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Title: The role of defective mismatch repair in the development of resistance to doxorubicin
Author: Mackay, Helen Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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In this present study we have examined the role of MMR in doxorubicin induced cell death in yeast and human tumour cell lines. In addition, we have explored the clinical relevance of loss of MMR protein expression in locally advanced breast cancer. Doxorubicin sensitivity was examined in matched cell lines of defined MMR status derived from A2780, a human ovarian cancer cell line. The cisplatin resistant derivative A2780/CP70 has lost MMR activity due to loss of expression of the mismatch repair protein MLH1 and is cross-resistant to doxorubicin. The hMLH1 gene is located on chromosome 3. Microcell-mediated transfer of a normal chromosome 3 into A2780/CP70 restores MLH1 expression and significantly increases doxorubicin sensitivity, as assessed by clonogenic assay (P<0.05). Whole chromosome transfer introduces multiple genes and thus mechanisms not associated with MMR could be influencing doxorubicin resistance in these chromosome transferrants. For instance, the gene encoding topoisomerase II b on chromosome 3. However no alteration in topoisomerase II activity was observed following chromosome 3 transfer into A2780/CP70. In order to examine doxorubicin sensitivity in cells with only MMR genes inactivated, the genetically tractable organism Saccaromyces cerevisiae was used. A significant 1.3-6 fold increase (p<0.05) in clonogenic resistance was seen following doxorubicin exposure in isogenic, haploid, strains with individual disruptions in the MMR genes MSH2, MSH3, MSH6, and MLH1 compared to the wild type strain. Furthermore, re-introduction of the MLH1 gene into the mlh1 mutant, using a high copy yeast expression vector, restored doxorubicin sensitivity to wild type levels. Together these observations in yeast and human tumour cell lines are consistent with a role for MMR in sensitivity to doxorubicin. To determine if doxorubicin exposure frequently results in loss of MMR in human tumour cell lines twenty cell lines were independently derived by repeated selection with increasing doses of doxorubicin from A2780 and, the human breast cancer cell line, MCF7. Resistance was confirmed by colony forming assay. All derived cell lines exhibited a significant increase (P<0.05) in clonogenic resistance to 24-hour exposures of 50nM (MCF7) and 15nM (A2780) doxorubicin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available