concentration of gp64 protein at one end used for virus attachment to cells. This virus can transduce and express a foreign transgene in a wide range of dividing and non-dividing mammalian cells in vitro and in vivo, but no viral gene expression or replication takes place in human cells. Our aim is to produce targeted reporter/therapeutic gene expression specifically in prostate cells.

A variety of membrane proteins are over-expressed in prostate cancer cells, some of which are not widely expressed elsewhere in the body. Insertion of the peptide ligand for the GnRH and neurotensin receptors into a copy of the gp64 coding sequence encoded within a customised transfer plasmid (also containing a human gene expression cassette) has resulted in a humanised prostate-tropic baculovirus. Since the native gp64 gene is also present, a mixture of modified and unmodified gp64 is expressed on the virus surface. The GnRH-targeted virus has a transduction advantage over unmodified Bv when the non-specific attachment molecule, heparan sulphate, is removed from the cell surface. Preliminary results also suggest that in vivo, the GnRH targeted virus results in a significantly higher number of transduced cells than the native baculovirus.

Putative motifs have been identified within gp64 that could confer heparan sulphate binding. As further modification of GP64 could compromise Bv infectivity and production, we have also generated a range of attachment modified soluble gp64 molecules, whose ability to specifically attach to prostate cancers (relative to normal and other tissues) is being assayed using a sandwich ELISA technique.

Most current prostate-specific promoters are too weak for use in a gene therapy vector and we are taking two strategies to address this. The first is the manipulation of the extended human prostate-specific transglutaminase promoter, to mutate 3 retinoic acid response elements both individually and in combination, that we believe could be responsible for the tight regulation of this promoter. The result will be a suitable 'synthetic' promoter for therapeutic use. Our second strategy is to amplify expression from the various androgen-regulated prostate-specific promoters. By linking the coding sequence for a ligand independent androgen receptor fragment (ARf) to EGFP via an enhanced human internal ribosome entry site (IRES) to EGFP, all under the control of an androgen-responsive promoter, we have created a positive feedback mechanism, where any translated ARf will act positively on the promoter. When reintroduced into human prostate cells, significantly elevated levels of ARf have been produced.

Double-targeted humanised baculovirus should offer a safer, higher capacity vector alternative to the currently available human virus vectors for cancer gene therapy.

Structure and functional studies of human papillomavirus E2 proteins
Dr. J.E. Burns, Ms. E. Hernandez-Ramon, Ms. C. Burn, Ms. H. Walker,
Prof. N.J. Maitland
Collaborators: Dr. A. Antson, Prof. K. Wilson, York Structural Biology Laboratory,
University of York
Dr. I. Morgan, Institute Of Comparative Medicine, University Of Glasgow
Dr S.V. Graham, Division of Virology, University of Glasgow
Dr. S. Allen, Dr. W. Zhang, School of Pharmaceutical Sciences, University of Nottingham

Papillomavirus E2 proteins are highly conserved proteins that regulate viral transcription and replication through binding to specific DNA sequences in the viral upstream regulatory region and direct interaction with the viral replication protein, E1, and several cellular proteins. They share a conserved tripartite structure comprising an N-terminal domain necessary for the transactivation and replication functions linked by a region of variable length and sequence to a C-terminal DNA binding and dimerisation domain.

The crystal structure of the N-terminal domain of HPV-16 E2 revealed a novel second dimerisation interface and we hypothesised that this could explain the formation of DNA loops observed with full-length E2 but not C-terminal proteins containing the DNA binding and major dimerisation domains. To test this we introduced mutations designed to disrupt N-terminal dimer formation without distorting the overall structure. We mutated E2 proteins from two HPV types: HPV-16: a high risk genital type found in over 50% of cervical cancers, and HPV-2a: a low risk cutaneous virus causing common warts but also implicated in a subset of oral cancers.

Both HPV-16 and HPV-2a dimer interface mutants failed to transactivate but retained the ability to stimulate viral DNA replication. In contrast, mutant E39A, which is outside the dimer interface, was able to transactivate but failed to replicate. Although the HPV-16 proteins retained the ability to bind to DNA containing E2 binding sites, atomic force microscopy revealed that only the wild type E2 protein was able to form DNA loops. Since HPV-2a, unlike HPV-16, is a highly replicative virus, the HPV-2a mutants were cloned back into the intact virus genome in order to study their effects on the virus life cycle. In organotypic culture of human keratinocytes stably transfected with mutant viruses, evidence of viral replication was seen with wild type and transactivation-deficient mutants but not with the replication-defective E39A mutant. This agrees with the recent report in the literature which suggest that the major function of E2 in vivo is in DNA replication.

Interaction of HPV E1 protein with human topoisomerase I
Dr. J.E. Burns, Mr. John Blase*, Prof. N.J. Maitland
*BSc project student, Dept. of Biology, University of York
Collaborators: Dr. D. Simmons, Department of Biological Sciences, University of Delaware, Newark, Delaware, USA,
Dr. J. Champoux, Dept. of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA

The E1 proteins of papillomaviruses are responsible for initiating replication of viral DNA. In order for the replication initiation complex to form at the viral origin of replication, E1 must interact with cellular proteins involved in DNA replication including replication protein A and DNA a-primase. Topoisomerase I (topo I) is recruited to the initiation complex where it functions to relieve torsional stress in DNA during unwinding. E1 shares functional and sequence homologies with SV40 T-antigen, which is known to bind to topo I during replication.

In order to investigate whether E1 similarly binds to topo I, we have purified E1 protein from HPV-2a and several truncated mutants of topo I. Analysis of these by far western blotting suggests the presence of two E1 binding sites in topo I situated in the N-terminal domain and core domain. Further experiments are underway to confirm these results.

Identification of cancer stem cells in human prostate tumours
Dr. A. Collins, Mr. P. Berry, Prof. N.J. Maitland
Collaborators: Mr. M. Stower, Dept. of Urology, York District Hospital, Wigginton Road, York
Prof. F. Hamdy, Urology Unit, Royal Hallamshire Hospital, Sheffield
Dr. K. Linton, Urology Unit, Royal Hallamshire Hospital, Sheffield

The goal of existing therapies for prostate cancer has been to eradicate the bulk of cells within a tumour. However, most patients go on to develop androgen-independent disease which remains incurable by current treatment strategies. The major unanswered question remains as to the origin of this resistance: we propose that it is that existing therapies fail to kill cancer stem cells effectively. Thus, the focus of this project is to identify and characterise the properties of the cancer stem cell.

Putative tumour stem cells have been isolated from a series of primary and metastatic prostate tumours using the markers CD44, a2b1 (Collins et al., 2001, JCS 114; 3865-3872) and CD133 (Richardson et al., 2004, JCS 117;3539-3545). Approximately 0.1% of cells express this phenotype and are clonogenic in soft agar/methylcellulose (33% colony forming efficiency; CFE), unlike their more differentiated progeny (1% CFE), and the committed basal population; (0% CFE), secretory luminal population; (0% CFE). Similarly, colonies formed from CD44+/a2b1hi/CD133+ stem cell population (on type I collagen) are significantly more likely to form secondary colonies than those founded by more differentiated cells. In the presence of stroma and androgens, spheroids generated from selected tumour cells are capable of differentiating into prostatic-like acini , depending upon the Gleason grade of the original tumour, but most importantly now express luminal differentiation markers associated with prostate cancer, namely androgen receptor, prostatic acid phosphatase and prostatic specific antigen.

The ultimate test of 'stemness' is the ability to grow and differentiate in vivo. On injection of as few as 500 CD44+/a2b1hi/CD133+ cells (with an established culture derived from a well differentiated prostate cancer; P4E6) into the ventral prostate of NOD/SCID mice, we have been able to demonstrate tumour formation in 5/5 mice. In contrast, injection of more differentiated phenotypes formed only rare tumours (e.g. CD44+/a2b1low/CD133- in 2/5 mice; CD57+/CD44- in 0/5 mice) even with an input of 106 cells.

In conclusion, the cancer cells displayed stem cell-like properties in that they were capable of generating new clones containing additional stem cells, as well as regenerating phenotypically mixed populations of non-clonogenic cells present in the original tumours.

The Role of Androgen Signalling in the Regulation of Human Prostate Epithelial Stem Cell Differentiation
Dr. A. Collins, Mr. P. Berry, Prof. N.J. Maitland
Collaborator: Mr. M. Stower, Dept. of Urology, York District Hospital, Wigginton Road, York

Androgens play a pivotal role in the pathogenesis of prostate cancer and benign prostatic hyperplasia, yet little is known of the processes that influence the initiation or progression of prostate disease. As stem cells are central to normal homeostasis and may be targeted in carcinogenesis, an understanding of the molecular basis of stem cell fate decisions will lead to insights into their disruption in prostate cancer and possible therapeutic targets. Stroma is the likely target and mediator of androgenic effects upon the epithelium, thus stem cell fate is likely to be regulated by the stroma.

Stroma is a complex mixture of cell types, the majority of which are fibroblasts and smooth muscle cells. We have successfully cultured stroma from a range of patient's samples with differing pathologies and have determined the androgen receptor-positive cells within this complex mixture. Androgen receptor (AR) is expressed in 16-55% of cells (n = 11) and the cells respond to androgen through a number of population doublings. Cells expressing AR also expressa smooth muscle actin, fibronectin and vimentin. Treatment for 6-8 hours with dihydrotestosterone (DHT) increased AR protein expression but did not increase the number of cells expressing AR. Moreover, treatment with DHT decreased the level of expression of smooth muscle actin and vimentin.

Integrins are important in cell motility, migration and invasion. We have also shown that stromal cells expressing AR also express integrins including a1, a2 a2, a3, a6. Our results suggest there is a higher proportion of AR positive integrin a1, or a2 positive cells, than a3 and a6. Further, there is a lower proportion of integrin positive AR negative cells than the levels observed fora actin, fibronectin or vimentin positive AR negative cells. Hence, isolation of AR positive cells by a1 immunoprecipitation and binding to MACS beads will allow us to sort AR positive cells from DHT treated cultures for Affymetrix GeneChip Microarray analysis to determine the androgen-responsive, paracrine factors affecting stem cell fate.

With this knowledge we can separate viable AR + stroma from the fibromusculer stroma by binding to MACS magnetic beads to provide a genome wide assessment of gene expression in androgenic stroma in prostate and identify the androgen responsive paracrine factors which affect stem cell fate.

Genotyping prostate cancer stem cells using SNP genechip arrays
Dr. S.D. Bryce, Dr. A.T. Collins, Prof. N.J. Maitland
Collaborator: Mr. M. Stower, Dept. of Urology, York District Hospital, Wigginton Road, York

An important prediction from the stem cell model of cancer development is that the cancer stem cells will harbour the genetic alterations which permitted the development of the malignant tumour. This project aims to confirm this hypothesis by detecting the underling genetic alterations in prostate cancer stem cells. Our existing research programmes aim to phenotypically characterise cancer stem cells from human prostate tumours. From this work we have been successful in isolating malignant cells which express high levels of integrin a2b1 and the marker CD133 that exhibit the properties of stem cells. We are now investigating the genetic alterations in these cells.

Oligonucleotide arrays which allow the simultaneous genotyping at over 10,000 single nucleotide polymorphism (SNP) loci have recently become available (Affymetrix 10K mapping GeneChips). These arrays allow us to map, to fine (sub-megabase) resolution, chromosomal sections showing marker loss of heterozygosity (LOH) and regions of local genome amplification, in the malignant cells. We have used this new technology to generate detailed genotypes from cancer stem cells and bulk unselected malignant cells along with peripheral blood lymphocytes from the same patient.

We are now beginning to analyse this data to look for consistent patterns of genetic change in the prostate cancer samples and if this pattern of change is reproduced in the cancer stem cells.

Alterations in the activation pathway of Protein Kinase B in the progression to metastasis of human prostate cancer
Dr. R.M. Sharrard, Ms. J. Spalton, Prof. N.J. Maitland
Collaborators: Prof. D. Alessi and Prof. C. P. Downes, University of Dundee; Prof. C. Cooper and Dr. J. Clark, Institute for Cancer Research, Sutton; Dr. H. Isaacs, University of York

Intracellular signalling via Protein Kinase B (PKB/Akt) is central to the regulation of cell growth and survival by growth factors (GFs) and cell-cell and cell-matrix contact. Activation of cell surface receptors stimulates phosphatidylinositol 3-kinase (PI3K) to generate PIP3, which recruits PKB/Akt to the membrane and leads to its phosphorylation and activation. Enhanced survival of metastatic prostate cancer (PCa) cells has been linked to deregulation of PKB activation. We examined the PI3K/PKB pathway in metastatic and non-metastatic prostate cell lines for alterations related to tumour progression.

Metastatic cells showed lowered expression of Insulin-like Growth Factor-1 Receptor (IGF1-R) and either absent or deregulated expression of the adaptor protein Insulin Receptor Substrate 1 (IRS1). IGF1 activated PKB even when expression of IGF1R or IRS1 was inhibited by siRNA, highlighting the potential for crosstalk between pathways and for signalling through alternative receptors or adaptor proteins.

The cell lines express different profiles of the three PKB isoforms (AKT1-3). We showed that the relative and absolute levels of these isoforms determine the activation status and total PKB activity in response to GF stimulation. Altered expression of individual PKB isoforms may determine specific patterns of escape from normal growth constraints, offering the exciting possibility that isoform-targeted drugs may increase therapeutic specificity in PCa treatment.

Deregulation of PKB activation in metastasis has been linked to loss of the tumour suppressor protein PTEN, which antagonises PI3K by degrading PIP3. We used kinetic analysis of dephosphorylation of PKB following PI3K inhibition to demonstrate that SHIP2 (SH2-containing inositolphosphatase 2) substitutes for PTEN in PKB regulation in PTEN-null metastatic PC3 cells, while PTEN may share this role with further PIP3-degrading mechanisms in other cell lines. These differences could profoundly affect the course of tumour progression. Analysis of the pattern of PIP3-regulating enzymes in individual tumours would thus supply valuable information for disease prognosis and provide a basis for improved design and selective administration of therapeutic agents.


YORKSHIRE CANCER RESEARCH P53 RESEARCH GROUP
Director of Unit: Professor A.J. Milner

The effect of phosphorylated p53 on site-specific histone H3 modifications
Dr. R.E. Adamson, Dr. S.J. Allison and Prof. J. Milner

Histone modifications play an essential role in the ordered regulation of chromatin-related processes such as transcription, replication and DNA repair. Such modifications result in alterations in chromatin structure. In many cancers, processes controlling histone modifications become altered leading to a loss of the tumour suppressor p53, which plays an essential role in regulating chromatin-related processes. In response to DNA damage, p53 activates specific gene expression necessary for DNA repair, cell growth arrest and apoptosis. It also interacts with histone modifying complexes influencing histone modifications.

Comparing isogenic clones of human p53+/+ and -/- epithelial cells we have previously shown that p53 influences histone H3 acetylation and phosphorylation. Loss of p53 results in significantly high S10 phosphorylation levels on histone H3, which may contribute to the development of abnormal chromosomes and aneuploidy in p53-deficient cancers.

In the present work we have identified p53 residues whose phosphorylation status influence histone H3 acetylation/phosphorylation levels. By transient transfection, we have expressed wild-type p53 and a number of p53 phosphorylation mutant proteins in human and murine cell lines. Histone H3 modifications in these transfected cells were characterised by Western blotting. Our data highlight the p53 regions that are involved in different histone modifications observed, in both murine and human cell lines.

Remodelling chromatin on a global scale: a novel protective function of p53
Dr. S.J. Allison and Prof. J. Milner

The tumour suppressor p53 has an essential role in maintaining the genomic integrity of the mammalian cell. This is achieved in part through its function as a transcription factor enabling it to induce either growth arrest or apoptosis in response to cellular stress. Changes in gene expression commonly require localized chromatin remodelling and p53 is known to interact in vivo with a variety of transcriptional co-activators and co-repressors with intrinsic histone modifying activities. Here we examine the links between p53 and chromatin structures associated with (i) transcriptional regulation of gene expression, (ii) with DNA repair as part of the process of nucleotide excision repair and (iii) with histone modifications which impact upon chromosomal condensation and ploidy.

p53 acetylation
Dr. J.R. Ford, Dr. M. Jiang and Prof. J. Milner

SIRT1 is a conserved NAD-dependent deacetylase which regulates lifespan in accord with nutritional provision. In mammalian cells SIRT1 also down-regulates stress-induced p53- and FoxO pathways for apoptosis, thus favouring survival under stress. Here we demonstrate that SIRT1 also enables survival under stress-free conditions, and suppresses apoptosis in human epithelial cancer cells. SIRT1 silencing (by RNA interference) activated p53 with Ser15 phosphorylation. This effect was cell-type specific. Gene knock-out and co-silencing revealed that SIRT1-regulated apoptosis is independent of p53, of Bax, and caspase 2, but appears to involve FoxO protein(s). Thus, despite p53 activation, SIRT1-regulated apoptosis is independent of p53. Moreover, our observations demonstrate that SIRT1 and Bcl-2 regulate separate apoptotic pathways within the same human cell lineage. Suppression of apoptosis by SIRT1 under non-stress conditions (this work) would explain high lethality in SIRT1-/- mice. Our discovery that SIRT1 suppresses p53 phosphorylation and activation under non-stress conditions identifies a new mechanism by which SIRT1 contributes towards longevity and survival. Our work also predicts that abnormal functioning of SIRT1 may have both p53-dependent and p53-independent oncogenic effects.

RNAi and apoptotic pathways
Dr. M. Jiang and Prof. J. Milner

To dissect apoptotic genes governing the survival of colorectal carcinoma cells, we employed RNAi to silence Bcl-2 and Bcl-xL in isogenic clones of p53+/+ and p53-/- cells, and of Bax+/- and
Bax-/- cells. We identify a novel proapoptotic function of p53 that does not require activation by genotoxic agents and that appears to be constitutively suppressed by Bcl-2. Silencing of Bcl-2 induced massive p53-dependent apoptosis. The "Bcl-2/p53 axis" requires Bax and caspase 2 as essential apoptotic mediators. This newly discovered Bcl-2/p53 functional interface represents a key regulator of apoptosis, which can be activated by targeting Bcl-2 in colorectal carcinoma cells.

Effects of p53 gene dosage upon the DNA damage responses
Mr. C. Lynch and Prof. J. Milner

p53 is a critical tumour suppressor often referred to as the guardian of the genome, and is found mutated or deleted in over 50% of human cancers. p53 couples detection of diverse stresses to global regulation of the cell cycle, programmed cell death and DNA repair. However, despite 25 years of intensive research, a complete description of p53's role in the cell and the precise mechanisms by which it operates, remains to be elucidated. For example, an extensive body of evidence demonstrates that p53 has a transcription-dependent gatekeeper role in tumour suppression. However, recent work by this laboratory indicates p53 also has a novel caretaker function in tumour suppression: directly facilitating DNA repair independent of transcription. This project examines the influence of p53 gene dosage on DNA repair and response to low levels of DNA damage, exploring a novel model for p53's cellular role as both gatekeeper and caretaker in tumour suppression.

p53 and DNA repair
Dr. C.P. Rubbi and Prof. J. Milner

We have recently shown that nucleolar function appears to be crucial for the maintenance of low levels of p53 and that impairment of this function abrogates the capacity of mdm2 to promote p53 degradation to its stabilisation. Others have also indicated that the link between p53 stabilisation and loss of nucleolar function can be mediated by small p53-binding molecules released from the nucleolus under stress. Thus, the nucleolus appears to be a sensor of cellular stress that can integrate a variety of stresses, including DNA damage, into a p53 response. With hundreds of proteins already identified in the nucleolar proteome there is great potential for the identification of targets for non-DNA damaging anti-cancer therapies.

p53 protein: structures and modifications
Dr. L.J. Warnock, Miss S.A. Raines and Prof. J. Milner

Antibody PAb240 detects a cryptic epitope in the DNA-binding region of p53 protein (aa 213-217) and as such is frequently used as a marker for the detection of mutant and denatured p53 protein. Following Western analysis the PAb240 epitope was detectable in only 1 out of 8 human epithelial colon carcinoma cell lines tested. Four cell lines in the study had a single p53 point mutation in the core domain and were MMR(+), (HT29, SW480, SW620, SW1116). The remainder were wild type for p53 and MMR(-), (HCT116, LoVo, RKO, SW48).

DNA sequencing confirmed expected mutations in the core region of p53 but was unable to detect further mutations in this region. Using an isogenic MMR(+) and MMR(-) RKO cell system we were able to support our initial finding that MMR status does not influence PAb240 epitope recognition. Interestingly, PAbDO-11 (aa 181-190) remained reactive with all four p53 mutant MMR(+) cell lines.

Further study is required to determine why the PAb240 epitope remains cryptic in the colorectal carcinoma cell lines under investigation.


Department of Biology
Head of Department: Professor A.H. Fitter

Regulation of initiation of mammalian DNA replication by Ciz1
Dr D Coverley

We have investigated the basic function of Ciz1 in DNA replication, demonstrating that it plays a positive role in both cell-based and cell-free experiments, and begun to describe the functional organisation of features within the Ciz1 protein. We have also demonstrated that Ciz1 promotes DNA replication independently of the cell-cycle inhibitor p21cip1, and shown that Ciz1 depletion by RNA interference, restrains cell proliferation. In addition, we have begun analysis of the complex issue of Ciz1 splice variant expression and their regulation during development and disease. Current work focuses on a polypyrimidine tract expansion that recurs in early passage cell lines from Ewings Sarcoma Family tumours that correlates with specific exon skipping events. Exclusion of this exon from Ciz1 reporter constructs alters the sub-cellular distribution of Ciz1 protein, raising the possibility that Ciz1 splicing influences the organisation of DNA replication.


Department of Chemistry
Head of Department: Professor P. H. Walton

Novel anti-tumour agents from marine natural products
Prof. R. J. K. Taylor, Mr. S. Martina

Natural products from the sea are providing many interesting new leads for cancer chemotherapy. The salicylihalamides, oximidines and lobatamides are all potent anti-tumour agents isolated from marine sources. Structurally, these compounds are based on large lactones and contain extremely unusual acyl enamide side chains. These new natural products possess a remarkable pattern of differential cytotoxicity and COMPARE pattern recognition analysis indicates that they have a unique mode of antitumour activity. They inhibit the growth of oncogene-transformed cells with unprecedented selectivity for mammalian versus non-mammalian vacuolar-type (H+)-ATPases (V-ATPases), and higher selectivity than previously known bafilomycin/concanamycin class of V-ATPase inhibitor. The aim of the project is to prepare a range of these compounds, plus novel synthetic analogues, for biological screening.

Our first target was the synthesis of a racemic truncated version of lobatamide A in order to validate new synthetic methods and to obtain novel analogues for bioassay. We have initially concentrated on a Still-Gennari approach to the synthesis which incorporates a novel use of the Nozaki-Kishi reaction. Novel methodological results on the Still-Gennari reaction were published in 2004, and a methodological paper on supported phosphonate reagents was published in 2004. Current research involves the completion of the synthesis of the macrolactone core and its elaboration to produce novel analogues of the lobatamides. All analogues will be screened as anti-cancer agents by Dr R. M. Phillips at the University of Bradford. The principle aim of this will be to establish the potency of the analogues relative to the parent compounds and to determine structure activity relationships which will drive further chemical synthesis.