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|Title:||The Effects of Mismatch Repair Proteins and Hypoxia on the Repair of Cisplatin-induced DNA Damage|
|Advisor:||Rainbow, A. J.|
|Abstract:||<p>Tumour resistance to the drug cisplatin poses a major impediment to successful<br />hemotherapy. Defects in the mismatch repair (MMR) system have been linked to<br />cisplatin resistance; however, conflicting reports have suggested that loss of MMR alone<br />may not be sufficient to confer resistance in some cell types. Rapidly growing tumours<br />often outstrip their blood supply resulting in a hypoxic tumour microenvironment which<br />has been shown to affect cellular responses to conventional cancer treatments. However, the effect of hypoxia on the repair of cisplatin-damaged DNA has not been investigated. We have used a host cell reactivation (HCR) assay to investigate the effects of MMR deficiency, p53 defects, SV40-transformation and hypoxia (or hypoxia coupled with acidosis) on the repair of a cisplatin-damaged reporter gene encoded by a recombinant adenovirus in a panel of murine and human cells. We have also examined HCR of a DVC-damaged reporter gene in human normal cells for comparison and clonogenic survival of murine fibroblasts following cisplatin treatment. In the isogenic murine fibroblasts we examined, loss of the MMR proteins MSH2, MSH6 or PMS2 alone were insufficient to enhance HCR of the cisplatin-damaged reporter gene. However, HCR was enhanced in SV40-transfonned MSH2-deficient cells relative to proficient controls. In the human tumour cells we examined loss of hMLHl alone, but not hMSH2, resulted in enhanced HCR of the reporter gene. These results indicate that loss of MMR alone may not be sufficient to confer cisplatin resistance in some cells, suggesting that other<br />concomitant alterations may also be necessary. We examined p53 defects and SV40-transformation changes as possible candidate alterations required for a cisplatin resistant phenotype in Li-Fraumeni syndrome cells and SV40-transformed human normal cells respectively. We found that SV40-transformation resulted in enhanced HCR of the cisplatin-damaged reporter gene relative to non-transfOlmed cells, but a similar repair enhancement was not seen for mutant p53-expressing Li-Fraumeni syndrome cells relative to control cells expressing wildtype p53. A comparison of repair of UVC- and cisplatin-induced DNA damage showed that HCR of the UVC-damaged reporter gene, but not the cisplatin-damaged reporter gene was reduced in SV40-transformed cells suggesting a differential effect of SV40-transformation-induced abrogation of p53 and/or pRb on nucleotide excision repair and homologous recombination repair in cells. The effects of hypoxia on the repair of cisplatin-damaged DNA vmied with cell type although reduced repair under hypoxic conditions was observed in both SV40-transformed human normal cells and in several human tumour cells when cells were examined at 40 h postinfection. However, differential effects of hypoxia on repair capacity were observed in some cell lines when the length of hypoxic treatment was shorter and cells were examined at an earlier time point indicating rate-dependent effects on the repair of cisplatin-damaged DNA in some cells. There were no detectable effects of hypoxia plus low pH treatment (24 h post-infection) on repair of either cisplatin-damaged or UVC damaged DNA in the human normal cells we examined suggesting that a longer duration of hypoxia and low pH treatment may be necessary to detect repair differences. Finally, clonogenic survival of SV40-transformed MSH2-deficient and MSH2-proficient murine cells treated with cisplatin revealed a hypoxia-enhanced survival of the MSH2-deficient cells. This enhancement of survival coupled with a reduction in repair capacity previously observed in these cells under hypoxic conditions provides a potential mechanism by which hypermutability may be established in hypoxic cells.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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