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A mitoxantrona (MXT) é um antineoplásico utilizado no tratamento de tumores como leucemias, linfoma não-Hodgkin e câncer de mama e próstata. Ela é classificada como uma antracenodiona, sendo um análogo estrutural das antraciclinas, como a doxorrubicina (DOX), cujo mecanismo de ação é baseado na inibição da enzima topoisomerase II (Topo II), através da formação dos complexos estabilizados Topo II-DNA. As antraciclinas e a MXT também são capazes de formar lesões do tipo adutos, pontes intercadeias de DNA (interstrand crosslink – ICL) e espécies reativas de oxigênio (ERO). Estudos têm demonstrado que a via de reparo por excisão de nucleotídeos (Nucleotide Excision Repair – NER) está envolvido na remoção de lesões no DNA induzidas pela DOX. Considerando as similaridades estruturais e de mecanismo de ação entre a MXT e a DOX, o objetivo deste trabalho foi avaliar a influência da via NER na citotoxicidade da MXT, a fim de elucidar possíveis mecanismos envolvidos na resistência tumoral a esta droga. Os resultados encontrados demonstraram que células deficientes na via NER (XPA, XPD, XPC e CSB) apresentam elevada sensibilidade a MXT comparadas a células proficientes em reparo (MRC5). Apesar disso, células CSB (deficientes na subvia associada à transcrição - Transcription coupled – TCR-NER) são mais sensíveis a MXT que células XPC (deficientes na subvia de reparo global do genoma – Global genome repair – GGR-NER) e também apresentam diferenças no perfil de ciclo celular, síntese de DNA e formação dos complexos Topo II-DNA após tratamento com MXT. Células XPC, da mesma forma que as células proficientes MRC5 apresentam parada de ciclo celular em G2/M, recuperação da síntese de DNA e sinal semelhante para formação dos complexos Topo II-DNA, enquanto células CSB apresentam acúmulo de células na fase S, diminuição na síntese de DNA e sinal mais intenso para formação dos complexos Topo II-DNA. Além disso, a complementação das células CSB com a proteína CSB recuperou a resistência das células a MXT e também diminuiu a intensidade do sinal dos complexos Topo II-DNA. Estes resultados indicaram que a via NER está envolvida na resistência das células ao tratamento com MXT e que a proteína CSB ou a subvia TCR-NER tem um papel chave no processamento dos complexos Topo II-DNA. / Mitoxantrone (MXT) is an antineoplastic drug used in treatment of tumors like leukemia, non-Hodgkin lymphoma and breast and prostate cancer. It is classified as an anthracenedione, being a structural analogue of anthracyclines, like doxorubicin (DOX), which action mechanism is based on topoisomerase II (Topo II) inhibition and formation of stabilized Topo II-DNA complexes. Anthracyclines and MXT also can form lesions like DNA adducts, interstrand crosslinks (ICL) and reactive oxygen species (ROS). Studies have shown that nucleotide excision repair (NER) pathway is involved in removal of lesions induced by DOX. Due to structural and action mechanism similarities between MXT and DOX, the aim of this work was to evaluate the influence of NER pathway in cytotoxicity of MXT, in order to elucidate possible mechanisms involved in tumor resistance to this drug. The results demonstrated that NER-deficient cells (XPA, XPD, XPC and CSB) show high sensitivity to MXT compared to repair proficient cells (MRC5). However, CSB cells (deficient in Transcription coupled repair – TCR) were more sensitive to MXT than XPC cells (deficient in Global genome repair – GGR) and also showed differences in cell cycle, DNA synthesis and Topo II-DNA complexes formation upon MXT treatment. XPC cells, in the same way as MRC5 proficient cells present G2/M cell cycle arrest, DNA synthesis recovery and similar signal for Topo II-DNA complexes formation, while CSB cells present accumulation of cells in S phase, reduced DNA synthesis and a more intense signal for Topo II-DNA complexes formation. Moreover, CSB cells complementation recovery MXT-resistance and also diminished Topo II-DNA complexes signal intensity. These results indicate that NER pathway is involved in cells resistance to MXT treatment and that CSB protein or TCR-NER sub pathway has a key role in processing of MXT induced Topo II-DNA complexes.

A mitoxantrona (MXT) é um antineoplásico utilizado no tratamento de tumores como leucemias, linfoma não-Hodgkin e câncer de mama e próstata. Ela é classificada como uma antracenodiona, sendo um análogo estrutural das antraciclinas, como a doxorrubicina (DOX), cujo mecanismo de ação é baseado na inibição da enzima topoisomerase II (Topo II), através da formação dos complexos estabilizados Topo II-DNA. As antraciclinas e a MXT também são capazes de formar lesões do tipo adutos, pontes intercadeias de DNA (interstrand crosslink – ICL) e espécies reativas de oxigênio (ERO). Estudos têm demonstrado que a via de reparo por excisão de nucleotídeos (Nucleotide Excision Repair – NER) está envolvido na remoção de lesões no DNA induzidas pela DOX. Considerando as similaridades estruturais e de mecanismo de ação entre a MXT e a DOX, o objetivo deste trabalho foi avaliar a influência da via NER na citotoxicidade da MXT, a fim de elucidar possíveis mecanismos envolvidos na resistência tumoral a esta droga. Os resultados encontrados demonstraram que células deficientes na via NER (XPA, XPD, XPC e CSB) apresentam elevada sensibilidade a MXT comparadas a células proficientes em reparo (MRC5). Apesar disso, células CSB (deficientes na subvia associada à transcrição - Transcription coupled – TCR-NER) são mais sensíveis a MXT que células XPC (deficientes na subvia de reparo global do genoma – Global genome repair – GGR-NER) e também apresentam diferenças no perfil de ciclo celular, síntese de DNA e formação dos complexos Topo II-DNA após tratamento com MXT. Células XPC, da mesma forma que as células proficientes MRC5 apresentam parada de ciclo celular em G2/M, recuperação da síntese de DNA e sinal semelhante para formação dos complexos Topo II-DNA, enquanto células CSB apresentam acúmulo de células na fase S, diminuição na síntese de DNA e sinal mais intenso para formação dos complexos Topo II-DNA. Além disso, a complementação das células CSB com a proteína CSB recuperou a resistência das células a MXT e também diminuiu a intensidade do sinal dos complexos Topo II-DNA. Estes resultados indicaram que a via NER está envolvida na resistência das células ao tratamento com MXT e que a proteína CSB ou a subvia TCR-NER tem um papel chave no processamento dos complexos Topo II-DNA. / Mitoxantrone (MXT) is an antineoplastic drug used in treatment of tumors like leukemia, non-Hodgkin lymphoma and breast and prostate cancer. It is classified as an anthracenedione, being a structural analogue of anthracyclines, like doxorubicin (DOX), which action mechanism is based on topoisomerase II (Topo II) inhibition and formation of stabilized Topo II-DNA complexes. Anthracyclines and MXT also can form lesions like DNA adducts, interstrand crosslinks (ICL) and reactive oxygen species (ROS). Studies have shown that nucleotide excision repair (NER) pathway is involved in removal of lesions induced by DOX. Due to structural and action mechanism similarities between MXT and DOX, the aim of this work was to evaluate the influence of NER pathway in cytotoxicity of MXT, in order to elucidate possible mechanisms involved in tumor resistance to this drug. The results demonstrated that NER-deficient cells (XPA, XPD, XPC and CSB) show high sensitivity to MXT compared to repair proficient cells (MRC5). However, CSB cells (deficient in Transcription coupled repair – TCR) were more sensitive to MXT than XPC cells (deficient in Global genome repair – GGR) and also showed differences in cell cycle, DNA synthesis and Topo II-DNA complexes formation upon MXT treatment. XPC cells, in the same way as MRC5 proficient cells present G2/M cell cycle arrest, DNA synthesis recovery and similar signal for Topo II-DNA complexes formation, while CSB cells present accumulation of cells in S phase, reduced DNA synthesis and a more intense signal for Topo II-DNA complexes formation. Moreover, CSB cells complementation recovery MXT-resistance and also diminished Topo II-DNA complexes signal intensity. These results indicate that NER pathway is involved in cells resistance to MXT treatment and that CSB protein or TCR-NER sub pathway has a key role in processing of MXT induced Topo II-DNA complexes.

A mitoxantrona (MXT) é um antineoplásico utilizado no tratamento de tumores como leucemias, linfoma não-Hodgkin e câncer de mama e próstata. Ela é classificada como uma antracenodiona, sendo um análogo estrutural das antraciclinas, como a doxorrubicina (DOX), cujo mecanismo de ação é baseado na inibição da enzima topoisomerase II (Topo II), através da formação dos complexos estabilizados Topo II-DNA. As antraciclinas e a MXT também são capazes de formar lesões do tipo adutos, pontes intercadeias de DNA (interstrand crosslink – ICL) e espécies reativas de oxigênio (ERO). Estudos têm demonstrado que a via de reparo por excisão de nucleotídeos (Nucleotide Excision Repair – NER) está envolvido na remoção de lesões no DNA induzidas pela DOX. Considerando as similaridades estruturais e de mecanismo de ação entre a MXT e a DOX, o objetivo deste trabalho foi avaliar a influência da via NER na citotoxicidade da MXT, a fim de elucidar possíveis mecanismos envolvidos na resistência tumoral a esta droga. Os resultados encontrados demonstraram que células deficientes na via NER (XPA, XPD, XPC e CSB) apresentam elevada sensibilidade a MXT comparadas a células proficientes em reparo (MRC5). Apesar disso, células CSB (deficientes na subvia associada à transcrição - Transcription coupled – TCR-NER) são mais sensíveis a MXT que células XPC (deficientes na subvia de reparo global do genoma – Global genome repair – GGR-NER) e também apresentam diferenças no perfil de ciclo celular, síntese de DNA e formação dos complexos Topo II-DNA após tratamento com MXT. Células XPC, da mesma forma que as células proficientes MRC5 apresentam parada de ciclo celular em G2/M, recuperação da síntese de DNA e sinal semelhante para formação dos complexos Topo II-DNA, enquanto células CSB apresentam acúmulo de células na fase S, diminuição na síntese de DNA e sinal mais intenso para formação dos complexos Topo II-DNA. Além disso, a complementação das células CSB com a proteína CSB recuperou a resistência das células a MXT e também diminuiu a intensidade do sinal dos complexos Topo II-DNA. Estes resultados indicaram que a via NER está envolvida na resistência das células ao tratamento com MXT e que a proteína CSB ou a subvia TCR-NER tem um papel chave no processamento dos complexos Topo II-DNA. / Mitoxantrone (MXT) is an antineoplastic drug used in treatment of tumors like leukemia, non-Hodgkin lymphoma and breast and prostate cancer. It is classified as an anthracenedione, being a structural analogue of anthracyclines, like doxorubicin (DOX), which action mechanism is based on topoisomerase II (Topo II) inhibition and formation of stabilized Topo II-DNA complexes. Anthracyclines and MXT also can form lesions like DNA adducts, interstrand crosslinks (ICL) and reactive oxygen species (ROS). Studies have shown that nucleotide excision repair (NER) pathway is involved in removal of lesions induced by DOX. Due to structural and action mechanism similarities between MXT and DOX, the aim of this work was to evaluate the influence of NER pathway in cytotoxicity of MXT, in order to elucidate possible mechanisms involved in tumor resistance to this drug. The results demonstrated that NER-deficient cells (XPA, XPD, XPC and CSB) show high sensitivity to MXT compared to repair proficient cells (MRC5). However, CSB cells (deficient in Transcription coupled repair – TCR) were more sensitive to MXT than XPC cells (deficient in Global genome repair – GGR) and also showed differences in cell cycle, DNA synthesis and Topo II-DNA complexes formation upon MXT treatment. XPC cells, in the same way as MRC5 proficient cells present G2/M cell cycle arrest, DNA synthesis recovery and similar signal for Topo II-DNA complexes formation, while CSB cells present accumulation of cells in S phase, reduced DNA synthesis and a more intense signal for Topo II-DNA complexes formation. Moreover, CSB cells complementation recovery MXT-resistance and also diminished Topo II-DNA complexes signal intensity. These results indicate that NER pathway is involved in cells resistance to MXT treatment and that CSB protein or TCR-NER sub pathway has a key role in processing of MXT induced Topo II-DNA complexes.

La division cellulaire est un processus fondamental des êtres vivants. À chaque division cellulaire, le matériel génétique d'une cellule mère est dupliqué et ségrégé pour produire deux cellules filles identiques; un processus nommé la mitose. Tout d'abord, la cellule doit condenser le matériel génétique pour être en mesure de séparer mécaniquement et également le matériel génétique. Une erreur dans le niveau de compaction ou dans la dynamique de la mitose occasionne une transmission inégale du matériel génétique. Il est suggéré dans la littérature que ces phénomènes pourraient causé la transformation des cellules cancéreuses. Par contre, le mécanisme moléculaire générant la coordination des changements de haut niveau de la condensation des chromosomes est encore incompris. Dans les dernières décennies, plusieurs approches expérimentales ont identifié quelques protéines conservées dans ce processus. Pour déterminer le rôle de ces facteurs dans la compaction des chromosomes, j'ai effectué un criblage par ARNi couplé à de l'imagerie à haute-résolution en temps réel chez l'embryon de C. elegans. Grâce à cette technique, j'ai découvert sept nouvelles protéines requises pour l'assemblage des chromosomes mitotiques, incluant la Ribonucléotide réductase (RNR) et Topoisomérase II (topo-II). Dans cette thèse, je décrirai le rôle structural de topo-II dans l'assemblage des chromosomes mitotiques et ces mécanismes moléculaires. Lors de la condensation des chromosomes, topo-II agit indépendamment comme un facteur d'assemblage local menant par la suite à la formation d'un axe de condensation tout au long du chromosome. Cette localisation est à l'opposé de la position des autres facteurs connus qui sont impliqués dans la condensation des chromosomes. Ceci représente un nouveau mécanisme pour l'assemblage des chromosomes chez C. elegans. De plus, j'ai découvert un rôle non-enzymatique à la protéine RNR lors de l'assemblage des chromosomes. Lors de ce processus, RNR est impliqué dans la stabilité des nucléosomes et alors, permet la compaction de haut niveau de la chromatine. Dans cette thèse, je rapporte également des résultats préliminaires concernant d'autres nouveaux facteurs découverts lors du criblage ARNi. Le plus important est que mon analyse révèle que la déplétion des nouvelles protéines montre des phénotypes distincts, indiquant la fonction de celles-ci lors de l'assemblage des chromosomes. Somme toute, je conclus que les chromosomes en métaphase sont assemblés par trois protéines ayant des activités différentes d'échafaudage: topoisomérase II, les complexes condensines et les protéines centromériques. En conclusion, ces études prouvent le mécanisme moléculaire de certaines protéines qui contribuent à la formation des chromosomes mitotiques. / Cell division is a fundamental process that continuously happens in all living organisms. In each cell division, genetic material of the parent cell duplicates and segregates to produce genetically identical daughter cells in a process called mitosis. Cells need to condense their genetic material to be able to partition them equally. Any subtle defects, either timing or compaction level, could lead to the unequal inheritance of genetic material, a phenomenon that is believed to be the leading cause of cancerous transformation. However, the precise molecular mechanisms underlying the coordinated changes of higher-order chromosome structure are poorly understood. In the last two decades, various approaches have identified several conserved factors required for chromosome condensation. To define the roles of known and novel factors in this process, I performed an RNAi based screen using high-resolution live imaging of the C. elegans one-cell embryo. Importantly, using an in vivo approach, I discovered seven novel factors required for mitotic chromosome assembly, including Ribonulceotide reducatase (RNR) and DNA topoisomerase II (topo-II). In this thesis, I report a structural role for topo-II in mitotic chromosome assembly and underlying molecular mechanisms. During chromosome condensation process, topo-II acts independently as a local assembly factor leading to global chromosome axis formation, contradicting models that chromosomes organize around preassembled scaffolds, thus representing a novel pathway for chromosome assembly in C. elegans. Furthermore, I also discovered a non-enzymatic role of RNR in the mitotic chromosome assembly process. During this process, RNR is involved in nucleosome stability, and thereby, it allows higher-order chromatin assembly. In this thesis, I also report preliminary data for other novel factors that I discovered in the RNAi based screen for factors involved in chromosome condensation. Importantly, my analyses revealed that the depletion of several proteins results in distinct chromosome condensation phenotypes, indicating that they function in discrete events during mitotic chromosome assembly. In sum, I conclude that metaphase chromosomes are built by the distinct scaffolding activities of three proteins: DNA topoisomerase II, condensin complexes and centromere proteins. Taken together, these studies provide underlying molecular mechanisms contributing to the mitotic chromosome formation.

Inhibitors of topoisomerase II (topo II) are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topo II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However, in clinical trials, modulating the multidrug-resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topo II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topo IIα and topo IIβ isozymes exhibit similar catalytic, but different biologic, activities. Whereas topo IIα is associated with cell division, topo IIβ is involved in differentiation. In addition to site specific mutations that can affect drug-induced topo II-mediated DNA damage, post-translation modification of topo II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topo II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topo II inhibitors. Overall, tumor cell resistance to inhibitors of topo II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.