Medical Biology - Theses
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ItemBIM is critical for DNA damage-induced apoptosis and enforces tumour suppression( 2013)Apoptosis is a highly regulated, ordered form of cell death that is critical for a wide variety of physiological processes. Loss of key apoptotic regulators or other defects in apoptosis can result in either excessive or insufficient removal of cells leading to severe consequences, such as embryonic lethality, autoimmunity or cancer. The BCL-2 family of proteins are key regulators of apoptosis that control the ‘point of no return’ integrating diverse upstream signalling pathways to determine whether a cell will live or die under conditions of stress. Due to their ability to regulate cell survival, abnormalities in the expression of the BCL-2 family members are frequently observed in human cancer. Furthermore due to their ability to initiate apoptosis signalling, the BH3-only subfamily of the BCL-2 protein family are required for mediating tumour cell killing following treatment with many chemotherapeutic agents. Accordingly, mutation or loss of the BH3-only proteins, or their upstream regulators, is associated with chemoresistance and poor treatment response. The tumour suppressor p53 is a critical direct transcriptional activator of the genes encoding the BH3-only proteins PUMA and NOXA. Mutations in p53, with consequent loss of its transcriptional activity, constitute the most frequent abnormality in human cancer and are associated with poor response to anti-cancer therapeutics, particularly those that cause DNA damage. Since p53 functions upstream of the BH3-only proteins in apoptosis signalling, therapies that act independently of p53 to induce the expression of BH3-only proteins are likely to be more efficacious for the treatment of patients that harbour p53-deficient tumours. In this thesis I describe the characterisation of the mechanisms by which DNA damage can induce apoptosis in the absence of p53 function, focussing on those that are relevant to the treatment of p53-deficient tumours. To do this I generated a panel of p53-deficient thymic lymphoma-derived cell lines and determined their ability to undergo apoptosis in response to DNA damaging agents that induce different types of DNA lesions, such as γ-irradiation, etoposide and cisplatin. I have examined changes in protein expression of members of the BCL-2 family following treatment with these DNA damaging agents and identified those members of the BH3-only sub-group that are up-regulated. By generating lymphoma-derived cell lines from mice that lack both p53 and select members of the BH3-only proteins, I have been able to determine which are essential for the induction of DNA damage-induced apoptosis in the absence of p53. Through this approach I have identified novel mediators of DNA damage induced-apoptosis both in lymphoma cell lines and also in primary non-transformed cells deficient for p53. I then sought to determine whether this newly identified pathway plays a critical role in the elimination of pre-leukaemic cells that have sustained physiological DNA damage in vivo (e.g. due to replication induced stress or the stress elicited by oncogene activation). To address this cohorts of mice deficient for p53 plus candidate transducers of this pathway were aged and the lymphoma incidence was compared to those lacking p53 alone. The additional loss of this novel DNA damage induced pathway provoked a pronounced acceleration in lymphoma onset in the p53-deficient mice and also resulted in a more aggressive tumour phenotype. The importance of this tumour suppressor pathway was further characterised by ageing p53-heterozygous mice deficient for pathway members and comparing their tumour incidence to that of mice lacking only a single allele of p53; revealing a profound acceleration of tumour development in this context. BIM has been shown to be a critical tumour suppressor in the Eμ-Myc mouse model of lymphoma and recent evidence has shown that the pro-survival BCL-2 family member BCL-XL is critical to promote survival of cells undergoing neoplastic transformation in this model. To gain further insight into the processes that govern cell survival during neoplastic transformation, I performed experiments to determine whether concomitant loss of BIM would be sufficient to abrogate the delay in lymphomagenesis observed in the absence of BCL-XL. Loss of a single allele of Bim was sufficient to revert this delay and loss of both alleles of Bim provoked a further acceleration in lymphoma onset. Thus through my investigations in vitro into the manner in which apoptosis can be induced in cells lacking p53 and my studies utilising various mouse models of lymphoma development I have identified a novel apoptotic pathway important for the elimination of in p53-deficient pre-leukaemic cells and malignant lymphoma cells. These data provide new mechanistic insights into DNA damage induced apoptosis in vitro and for tumour suppression in vivo.