The DNA-damaging compound, cisplatin (cis-diamminedichloridoplatinum (II)), is a highly successful anti-tumour agent widely used in the treatment of a range of human cancers. Despite the clinical efficacy of cisplatin, its curative potential is largely restricted by the occurrence of drug resistance and several dose-limiting toxic side effects. Attempts to ameliorate these deficiencies have met with limited success. However, it is ultimately hoped that such efforts will benefit from a more comprehensive understanding of the mechanism by which cisplatin elicits tumourspecific cytotoxicity. The overall aim of this project was to further elucidate the mechanism of action of cisplatin. For this purpose, different experimental approaches were employed to examine the interaction of cisplatin with DNA, and the drug???s effect on human gene expression. Firstly, chromatin structures native to DNA in human cells were simulated in vitro via reconstitution techniques. Their effect on cisplatin-mediated DNA damage could then be evaluated. Various cisplatin analogues and other DNA-damaging compounds were also investigated in this model system. Secondly, the transcriptional response of human cells to cisplatin treatment was analysed using microarrays and gene expression profiling techniques. Transcript profiles compiled for cisplatin and its clinically ineffective isomer, transplatin, were compared to establish a possible relationship between compound-specific responses and therapeutic efficiency. To assess the relative influence of individual chromatin elements on cisplatin- DNA interactions, the first part of this study employed the octamer transfer method of nucleosome reconstitution to create a defined experimental construct. Standard footprinting techniques allowed the precise location of two positioned nucleosomes to be established with respect to the DNA sequence. The construct was then subjected to drug treatment and the resulting DNA damage was quantitatively analysed using a Taq DNA polymerase stop assay. At sites of damage, densitometric comparisons between purified and reconstituted DNA were used to evaluate the influence of nucleosomal core proteins on specific drug-DNA interactions. For cisplatin and most of the other DNAdamaging agents studied, this method revealed regions of the construct that were relatively protected from drug-induced damage. These regions corresponded to the sites of the positioned nucleosome cores and indicated that the preferred site of DNA binding for these compounds was in the linker region of the construct. Statistical analyses confirmed the significant level of damage protection conferred by the nucleosome cores and exposed subtle differences between the agents examined. Most prominent among the trends observed, was the negative correlation between compound size and the relative propensity for damaging nucleosomal core DNA. Larger compounds generally displayed a greater tendency to target the linker region of the nucleosomal DNA. In contrast, the access of smaller molecules was not impeded as significantly. In the second stage of the project, the effect of cisplatin on human gene expression levels was described using 19000-gene microarrays and transcription profiling methods. In cultured human cells, cisplatin treatment was shown to significantly up- and down-regulate consistent subsets of genes. Many of these genes responded similarly to treatment with transplatin, the therapeutically inactive isomer of cisplatin. However, a smaller proportion of these transcripts underwent differential expression changes in response to the two isomers. Some of these genes may constitute part of the DNA damage response induced by cisplatin that is critical for its anti-tumour activity. Ultimately, the identification of gene expression responses unique to clinically active compounds, like cisplatin, could thus greatly benefit the design and development of improved chemotherapeutics.
| Identifer | oai:union.ndltd.org:ADTP/234160 |
| Date | January 2006 |
| Creators | Galea, Anne Marie, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Anne Marie Galea, http://unsworks.unsw.edu.au/copyright |
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