Elucidating the role of altered DNA damage response in Nup98-associated leukaemia
| Autor: | Nilles, Nadine |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: | |
| Druh dokumentu: | Doctoral Thesis |
| Popis: | Acute myeloid leukaemia is a heterogeneous disease characterized by uncontrolled proliferation of neoplastic haematopoietic precursor cells, which leads to the disruption of normal haematopoiesis and bone marrow failure. Impaired haematopoiesis is often associated with balanced chromosomal translocations that involve the nucleoporin Nup98 fused to around 30 different partner genes, such as the homeobox genes HOXA9 and PMX1. Nup98-associated AML is characterized by poor prognosis and poor treatment outcome for the patients. The aim of the study was to elucidate the mechanisms underlying chemotherapy-resistance. Previous experiments showed that the expression of Nup98 fusion proteins leads to changes in nuclear organization. Based on these observations, we hypothesize that the expression of Nup98 fusion proteins affect DNA double-strand break (DSB) repair. Our work shows that the expression of Nup98-HoxA9 and Nup98-HHEX in U2OS cells does not induce any DSBs. Further, we examined the repair phenotype of exogenously induced DSBs. Experiments carried out using etoposide (ETO) or neocarzinostatin (NCS) revealed that Nup98 fusion proteins affect non-homologous end joining (NHEJ). The second major DSB repair pathway, homologous recombination (HR), remains unaffected by Nup98 fusion proteins. The repair phenotype showed that at most timepoints analyzed, cells expressing Nup98 fusion proteins present less DSBs that control cells. We further performed single cell gel electrophoresis assays, also called COMET assay. This assay determines the amount of broken DNA at the single cell level. COMET assays showed that cells expressing Nup98-HoxA9 get equally damaged as control cells. Taken together, these results show that Nup98-HoxA9 induces faster DNA repair by affecting NHEJ. Additional experiments, pointed toward a role of p53 in the effect of Nup98 fusion proteins on DSB repair. Monitoring the repair phenotype in a wild-type and p53 depletion background, revealed that the effect of Nup98-HoxA9 on NHEJ is partially p53 dependent. A further search for the potentially implicated factor in the accelerated NHEJ remained inconclusive so far. In conclusion, Nup98-HoxA9 induces accelerated NHEJ in a partially p53-dependent manner. Option Biologie moléculaire du Doctorat en Sciences info:eu-repo/semantics/nonPublished |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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