research reports

University of York

2001/2

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

Gene therapy for prostate cancer
Prof. N.J. Maitland
Dr R.M. Sharrard
Ms J. Hall
Ms H. Rippon
Mr H. Guan
Mr R. Birnie
Mr V. Dussupt
Mrs J. Knight
Collaborators:
Prof. O. Cussenot and Dr P. Berthon, Hopital St Louis, Paris, France
Dr C .Bangma, University of Rotherdam, Rotterdam, Netherlands
Prof. P. Mannoni, University of Marseille, Marseille, France
Prof. T. Totterman, University of Uppsala, Uppsala, Sweden
Dr C. Wrighton, Cobra Therapeutics, University of Keele
Prof. S. Bettuzzi, University of Parma, Italy

Joint funding from YCR and the European Community has provided support for this project which will develop viruses capable of directing the expression of therapeutic genes into prostate cancers. In York we have developed and compared the efficiency of two commercial kits for the generation of recombinant adenoviruses, Adeno-X and Ad-Easy, into which we have incorporated the control sequences of genes expressed at high levels in the prostate to drive indicator (Green Fluoresecent Protein) and potentially therapeutic genes. The resultant viruses infect human prostate cells, tissues and tissue reconstructions with high efficiency, expressing the GFP indicator with a high degree of cell type specificity.

Additionally, we have developed the use of insect virus vectors, to target prostate epithelium, by inserting new attachment proteins into the viral envelope (based on earlier YCR-funded studies of the cell surface phenotype of prostate cancer cells). The generation of prostate-specific baculoviruses (Bv) which only express the therapeutic genes is achieved by inserting the same prostate-specific gene expression control sequences as inserted into the adenoviral vectors. In a critical study the failure of the native Bv promoters to drive the expression of Bv genes, was confirmed by RT-PCR in infected cells and tissues. These vectors could provide a novel and safer alternative to the conventional adenoviral vectors.

We have continued to expand the number of gene control sequences available for the construction of these vectors, by cloning and analysis of the control sequences from prostate stem cell antigen and prostatic transglutaminase. Each promoter shows a specific pattern of activation, which should enable the construction on an empirical basis of a spectrum of vectors to target the different types of prostatic carcinoma. An increasing knowledge base of gene control in the prostate should allow us to achieve our ultimate aim, the construction of a prostate cancer targeted synthetic promoter, unaffected by normal cellular controls.

Structure and functional studies of human papillomavirus E2 proteins
Dr J.E. Burns
Ms E. Hernandez-Ramon
Ms C. Burn
Ms H. Walker
Mr A. Kalinska
Prof. N.J. Maitland
Collaborators: Dr A. Antson, Ms O. Moroz, Dr I. Bronstein, Prof. K. Wilson and Prof. G. Dodson, York Structural Biology Laboratory, University of York
Prof. M Wells, Dept of Pathology, University of Sheffield
Dr D. Hicks and Dr S. Bates , Royal Hallamshire Hospital, Sheffield
Dr D. Jenkins, Dept of Pathology, University of Nottingham Medical School,
Dr I. Morgan, Institute Of Comparative Medicine, University Of Glasgow

Human papillomaviruses (HPV) cause a variety of hyperproliferative lesions in epithelial tissues. The majority are benign but a subset of "high risk" viruses is implicated in the development of malignant tumours. We are interested in high risk HPV type 16 which is closely linked to cervical malignancy and also in HPV2a, a low risk type causing hand warts.

The E2 proteins of HPV are the major virally encoded regulators of viral gene expression and replication. They bind to specific DNA sequences within the viral genome and regulate expression of the viral oncogenes E6 and E7. Malignant progression usually involves loss of functional E2, allowing uncontrolled expression of E6 and E7 and loss of cell cycle controls. E2 proteins share a highly conserved structure comprising an N-terminal transactivation and replication domain, linked by a flexible hinge region to a C-terminal dimerisation and DNA-binding domain.

Previously we have identified a second dimerisation interface within the N-terminus. We are currently generating single amino acid mutants which should disrupt this dimerisation without causing major distortions in the E2 structure. These are being analysed in vitro by functional assays for replication, transactivation and DNA binding.

Phosphorylation of HPV16 E2 occurs when the protein is expressed in insect and probably mammalian cells and has been proposed as a mechanism for functional regulation of E2.We are mutating potential phosphorylation sites with the aim of identifying the sites and enzymes involved.

Our ability to generate highly specific antibodies against the E2 protein has resulted in a number of studies of the expression of E2 in clinical samples, most recently in cervical smear material, where expression of E2 might provide a useful enhancement over current technologies. Larger studies of E2 expression are now proposed.

Expression, purification and functional studies using HPV2A E1 protein
Ms A. Lloansi Vila
Dr J.E. Burns
Prof. N.J. Maitland
Collaborators: Dr I. Bronstein, Structural Biology Laboratory, University of York,
W.C. Phelps, W.J. Rocque, Glaxo Smith-Kline Beecham Laboratories, North Carolina, USA

The E1 proteins of HPV are essential for virus replication and thus represent a potential target for anti-viral therapy. They are the most highly conserved HPV proteins with structural and functional similarities to SV40 T-antigen, including DNA binding, ATPase and helicase activities. Unlike high risk HPV which lose functional E1 during malignant progression, HPV2a causes warts and represents a highly replicative system in which E1 is active. We have cloned HPV2a E1 into a variety of expression vectors and overexpressed it in bacteria and baculovirus-infected insect cells.

In vivo E1 interacts with E2 during formation of the replication initiation complex and also interacts with cellular replication proteins such as DNA polymerase. Partially purified E1 retains DNA binding activity and can be shown to interact with purified HPV16 E2 and Topoisomerase I. We are also combining E1 and E2 mammalian cell expression vectors to develop an in vitro replication assay to assess the effects of site directed mutants or E1 fragments on viral replication.

The role of protein kinase B (PKB/Akt) and PTEN in progression to metastasis of human prostate cancer
Dr R.M. Sharrard
Ms J. Spalton
Ms K. Hedley
Prof. N.J. Maitland
Collaborators: Prof. P. Downes and Dr D. Alessi, University of Dundee

A key event in the progression of epithelial tumours to invasiveness and metastasis is the development of ability to survive and proliferate in conditions of aberrant growth factor stimulation and cell-cell and cell-matrix contact. A major pathway which regulates these responses involves activation of Protein Kinase B (PKB/Akt) by the phospholipid PIP3 generated by phosphatidylinositol 3-kinase (PI3K). This pathway is regulated by the tumour suppressor protein PTEN, which antagonises PI3K by destroying PIP3 and which is characteristically lost in late-stage, metastatic prostate cancers.

We have investigated the activation of PKB using specific antibodies to determine its phosphorylation status at its primary (thr308) and secondary (ser473) activation sites. In non-tumour prostatic cells, activation at both sites is dependent upon soluble growth factors (especially IGF-I) and adhesion to substrate, while tumour cells are growth factor-independent and show reduced dependence on adhesion. Cell-cell contact was found to modulate the expression of multiple forms of thr308-phosphorylated PKB in non-tumour cells, an effect which was reduced or absent in metastatic tumour cells. Confocal microscopy revealed dramatic differences in the intracellular location of activated PKB between cell lines: non-tumour cells expressed the majority of ser473-phosphorylated PKB in a punctate intracytoplasmic pattern which was insensitive to the PI3K inhibitor LY294002, which mimics the action of PTEN; tumour cells expressed activated PKB largely or entirely at the plasma membrane and were highly sensitive to LY294002.

Our results support the hypothesis that loss of PTEN facilitates survival of metastatic cells by enhancing PKB activation independent of the growth factors, cell-cell contacts and cell-matrix adhesion which are required for survival of normal epithelial cells and which thereby maintain normal tissue architecture. PTEN gene therapy, or drugs which modulate PI3K activity, may thus have therapeutic potential against metastatic prostate cancer.

Three dimensional in vitro modelling of the prostate
Dr S. Lang
Mrs K. Hyde
Prof. N.J. Maitland
Collaborators: Dr A. Collins, Prostate Research Group, Dept. Surgery, The Medical School, University of Newcastle
Mr N. Clarke, Department of Surgery, Christie Hospital NHS Trust and Department of Urology, Withington Hospital, Manchester
Mr M. Stower, Dept. of Urology, York District Hospital, Wigginton Road, York
Prof. D. Edwards and Mr A. Riddick, Dept of Biological Sciences, University of East Anglia
Prof. A. Turner, Dr B. Usmani and Ms L. Dawson, School of Biochemistry and Molecular Biology, University of Leeds

We have established a three dimensional model of the prostate, in vitro, using co-cultures of epithelia and stroma derived from human tissue. Cells co-cultured in Matrigel gave rise to acinus-like spheroids which contain a lumen surrounded by epithelial bilayers. These 'acini' show expression of markers specific for prostatic function in an organised manner similar to human tissues. We have extended these studies to try and find an immortalised prostate epithelial cell line which is also capable of producing in vitro acini. We found that both the PC-3 and Shmac 5 (generated in this laboratory) cell lines, can produce spheroids which contain lumen surrounded by single or double epithelial layers. In particular, Shmac 5 cells are also responsive to stromal co-culture in a manner similar to primary epithelial cells. Shmac 5 and stromal co-cultures cause further epithelial morphological and differentiation, not seen in PC-3 cells.

Our present studies are trying to understand how the state of epithelial differentiation contributes to acinus formation. In collaboration with Dr A. Collins we are determining whether or not stem cells or early epithelial progenitors are required to generate spheroids. Studies are also trying to establish the exact nature of the growth factors which are important for the epithelial: stromal interactions. An understanding of this mechanism is important to understand the normal controls of prostatic growth and differentiation and how these interactions alter during tumour formation.

The influence of prostatic stroma on the ability of prostatic epithelium (of both normal and tumour origin) has been assessed in a complementary model, in which the epithelial cells are layered on to a collagen gel populated with stromal cells. The results confirmed the distinct biological properties of tumour stroma, inferred by earlier animal studies, and provide a new model to study gene interaction and the potency of therapies to combat tumour invasion.
The phenotype of the component cells is also being studied in our collaborations with the Universities of Leeds and East Anglia.

In collaboration with Mr N Clarke we are also investigating the interaction of prostatic epithelia with bone marrow cultures. These studies aim to understand why prostatic cancers frequently spread to the bone marrow. We have successfully established human bone marrow endothelial cultures and can measure the ability of prostatic epithelia to bind to and invade these cell layers. Such studies will help to find the important molecules responsible for prostatic tumour growth within the bone marrow allowing therapies to be established.


York Structural Biology Laboratory and Yorkshire Cancer Research Unit, Department of Biology

Structural and functional analysis of Mts1 and related S100 proteins
Dr. O.V. Moroz
Prof N.J. Maitland
Dr. G.G. Dodson
Prof I.B. Bronstein

S100A12 is a member of the S100 subfamily of the EF-hand calcium-binding proteins. S100A12 has been shown to be one of the ligands of RAGE, "receptor for advanced glycation end products", for which involvement in tumour growth and spread has been reported. S100 proteins have intracellular and extracellular regulatory activities. Most of S100 proteins exist in the form of dimers within cells; they can form oligomers in the extracellular space. Oligomer formation is thought to be significant for the extracellular activities of S100 proteins. We determined the structure of S100A12 in the presence of calcium in two crystal forms: In one crystal form S100A12 is a dimer. In the other crystal form dimers of S100A12 are arranged as a hexamer. We suggest that the S100A12 hexameric assembly might interact with three RAGE extracellular domains bringing them together. Receptor oligomerization is a well-known mechanism of signal transduction in many signaling pathways. We have cloned and expressed a fragment of RAGE extracellular domain. Far-Western blot experiments indicate binding of this fragment to S100A12. The structure of S100A12 hexamer was published in Acta Cryst D58, pp. 408-413, 2002. Our next aim is to co-crystallize and solve the structure of the complex of extracellular domain of RAGE with S100A12. Understanding the atomic interactions of RAGE-S100 signaling complexes will provide a structural basis for anti-cancer drug design.

S100A12 contains a Zn/Cu - binding motif. We have recently obtained the crystals of S100A12 copper complex and solved the structure at 3 resolution. Copper ions are bound at the predicted binding sites, and binding of copper leads to several clearly visible changes in the structure. There is data emerging confirming significant role of copper in the processes where S100 are implicated (prolifiration of human endothelial cells, angiogenesis), therefore detailed structural information about S100A12-copper complex could explain its several extracellular activities.

In collaboration with Prof. E. Lukanidin (Danish Cancer Society) we tested extracellular functions of S100A4 (Mts1) and S100A12 proteins and have demonstrated neurite outgrowth and angiogenic activities for both proteins.


Yorkshire Cancer Research P53 Research Laboratory
Director: Professor J. Milner

Cell biology of the tumour suppressor p53
Dr. C. Rubbi and Prof. J. Milner

Nucleotide Excision Repair (NER) removes bulky DNA lesions and is thus crucial for the protection against environmental carcinogens and UV light exposure. Deficiencies in NER cause increased mutation rates and chromosomal aberrations. Current methods for studying NER are mostly based on either quantitation of lesion removal or detection of repair DNA synthesis. Both have their limitations: lesion removal is inaccurate at very short times post-lesion, where the fraction of removal is low. Repair synthesis is difficult to apply to normally cycling cells due to the need to discriminate repair from replicative DNA synthesis. To overcome these problems we developed a method for analysis of nucleotide excision repair based on detection of transient single-stranded (ss) DNA stretches generated at the nucleotide excision step. Cells are metabolically labelled with BrdU, exposed to UV-irradiation and the ssDNA transients generated during excision repair are detected using anti-BrdU antibody. The method allows single-cell microscopic analysis of the distribution of DNA repair response. Studies using various DNA repair-deficient cell lines indicate that the detection method integrates a number of pre-synthesis nucleotide excision repair stages. Thus, assembled repair sites can be detected even when they may not lead to complete resolution of the DNA lesion. Using this approach, we show that repair helicase-deficient cells differ from endonuclease-deficient cells.

Molecular biology of the tumour suppressor p53
Dr. A. Okorokov, Dr. L. Warnock and Prof. J. Milner

Balanced regulation of DNA double strand break (DSB) repair is crucial for genetic integrity and cell survival. Cells perform DSB repair either by homologous recombination (HR) or by non-homologous end joining (NHEJ). Either option carries risk for DNA instability. The presence in the cell of the tumour suppressor p53 has been shown to suppress the levels of HR, however, the effect of p53 on DNA EJ is less well understood. Here we demonstrate dramatically increased DNA EJ activity in cell-free extracts from p53-/- mouse embryo fibroblasts (MEFs) compared to p53+/+ MEFs. The addition of wild type (wt) p53 to p53-/- MEFs extracts inhibited DNA end-joining in a dose dependent manner. Binding of wt p53 to DNA ends in vitro protected them from exonuclease attack and inhibited T4 DNA ligase dependent end-joining. This inhibitory effect was markedly enhanced for p53 R175H, a cancer derived mutant of p53. In contrast, inhibition was negated in the presence of p53 S15D, a phosphorylation-mimicking mutant protein. Interestingly, p53 S15D stimulated in vitro DNA end-joining of the blunt ended DNA by T4 DNA ligase. Here we discuss the possibility that, in conjunction with its ability to control levels of HR, p53 may also serve to suppress DNA EJ in cells under normal conditions. This suppression may be associated with DNA-PK or ATM kinases, providing potential crosstalk between major cellular pathways of DNA repair and cell cycle checkpoint mechanisms.

Activation of p53 following exposure to DNA damage
Dr. M. Jiang and Prof. J. Milner

The tumour p53 is a multifunctional protein important for the maintenance of genomic integrity. It is able to form molecular complexes with different DNA targets and also with cellular proteins involved in DNA transcription and DNA repair. In mammalian cells the biochemical processing of DNA occurs on a nuclear substructure termed the nuclear matrix. Previously Deppert and co-workers have identified p53 in association with the nuclear matrix in viral- and non-viral transformed cell lines. In this study we demonstrate, for the first time, that the p53 is bound to the nuclear matrix in primary cultures of normal mammalian cells and that this binding increases following DNA damage. Analysis of cell lines expressing structural mutants of p53 revealed that association with nuclear matrix is independent of the tertiary and quaternary structure of p53. However, the proline-rich domain towards the N-terminus of p53 (residues 67-98) appeared important for binding to the nuclear matrix. This was demonstrated by TET-ON regulated expression of p53-derived constructs in p53-/- murine fibroblasts (MEF p53-/-). The protein-rich domain of p53 has potential for SH3 protein-protein interaction, and has a role in p53-mediated apoptosis and possibly base-excision repair of DNA damage. We discuss our observations in relation to the ability of p53 to facilitate DNA repair and also review evidence indicating that matrix-bound p53 in SV40-transformed cells may facilitate the transforming potential of SV40 large T antigen.

Transactivation-independent functions of p53
Dr. F. Chan and Prof. J. Milner

The tumour suppressor p53 is thought to have a role in DNA repair through is ability to upregulate the expression of factors required in DNA repair, example GADD45 and p48. However, work in the laboratory by Dr C Rubbi has suggested that p53 may have a direct role in the repair of UV damaged DNA through its ability to act as a chromatin accessibility factor. When DNA is damaged it can be repaired via the BER or NER pathway. The latter pathway consists of the Transcription Coupled Repair (TCR) or Global Genomic Repair (GGR). In GGR the tightly packed chromatin has to be relaxed in order to allow repair factors to bind onto the damaged DNA before it can be repaired. We suggest that p53 may have a role in the relaxing or "opening" of the highly packed chromatin through the recruitment of acetyltransferases to allow the repair factors to assemble onto the damaged DNA. Confocal work has shown that micro-injection of anti-p53 antibody, DO-1 inhibits DNA repair after UV irradiation and that p53 colocalises to DNA damage repair sites. Thus, to show that p53 has the ability to bind onto damaged DNA, which leads to the acetylation of histones, Chromatin Immunoprecipitation (ChIP) is being established. The ChIPed DNA products can then be tested for UV induced DNA damaged products (ie CPDs and 6-4 PPs) via south-westerns and to assess if p53 binds the damaged DNA via its promoter sites only or to all DNA damaged sites by PCR.

Selective inhibition of oncogenic functions of mutant p53
Dr. S. Allison and Prof. J. Milner

The p53 gene is mutated in over half of all human cancers resulting in the loss of its tumour suppressor functions. In contrast to other tumour suppressors the majority of these mutations are missense suggesting that mutant p53 proteins can confer some selective advantage in carcinogenesis. Indeed dominant mutants of p53 inhibit wild type p53 function through hetero-oligomerisation and sequestration into an inactive complex. In addition, some p53 mutants can promote tumour progression in a p53-null background. I am particularly interested in such "gain of function" mutants, their tumour promoting properties and the molecular bases for their oncogenicity.

With the advent of DNA chip technology it is possible to characterise p53 mutations in tumour cells of cancer patients. This is important since different missense point mutations have different effects on tumour progression. This project concerns mutations which convert p53 from a suppressor to promoter of tumour progression. The mutant protein exhibits novel properties. It binds DNA elements which attach chromatin to the nuclear matrix (MAR elements) and disrupts higher order chromatin structure and may thus promote genomic instability. By mapping MAR binding sites on mutant p53 we aim to identify small peptides capable of selectively blocking mutant p53-MAR binding in vitro. Subsequently, the in vivo effects of these "blocking peptides" on cell growth and functional organisation of chromatin will be assessed. The long term objective is to identify small peptides capable of suppressing genomic instability and tumour progression in human cells expressing oncogenic mutants of p53.

P53 acetylation
Dr. J. Ford and Professor J. Milner

We have developed an in vitro assay to analyse the consequences of p53 acetylation as regards sequence-specific DNA binding and E6-mediated degradation. P53 was translated in vitro using the rabbit reticulocyte translation system, and was subsequently incubated with butyric acid [a deacetylase inhibitor] to enhance acetylation, and with short double-stranded oligonucleotides representing cognate binding sequences and with the 'activating' MAb 421. HPV type 16 E6 was translated in vitro by the same system and after treatment of the p53, aliquots of p53 and E6 were mixed and sampled at timepoints to assess degradation. Using this system we determined: 1] Enhanced acetylation does not alter the kinetics of E6-mediated degradation of either human [hp53] or murine[mp53] p53. 2] The presence of cognate binding sites in the form of short double-stranded oligonucleotide duplexes provided protection for hp53 and mp53 from E6-mediated degradation. 3] Protection was not equal between different cognate sequences, with more protection gained from interaction with the p21CIP1 promoter than the GADD45 promoter; least protection was gained from the MDM2 promoter. 4] Protection is a dynamic event, largely restricted to the first 30 minutes following exposure of p53 to E6. 5] A dimeric mutant of mp53 is more intrinsically resistant to E6-mediated degradation than wild-type mp53, but it does not gain any protection from the presence of cognate binding sites. 6] Presence of MAb 421 enhanced protection for mp53 but did not greatly enhance protection for hp53. These data suggest structural differences between mp53 and hp53 in complex with cognate sequences as regards interaction with the HPV E6 protein.


Department of Chemistry
Head of Department: Professor R.N. Perutz

Preussomerins, Deozypreussomerins and Diepoxins: Natural product leads for the synthesis of novel anti-tumour agents
Prof. R.J.K. Taylor

The title compounds are naturally occurring ras farnesyl transferase inhibitors. The aim of the project is to prepare a range of these compounds, plus novel synthetic analogues, for biological screening.

We have established the basic methodology needed to prepare simple compounds of this type and successfully used this chemistry to prepare palmarumycin and deoxypreussomerin natural products. Recent studies have been concerned with the development of novel chemistry to enable the more complex members of the preussomerin and diepoxin family to be prepared. Organosulphur derivatives are being studied in Grignard / Barbier-type reactions in order to provide a more efficient approach to these systems. Parallel investigations into the use of the palladium-catalysed Stille coupling are also being conducted. We are also investigating the synthesis, and subsequent acid catalysed dimerisation, of activated 8-hydroxytetralone derivatives to provide an alternative synthetic route to the preussomerin scaffold. Novel compounds will be screened at the University of Bradford (Dr. R.M. Phillips).

Novel anti-tumour agents from marine natural products
Prof. R.J.K. Taylor

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. The aim of the project is to prepare a range of these compounds, plus novel synthetic analogues, for biological screening. This project has only just commenced (10/01) but already interesting new phosphorus and tin chemistry has been developed which will be applied to the synthesis of analogues of lobatamide analogues.

All analogues will be screened as anti-cancer agents by Dr. R.M. Phillips at the University of Bradford. These procedures will involve chemosensitivity studies against a panel of cell lines derived from a range of human tumours. 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 may drive further chemical synthesis. The novel mode of action of these compounds will also be probed.