No description available.

XR5944 and XR11576 are two potent DNA interactive agents, previously shown to be capable of stabilising topoisomerase I and II cleavable complexes in vitro. This thesis investigated the possibility that the mechanism(s) of action of these compounds may be unrelated to that of topo inhibition. These studies made use of a wide range of drug-sensitive and resistant cancer cell lines. It was demonstrated that both XR5944 and XR11576 retain potent cytotoxicity in cancer cell lines presenting with atypical drug resistance to single topoisomerase poisons. XR5944 showed less potency in cell lines expressing ABC-transporter proteins, but this may not be sufficient to compromise the activity of this potent anti-tumour agent in the clinical setting. The mechanism of XR11576 induced cytotoxicity was not affected by any of these transporters and from this and other observations it may possess a different mechanism of action from XR5944. An apoptotic response was observed in XR5944 and XR11576 treated cells. Factors such as Bax and t-Bid were expressed in increasing amounts in response to treatment and implicated the mitochondrial route of apoptosis in these compounds' mechanism of cell kill. Both XR5944 and XR11576 induced appreciable levels of DNA-protein crosslink formation and induced a p53 DNA damage response in drag treated cells. Differences in the timing of onset and extent of DNA damage and p53 induction were noticed between the two compounds. Overall, XR5944 was slower at causing these DNA interactive effects in line with the onset of cytotoxicity. However, this did not appear to pose any particular problems, such as scheduling of dosing, with regard to its in vivo activity, as shown by the work of others. These findings suggest that both XR5944 and XR11576 promote potent cytotoxicity in cancer cell lines showing multiple mechanisms of drug resistance and, therefore, should be of use in the clinic for the treatment of drug resistant tumours. The data presented in this thesis suggest that neither agent exerts its cytotoxicity via a topo directed effect. Furthermore, some of the data generated suggests that these two compounds may differ from each other in their mechanisms of action.

No description available.

This work is concerned with the synthesis and potential anti-tumour properties of novel Diaziridinylquinones. These types of compounds are usually activated by bioreduction to form cytotoxic species, which alkylate DNA and block cells in the G 2 /M phase of the cell cycle. However, the present studies have concentrated on compounds which appear to function by other mechanisms. Certain phenol/ester derivatives of PDZQ (2,5-diaziridinyl-3-phenyl-l,4-benzoquinone) were significantly more cytotoxic than PDZQ in all of the cell lines investigated (Chapter 3). The esters were cleaved by esterases to form a highly cytotoxic stable meta-phenol or an unstable para-phenol. The compounds were studied in detail using DNA cross-linking, clonogenic, apoptosis and flow cytometry assays. Preliminary studies on the protein tyrosine kinase (PTK) activity of the epidermal growth factor receptor (EGF-R) showed that the |iM concentrations of the meta-phenol can reduce the PTK activity of purified EGF-R by 50%. The overall proposed mechanism is that the cytotoxic esters are cleaved by esterases to form reactive phenols. However, the enhanced toxicities of these compounds are not simply due to the differences in DNA cross-linking efficiencies. It is proposed that the phenols cross-link DNA and inhibit one or more tyrosine kinases. Preliminary tumour xenograft studies suggest that some acridine derivatives of PDZQ have a very high therapeutic index (Chapter 4). These compounds cross-link DNA but there is no DNA-intercalation (from fluorescence, absorbance, DNA-unwinding and T m studies). It is proposed that these compounds can be reduced within a cell and interact with Topoisomerases. RH1 is scheduled for Phase I/II clinical trials and the final chapter of this thesis reports on the induction of apoptosis by this diaziridinylquinone. These studies used many different biochemical/visual techniques to measure apoptosis. The general conclusion from this study is that although RH1 can induce apoptosis, the extent is strongly dependent on the cell line and it is only significant at relatively high concentrations of drug.

The contention that cancers are immunogenic is still being debated: one view states that cancers evolve to evade immune surveillance, while the other claims that cancers actively induce tolerance. A unifying hypothesis is that immune surveillance operates early on in carcinogenesis, but eventually tumours adapt to shift the balance from activation to tolerance. Cancer vaccines therefore, must stimulate a tumour specific response and break active tolerance mechanisms. Cancer vaccines were initially comprised of tumour associated antigens (TAAs), but since these were poorly immunogenic, they were replaced with dendritic cell (DC) vaccines loaded with TAAs (adopted because DC prime naive T cells). However, such DC-based vaccines are impractical to produce and have not proved particularly effective. These disappointing results have led to the search for more innovative vaccine strategies. Viral vectors encoding TAAs are promising vaccine candidates because they can infect antigen presenting cells (APCs) in vivo they may also signal "danger" to the immune system through pathogen recognition pathways. This thesis is concerned with the development of vaccines based on lentiviral vectors (lentivectors) such vectors are readily used for gene therapy because they can stable modify non-dividing cells. The aim of this project was to assess their ability to deliver antigens to dendritic cells in vivo and to stimulate effective T cell responses. The findings of this thesis are that: Directly injected lentivectors can stimulate CD4+ (and CD8+) T cell responses to their encoded antigen if it is targeted into the endocytic pathway. DC stably present antigen in vivo following vector immunisation. DC can be induced to mature by including an NF-kB activator (vFLIP) in the vector. These results support the development of lentivirus-based cancer vaccines.

Cyclin-dependent kinase inhibitor p16INK4A is an important tumour suppressor and inducer of cellular senescence often inactivated during the development of cancer. I investigated the mechanism by which EBV latency-associated nuclear antigens EBNA3A and EBNA3C repress p16INK4A expression. Using lymphoblastoid cell lines (LCL) expressing a conditional EBNA3C, I demonstrate that EBNA3C inactivation resets the epigenetic status of p16INK4A to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16INK4A transcription and allows proliferation. LCL lacking EBNA3A express relatively high levels of p16INK4A and have a similar pattern of histone modifications on p16INK4A as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP was linked to the oncogenic activity of EBNA3C and EBNA3A, LCL with viruses encoding EBNA3C- and/or EBNA3A-mutants that no longer bind CtBP were established. These novel LCL revealed that the epigenetic repression of p16INK4A requires the interaction of both EBNA3C and EBNA3A with CtBP. Epigenetic repression of p16INK4A by latent EBV may facilitate p16INK4A DNA methylation during lymphomagenesis. Furthermore, by transforming the peripheral blood lymphocytes (PBL) from an individual homozygous for a deletion in CDKN2A locus with recombinant EBV viruses expressing conditional EBNA3C, we developed a system that allows inactivation of EBNA3C in LCL lacking functional p16INK4A protein (p16-null LCL 3CHT). EBNA3C inactivation has no impact on the proliferation rate of p16-null LCL, proving that the repression of p16INK4A is the main function of EBNA3C in EBV-driven LCL proliferation. The p16INK4A locus is epigenetically modified by EBNA3C despite the absence of functional p16INK4A protein. Since the selection pressure based on faster outgrowth of advantageously modified subset of cells is removed, the gradual and relatively slow kinetics of H3K27me3 restoration at p16INK4A following EBNA3C reactivation in p16-null LCL 3CHT seems to be genuinely related to the mechanism of EBNA3C-mediated p16INK4A regulation. The p16-null LCL 3CHT system further allows distinguishing genes regulated specifically by EBNA3C, rather than as a consequence of activation of p16INK4A/Rb/E2F1 axis. Lastly, new cellular targets of EBNA3C and/or EBNA3A from the group of microRNAs are identified in this work. Most notably, both EBNA3C and EBNA3A are shown to repress the tumour supressor miR-143/145 cluster and their precursor long non-coding RNAs in LCL.

Understanding the effect of drugs on solid tumours presents a number of challenges owing to the complexity of the blood flow and drug transport, as well as the highly complex biochemical networks involved in the cellular response with characteristics of high non-linearity of signal transduction. It is imperative to develop a bottom-up systematic computational framework that is able to disentangle the multiple layers of complexity, to present mechanistic and predictive descriptions of drug effect on tumour cells and to allow the incorporation of other complexities. This study represents the first effort towards developing such a framework and includes the most essential modelling components: extracellular drug transport, intracellular signalling dynamics (target cellular apoptosis pathway) and the effect on the population density of cells. The integration of these components is also addressed in this study, where the strategy is to bypass the detailed and complex network of apoptosis by focusing on capturing its key qualitative features. The framework is further expanded by incorporating a drug-induced resistance module, which is modelled by applying negative feedback regulation to the target cellular apoptosis pathway. Two simplified but representative resistance modules are extracted from the literature and examined in the spatially distributed framework. Another feature of the model presented in this thesis is the integration of blood flow, which contributes to more detailed and heterogeneous drug distribution. As a bottom-up approach, the simplest geometry of tumour vasculature is adopted to capture the basic features of tumour blood flow and to allow further investigation into the interaction between drug transport and intracellular signalling within this context. It is demonstrated that a skeletal systems framework has been created which can serve as the basis of future systematic expansion to include additional aspects of cellular and tissue complexities.

Cancer-related pain is a major clinical problem. The World Health Organisation recommends morphine first-line for moderate to severe cancer-related pain, however several other strong opioids are available. There is wide inter-individual variation in morphine response, in terms of analgesic efficacy and side effects. Switching to an alternative opioid such as oxycodone has become common clinical practice to improve outcomes. There has been growing interest in potential genetic factors behind such differences in clinical response. This thesis aimed to compare the overall response rates of morphine and oxycodone, explore the effects of opioid switching in non-responders, and to redefine opioid response phenotypes to allow predictive modelling. Data from two opioid response studies is presented: the first a prospective observational study of morphine in cancer-related pain (n=298), the second a large open-label randomised controlled trial of oral morphine versus oral oxycodone in cancer-related pain (n=200). Single nucleotide polymorphisms from 15 candidate genes were tested using sequence specific primer polymerase chain reaction. Principal Component Analysis (PCA) was used to mathematically define opioid response phenotypes. Multivariate regression analysis of Component scores was used to build predictive models from genetic and clinical variables. There was no difference between the response rates of oral morphine and oral oxycodone when used first-line in cancer-related pain. Analgesia and adverse reaction profiles were similar. Switching from morphine to oxycodone and vice versa improved outcomes in the majority of non-responders. Opioid response appears to have three main domains identified by PCA in the two studies: analgesic response, upper gastrointestinal adverse reactions and central adverse reactions. Morphine and oxycodone although had similar response phenotypes had different clinical and genetic factors contributing to each, suggesting different mechanisms. Future work includes collaboration to enable larger studies to be conducted and to investigate the potential role of metabolites in oxycodone response.

No description available.

Our studies on the synthesis and biological evaluation of novel anticancer drugs consist of three research areas; namely, synthesis of Mitogen Activated Protein (MAP) kinase inhibitors, Checkpoint (Chk1) inhibitors and nordihydroguaiaretic acid (NDGA) analogues. The first research area involved synthesis of MAP kinase inhibitors. MAP kinases are a family of serine I and threonine II kinases which can act together to generate a process of phosphorylation events within the cell signalling pathway leading eventually to cell division. The compounds made in this project were specifically designed to target the stress related kinases, a MAP kinase pathway which controls the expression of genes involved in cell proliferation. The stress related kinases are known to have serine or threonine joined to a proline III residue. In an attempt to prepare selective inhibitors of stress related kinases, compounds of types IV and V were deigned in which a conformationally restricted serine analogue is joined to L-proline via an amide link in one of two possible ways. Examples of these two sets of compounds were synthesised and those that were tested by Professor David Gillespie at the Beatson Institute for Cancer Research, Glasgow were shown not to be inhibitors of these kinases. (Fig. 1144A) The second research area concentrated on the checkpoint signalling pathway. Components in the DNA damage checkpoint signalling pathway such as ChK1 could be potential targets for chemical intervention. Caffeine VI and pentoxifylline VII have been shown to sensitise p53-deficient tumour cells to killing by DNA damage. We envisaged that the xanthine derivatives, caffeine VI and pentoxifylline VII might also disrupt the G2 checkpoint by preventing activation of Chk1. To test his hypothesis, a range of xanthine derivatives shown below were prepared by alkylation of theophylline VIII or theobromine IX. (Fig. 1144B) The biological evaluation of these xanthine derivatives by Professor Gillespie revealed that three of these compounds, X, XI and XII, suppressed G2/M arrest very effectively. All three active compounds possess a long aliphatic chain that provides a large degree of flexibility to the structures. The long aliphatic chains could bind to a hydrophobic pocket in the enzyme’s active site that might confer selectivity on the compounds. (Fig. 1144C) The third area, synthesis of NDGA analogues, was the major part of the synthetic work. NDGA XIII is known to be a selective inhibitor of lipoxygenase and blocks small cell lung cancer growth in vitro and in vivo. In addition to its lipoxygenase activity, NDGA was demonstrated to inhibit c-kit, a tyrosine kinase that has been observed preferentially in SCLC. The main drawbacks to the use of NDGA in cancer treatment are its poor solubility and moderate potency. Therefore chemical modifications are required to provide better compounds for clinical use. Preliminary work in our group was performed by McDonald and Macleod. They synthesised a range of analogues of NDGA which were tested for their activity in vitro by Professor Michael Seckl at the Medical Oncology Department of Hammersmith Hospital, London. Improved potency over NDGA for new analogues with 4-6 atoms between the two aromatic rings was observed. Furthermore introduction of an amide linkage between the two aromatic residues resulted in NDGA analogues which are more active than NDGA. Based on these preliminary results, the structural modifications proposed for this project focused on three areas. The main programme of research was drug solubilisation of new analogues which have higher potency than NDGA for in vivo work. The second area of study sought to introduce position variations of the amide linkage between the two aromatic residues. The third area of work involved modification of the substituents on the two aromatic rings. (Fig. 1144D) A range of NDGA analogues was successfully synthesised and evaluated for anticancer activity in vitro. Compounds XIV and XV were confirmed as lead compounds which are ten times more active than NDGA. Compound XIV was successfully transformed into a water soluble form XVI which is now available for in vivo work. In addition NDGA was converted into a water soluble form which was more potent than NDGA in vitro. Moreover a NDGA analogue XVII with no free hydroxy groups was found to be as active as NDGA, which was an unexpected discovery. (Fig. 1144E)