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Title: Genetic analysis of caspase-6 and caspase-7 function in vertebrate DT40 cell line
Author: Korfali, Nadia
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Apoptosis, also known as programmed cell death, is an evolutionarily conserved mechanism by which the organism removes unwanted cells. The process is common to somatic as well as germ cells. It is actively involved in development and morphogenesis, cell number control and removal of infected, mutated or damaged cells. Many insights on the activation of final triggers and pathways in apoptosis came from genetic studies of apoptosis using the worm, C.elegans This work showed that the worm Ced-3 is a protease of pivotal role in apoptosis of C.elegans. The mammalian counterparts of CED-3 have been identified as members of a family of intracellular proteases that form the core of the apoptotic machinery. Since these are cysteine proteases that cleave cellular substrates at specific aspartate residues, they were all termed caspases. In the last ten years natural and synthetic inhibitors and genetic approaches have been used to study elucidate specific caspase function. An alternative system is gene disruption in the chicken DT40 cell line. These cells have a high homologous recombination rate that facilitates the isolation of knockout alleles. The aim of my project is the genetic analysis of caspase-6 and -7 function. My work started with an ongoing caspase-6 knockout project along with a senior postdoctoral scientist in our lab, Dr Sandrine Ruchaud. Subsequently, I concentrated my work on the caspase-7 knockout poject. I generated DT40 cell line where both alleles of caspase-6 and -7 were disrupted independently. Caspase-6: No obvious morphological differences were observed in the apoptotic process in caspase-6 deficient cells compared to the wild type. However, examination of apoptosis in a cell free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6 deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor zVEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present, it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution. Caspase-7: Viability assays showed that caspase-7' clones are more resistant to common apoptosis-inducing drugs such as etoposide and staurosporine. Further examination of the apoptotic process revealed that caspase-7 cells show a delay in phosphatidylserine externalization and DNA fragmentation as well as cleavage of the caspase substrates PARP and lamins BI and B2. Caspase affinity labeling and activity assays indicated that deficient cells exhibit a delay in caspase activation when compared to wild type DT40 cells, providing an explanation for the differences in apoptotic execution between caspase-7 null and wild type DT40 cells. These results strongly suggest that caspase-7 is involved earlier than other effector caspases in the apoptotic execution process in DT40 B lymphocytes. My results have provided important new insights into the function of caspase-6 and caspase-7 during apoptotic execution.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available