Browsing by Author "Magan, Natisha"

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    Basal transcription of human topoisomerase II : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 2002) Magan, Natisha
    Topoisomerase II is a ubiquitously expressed enzyme, which is required for cell survival. It has the ability to alter the topological states of DNA by introducing transient double-stranded breaks in DNA. Humans have two topoisomerase II isoforms, ɑ and β, and both are differentially expressed and localized. Tissues with rapidly proliferating cells exhibit elevated topoisomerase IIɑ gene expression whereas the β isoform is ubiquitously expressed amongst tissues. In addition to a role in cell survival, a number of anti-cancer drugs have been shown to target human topoisomerase II in vivo. However, the development of drug resistance is a major clinical problem; for example, approximately 60% of breast cancers treated with the topoisomerase II poison doxorubicin become resistant to this drug. Down-regulation of topoisomerase II is thought to be one of the factors involved in the development of drug resistance, where the relative levels of topoisomerase IIɑ and topoisomerase IIβ in cells is thought to effect drug efficacy. The expression of topoisomerase IIɑ and β is regulated at the transcriptional level, through binding of transcription factors to specific elements within the promoter sequence. Therefore investigating the transcriptional regulation of both isoforms could lead to an understanding of the mechanisms involved in the development of drug resistance. The initial aim of this study was to isolate a fragment of the upstream regulatory sequence of the topoisomerase IIβ gene and carry out systematic analysis of this sequence. However, this could not be pursued, as the clones that were examined did not contain the required topoisomerase IIβ sequence. This study progressed to examine the relevance of three elements (GC1, ICB1 and GC2) within the topoisomerase IIɑ minimal promoter and the importance of the cognate transcription factors NF-Y, Spl and Sp3 in regulating the expression of the topoisomerase IIɑ gene. Electrophoretic mobility shift assays and transient transfection assays were used to study protein/DNA interactions and the functional significance of these interactions, respectively. Both NF-Y and Spl were shown to activate the transcriptional regulation of topoisomerase IIɑ by binding to their respective elements; in addition functional interactions between the two proteins bound to the promoter was observed.
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    Protein interactions at the human topoisomerase II[alpha] promoter : a thesis presented to Massey University in partial fulfilment of the requirement for the degree Doctor of Philosophy in Biochemistry
    (Massey University, 2009) Magan, Natisha
    Among women in the 45 to 64 age group, over half of the recorded deaths are from cancer, breast cancer being the most common. Just over 30% off all deaths in New Zealand women is caused by breast cancer. Treatment of cancer is difficult, not only due to the physiological and immunological similarities between a cancer cell and a normal cell, but also due to the high cardiotoxicity of many treatments, and also the problems related with the development of resistance. Approximately 40% of the cancer cells treated with the chemotherapy drug doxorubicin will become resistant to treatment. Drug efficacy is strongly associated with the proliferation status of a cell, as cancer cells divide rapidly, this can often be the defining factor between effective treatments or the development of resistance. Central to this proliferation status is an enzyme known as topoisomerase IIa. This essential enzyme is expressed in all cells and is required to relieve the torsional stress in DNA that is created during normal cellular processes. A number of commonly used anti-cancer drugs have been found to target topoisomerase IIa in cancer cells and significantly, during the development of drug resistance levels of topoisomerase IIa enzyme have been found to be reduced in some cell lines and tumours. There are a number of factors that can modulate the amount of topoisomerase IIa enzyme found in a cell, and one of the ways to understand this is to examine the regulation of the topoisomerase IIa gene, most importantly the proteins that interact with the promoter region to direct transcription. The human topoisomerase IIa promoter has been found to be regulated by a number of transcription factors that can bind to their cognate sequences. The introduction of mutations within specific sequences of the topoisomerase IIa promoter has enabled the identification of a key regulatory region within the promoter, a sequence of DNA that encompasses both the ICB1 and GC1 regulatory elements. Transcription factor NF-Y is found to bind to ICB1 element, whereas transcription factors Sp1 and Sp3 have been found to associate with the GC element. However this region of the promoter was also found to bind a fourth uncharacterised component. This research aims to further define the protein components that are found to bind to this important ICB1/GC1 regulatory region and distinguish the protein-protein and protein-DNA interactions that are important for the regulation of the human topoisomerase IIa promoter.