The interaction of cisplatin with telomeric DNA sequences

The anti-tumor drug cisplatin (cis-diamminedichloroplatinum(II)) has been in clinical use for nearly forty years and is highly effective in the treatment of a variety of human cancers. However, toxic side effects and drug resistance limit its use. It is therefore important to gain a comprehensive understanding of the mechanisms by which cisplatin is able to target and inhibit the growth of cancerous cells so that more effective cisplatin analogues can be developed.Determining the sequence selectivity of DNA damaging agents could elucidate how the drugs work. The implementation of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has given rise to sensitive and precise DNA analysis. The first part of this project is involved with successfully using the ABI3730 DNA Analyzer to determine the sequence specificity of cisplatin on purified DNA samples, replacing radioactive labeling and time-consuming slab gels. Optimizing the protocol for this method was hindered by the presence of artifact bands, which was overcome to provide very accurate analysis of cisplatin-DNA adducts; both precision of damage site location and quantification was greatly improved compared with manual analysis by slab gel electrophoresis.Telomeres are G-rich DNA sequences located at the end of chromosomes and mainly provide genetic stability by protecting the ends from degradation or end-to-end fusion. In most cancer cells, they are continuously extended and are a likely target for cisplatin, which primarily forms DNA adducts between two consecutive guanine nucleotide bases. Using CE-LIF, a clear comparison was made of cisplatin targeting in telomeric iiiDNA sequences against bulk DNA. It was found that cisplatin appeared to preferentially target telomeric DNA sequences.Because of drug resistance and toxic effects, the development of cisplatin analogues is of great interest. The aim is to develop analogues that have a sequence selectivity shift away from that of cisplatin, and could possibly intercalate with a different telomere- specific motif, such as the G-quadruplex. The four analogues tested in this project show potential in fulfilling the aim, as there is a clear sequence specificity shift. Information gathered could greatly benefit the development of new and improved chemotherapeutics for cancer treatment