No description available.

No description available.

No description available.

It is increasingly apparent that target-based drug discovery is not delivering adequate numbers of clinical candidates for treatment of malignancies. The main problems encountered are target identification, validation and late stage attrition. New complementary techniques are required to increase the success rate of cancer drug discovery. Phenotype-guided discovery, using chemicai genetic screens, provides an alternative strategy that circumvents many of the issues associated with target-based discovery. Zebrafish is a vertebrate model uniquely suited to such high throughput screens, and establishing relevant neoplasia models would enable screening for novel anti-cancer therapeutics.

Indole-3-carbinol (I3C), a dietary constituent derived from cruciferous vegetables has been shown to exert anti-tumour and chemopreventive activity in vitro and in vivo. 3,3'-diindolylmethane (DIM), an acid condensation product of I3C, exerts promising activity and is thought to be partially responsible for the chemopreventive activity of orally administered I3C. In order to aid the development of I3C or DIM as cancer chemopreventive agents, the pre-clinical pharmacology and cancer chemopreventive activities were explored. An HPLC method was developed and validated to enable accurate pharmacokinetic studies of both compounds. The tissue distribution and pharmacokinetics of I3C, DIM and an absorption-enhanced DIM formulation [(BioResponse-DIMâ; BR-DIM)] were investigated. I3C was rapidly absorbed, distributed and eliminated, with peak levels in plasma and tissues occurring within the first 15 min and falling below the limit of detection by 1 h. Acid condensation products and oxidative metabolites were identified in mice that received I3C. Following administration of crystalline DIM or BR-DIM, absorption was rapid with maximal plasma and tissue concentrations occurring within 0.5-1 h. In contrast to I3C, DIM could be quantified in the plasma and most tissues at all time points up to and including 24 h. A physiologically based pharmacokinetic model was developed to characterise the pharmacokinetics of BR-DIM and crystalline DIM. BR-DIM exhibited approximately 50% higher bioavailability than the crystalline form. In vitro, DIM was more potent at inhibiting the growth of the MDA MB468 breast cell line than the normal derived HBL100 line. DIM induced apoptosis and inhibited phosphorylation of protein kinase B in MDA MB468 cells with higher potency than I3C. Neither I3C nor DIM, whether administered in the diet or by repeated oral gavage, was able to inhibit the growth of MDA MB468 xenografts in nude mice. In conclusion, these results further our understanding of the in vivo pharmacology and cancer chemopreventive activity of I3C and DIM.

TP53 is the most commonly mutated gene in all human cancers [Vogelstein, 1990] and co- ordinates many of the cellular responses to DNA damage. It is vitally important therefore that we gain a better understanding of the function and regulation of p53. Since a major consequence of p53 mutation is cancer, a disease of complex multicellular organisms, the best system to study the real complexities of p53 function and regulation is in vivo. Accordingly we have proposed to use gene targeting in ES cells to create genetically altered strains of mice to enable a range of studies of p53 regulation and function. Given the importance of p53 in human disease processes, it is perhaps surprising that there have been relatively few p53 reporter mouse strains described. Heretofore few p53 reporter mouse strains have been described and these have been based upon un-targeted, often artificial, repeated p53 response elements in constructs that integrate at random [Komarova et aI., 1997, Gottlieb et aI., 1997, Vasey et aI., 2008, Briat and Vassaux, 2008]. Such random integration is known to be associated with loss of fidelity resulting from positional effects and thus it is unlikely that any of these provide authentic reporting of p53 activity. The ideal solution might be to target a p53 responsive gene using a knock-in approach, preferably in a gene that was exclusively activated by p53, eliciting a specific response program (cell cycle arrest/senescence/apoptosis) and which also does not display any haploinsufficiency. This project was based on such a strategy. In order to study p53 activation and consequence(s) in the gastrointestinal tract we aim to generate novel transgenic mice which would contain fluorescent reporters in the downstream target genes of p53 activation in both of p53s main responses to DNA damage (apoptosis and cell cycle arrest). The resulting dual apoptosis/cell cycle arrest p53-reporter mice would allow a unique opportunity to visualise not only that p53 was activated but what responses p53 had induced. Furthermore, as visual technology advances these mice, or second generation mice with newer fluorescent proteins with improved bioimaging properties (such as increased tissue penetration and brightness), could also be used in real-time analyses of p53 responses to DNA damage or other stimuli, as well as for long term monitoring of gene-regulation during cancer development. In addition, we aim to address fundamental questions regarding the regulation of p53 again using such gene targeting technology for in vivo studies. Importantly it is not yet known whether the p53-mediated up-regulation of its essential negative regulator Mdm2 is absolutely required for viability. Therefore we intend to make two transgenic mouse strains which lack either the Mdm2 P1 (constitutive) promoter or the p53 RE sites within the Mdm2 P2 (inducible) promoter. Studies of these mice are likely to have a profound impact on the field of cancer biology and normal development.

Improvement of cancer treatments is a priority health issue. One approach is to use multivalent scaffolds to prepare drug delivery systems in order to make anti-cancer agents more specific to their target tumours. The aim of this thesis was to design a lectin specific drug delivery system through the preparation of a series of calix[4] sugars suitable for binding with the cell membrane asialoglycoprotein receptor (ASGP-R). Chapter 1 describes the background to this work and focuses on the synthetic methods available for the synthesis and derivatisation of calixarenes with particular emphasis on the preparation of calix[4]sugars. Chapter 2 describes investigations into the incorporation of amino sugars at the upper-rim of calix[4]arene for the synthesis of a series of linked calix[4]sugars. The synthesis of the key intermediate tetra carboxylic acid functionalised calix[4]arene is discussed in detail.

The introduction (Chapter 1) reviews progress made in the last eight years in the field of multicomponent palladium-catalysed reactions, with particular attention paid to those processes which may be easily applied to the synthesis of compound libraries. The Results and Discussion (Chapters 2 to 7) provides an account of the author's work in the development of palladium-catalysed reactions of allene, and the application of such reactions to the synthesis of novel histone deacetylase inhibitors for the treatment of cancer.

No description available.

Purpose: The epidermal growth factor receptor (EGFR) is commonly expressed in human tumours and provides an important target for therapy. Several classes of agents including small molecule inhibitors and antibodies are currently under clinical development. These agents have shown interaction with chemotherapeutic agents in vitro and in vivo. However, the mechanisms of these interactions are not clearly understood. The purpose of this study was to investigate mechanisms for this modulation. Experimental Design: The synergistic effects of the EGFR inhibitor gefitinib (Iressa, ZD 1839) in combination with a variety of chemotherapeutic agents was determined in several cancer cell lines and analysed pharmacologically. Using the alkaline single-cell gel electrophoresis (comet) assay, the kinetics of DNA damage and repair following treatment with the chemotherapeutic drugs combined with gefitinib were investigated. The modulation of DNA-PK activity by gefitinib was quantitated using a variety of techniques including immunoprecipitations, immunoblotting, cellular fraction extractions and immunohistochemistry. The effects of inhibiting the DNA repair protein DNA-PK, with LY294002, wortmannin and siRNA to its catalytic subunit (DNA-PKCs) were investigated in the same cancer cell lines and compared with the effects of gefitinib. Results: Synergistic effects of EGFR inhibitors and chemotherapy were found with a variety of cell lines. The synergy was found with etoposide, doxorubicin and cisplatin but interestingly not with melphalan. Experiments on DNA repair using the comet assay demonstrated delay in repair of DNA strand breaks and inter-strand cross links (ICLs) by gefitinib following treatment with etoposide and cisplatin respectively. The mechanism of this interaction was investigated and found that interference with the DNA-PK pathway mimicked the effects of EGFR inhibition. Additionally, gefitinib treatment induced a physical interaction between EGFR and DNA-PK and a cellular re distribution of DNA-PK was associated with a fall in DNA-PK activity. Conclusions: These results highlight the important effects EGFR inhibitors have on DNA repair and its associated machinery when added in combination with certain chemotherapeutic agents. These results will form the design of further clinical schedules combining these classes of agents and point the way to further studies to investigate the molecular mechanisms of these interactions.