Dans les cellules somatiques, les télomères s'érodent à chaque division cellulaire. Ce processus appelé « Sénescence Réplicative» est contrebalancé de manière basale chez la levure bourgeonnante S. cerevisiae par l'action de la télomérase qui, alors qu'elle est inactive dans les cellules somatiques des eucaryotes supérieures, est activée dans 85% des cancers. Un autre mécanisme impliqué dans les 15% des cas de cancer restants et est appelé Alternative Lengthening of Telomere (ALT). Dans ce processus, le maintien des télomères est assuré par des mécanismes de recombinaison télomérique induisant des échanges de séquences télomériques de chromatides sœurs (T-SCE).Nous avons évalué l'existence d'ALT dans la LLC-B connue pour rarement exprimer la télomérase. Nous avons montrer que 90% des patients LLC-B présentent une diminution de l'expression de TopoIIIα corrélée à une méthylation plus importante des îlots CpG de la région promotrice du gène suggérant que dans les LLC-B le maintien des télomères est défectueux.Nous avons étudié l'implication de la SUMOylation de TopoIIIα/Top3 dans les mécanismes de régulation du ALT. Nous avons montré que TopoIIIα était SUMOylée in vitro et in vivo au sein des cellules U2-OS ALT. Nous avons aussi observé chez S. cerevisiae que Top3 ne serait SUMOylée qu'en absence d'une activité télomérase. Nos résultats suggèrent que la SUMOylation de TopoIIIα augmenterait son activité in vitro et in vivo en diminuant son affinité pour les télomères une fois la recombinaison achevée et qu'elle serait requise pour son accumulation dans les APBs mais pas pour leur formation. / In somatic cells, telomeres erode with each cell division. This process named « Replicative Senescence » is basically counterbalanced in the budding yeast Saccharomyces cerevisiae by the action of telomerase which, while it is inactive in somatic cells of higher eukaryotes is activated in 85 % of cancer cases. Another process of telomere maintenance is involved in 15% of remaining cancer cases and is called Alternative Lengthening of Telomere (ALT). In this process, telomere maintenance is provided by telomeric recombination mechanisms inducing exchange of telomeric sister chromatid (T-SCE).We assessed the existence of an ALT mechanism in B-CLL known to rarely express telomerase. We have shown that 90% of B-CLL patients have a decreased expression of TopoIIIα correlated with largest methylation of CpG islands of the gene promoter region. Our results suggest that in B-CLL, telomere maintenance is defective either by telomerase or ALT mechanism.We investigated the involvement of post- SUMOylation of TopoIIIα/Top3 in mechanisms regulating ALT phenomenon. We have shown that TopoIIIα was SUMOylated in vitro and in vivo in U2-OS ALT cells. We also observed in S. cerevisiae that Top3p might be SUMOylated in absence of telomerase activity. Our results suggested that the SUMOylation of TopoIIIα increased its activity in vitro and in vivo by reducing its affinity for telomeres once recombination occurred and would be required for its accumulation in APBs but not for their formation.

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...

Chez les plantes, les stress biotiques et abiotiques causent la surproduction d’espèces réactives de l’oxygène (ROS) dans les chloroplastes et les mitochondries. Ces ROS provoquent des dommages parfois irrémédiables pour la cellule ou déclenchent des voies de signalisation rétrogrades qui induisent la tolérance au stress. En parallèle, le génome nucléaire contrôle le développement et l’activité des organites grâce à la signalisation antérograde. Dans ce contexte, le complexe nucléaire Topoisomérase VI (Topo VI) a été étudié chez Arabidopsis thaliana. Topo VI est un régulateur des gènes de réponse à la signalisation rétrograde de la ROS oxygène singulet ; toutefois, les mécanismes qui régissent cette régulation sont inconnus. Afin de les déterminer, des interacteurs de la Topo VI ont été mis en évidence dont la sous-unité alpha du facteur général de transcription TFIIF (TFIIFα). Après caractérisation de l’expression du gène TFIIFα et des isoformes protéiques codées, des analyses transcriptomiques réalisées en condition standard de croissance et en condition de stress photo-oxydant ont montré que Topo VI et TFIIFα co-régulent la majorité des gènes de réponse à ce stress. De plus, ces interacteurs ont un rôle opposé dans la régulation de l’expression de gènes « PentatricoPeptide Repeat » (PPR). Les protéines PPR sont adressées aux chloroplastes et aux mitochondries dans lesquels elles participent à la régulation post-transcriptionnelle ; chez des mutants tfIIfα, des gènes PPR impliquées dans l’édition des ARN sont réprimés et l’édition de sites cibles associés est parfois altérée. Les conséquences de ces altérations sont discutées par l’analyse de fonctions chloroplastiques. / Under biotic and abiotic stress, plants overproduce reactive oxygen species (ROS) in plastids and mitochondria. ROS can cause damages sometimes definitive for cells or trigger retrograde signalling pathways to the nucleus that provide stress tolerance. Concurrently, the nuclear genome controls the development and the activity of organelles through anterograde signalling. In this context, the nuclear complex Topoisomerase VI (Topo VI) is studied in Arabidopsis thaliana. Topo VI is a regulator of stress responsive genes in the retrograde signalling triggered by the singlet oxygen ROS; nevertheless, the mechanisms controlling this regulation are still unknown. To determine them, Topo VI interactors were found out, including the alpha subunit of the general transcription factor TFIIF (TFIIFα). TFIIFα gene expression and the encoded protein isoforms were first characterized and then, transcriptomic analysis in standard growth conditions and under photo-oxidative stress showed that Topo VI and TFIIFα co-regulate most of stress responsive genes. Moreover, these interactors have an opposite role in the expression of "PentatricoPeptide Repeat" (PPR) genes. PPR proteins are targeted to plastids or mitochondria in which they contribute to post-transcriptional regulation mechanisms; in tfIIfα mutants, PPR genes involved in organellar RNA editing are down-regulated, and the editing of associated target sites was sometimes impaired. Possible consequences of this impairment were discussed through the analysis of plastidial functions.

Topoisomerases são enzimas nucleares que participam na regulação da estrutura do DNA nas células eucarióticas. A topoisomerase III é o mais novo membro da família das topoisomerases. Seu papel no desenvolvimento dos tumores mamários ainda necessita ser investigado. O objetivo do presente estudo foi avaliar a imunoexpressão da topoisomerase III nos carcinomas mamários, e comparar sua expressão com dados clinicopatológicos e marcadores imunohistoquímicos clássicos de importância prognóstica nos carcinomas mamários. Utilizando-se tissue microarrays contendo 171 casos de carcinomas ductais mamários primários, foi analisada a expressão imunohistoquímica de topoisomerase III, receptor de estrógeno, receptor de progesterona, HER-2, Ki67, p53 e BRCA-1. Positividade para topoisomerase III foi encontrada em 33,9% dos casos, e sua expressão relacionou-se com metástases à distância (p=0.036) e óbito (p=0.006). Negatividade para topoisomerase III relacionou-se com negatividade para HER=2 (p<0.001), p53 (p<0.001) e BRCA-1 (p=0.001), e com baixa expressão de Ki-67 (p<0.001). Na Análise de Riscos Múltiplos de Cox, a expressão de topoisomerase III foi um significante preditor de sobrevida [razão de risco 3.006 (intervalo de confiança a 95%: 1.582-5.715); p=0.001]. Concluindo, a topoisomerase III pode ser útil na avaliação do prognóstico de pacientes com câncer de mama, além de ser um fator independente de predição da sobrevida. / Topoisomerases are ubiquitous nuclear enzymes that regulate DNA structure in eukaryotic cells. The role of topoisomerase III, the newest member of the topoisomerase family, in the clinical outcome of breast cancer is still poorly understood. This study aims to investigate the immunoexpression of topoisomerase III in breast cancer and its relationships with clinicopathological features and immunohistochemical markers of prognostic significance in breast pathology. Using tissue microarrays containing 171 cases of primary invasive breast cancer, we analyzed the immunoexpression of topoisomerase III, estrogen receptor, progesterone receptor, HER-2, Ki67, p53 and BRCA-1. Immunostaining for topoisomerase III was found in 33.9% of breast carcinomas, and immunopositivity was related with distant metastasis (p=0.036) and death (p=0.006). Decreased expression of topoisomerase III was related with negativity with HER-2 (p<0.001), p53 (p<0.001) and BRCA1 (p=0.001) and low expression of Ki67 (p<0.001). In the multivariate analysis, topoisomerase III expression was a significant predictor of survival [hazard ratio 3.006 (95% confidence interval 1.582-5.715); p=0.001]. In conclusion, topoisomerase III expression can be a useful marker in assessing the prognosis of patients with breast cancer and is an independent predictor of survival.

In order to evaluate the suitability of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) as a potential drug target for an anti-parasite therapy, we are studying its role in the bloodstream form of Trypanosoma brucei brucei, the eukaryotic parasite that causes African Sleeping Sickness. Eukaryotic TDP1 removes covalently trapped topoisomerase IB and other adducts from the 3’ end of the DNA at DNA strand breaks. Covalent topoisomerase IB stalling is caused by endogenous DNA damage and by anti-cancer drugs such as camptothecin (CPT). A potential approach could be to use TDP1 inhibitors synergistically with CPT in a combined anti-parasite therapy. T. brucei TDP1 knock out cells are hypersensitive to CPT and accumulate in the late S phase of the cell cycle upon treatment with the drug. The CPT hypersensitivity of the TDP1-/- cells can be fully rescued through ectopic expression of wild type TDP1. The catalytic activity of TDP1 is required for complementation of the CPT sensitivity since overexpression of a catalytically inactive mutant form of TDP1 further sensitises TDP1-/- cells to CPT. In this context, expression of the mutant H358N, which shows reduced activity, also increases sensitivity of TDP1-/- cells to the drug. Surprisingly, expressing TDP1 carrying an analogous mutation to the one that causes SCAN1, a human neurodegenerative disease, does not sensitise TDP1-/- cells further. With this unique set of mutant TDP1 proteins in a TDP1-/- background we hope to answer questions concerning TDP1 function that have so far been elusive.

La famille des GTPases Rho, comprenant 20 membres, contrôle la dynamique du cytosquelette d'actine et différents processus cellulaires comme la migration. En plus de leurs rôles bien établis, certaines GTPases Rho, notamment RhoB et Rac1, ont émergé en tant que gènes de réponse précoce aux dommages à l'ADN. En effet, RhoB est induite en réponse à divers stress génotoxiques, y compris la camptothécine (CPT), les UV et le cisplatine, et protège principalement les cellules de l'apoptose. Le rôle des autres GTPases Rho en réponse précoce aux génotoxiques reste largement méconnu. Dans ce projet, nous avons utilisé la camptothécine, un inhibiteur de la topoisomérase I (TOP1), qui stabilise sélectivement les complexes de clivage TOP1-ADN (TOP1cc) sur la chromatine, afin de cribler les GTPases Rho induites de façon précoce par les dommages à l'ADN. En plus de RhoB, nous avons identifié RND1 comme un gène rapidement induit par la CPT. L'induction de RND1 est réversible et étroitement corrélée à la présence de TOP1cc induit par la CPT. En accord avec ces observations, les rayons UV et le péroxyde d'hydrogène, qui stabilisent indirectement les TOP1cc, induisent également RND1. La CPT augmente la transcription de RND1 indépendamment de l'activité de son promoteur minimal. De plus, la CPT augmente l'activité de la poly ADP-ribose polymérase (PARP1), dont l'inhibition prévient la transcription de RND1. La surexpression de RND1 augmente également l'expression de PARP1, suggérant une régulation positive entre PARP1 et RND1 en réponse aux TOP1cc. Ainsi, nous proposons qu'en réponse à la CPT, les TOP1cc activent PARP1, qui à son tour favorise la transcription de RND1, initiant ainsi une boucle de rétrocontrôle positive. Enfin, nous avons montré que RND1 protège les cellules contre l'apoptose induite par la CPT et entraîne leur résistance à la CPT. L'ensemble de ces résultats ont permis d'identifier RND1 comme nouvelle GTPase Rho impliquée dans la réponse au stress et proposent un nouveau mécanisme de régulation de la transcription des gènes en réponse aux TOP1cc via l'activation de PARP1. Ces résultats suggèrent par ailleurs qu'inhiber la signalisation de RND1 pourrait sensibiliser les cellules tumorales aux dérivés cliniques de la CPT. / Rho GTPase family comprises 20 members that regulate key cellular functions such as actin cytoskeleton organization and migration. Beside their canonical functions, certain Rho GTPases, including RhoB and Rac1, emerged as early DNA damage-inducible genes. Indeed, RhoB is readily induced in response to various genotoxic stress, including camptothecin (CPT), UV and cisplatin, and primarily protect cells against apoptotic cell death. Whether other Rho GTPases also respond early to genotoxics is largely unknown. In this project, we used camptothecin, a topoisomerase I (TOP1) inhibitor that selectively stabilized TOP1-DNA cleavage complexes (TOP1cc) onto chromatin, to screen for early DNA damage-inducible Rho GTPases. Besides RhoB, we identified RND1 as a gene rapidly induced by CPT. RND1 induction is reversible and closely associated with the presence of TOP1cc induced by CPT. Consistently, UV light and hydrogen peroxide, which indirectly stabilized TOP1cc, induce RND1 as well. CPT increases minimal promoter-independent RND1 transcription. Additionally, CPT increases poly ADP-ribose polymerase (PARP1) activity, whose inhibition prevents RND1 transcription. Overexpression of RND1 also increases PARP1 expression, suggesting a positive regulation between PARP1 and RND1 in response to TOP1cc. Thus, we propose that in response to CPT, TOP1cc activate PARP1, which in turn promotes RND1 transcription resulting in a positive feedback loop. Finally, we found that RND1 protects cells against CPT-induced apoptosis and leads to resistance to CPT. Together, these results highlight RND1 as a new Rho GTPase involved in the response to stress and propose a new mechanism for TOP1cc-induced gene transcription through PARP1 activation. These findings further suggest that inhibiting RND1 signaling could sensitize tumor cells to CPT derivatives.

Topoisomerases são enzimas nucleares que participam na regulação da estrutura do DNA nas células eucarióticas. A topoisomerase III é o mais novo membro da família das topoisomerases. Seu papel no desenvolvimento dos tumores mamários ainda necessita ser investigado. O objetivo do presente estudo foi avaliar a imunoexpressão da topoisomerase III nos carcinomas mamários, e comparar sua expressão com dados clinicopatológicos e marcadores imunohistoquímicos clássicos de importância prognóstica nos carcinomas mamários. Utilizando-se tissue microarrays contendo 171 casos de carcinomas ductais mamários primários, foi analisada a expressão imunohistoquímica de topoisomerase III, receptor de estrógeno, receptor de progesterona, HER-2, Ki67, p53 e BRCA-1. Positividade para topoisomerase III foi encontrada em 33,9% dos casos, e sua expressão relacionou-se com metástases à distância (p=0.036) e óbito (p=0.006). Negatividade para topoisomerase III relacionou-se com negatividade para HER=2 (p<0.001), p53 (p<0.001) e BRCA-1 (p=0.001), e com baixa expressão de Ki-67 (p<0.001). Na Análise de Riscos Múltiplos de Cox, a expressão de topoisomerase III foi um significante preditor de sobrevida [razão de risco 3.006 (intervalo de confiança a 95%: 1.582-5.715); p=0.001]. Concluindo, a topoisomerase III pode ser útil na avaliação do prognóstico de pacientes com câncer de mama, além de ser um fator independente de predição da sobrevida. / Topoisomerases are ubiquitous nuclear enzymes that regulate DNA structure in eukaryotic cells. The role of topoisomerase III, the newest member of the topoisomerase family, in the clinical outcome of breast cancer is still poorly understood. This study aims to investigate the immunoexpression of topoisomerase III in breast cancer and its relationships with clinicopathological features and immunohistochemical markers of prognostic significance in breast pathology. Using tissue microarrays containing 171 cases of primary invasive breast cancer, we analyzed the immunoexpression of topoisomerase III, estrogen receptor, progesterone receptor, HER-2, Ki67, p53 and BRCA-1. Immunostaining for topoisomerase III was found in 33.9% of breast carcinomas, and immunopositivity was related with distant metastasis (p=0.036) and death (p=0.006). Decreased expression of topoisomerase III was related with negativity with HER-2 (p<0.001), p53 (p<0.001) and BRCA1 (p=0.001) and low expression of Ki67 (p<0.001). In the multivariate analysis, topoisomerase III expression was a significant predictor of survival [hazard ratio 3.006 (95% confidence interval 1.582-5.715); p=0.001]. In conclusion, topoisomerase III expression can be a useful marker in assessing the prognosis of patients with breast cancer and is an independent predictor of survival.

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.

<p>Breast cancer mortality in Sweden is now in decline, thanks to early detection and the wide use of adjuvant endocrine therapy and chemotherapy. </p><p>While hormone receptor status is predictive of response to endocrine treatment, there is no clinically useful predictive marker of a patient’s response to chemotherapy. Consequently, patients receive chemotherapy with considerable toxicity but minimal benefit. The aim of this thesis was to investigate a number of methods with the potential to predict response to chemotherapy and thus enhance treatment efficacy in breast cancer patients.</p><p>We found that topo IIα, the key target enzyme of topo II inhibitors, is significantly expressed in nonproliferating breast cancer cells. This finding may explain why topo II inhibitors are effective in patients with slow growing tumors and a low proliferation rate.</p><p>Topo IIα gene amplification was suggestive of increased response to anthracyclines in advanced breast cancer, whereas the oncogene HER2 had no predictive value by itself. These findings are in accordance with current knowledge.</p><p>Cyclin A, a marker of cell proliferation, showed good prognostic value but did not predict response to chemotherapy in advanced breast cancer.</p><p>In vitro chemosensitivity testing with FMCA predicted tumor response in patients with advanced breast cancer with a sensitivity of 89% and a specificity of 53%. Our results are consistent with the results from similar assays, which predict drug resistance with good accuracy while clinical drug sensitivity is less reliably predicted. The use of FMCA and similar assays is not yet recommended outside clinical trials; their main utility is in preclinical testing of new anti-cancer drugs, including targeted therapies.</p><p>The combination of epirubicin, capecitabine, and cisplatin (EXC) demonstrated high clinical response rate (74%) and pathological complete response rate (22%) in locally advanced breast cancer, but with cumbersome toxicity. The fluoropyrimidine biomarkers TS, TP, and DPD did not predict response to the EXC regimen.</p>

<p>Antibiotic-resistant bacteria are increasingly a major healthcare problem but very few new classes of antibiotics have been discovered or launched in recent decades. Approaches to dealing with the problem include learning how bacteria evolve to resistance and improving dosing regimens with current antibiotics so as to reduce the selection of resistant bacteria. </p><p>This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in <i>Escherichia coli</i>. It also addresses the genetics of fluoroquinolone resistance in <i>E. coli</i> in relation to fitness costs for the resistant bacteria, and the evolution of <i>E. coli</i> to reduce the costs of resistance.</p><p>The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection <i>E. coli</i> strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an <i>in vitro</i> kinetic model showed that an AUC/MPC >22 for ciprofloxacin was the single best pharmacodynamic index that predicted prevention of resistance emergence in the wild-type. Simulating currently approved dosing regimens for three different fluoroquinolones it was found that only a few were effective in preventing the selection of a small sub-population of pre-existing mutants. </p><p>Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness<i> in vitro</i> and <i>in vivo</i>. Systematic construction of isogenic resistant strains confirmed this result and revealed that some combinations of resistance mutations mutually compensate and increase both resistance and fitness. It was discovered that mutations altering RNA polymerase could ameliorate the fitness costs of fluoroquinolone resistance. Thus, the major fitness cost of fluoroquinolone resistance is due to defective transcription. </p><p>The finding that fluoroquinolone resistance mutations can increase resistance while mutually compensating their fitness costs, shows that resistance to fluoroquinolones can continue to evolve in the absence of antibiotic selection.</p>