Defective Mismatch Repair and Cisplatin Resistance in Ovarian Cancer Cells

Anthoney, David Alan (1997) Defective Mismatch Repair and Cisplatin Resistance in Ovarian Cancer Cells. PhD thesis, University of Glasgow.

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The development of resistance to anticancer drugs poses one of the major obstacles to improving the survival from malignant disease. Most clinically effective anticancer agents act upon DNA. Resistance has been shown to arise by mechanisms that reduce the amount of drug reaching the DNA. However, drug resistance developing through alterations in the capacity of tumour cells to repair or respond to DNA damage, may be as important. Microsatellite instability (RER+) is generally taken as an indication of defective DNA mismatch repair. Ovarian and breast carcinoma cell lines, selected for resistance to cisplatin and doxorubicin, were shown to display an RER+ phenotype as shown by multiple mutations at microsatellite loci in comparison to the parental line. The ovarian carcinoma cell line A2780 was shown to be RER- by microsatellite analysis of random sub-clones from the cell line. A2780/cp70, a cisplatin resistant cell line derived from A2780, displayed an RER+ phenotype when subject to the same analysis. Ten independently derived, cisplatin resistant, A2780 cell lines were developed by selection with increasing concentrations of cisplatin to a final concentration of 15muM. These were shown to display stable resistance to cisplatin of between 4 fold (as determined by clonogenic assay) and 7 fold (s determined by MTT assay), when compared to the parental line. The RER+ phenotype was detected in all but one of these lines. An association between the RER+ phenotype and cisplatin resistance was further strengthened by the finding of microsatellite instability in resistant A2780 cell lines selected by a single exposure to 15muM cisplatin. Mutations in genes involved in mismatch repair have been shown to be responsible for microsatellite instability. The biochemical basis of the RER+ phenotype in drug resistant A2780 derived cell lines was determined by in vitro repair assay. An inability to correct DNA mismatches, when repair was directed from 3' to the lesion, was observed, and hMutLalpha was identified as the defective component of mismatch repair by in vitro complementation. Further studies revealed the absence of hMLH1 and hPMS2 proteins, which constitute hMutLalpha, within the RER+ resistant cell lines. This arises from the failure of these cells to express hMLH1 mRNA. No defect in the expression of the other known mismatch repair genes was observed. P53-dependent DNA damage responses were characterised in the RER+, drug resistant cell lines. A correlation was observed between RER+ status and loss of radiation induced G1 arrest, reduced expression of the CIP 1 gene and decreased levels of apoptosis after cisplatin exposure. This association between DNA mismatch repair and the DNA damage response pathway suggests that the mismatch repair system might act as a sensor for DNA damage. This supports the hypothesis that mismatch repair may be involved in determining the cellular sensitivity to cisplatin; defects in such repair leading to cisplatin resistance.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Robert Brown
Keywords: Oncology, Cellular biology, Biochemistry
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75499
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:38
Last Modified: 19 Nov 2019 19:38

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