Investigation of DNA adducts formed in cells and clinical tumour biopsies following exposure to platinum-containing anticancer drugs

摘要

Platinum-based anticancer drugs are believed to exert their action through chemical reactions with genomic DNA, forming adducts with DNA bases. Although the pharmacology of such adducts has been widely studied, the cytotoxic mechanism remains unclear. The possibility that non-DNA molecules have the potential to alter the types of adducts formed has received very little attention, and limited information is available on the levels of adducts formed in clinical tumours. Further understanding of platinum-DNA adduct formation may be important in explaining the efficacy of platinum-based drugs in different tumour types, providing insights into both the cytotoxic mechanism and the development of clinical resistance. The aims of the work described in this thesis were: a) to analyse the nature of DNA adducts formed by three clinically used platinum-based anticancer drugs and to investigate the potential intracellular formation of additional types of adducts to those previously characterised on pure DNA; b) to determine platinum-DNA adduct levels formed in solid ovarian cancer tissue following treatment of patients with carboplatin and test the hypothesis that these levels are comparable to the levels of DNA adducts formed in blood cells; and c) to determine whether sodium thiosulfate (STS), which is currently in clinical trials to protect against cisplatin-induced normal tissue toxicity, impacts on DNA adduct formation. Analysis of the properties of all DNA adducts formed in cells was made possible by analysing enzymatically digested DNA using anion exchange chromatography together with inductively-coupled plasma mass spectrometry (ICP-MS). Putative adducts involving deoxyguanosine monophosphate cross-linked via cisplatin to glutathione were prepared and the chromatographic properties determined. Studies were carried out to characterise the types of adducts formed following incubations of cisplatin with four cancer cell lines. No additional types of adducts were observed compared to those formed by the reaction of cisplatin with pure DNA. The chromatographic behaviour of adducts formed in cells incubated with carboplatin and oxaliplatin were comparable to those formed by cisplatin. This study is the first to investigate carboplatin-DNA adduct levels induced in solid tumours during therapy in patients. Total DNA adduct levels in tumour biopsies and blood cells were measured using ICP-MS with thallium as an internal standard. Tumour biopsies from all four patients studied showed clearly detectable levels of treatment-induced DNA adducts ranging from 1.9 - 4.2 nmoles Pt/g DNA. Blood cell adduct levels ranged from 0.15 – 3.5 nmoles Pt/g DNA. Both tumour and blood cell adduct levels were significantly above background measurements. No correlation was observed between adduct levels in DNA from biopsies and levels in DNA from peripheral blood cells. Concurrent incubation of four human tumour cell lines with cisplatin and STS caused greater than 2-fold decreases in total DNA adducts. Delayed administration of STS had no effect of adducts levels. STS did not appear to affect the chromatographic behaviour of DNA adducts formed in cells following incubation with cisplatin.