Development of More Effective Anti-cancer Drugs Based on Cisplatin

The overall aim of the lab is to develop more effective cancer chemotherapeutic agents based on cisplatin and bleomycin. These drugs are widely used in clinical applications: cisplatin is used to treat testicular and ovarian cancer; bleomycin is used to treat germ cell tumours, certain types of lymphoma, and squamous cell carcinomas. Both compounds are thought to act by damaging DNA inside tumour cells. Cisplatin preferentially targets G-rich DNA sequences, while bleomycin targets GT and GC DNA sequences. There are several hypotheses concerning the precise cellular DNA target(s) for cisplatin and bleomycin.

  • Telomeres: Human telomeres contain thousands of tandemly repeated copies of the G-rich sequence, (GGGTTA)n. Since the telomeric repeat contains a GGG repeat sequence, it is expected to be a major target site for cisplatin adduct formation. The formation of cisplatin lesions at the telomeric regions of chromosomes would be expected to severely inhibit DNA replication and hence, cell division. Since the telomeric repeat contains a GT repeat sequence, it is also expected to be a major target site for bleomycin cleavage.
  • Guanine-rich promoter sequences: A large number of human promoters contain G-rich regions (CpG islands) with GC and GT DNA sequences. Cisplatin or bleomycin damage in these regions would severely alter gene expression and lead to cell death. As well as inhibiting DNA replication, transcription would also be inhibited but its extent would vary from gene to gene. This could give rise to different levels of cell killing, dependent on the gene expression profile of the individual cell.
  • Effect of chromatin structure: The more open nature of chromatin in transcribed genes can lead to more cisplatin and bleomycin damage in these regions. We have previously shown that nucleosome cores inhibit the ability of cisplatin to damage DNA. Bleomycin preferentially cleaves chromatin in the linker region of the nucleosome.
  • Twenty or more consecutive guanine bases: There are at least 50 sites in the human genome that have 20 or more consecutive guanines. These sites would expected to be major sites of cisplatin adduct formation. We have initiated experiments to look at the interaction of these long runs of consecutive Gs with cisplatin (in plasmid constructs), in order to investigate the properties of these unusual DNA sequences.

There are two main aims of this project. First, by constructing plasmid clones containing various genomic elements (e.g. telomeric DNA sequences, promoter DNA sequences and consecutive guanine sequences) the relative targeting of these sequences by cisplatin and bleomycin can be assessed. Second, by utilising genome-wide DNA sequencing of human cells treated with cisplatin or bleomycin, we will pinpoint the precise DNA sequences targeted by cisplatin and bleomycin in human cells. In this latter aim, we will utilise the immense power of Illumina next-generation DNA sequencing techniques to determine the precise targets of cisplatin and bleomycin in the human genome, in a systematic and unbiased way.

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