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Title: The role of nucleotide excision repair in providing resistance to the nucleoside analogue gemcitabine
Author: Robinson, Helena
ISNI:       0000 0004 6498 7609
Awarding Body: Prifysgol Bangor University
Current Institution: Bangor University
Date of Award: 2017
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Gemcitabine is a clinically important chemotherapy drug, used to treat a variety of solid tumours. It is a cytidine analogue and when inserted into DNA in place of cytidine serves to inhibit further chain extension, causing replication stress and ultimately cell death. The nucleotide excision repair (NER) pathway is known to repair a variety of bulky DNA lesions but is not known to have a role mitigating replication stress. However, it was found in a S. pombe screen that mutants lacking homologues of xpa, xpc, xpf and ercc1 showed sensitivity to gemcitabine. Further to this, patient fibroblasts mutated in NER genes were shown to be sensitive to gemcitabine when compared to NER proficient fibroblasts. The sensitivity of these mutants to gemcitabine is not explained by the current understanding of NER and this project set out to unearth a new role for NER factors in gemcitabine resistance in human cells. However, data presented here show large differences in sensitivity between two NER proficient fibroblast lines MRC-5 and GM637, which confounded the previous work showing NER contributed to gemcitabine resistance in human cells. Different experimental strategies which enabled the use of controls with the same genetic background were then employed to circumvent this problem. Two ERCC1 knockout cell lines, generated via CRISPR-Cas9 in MRC-5 and HEK293 backgrounds, were characterised as part of this project. ERCC1 was shown to have a role in gemcitabine resistance in MRC-5 cells, but not HEK293 cells. The role of XPA was investigated by using a human lymphoblast (TK6) knockout cell line and complementation of an XPA deficient fibroblast line with a functional copy of the gene and neither approach showed any effect of XPA on gemcitabine resistance. XPC and XPG were also investigated by complementation of deficient fibroblasts which resulted in a small rescue of gemcitabine sensitivity and a small sensitisation respectively. A role for ERCC1 in gemcitabine resistance in human cells has not been previously reported and may have both clinical implications and implications for understanding the processes which occur at stalled replication forks. It is unclear from these results whether this role is related to NER or the NER-independent functions of this gene, but there is a suggestion that a second NER factor, XPC may also be involved. The cell line dependence of the role of ERCC1 also suggests that the DNA repair response to gemcitabine and the role of NER factors in this process differs between cell lines, and this should be an important consideration for future work on this topic.
Supervisor: Hartsuiker, Edgar Sponsor: Tenovus
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available