P53 responses to fludarabine in human B-lymphoid cancers

The tumour suppressor protein p53, is a transcription factor responsible for regulating the cell cycle and/or apoptosis in proliferating cells subjected to a variety of stressful events, including DNA damage. Mutations of p53 occur in >50% of human tumours, the most frequently altered gene in cancers. This thesis looks at the responses of the p53 interactome in B-lymphoid cancers after fludarabine (2-FaraA) induced DNA damage. The aims are 1) analyse the response of p53 and family proteins p63 and p73, after 2-FaraA induced DNAdamage, 2) compare the p53 interactome following DNA damage in cells with and without p53mutations 3) study the DNA-damage phosphorylation cascades in the p53 interactome and 4) analyse the effects of p53 knock-down on fludarabine responsive cells. 1D and 2D Western blotting and quantitative PCR has demonstrated that in the 2-FaraAsensitive cells, Raji and IM9, p53 accumulates in nuclei, mitochondria and cytosol. Raji cells accumulate p53 in mitochondria earlier than in the nucleus suggesting that initiation of apoptosis in these cells is dependent on the roles of p53 at mitochondria, prior to effects on gene expression in the nucleus. 2-FaraA also induces phosphorylation and accumulation of lower molecular weight derivatives of p53, p63 and p73 in the nuclei of Raji cells that may have roles in inducing apoptosis. Qualitative mass spectrometry analysis of the p53 interactome indicated that MEC1 may have a gain of function p53 mutation that may contribute to 2-FaraA resistance. In 2-FaraA responsive cells, treatment dissociates HSP90 from the p53 interactome suggesting that HSP90 binding may play a role in regulating p53 activity and sensitivity to 2-FaraA treatment. Quantitative phospho-proteomics of the p53 interactome showed that in response to 2-FaraA, HSP90 is phosphorylated at serine 254 by casein kinase, that may be associated with its dissociation from p53. p53 knockdown in Raji and IM9 with siRNA induced up-regulation of transcription the factor YY1, and the surface antigen ICAM1, that may have roles in altering the biochemistry of the cells and allow them to become resistant to 2-FaraA-induced apoptosis in p53 knock-down cells. Taken together the novel findings of this thesis provide greater insight into the p53-centric mechanism of 2-FaraA against B-lymphoid cancers and suggest new treatment strategies for drug resistant chronic lymphocytic leukaemia and lymphoma.