Made available in DSpace on 2015-05-14T12:59:25Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 799830 bytes, checksum: e9ce4907eb8193bacb26ea198ef6011e (MD5) Previous issue date: 2011-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Topoisomerase II is involved in several vital cellular processes such as replication and transcription of DNA, in addition to the segregation of chromosomes. This enzyme catalyzes changes in DNA topology by temporary disruptions and subsequent rearrangement of the double helix and is essential for cell proliferation to occur. Many compounds that interfere with the catalytic activity of this enzyme are effective in chemotherapy of cancer. The development of new inhibitors from plant sources can be a valuable strategy, serving also as a tool for the production of semi-synthetic active agents that do not cause serious damage to the body and with a lower cost. Has already been reported that some lignoids derived from plants have inhibitory action on DNA Topoisomerase II. Plants of the Lauraceae family are sources of bioactive lignoids. Among different species, there are the Licaria aurea and Licaria chrysophylla. In this study, 12 lignoids derived from both species were evaluated for their inhibitory effect on human DNA Topoisomerase II, using etoposide as action control of substances. Two of these compounds showed inhibitory activity against the human enzyme Topoisomerase II. These compounds are the lignan (2S,3S)-Dihydro-dehydrodiconiferyl alcohol-3-(-D-glycopiranosyl)-hexaacetate and the neolignan 1,4-Bis-(3, 4,5-trimethoxyphenyl)-2,3-dimethylbutane. / A Topoisomerase II está envolvida em diversos processos celulares vitais, como a replicação e a transcrição do DNA, além da segregação dos cromossomos. Essa enzima catalisa mudanças na topologia do DNA através de rupturas temporárias e posterior rearranjo da dupla hélice, sendo fundamental para que ocorra a proliferação celular. Muitos compostos que interferem na atividade catalítica dessa enzima são eficientes no tratamento quimioterápico de câncer. O desenvolvimento de novos inibidores a partir de fontes vegetais pode ser uma estratégia valiosa, podendo servir também como um instrumento adequado para a produção de agentes ativos semi-sintéticos que não causem danos graves ao organismo e que apresentem um menor custo. Já se tem reportado que alguns lignóides derivados de plantas têm ação inibitória sobre a DNA Topoisomerase II. Plantas da família Lauraceae são fontes de lignóides bioativos. Dentre as diversas espécies, encontra-se a Licaria aurea e a Licaria chrysophylla. No presente estudo, 12 lignóides derivados destas duas espécies foram avaliados quanto à sua capacidade inibitória sobre a DNA Topoisomerase II humana, utilizando a etoposida como controle de ação das substâncias. Dois dos compostos estudados apresentaram atividade inibitória sobre a enzima Topoisomerase II humana. Esses compostos são a lignana (2S,3S)-Dihidro-dehidrodiconiferil alcool-3-(-D-glicopiranosil)-hexaacetate e a neolignana1,4-Bis-(3, 4,5-trimetoxifenil)-2,3-dimetilbutano.

A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Aneta Karešová Supervisor: PharmDr. Anna Jirkovská, PhD. Title of diploma thesis: DNA-protein covalent complexes detection as the means for the assessment of the DNA damage induced by topoisomerase poisons. Topoisomerase II is essential cellular enzyme, which modifies the secondary structure of DNA. By introducing a temporary double strand break to DNA it relieves a structural tension raised during transcription and translation. Absolutely indispensable is the role of topoisomerase II in the separation of sister chromatids synthesized in the S-phase of the cell cycle. The mechanism of DNA cleavage involves a covalent bond formed between active site tyrosine and 5' phosphate on both of the DNA strands and through the formed break the other strand or the other DNA molecule can pass. After that, the DNA strands are rejoined and topoisomerase II is detached. The indispensability of topoisomerase II mainly for proliferating cells makes it a great target for the antineoplastic drugs and the molecules belonging to the class of topoisomerase II inhibitors (etoposide, anthracyclines) are amongst the most useful anticancer drugs in the clinical practice. These clinically used "topoisomerase...

Charles University Faculty of Pharmacy in Hradec Králové Department od Biochemical Sciences Candidate: Bc. Robin Klieber Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of thesis: The use of RNA interference for the modification of DNA topoisomerase II levels in cancer cells and its influence on the antineoplastic effect of anthracyclines. Topoisomerase II (TOP II) is an enzyme that alters the topological state of the DNA double helix during physiological processes through the formation of transient DNA double strand breaks. Two TOP II isoforms are known: TOP IIα is essential for proper separation of chromosomes in mitotic cells, whereas TOP IIβ is primarily associated with gene transcription. Anthracycline antibiotics (ANT) belong to the group of topoisomerase poisons that stabilize the covalent complex of TOP II and DNA. This prevents the religation of the DNA double strand breaks and thus causes irreversible DNA damage leading to programmed cell death. Although ANTs are frequently administered in various antineoplastic protocols (hematooncological malignancies, hormone-dependent tumors and others), the therapy still possess a high risk of irreversible cardiotoxicity. The mechanism of cardiotoxicity remains unraveled. However, it has been previously discussed that TOP IIβ inhibition could play a...

Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and multiple myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions exhibit a substantial export of endogenous topoisomerase II alpha from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II alpha may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. In the current report, we have defined nuclear export signals for topoisomerase II alpha at amino acids 1017-1028 and 1054-1066, using FITC labeled BSA-export signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals (1017-1028 and 1054-1066) was provided by transfection of human myeloma cells with plasmids containing the gene for a full-length human FLAG-topoisomerase fusion protein, mutated at hydrophobic amino acid residues in the export signals. Of the six putative export signals tested, the two sites above were found to induce export into the cytoplasm. Export by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM-1 dependent pathway mediates export. Site-directed mutagenesis of two central hydrophobic residues in either export signal in full-length human topoisomerase blocked export of recombinant FLAG-topoisomerase II alpha, indicating that both signals may be required for export. Interestingly, this pair of nuclear export signals (1017-1028 and 1054-1066) also defines a dimerization domain of the topoisomerase II alpha molecule.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

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.

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Includes bibliographical references (leaves 213-243). Issued in print and online. Available via ProQuest Digital Dissertations.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24521号 / 医博第4963号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 萩原 正敏, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM