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    Transcriptional regulation of human topoisomerase II beta : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry
    (Massey University, 2006) Mawson, Claire
    Topoisomerase II has an essential role in maintaining the DNA in the correct topological state required for various cellular processes. Its mechanism of action involves the introduction of a double-Dstranded break into the DNA, passage of a different piece of DNA through the break, followed by the religation of the DNA. Topoisomerase II, in humans, exists as two different isoforms: topoisomerase II alpha, which is cell cycle-Dregulated and highly expressed in rapidly proliferating cells, and topoisomerase II beta, which is ubiquitously expressed and it is not under the influence of the cell cycle. Several chemotherapeutic drugs have been designed to interfere with the catalytic mechanism of the topoisomerase II enzyme. By either stabilising the DNA cleavage complex or interfering with another step of the mechanism, these topoisomerase II targeted drugs promote the entry of the cell into cell death pathways. An increasing problem in the treatment of cancer with these drugs is the rising number of patients with inherited or developed drug-resistance. It has been shown that drug-resistance, at least in part, results from the down-regulation of topoisomerase II expression. The expression of a gene is a highly regulated process and the initiation of transcription represents a major point of regulation. Prior to this study little was known regarding the regulation of transcription of topoisomerase II beta. Understanding the processes surrounding the regulation of this enzyme would provide some insight as to how it is down regulated in drug-resistance. The focus of this study was to examine the role of three elements in the topoisomerase II beta promoter, GCI, ICB1, and ICB2 and the transcription factors that bind to them. Electrophoretic mobility shifts assays revealed that Sp1, Sp3, NF-Y and two uncharacterised proteins are capable of binding to the promoter in vitro. Transient transfection assays showed in vivo that Sp1 was able to activate transcription and that Sp3 inhibited transcription driven by the topoisomerase II beta promoter. In addition the key activating elements appear to be ICB2 and GC1, while ICB1 is inhibitory.
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    The role of NF-Y in the transcriptional regulation of human topoisomerase II¯ : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Master of Science in Biochemistry
    (Massey University, 2001) Hintz, Patricia Ann
    DNA topoisomerases are ubiquitous enzymes that catalyse reactions that alter the topological state of DNA during the various processes of DNA metabolism including transcription, recombination, replication and chromosome segregation. Human cells exhibit a Type II enzyme termed DNA topoisomerase IIα. This enzyme is expressed at higher levels in proliferating cells due to an increased demand for chromosome separation. This is advantageous with respect to some of the drugs used in chemotherapy. These drugs can specifically target cancer cells by only being effective at high levels of topoisomerase IIα gene expression. However, the use of such drugs has been limited by both toxicity and the development of resistance. This resistance has been associated with a decrease in topoisomerase IIα at both protein and mRNA levels. The topoisomerase Ila minimal promoter is 650 base pairs in length and includes promoter elements such as inverted CCAAT boxes (ICBs) and GC rich regions. It has been determined that the ICB elements are of the most interest in terms of regulation of the topoisomerase IIα gene expression. Several studies have shown that the transcription factor NF-Y binds to ICB1-4 of the topoisomerase IIα promoter and regulates transcription through these elements. This study aimed to determine the importance of NF-Y in the transcriptional regulation of topoisomerase IIα and to investigate the molecular mechanisms by which NF-Y associates with the topoisomerase IIα promoter with a particular focus on the inverted CCAAT box elements. The binding of NF-Y to oligonucleotides containing selected consensus elements of the topoisomerase IIα promoter was analysed (in vitro) by electrophoretic mobility shift binding assays. The importance of NF-Y in the regulation of topoisomerase IIα expression was analysed by functional assays, using reporter gene constructs in transiently transfected HeLa cells. The binding studies indicated that the flanking sequences affect the affinity of the transcription factor NF-Y for ICB1 and ICB2 and that a regulatory element flanking ICB2 may aid in NF-Y binding to that element. Functional assays showed that NF-Y appears to act a negative regulator of topoisomerase IIα with its effect being entirely -due to interaction with ICB2.
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    Regulation of the human topoisomerase IIℓ: a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry
    (Massey University, 2004) Willingham, Melanie
    Eukaryotic DNA topoisomcrase II is a ubiquitous nuclear enzyme essential for maintaining the correct conformation of DNA. The enzyme acts to catalyse changes in the tertian structure of DNA. via the introduction of transient double-stranded breaks. Mammalian cells express two isoforms of type II topoisomerase. designated topoisomerase IIα and topoisomerase IIβ, which display differential expression and intracellular localisation. Levels of topoisomerase IIα gene expression are elevated in rapidly proliferating cells, whereas the β isoform is expressed at approximately equal levels throughout the cell cycle. Protein products of the two isoforms of topoisomerase II found in human cells are the primary intracellular targets of many common, effective chemotherapy drugs. The development of drug resistance, however, is a major clinical problem caused by both enzymes. The levels of topoisomerase IIα and topoisomerase IIβ are important determinants for the sensitivity of cells to the cytoxicity of drugs, with down-regulation of topoisomerase II thought to be a major factor involved in drug resistance. The rate of transcription is the main mechanism for controlling topoisomerase II expression and activity, and this is achieved by the binding of transcription factors to specific regulatory elements within the promoter sequence. Molecular mechanisms responsible for the regulation of expression of the topoisomerase II enzymes are thought to be associated with resistance to chemotherapy drugs, and therefore understanding these mechanisms is an important research focus. This study reports the cloning and characterisation of a 1.5 kb fragment of the 5'- flanking and untranslated region of the topoisomerase IIβ promoter (-1357 to +122). Analyses of 5'-serially and internally deleted lueiferase reporter constructs revealed a region upstream of the transcription start site (-1357 to -1228). which could have a negative regulatory role, and suggested 55% of topoisomerase IIβ promoter activity could be attributed to the region between -654 and -456. Mutational analysis of putative regulatory elements indicated that the two inverted CCAAT box (ICB1 and ICB2) within the latter region were important for regulation of topoisomerase IIβ promoter activity. Gel mobility shift assays indicated that both inverted CCAAT boxes in the promoter bound the transcription factor NF-Y. while ICB2 and a GC element were capable of binding transcription factors Sp1 and Sp3.
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    Regulation of Topoisomerase IIa expression in humans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2006) Senior, Kelly
    In mammalian cells, the loss or down-regulation of tumour-suppressor genes and/or the mutation or overexpression of proto-oncogenes, whose products promote unregulated proliferation in cells, characterise the process of malignant transformation. This generates mitogenic signals that promote abnormal cell growth resulting in tumour progression. Topoisomerase IIα (topo IIα) is an enzyme present in elevated concentrations in highly proliferating cells due to the requirement for untwisting and unknotting of the DNA which is essential for replication. Because of this requirement, a number of anti-cancer drugs have been designed with topo IIα as their primary target. The effectiveness of these drugs however is limited by the development of resistance. One factor linked to drug resistance is the down-regulation of topo IIα at the transcription level. Expression of topo IIα appears to be regulated through various transcription factors with members of the Spl family having a major contribution. Previous work has shown down regulation of topo IIα can occur at the level of transcription. Nucleotide sequencing of the topo IIα promoter in drug-resistant cell lines has not revealed any mutations thus far. Three known proteins and one uncharacterised protein are capable of interacting with the proximal topo IIα promoter region. The uncharacterised protein may act as a co-activator or a co-repressor depending on the complement of transcription factors associated with the DNA in this region. Because drug resistant cell lines showed modulated expression of these transcription factors, it is important to identify the unknown protein and characterise its role in regulating topo IIα expression. This research aimed to identify the minimal binding site and DNA elements required for the uncharacterised protein to bind, as well as introduce mutations into this proximal region and examine their functional significance. The results of this study could provide insights into the molecular mechanisms responsible for the development of drug resistance, contributing to more efficient and effective methods for the treatment of cancer.
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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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    An investigation into the regulation of the topoisomerase IIα promoter in breast cancer cells exposed to doxorubicin : a thesis presented to Massey University in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 2003) Allen, Kirsty Ann
    Chemotherapeutic drugs, such as doxorubicin, are some of the most effective agents for the treatment of breast cancer. Acquired resistance to these drugs often develops, however, and may preclude effective treatment. Such resistance is multifactorial in origin, but may include down-regulation of topoisomerase IIα (topo IIα) - an essential enzyme involved in normal DNA metabolism and a target for some of the anticancer drugs. A reduction in the levels of this enzyme is thought to reduce DNA damage induced by the drug-topo IIα complex and so increases the chances of survival. The mechanisms involved in this down-regulation and the development of resistance to doxorubicin are the focus of this study. Stable breast cancer cell lines, containing deletion constructs of the topo IIα promoter linked to the hGH reporter gene, were exposed to doxorubicin and both the reporter and endogenous gene expression were analysed in the surviving cells. It was shown that the reporter and endogenous topo IIα gene expression in the cell line containing the full length topo IIα promoter construct was no longer correlated in the surviving cells negating the use of this experimental system. Instead the endogenous expression of topo IIα and putative regulatory factors were investigated. Data suggest that specific cell lines show a down-regulation in the levels of the topo IIα protein. These changes were not due to changes in cellular proliferation rates, cell cycle profile or promoter sequence. Selected cell lines were analysed for changes in the relative amounts of specific transcription factors with putative roles in topo IIα gene regulation and for the expression of proteins proposed to have a role in the development of drug resistance. In specific cell lines, a reduction in topo IIα protein levels correlated with alterations in the relative amounts of NF-YA and/or Sp1. It was shown that the drug efflux pumps MDR1 and MRP1, as well as the heat shock factor Hsp70 were not involved in the survival of cells that were exposed to the drug. In vivo footprinting was attempted to detect changes in the in vivo binding of proteins to the topo IIα promoter after short term drug exposure.