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|Title:||Cellular Responses to the Anti-Cancer Drug, Cisplatin.|
|Authors:||Bulmer, Todd Jason|
|Advisor:||Rainbow, A. J.|
|Abstract:||<p>cis-Diamminedichloroplatinum (cisplatin) and other platinum-based drugs, represent the best hope for cancer patients with ovarian tumours and many other types of solid tumours. Sadly, the clinical efficacy of cisplatin is often limited by the emergence of drug-resistant tumours. The research presented here is intended to expand understanding of the molecular characteristics that make cells resistant to cisplatin. Cisplatin kills cells by damaging the genome and inducing cell suicide (apoptosis). Despite having been used for more than twenty years, the cellular responses responsible for this drug's cytotoxic effect remain fundamentally unknown. When cisplatin reacts with DNA, a spectrum of addition products (adducts) are formed. Most of these adducts can be repaired by nucleotide excision repair (NER) and cells with defects in NER are extremely sensitive to the drug. In this thesis, we show that human and Chinese hamster ovary (CRO) cells with defects in NER are reduced in their capacity to repair cisplatin-damaged adenovirus (Ad) DNA. Excision repair cross complementing gene-1 (ERCC 1) is required for NER and many researchers have proposed that clinical cisplatin-resistance results from enhanced ERCC1 expression. To study the role of this gene in cisplatin resistance and DNA repair, we have constructed Ad viruses that express ERCC 1. Cells infected with these viruses express very high levels of the protein. We report here that these very high expression levels actually impede DNA repair and make some human tumour cells cisplatin-sensitive. The response to DNA damaging agents like cisplatin is dynamic. Upon exposure to cisplatin, many mechanisms that affect cell survival are induced. We show here that some human tumour cells are much more sensitive to low doses of cisplatin (per unit of dose) than they are to higher doses. This hypersensitivity suggests that, at some critical dose of the drug, a mechanism that protects cells from death is triggered. Treating cells with other DNA damaging agents (including ultraviolet light (UV) and ionising radiation) before cisplatin exposure can induce this resistance. We have also induced resistance to low doses of cisplatin by infecting cells with UV -irradiated Ad suggesting that it is the DNA damage per se that triggers the mechanism of cisplatin resistance. The Jun N-terminal kinase (JNK) is a family of mitogen-activated protein kinases (MAPKs) that responds to a broad range of cellular stresses. While the contribution of JNK activity to determining cell fate is complex, it is generally considered a proapoptotic factor. Several research groups have proposed that JNK activity is important in cisplatin-induced cell death. By studying cisplatin-induced JNK activity in human xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells with well known DNA repair defects, we have shown that it is the persistence of cisplatin-DNA lesions that is associated with prolonged JNK activation. This is the first direct evidence that it is DNA lesions that promote JNK activity in cisplatin- reated cells. Further, we have used human cells with defective transcription-coupled repair (TCR) to show that damage in actively transcribing genes causes persistent JNK activity while persistent DNA damage in untranscribed regions does not. Our results support a role of JNK activation in cisplatin-induced cell death by showing that cells with defects in TCR are extremely sensitive to cisplatin while cells with defects in global genome repair are not.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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