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Title: An analysis of the role of glutathione and p53 in the response to oxidative injury
Author: Coe, Jonathan P.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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The response to oxidative stress, a process that can lead to genotoxic injury, is thought to involve the abundant cellular antioxidant, glutathione, and the stress response transcription factor, p53. Glutathione (GSH) biosynthesis occurs through a two-step pathway, the first reaction of which is rate limiting and is catalysed by the enzyme gamma glutamylcysteine synthetase (γGCS). γGCS is a heterodimer, composed of a heavy (γGCSh) and a light (γGCS1) subunit. The heavy subunit contains the active site, whereas the light performs a regulatory function on the heavy by means of a redox-sensitive inter-subunit disulphide bridge. The hypothesis that GSH mediates protection against oxidative stress was investigated by gene targeting of γGCSh in murine embryonic stem (ES) cells. Mouse γGCSh cDNA sequence was isolated by RT-PCR, cloned, characterised and used to screen a mouse genomic λ library. Characterisation of the resultant clone confirmed that it contained γGCSh gene sequences. This information was used to design and construct a replacement targeting vector which was subsequently electroporated into ES cells to delete a segment of the endogenous locus. A total of 285 clones were isolated and analysed for a correct gene targeting event. Unfortunately, no positive clones were identified. The role of GSH and p53 in the response of ES cells to oxidative stress was also examined via a series of in vitro assay strategies measuring cellular viability, apoptosis and intracellular GSH levels. ES cells were shown to express γGCSh. Agents known to induce oxidative stress or lower GSH levels in other cell lines were then tested for toxicity and their potential to modulate GSH levels in ES cells. On the basis of these experiments, the quinone menadione (MQ) and the γGCS inhibitor, buthionine sulphoximine (BSO), were investigated further. Treatment with MQ was associated with a transient elevation of GSH, a strong apoptotic response and reduced clonogenic survival. Addition of BSO depleted GSH levels and prevented the MQ-induced increase in GSH, sensitising cells to oxidative insult. In order to address the role of p53 in the response to oxidative stress, karyotypically normal p53-/- ES cells were compared to wild-type cells. This showed that both maintenance of basal GSH levels and MQ-induction of GSH were independent of p53 status. However, a role for p53 in this response was demonstrated as the kinetics of MQ-induced apoptosis were delayed in the absence of p53. Taken together, these findings suggest that the pathways involving p53 and GSH act independently to protect against the deleterious effects of oxidative damage. Consistent with studies using a wide spectrum of other DNA damage inducing agents, loss of p53 conferred an immediate survival advantage post oxidative stress. However, the long-term clonogenic survival of p53-/- ES cells was found to be lower than cells with an intact p53 pathway. This suggests that compensatory mechanisms exist to ensure that, in the absence of functional p53, cells bearing genetic lesions are less likely to be propagated, and furthermore that the ability to engage apoptosis does not necessarily predict long term clonogenic survival. In summary, an attempt was made to address the in vivo significance of GSH by creating a γGCSh null strain of mice. To this end a targeting vector was generated and used in ES cells, but unfortunately this failed to produce a mutant γGCSh allele. This thesis has also explored the relationship between oxidative damage and the cellular responses of GSH and p53 in vitro. Evidence is presented to demonstrate that, within embryonic tissues, multiple pathways operate in response to oxidative stress, and that in the absence of p53 cells are prevented from propagating.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available