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Subject: search.filters.subject.Doxorubicin

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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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    Differentially regulated proteins in breast cancer chemotherapy : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 2005) Koehn, Henning
    Intrinsic or acquired drug resistance of tumours is a major problem for successful therapy of breast cancer patients. The efficacy of doxorubicin, one of the most important and commonly used drugs in chemotherapy, can be severely compromised by a variety of unspecific mechanisms rendering tumours drug resistant. Little is known however, about the specific events taking place in response to doxorubicin treatment, which may repair doxorubicin-induced damage, leading to drug resistance. Doxorubicin is a topoisomerase II poison, which interferes with topoisomerase II enzymes during DNA replication, resulting in DNA double-strand breaks. Topoisomerase II enzymes mediate the passage of DNA strands by introducing transient DNA breaks, and are essential for changes in DNA topology during replication. The DNA lesions induced by the combination of topoisomerase II and doxorubicin can be repaired by either non homologous end-joining or homologous recombination repair, as both pathways are specifically responsible for the repair of DNA double-strand breaks. The DNA-dependent protein kinase catalytic subunit in non homologous end-joining and Rad51 in homologous recombination repair are essential for each of these pathways. If it was possible to specifically target these proteins or other antagonistic mechanisms of doxorubicin-induced cell death, which may be activated in response to doxorubicin treatment, chemosensitivity of tumours could be restored and chemotherapy made more effective. Hence it was the purpose of this study to investigate proteome-wide changes in protein expression in response to drug treatment, as well as specifically analysing alterations in the protein levels of the DNA-dependent protein kinase catalytic subunit and Rad51. Global changes in protein regulation of breast and breast cancer cells were investigated using mass spectrometric and electrophoretic analysis techniques. These experiments however, could not reproducibly identify any genuine drug-induced changes in protein levels, as only proteins of relatively high abundance could be analysed. Immunoblotting results however, showed that Rad51 was differentially regulated in a cell line- and drug dosage-dependent manner, while levels of the DNA-dependent protein kinase catalytic subunit remained largely unchanged. Furthermore, increased levels of topoisomerase II alpha protein were also detected. In addition, immunohistochemical analysis demonstrated that both Rad51 and the DNA-dependent protein kinase catalytic subunit could be independently overexpressed in breast tumours and therefore may represent potential targets for selectively enhancing chemosensitivity of breast cancers.