Year: 2014
Type: dissertation
Abstract: Caspases are a family of cysteine proteases that have essential functions in the regulation of apoptosis and inflammation. Despite being the most evolutionarily conserved caspase, the physiological functions of caspase-2 remain poorly defined. This is partly because caspase-2 knockout (Casp2⁻ʹ⁻) mice show no overt phenotype and only limited, tissue-specific apoptotic defects. Previous work from our laboratory has provided the first direct evidence demonstrating a role for caspase-2 in tumour suppression and protection against cellular transformation. However, the molecular mechanisms by which caspase-2 exerts these functions were not clearly defined. In order to characterise the tumour suppressor function of caspase-2, the processes and pathways disrupted in caspase-2-deficient cells were investigated. Analysis of serially-passaged mouse embryonic fibroblasts (MEFs) demonstrated that caspase-2-deficiency promoted escape from replicative senescence which coincided with impaired induction of cyclin-dependent kinase inhibitors. Consistent with the increased proliferation rate of primary Casp2⁻ʹ⁻ MEFs, spontaneously-immortalized Casp2⁻ʹ⁻ MEFs that had escaped replicative senescence also displayed an enhanced proliferative capacity compared to their wild type counterparts. These findings suggest that caspase-2 regulates cell proliferation which may contribute to its ability to protect against cellular transformation. Furthermore, serially-passaged Casp2⁻ʹ⁻ primary MEFs and Eμ-Myc/Casp2⁻ʹ⁻ lymphomas showed enhanced aneuploidy, demonstrating that loss of caspase-2 promotes genomic instability (GIN). Treatment of Casp2⁻ʹ⁻ MEFs with ionizing radiation (IR) resulted in persistent DNA damage and defective cell cycle checkpoint regulation, suggesting that caspase-2-deficient cells have an impaired ability to efficiently respond to and repair DNA damage. Further analysis revealed that caspase‐2‐deficient MEFs and Eμ-Myc lymphomas displayed defective activation of p53 and its downstream targets following IR treatment. Therefore, an attenuated p53 response may contribute to defective DNA damage response (DDR) signalling and GIN in caspase-2-deficient cells. In order to further investigate the extent and specificity of caspase-2 function in tumour suppression using an independent tumour model, Atm⁺ʹ⁻ and Casp2⁻ʹ⁻ mice were inter-crossed to generate Atm⁻ʹ⁻Casp2⁻ʹ⁻ mice. Initial characterization revealed that caspase-2-deficiency enhanced growth retardation and caused perinatal lethality in Atm⁻ʹ⁻ mice. A comparison of tumour susceptibility demonstrated that Atm⁻ʹ⁻Casp2⁻ʹ⁻ mice developed lymphomas with a dramatically increased onset and penetrance compared to Atm⁻ʹ⁻ mice, providing additional evidence supporting a tumour suppressor function for caspase-2. Furthermore, Atm⁻ʹ⁻Casp2⁻ʹ⁻ lymphomas showed an increased proliferation rate and enhanced oxidative damage compared to Atm⁻ʹ⁻ lymphomas. Moreover, lymphomas and pre-malignant lymphocytes derived from Atm⁻ʹ⁻Casp2⁻ʹ⁻ mice displayed enhanced aneuploidy, linking the function of caspase-2 in the maintenance of genomic stability to its tumour suppressive activity. Overall, this thesis provides novel insights into the physiological functions of caspase-2, highlighting its roles in the regulation of cell proliferation, the DDR, maintenance of genomic stability and tumour suppression.
Author: Joseph Puccini
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2562910963