Most chemotherapy drugs work through the induction of apoptosis in tumour cells. Mutations in apoptotic pathways, notably p53 and Bcl-2, are common in cancer and associated with increased resistance to apoptosis and therefore to chemotherapy. The sensitivity of two types of cells have been compared, A 172 human glioblastoma cells and MDA-MB-23l human breast cancer cells, for their sensitivity to the chemotherapy drugs cisplatin, doxorubicin, gemcitabine, vincristine and temozolomide. Data shows that there were differences in the sensitivity of breast cancer and glioma cells to a variety of chemotherapy drugs. There were differences in nitric oxide synthase (NOS) expression between the two different types of cancer cells and yet a role was not observed for nitric oxide in changing the expression of the Bcl-2 family of proteins (both pro- or antiapoptotic). Additionally, NOS expression changed following treatment with chemotherapy drugs, suggesting that this may be the mechanism by which the cells become resistant.

Antibody directed enzyme prodrug therapy (ADEPT) is a two-step targeting system, which utilises pretargeting of antibody-enzyme to tumour followed by administration of a prodrug, which is converted to an active cytotoxic by the enzyme at tumour sites. This system aims to overcome cancer resistance mechanisms by the tumour specific generation of large quantities of a cytotoxic agent. ADEPT is a complex therapeutic strategy as it involves multiple components, each of which has their own unique requirements for the system as a whole to be successful. It is hypothesised that by measuring mechanism in a clinical trial setting, a greater understanding of ADEPT can be achieved. Additionally, rational modifications can be determined and implemented, thereby accelerating the development process. This thesis demonstrates how mechanistic data attained from one phase I/II ADEPT clinical trial lead to the development of a novel genetically engineered antibody-enzyme fusion protein for ADEPT, which was shown in a second clinical trial to have enhanced features for tumour targeting in ADEPT. The metabolic assessment of tumours using F-18 FDG PET was assessed during both ADEPT trials and shown to provide additional information when compared with conventional response assessment, particularly in the assessment of patients with radiological stable disease. As the data generated on ADEPT from both preclinical and clinical development is large, and the possibilities for modification to the system are numerous, an Ontology and Conceptual Model have been developed to organise the data attained on ADEPT. This thesis demonstrates that although ADEPT is a complex therapeutic strategy it can be successfully studied in a clinical setting using mechanistic clinical trial design, rational modifications can be made and data can be organised to facilitate development for the future.

DNA quadruplexes are involved in the regulation of gene expression, recombination and chromosomal alignment. They have been extensively studied using telomeric DNA sequences. Their overall conformation depends on the number of DNA strands (one, two or four) and on ionic conditions and it has been suggested that this behaviour may relate to regulatory mechanisms. Several proteins, including telomerase, have high binding affinities for quadruplexes. Telomerase function has been implicated with the immortality of cancer cells. Stabilising quadruplexes by small ligands has been used to inhibit telomerase function, thus providing a target for anti-cancer therapies. Chapters 3 and 4 describe detailed small angle scattering studies of K+ and Na' concentration effects on monomeric, dimeric and tetrameric quadruplexes in solution, both in the absence and presence of a small ligand. The results of Chapter 3 show that K+ and Na+ have markedly different effects on quadruplex structure in solution, further supporting the idea that the balance of these ionic species may be important in regulatory processes. The results of Chapter 4 extend these observations and show that the interaction of the acridine drug depends critically on the type of quadruplex involved - with clear evidence to suggest that the drug interaction is more favoured for quadruplex structures having extended loops. Chapter 5 describes solution scattering studies of the melting/annealing of monomeric and tetrameric quadruplexes as a function of temperature. Ab initio shape determination was used to study folding pathways, and models for the denatured, fully formed and intermediate quadruplex species are presented. These results show that the tetrameric quadruplex does not melt in the presence of K+, emphasising the clear structural differences in the two ionic environments and raise the intriguing possibility that this difference in behaviour for the two salts could be implicated in biological control/switching processes.

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