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Title: The cellular response to alkylating agents : a complex interplay between DNA repair, oxidative signalling and energy metabolism pathways
Author: Jbbar, Ali
ISNI:       0000 0004 6423 3153
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2017
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It is estimated that 10,000 lesions arise in the genome of a cell every day. Cells have therefore also evolved ways to protect the integrity of their genomes using direct DNA repair enzymes and multi-step pathways including base excision repair and nucleotide excision repair. Alkylating agents are reactive chemicals that transfer alkyl groups to biological molecules, including DNA. The base excision repair pathway mainly repairs non-bulky lesions produced by alkylation, oxidation or deamination of bases. This pathway is initiated by alkyladenine DNA glycosylase (Aag). Antioxidants neutralise free radicals including reactive oxygen specied (ROS), and have been widely reported to protect against disease. However, some studies have also reported that anti-oxidants may instead make disease progression worse. This thesis aims at evaluating the role of antioxidants in the cellular response to the alkylating agent, methylmethane sulfonate. WT and Aag-deficient mouse embryonic fibroblasts (MEFs) were pre-treated with the antioxidant N-acetylcysteine (NAC) and exposed to MMS. NAC increased MMS-induced cell death in both Aag-deficient and wild-type (WT) MEFs. These were further confirmed with embryonic stem cells (ESc) being also sensitized to MMS-induced cell death by the anti-oxidant 2-mercaptoethanol (2-ME); and with 661W photoreceptor cells being sensitised to MMS-induced cell death by a commercial antioxidant mixture and NAC. MEFs exhibited ROS generation when exposed to MMS, which was abrogated with NAC. The mitochondrial superoxide probe MitoSox proves that the MMS-induced ROS generation did not originate from the mitochondria. The NADPH oxidase inhibitor Diphenyleneiodonium (DPI) abrogated MMS-induced ROS generation and also sensitised cells to MMS in a similar fashion to NAC. Collectively, we conclude that cells generate ROS as a response to MMS treatment, and that this ROS generation is essential for cell survival. We also show by using different glucose concentrations, ATP levels appear to be irrelevant to MMS-induced cell death, and that higher basal NAD levels correlates with higher amount of MMS-induced cell death.
Supervisor: Elliott, Ruan ; Meira, Lisiane Sponsor: University of Surrey
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available