Browsing by Author "Allen, Kirsty Ann"

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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.
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    The responsiveness of the bovine lactoferrin promoter to cytokines and glucocorticoids : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1998) Allen, Kirsty Ann
    Lactoferrin is an iron-binding protein found in many bodily secretions and in the secondary granules of polymorphonuclear leukocytes. While there are many proposed functions for the lactoferrin protein - e.g. for iron storage, antibacterial properties, or a role in inflammation, the specific function(s) of lactoferrin have yet to be elucidated. Evidence that lactoferrin may be involved in inflammation was observed by Harmon et al. (1976) where after the induction of bovine mammary infections, a significant increase in secreted lactoferrin protein was seen during the early phase of the infection. As this increase was during the period of the acute phase response, this suggested that lactoferrin, as was the case with other proteins induced during this time, may have a role in the inflammatory response. The bovine lactoferrin (bLf) promoter contains many putative binding sites for inflammatory modulators, which suggests that the increases in lactoferrin seen during inflammation may be due to activation of lactoferrin gene transcription by these specifically-induced transcription factors. Substantiation of this suggestion would provide further evidence for a specific role for lactoferrin during inflammation. To investigate the cytokine-responsiveness of the bLf promoter, constructs corresponding to various lengths of the putative bLf promoter were linked to the luciferase reporter gene and introduced, by transient transfection, into RL95-2 human endometrial carcinoma cells. Cytokines, glucocorticoids or expression vectors for transcription factors were added to the cells, or potential 'masking' factors in the media such as phenol red or insulin were removed. The luciferase activity of the transfected cells was monitored for significant variation from the basal levels. The addition of cytokines with or without phenol red or insulin did not cause any significant changes in bLF promoter activity. In phenol red-free media, increases in luciferase reporter gene activity were observed after the co-transfection of an expression vector for NF-IL6, the addition of dexamethasone and also the addition of dexamethasone together with the co-transfection of a glucocorticoid receptor expression vector. These data provided evidence that lactoferrin transcription may be induced by inflammatory factors which support the suggestion that lactoferrin has a role in the inflammation process.