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Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/β-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G 1 cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo . However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance.

Increased generation of reactive oxygen species (ROS) in hyperglycaemia is linked to endothelial cell DNA damage in diabetes. Nuclear and/or mitochondrial DNA (mtDNA) damage may accelerate ageing of endothelial cells and, in part, account for the endothelial dysfunction associated with the pathogenesis of many cardiovascular diseases. The aim of this thesis was to investigate cell ageing in human endothelial cells, by studying the role of ROS in telomere attrition after exposure to increased glucose levels. This thesis also analysed the effects of mtDNA depletion on the pro-inflammatory phenotype of endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with high glucose (HG; 22mM) or alternating ‘normal’ (5.5mM) /HG (AG; to mimic post-prandial fluctuations in glucose). Telomere attrition rate, measured globally across all chromosomes using Southern blotting and specifically on the XpYp chromosome using single telomere length analysis (STELA) were increased 3-6-fold in HUVECs cultured in HG or AG. This was preceded by increased ROS generation and largely blocked by an antioxidant and inhibition of mitochondrial electron transport. Moreover, mtDNA content was lower in HUVECs cultured in HG. It was hypothesised that changes in mtDNA mediated some of the effects of HG on accelerated vascular ageing in cardiovascular disease. This was supported by experiments showing that mtDNA depletion, using ethidium bromide or dideoxycytidine, resulted in a shift towards a pro-inflammatory phenotype; namely that levels of endothelial nitric oxide synthase protein were decreased and the expression of the adhesion molecule, ICAM-1, was increased. The data confirmed that exposure to high glucose raised ROS production in HUVECs and resulted in increased telomere attrition. This thesis also highlighted the possible importance of mtDNA integrity as a determinant of endothelial cell function and showed that agents which affected mtDNA content/integrity accelerated the process of endothelial dysfunction through phenotypic modulation.