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

Abstract

Pages 24 and 25 are missing from the original copy.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