1,721,097 research outputs found
Molecular pathways of telomere maintenance
Full text linkTelomeres are the physical ends of the linear eukaryotic chromosomes, protecting them against being recognised as double-stranded DNA breaks (DSBs) and preventing them from fusing with each other. Due to the progressive shortening of telomeric DNA during each round of DNA replication, telomeres eventually lose their protective capacity. This “end replication problem” is counteracted by telomerase. Nevertheless, telomerase is not expressed at sufficient levels to prevent telomere attrition in most human somatic cells, which impacts two critical features of human health. On the one hand, progressive telomere shortening results in replicative senescence, which is a hallmark of ageing. In contrast, reactivation of telomerase is a characteristic in most cancer cells, allowing them to divide indefinitely. For these reasons, uncovering molecular details in different aspects of telomere biology is highly relevant.My work covers parts of three main features of telomere biology: telomere protection, the end replication problem, and telomere replication stress. Firstly, in terms of telomere protection, we investigated the role and consequences of telomeres depleted of the major protein associated with double-stranded telomeric DNA in budding yeast, Rap1.Secondly, to investigate how telomerase is recruited to telomeres to solve the end replication problem, we determined that Rap1-depleted telomeres dramatically increases the telomere extension frequency. Finally, besides the end replication problem, there is a second and less-recognised problem where the DNA replication forks often stall and collapse while traversing telomere sequences. To investigate these characteristics, we examined the properties of interstitial telomeric sequences (telomere sequences located internally in the genome)
Identifying aneuploidy-tolerating genes
Full text linkWhen cells mis-segregate chromosomes during cell division, this can lead to cells having an abnormal number of chromosomes, a state called aneuploid. Aneuploidy imposes a metabolic burden, reduces cellular fitness and induces a cellular stress response. Despite this aneuploidy-induced stress, two-third of the cancers are aneuploid. Apparently aneuploid cancer cells have mechanisms to tolerate the disadvantages imposed by aneuploidy. The overall aim of this thesis is to identify aneuploidy-tolerating mechanisms. Specifically, our research shows that aneuploidy combined with p53 deficiency in murine T cells deficiency accelerates T cell lymphoma development. These aneuploid murine T cell lymphomas have overexpression of Prmt5 (protein methyltransferase 5). We therefore studied the role of PRMT5 in aneuploid cells using different approaches. We identified that PRMT5 is a sensor for the amino acid methionine to activate mTORC1, which is essential for cell growth and metabolism, which could explain the need of Prmt5 overexpression in aneuploid cancer cells. To identify aneuploidy-tolerating genetic changes in a more unbiased fashion, we performed an in vivo genetic screen, in which we combined transposon-mediated mutagenesis with aneuploidization of the haematopoietic system. Combination of aneuploidy and transposon mutagenesis reduced tumour latency significantly compared to transposon mutagenesis alone, confirming that aneuploidy also accelerates tumorigenesis in this setting. Preliminary analysis of the transposon common insertion sites in these tumours is suggesting that Foxp1 and Notch1 are potential genes involved the aneuploidy tolerization. Altogether, we have found new mechanisms that facilitate tolerance of aneuploidy, which can be potential targets in the treatment of aneuploid cancer
Causes and consequences of mitotic DNA damage
Full text linkIn order to divide, a cell has to successfully duplicate its DNA during S-phase, followed by the division of genomic content during mitosis. Faithful cell division in cancer cells can be challenged in various ways. First, an increase in the formation of DNA lesions can challenge correct DNA duplication, for instance, due to oncogene overexpression or the inability to resolve DNA lesions due to defective DNA repair mechanisms. Second, the faithful division of genomic content over the emerging daughter cells can be impaired due to mitotic aberrations. The overall aim of this thesis was to dissect the mechanisms by which replication-born DNA lesions affect mitotic behavior of cancer cells, to ultimately improve treatment of genomically instable cancers. We describe how replication-born DNA lesions induced by BRCA1/2 inactivation combined with PARP and ATR inhibitor treatment affect mitotic behavior. We show that ATR inhibition potentiates PARP inhibitor treatment in homologous recombination-deficient cells, which was related to the role of ATR in regulating cell cycle control. Furthermore, we studied how overexpression of the CCNE1 oncogene (encoding Cyclin E1) affects mitotic behavior. We found that overexpression of Cyclin E1 caused mitotic aberrancies, which could be exacerbated by the inhibition of the G2/M checkpoint kinases ATR or WEE1. Moreover, we found Cyclin E1 overexpression to result in the activation of RAD52-dependent Mitotic DNA synthesis (MiDAS) pathway. Inhibition of the MiDAS pathway resulted in an increase in residual DNA damage following mitosis, implicating a possible crucial role for MiDAS in dealing with increased levels of DNA lesions in mitosis
Replication-stress induced mitotic aberrancies in cancer biology
Full text linkOne of the most important challenges in the treatment of cancer is to obtain successful tumor clearance, while at the same time making sure that healthy tissues remain unharmed. To achieve this, a better understanding of tumor-specific properties is required to uncover tumor-selective treatments. One example of such a strategy is the specific cytotoxic effect of PARP inhibitors in tumors harboring mutations in the BRCA1 or BRCA2 genes. However, how PARP inhibitors exert their effects remains unclear. In this thesis, we show PARP inhibitors to impair replication fork stability in homologous recombination (HR)-deficient cancer cells. Furthermore, we show that replication-born lesions are transmitted into mitosis, resulting in mitotic defects, including the formation of chromatin bridges during anaphase. If left unresolved, chromatin bridges lead to cytokinesis failure. Our findings indicate that mitotic progression is essential for PARP inhibitor cytotoxicity. Furthermore, we show that ATR inhibition potentiates PARP inhibitor treatment in HR-deficient cells, which was related to a role for ATR in regulating cell cycle control. Indeed, ATR inhibition induced premature mitotic entry, enhancing chromatin bridges and lagging chromosomes. Mitotic aberrancies are not only observed following PARP inhibitor treatment. We observed that also oncogene-induced replication stress impaired the ability of cells to properly distribute chromosomes over daughter cells. Interestingly, cells harboring oncogene-induced mitotic aberrancies were highly sensitive to inhibition of checkpoint kinases ATR and WEE1. For this reason, the findings presented in this thesis could aid in the selective killing of cancer cells by specific targeting of cells with replication stress-induced mitotic aberrancies
Evolution of karyotype landscapes in cancer
Full text linkChromosomal instability (CIN) is a phenomenon where cells mis-segregate chromosomes more frequently than normal. As a result of CIN, cells can gain and lose whole chromosome and acquire an aneuploid karyotype. Despite their association with detrimental cell growth, both aneuploidy and CIN are commonly observed in proliferating cancer cells. It is suggested that CIN provides adaptive capacity to cells by enabling rapid emergence of genomic alterations that enhance tumorigenesis or metastatic potential. CIN has been shown to contribute to intra-tumour heterogeneity, metastasis, and therapy resistance. However, a better understanding of the dynamics of karyotype evolution is still needed to understand how CIN drives tumour progression. Most existing methods used to map tumour karyotypes cannot fully dissect intra-tumour karyotype heterogeneity. The overall aim of this thesis is to better understand karyotype evolution in tumour cells that undergo continuous CIN using single-cell sequencing and state-of-the-art mouse models. A new bioinformatics method, Aneufinder, was developed to determine single-cell karyotypes in CIN-driven tumour cells. Using single-cell sequencing and Aneufinder in mouse models for T-cell lymphoma, we show that tumours quickly select for recurrent karyotype aberrations to promote tumour growth. These findings were supported using an in silico model and further investigated in various human cancers, including B-cell leukaemia, basal cell carcinoma, and paediatric cancers. Together these data underline a role for CIN driving tumour heterogeneity and adaptivity, that can be used to further understand tumour progression and thus help improve clinical prognosis
CINister thoughts
No full textChromosome instability (CIN) is the process that leads to aneuploidy, a known hallmark of human tumours for over a century. Nowadays, it is believed that CIN promotes tumorigenesis by shuffling the genome into a malignant order through translocations, amplifications, deletions (structural CIN), and gains and losses of whole chromosomes (numerical CIN or nCIN). The present review focuses on the causes and consequences of nCIN. Several roads can lead to nCIN, including a compromised spindle assembly checkpoint, cohesion defects, p53 deficiency and flawed microtubule-kinetochore attachments. Whereas the link between nCIN and tumorigenesis is becoming more evident, indications have emerged recently that nCIN can suppress tumour formation as well. To understand these paradoxical findings, novel reagents and more sophisticated mouse models are needed. This will provide us with a better understanding of nCIN and eventually with therapies that exploit this characteristic of human tumours
Aneuploidy in the human brain and cancer: Studying heterogeneity using single-cell sequencing
Full text linkWanneer een cel niet het normale aantal chromosomen bevat is de cell aneuploid. Er zijn meerdere methoden om aneuploidy te bestuderen. In dit proefschrift wordt een overzicht gegeven van de meest gebruikte methoden met de voor- en nadelen van elke methode. In het ontwikkelend en volwassen menselijk brein zijn met fluorescentie in situ hybridisatie (FISH) aneuploide cellen gevonden en zelfs nog hogere aantallen in brein dat is aangedaan door een neurodegeneratieve ziekte. In tegenstelling tot deze studies met FISH, worden met single cell sequencing veel lagere aantallen aneuploide cellen gevonden. Om meer inzicht te krijgen in de aanwezigheid van aneuploïde cellen in normaal hersenweefsel en daarmee de mogelijke rol van aneuploïdie in de ziekte van Alzheimer hebben we individuele cellen gesequenced van normaal brein en brein in verschillende stadia van de ziekte van Alzheimer. In tegenstelling tot de met FISH verkregen data, is er uit ons onderzoek gebleken dat er heel weinig aneuploïde cellen in normaal brein aanwezig zijn. Bovendien hebben we vergelijkbare hoeveelheden aneuploïde cellen gevonden in normaal brein als in brein van patiënten met de ziekte van Alzheimer. Aneuploidie speelt waarschijnlijk geen belangrijke rol in de ontwikkeling en progressie van de ziekte van Alzheimer. In tegenstelling tot het brein, is het bekend dat aneuploïdie een belangrijke rol speelt in kanker. Aneuploïde tumoren zijn geassocieerd met een slechtere prognose voor de patiënt. De heterogeniteit van een tumor kan worden bepaald met single cell sequencen. Wij hebben met behulp van single cell sequencing gekeken naar de rol van heterogeniteit in kleincellige longkanker in de primaire tumor en verschillende metastasen. Hieruit is gebleken dat er grote verschillen zijn tussen de heterogeniteit van de primaire tumor en de metastasen en er binnen één patiënt zowel monoklonale als polyklonale metastasen kunnen ontstaan
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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