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Title: Mechanisms of resistance to cisplatin in ovarian cancer cells
Author: Stephens, Imogen F. D.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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Three ovarian carcinoma continuous cell lines, A2780, 2780AD and 2780CP, were found to have a stable, 80-fold, range of cisplatin sensitivity, using a modified MTT chemosensitivity assay. This experimental model was used to investigate various theoretical mechanisms of platinum resistance. Inductively coupled plasma mass spectrometry (ICPMS), a highly sensitive technique for platinum analysis, was used to develop a new assay for the direct measurement of intrastrand platinum-DNA adducts. Highly platinated calf thymus DNA was disaggregated enzymatically; nucleotides and platinum-containing oligonucleotides were separated by HPLC using an anion exchange column. Purified adduct standards were used to calibrate the chromatogram; platinum analysis of eluate fractions revealed two peaks coinciding with the eluate positions of the two major intrastrand adducts. Electrothermal vaporisation, a modification of ICPMS, was used to improve analytical sensitivity prior to the direct investigation of intrastrand adducts in this cellular model of induced platinum resistance. Accumulation of platinum, both intracellular and DNA-bound, was measured by ICPMS; a linear relationship was observed between intracellular platinum levels and chemosensitivity for all three cell lines. Platinum accumulation was reduced in both resistant sublines, although to a similar degree in each. Uptake did not appear to be energy-dependent; nor did the P glycoprotein efflux pump appear to be a significant factor. Induced cisplatin resistance was found to be associated with elevated levels of reduced glutathione (GSH). The inhibitor of glutathione synthesis, buthionione sulfoxidine(BSO), was used to achieve up to 100% reduction in GSH levels; however no effect on cisplatin sensitivity was observed under these experimental conditions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available