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The role of asparagine synthetase in leukemic cells
Šafrhansová, Lucie ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
This thesis focuses on the detection of mutations in the enzyme asparagine synthetase on leukemia cell metabolism and the role of this enzyme in the context of L-asparaginase- based chemotherapy. The experimental part of the work is divided into two separate sections. Given the lack of asparagine synthetase gene sequencing data in leukemias, the first objective was to determine whether mutations are present in the leukemia cell line that could affect ASNS function and thus play a role in the resistance of leukemia cells to ASNase therapy. No mutations that could affect the activity of the enzyme were detected by next-generation sequencing. In the second part, a model of RS4;11 that expresses ASNS was established. The effect of ASNS on glycolysis was then studied to sensitize these cells to the effects of L-asparaginase and to the depletion of asparagine and glutamine. It was observed that ASNS expression increased the level of glycolysis and increased the resistance of these cells to asparagine and glutamine depletion and their resistance to asparaginase. Key words: ASNS, aspragine, leukemia, L-asparaginase, chemotherapy, drug resistance
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Molecular mechanisms of mutagenesis and resistence in CML cell lineages
Karasová, Dominika ; Čuřík, Nikola (advisor) ; Savvulidi Vargová, Karina (referee)
Chronic myeloid leukemia is a clonal haematopoietic disease, with characteristic BCR-ABL1 fusion gene. Despite the significant improvement in patient treated with tyrosine kinase inhibitors (TKI), 20-30 % of patients develop resistance. One of the main causes of treatment failure are mutations in the BCR-ABL1 kinase domain (KD). The aim of this work was to elucidate the molecular mechanisms of resistance and mutagenesis development in CML using an in vitro CML model KCL-22. The main part of this work was focused on the identification of genes involved in DNA damage response and repair, that could play a role in the process of mutagenesis of BCR-ABL1. We used the RT2 Profiler PCR Arrays method for the group of selected genes regulating DNA damage response and repair. We identified the genes XRCC6 and PARP1 whose gene expression was significantly and specifically decreased during KD BCR-ABL1 mutagenesis. Products of these genes are involved in repairing DNA double-strand breaks through non-homologous end joining (NHEJ). During study of the KD BCR-ABL1 mutagenesis we also found that clones, which developed mutations, did not show the increased BCR-ABL1 expression in the beginning of the culture compared to the clones in which mutations have not evolved. Key words: myeloid leukemia, mutation,...
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Study of translation initiation factors of eIF4E protein family in relation to acute lymphoblastic leukemias and ontogenesis of Tribolium castaneum
Mrvová, Silvia ; Mašek, Tomáš (advisor) ; Doležel, David (referee) ; Čuřík, Nikola (referee)
Translation initiation is one of the key moment in the cell at which many translation initiation factors participate. This step must be carefully regulated not to waste energy. Also excess or lack of many proteins can lead to different diseases. Translation initiation factor eIF4E is important mainly because of its ability to bind a cap and eIF4G and therefore its role in translation initiation. There are three isoforms in human cells - eIF4E1, eIF4E2, eIF4E3. eIF4E1 has been the most studied isoform; however the attention nowadays is drawn to the other two and their functions in cells as well. Upregulation of eIF4E1 is connected with different types of tumors or malignancies. The role of eIF4E isoforms comes to the fore, there has been a research in many model organisms, for example D. melanogaster or C. elegans where the individual isoforms have a role in maturation of oocytes, in forming body axes etc. I focused on all three isoforms of eIF4E, their 3′-UTR regions in lymphoblastic leukemic cells and the utilization of polyadenylation signals of these transcripts because there were shown several types of post-transcriptional regulations, for example stabilization of transcripts by HuR protein. I characterized 3′-UTR regions of eIF4E1, eIF4E2 and eIF4E3 transcripts and confirmed utilization of...
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Transcription factor PU.1 is a target of 5-azacitidine during differentiation therapy of myelodysplastic syndrome
Čuřík, Nikola
PU.1 is a key hematopoietic transcription factor. Knock-out of PU.1 in mouse is embryonic lethal due to complete depletion or several disruption of differentiation of multiple blood cell lineages. Low level of PU.1 and the disruption of its regulation are associated in vivo with acute myeloid leukemia and other hematologic malignancies. Myelodysplastic syndrome (MDS) is hematopoietic stem cell disorder with extremely heterogeneous features and outcome. It is characterized by improper differentiation of blood cells resulting in loss of function, dysplasia and blasts accumulation in bone marrow. About one third of MDS cases transforms into AML. MDS is also characterized by silencing of gene expression caused by aberrant DNA hypermethylation. Using DNA Methyltransferase inhibitors (DNMTi) such as 5-azacitidine (AZA) has good clinical results for the MDS patients with higher risk of disease. Indeed, AZA became standard therapy of high risk MDS in recent years. Nonetheless, our understanding of molecular mechanisms of AZA remains incomplete. This PhD thesis reports about the role of transcription factor PU.1 in MDS. We found that significant subset of high risk MDS patients express low level of PU.1 due to DNA hypermethylation of PU.1 upstream regulatory element (URE). We also found significant...
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Transcription factor PU.1 is a target of 5-azacitidine during differentiation therapy of myelodysplastic syndrome
Čuřík, Nikola ; Stopka, Tomáš (advisor) ; Kleibl, Zdeněk (referee) ; Trka, Jan (referee)
PU.1 is a key hematopoietic transcription factor. Knock-out of PU.1 in mouse is embryonic lethal due to complete depletion or several disruption of differentiation of multiple blood cell lineages. Low level of PU.1 and the disruption of its regulation are associated in vivo with acute myeloid leukemia and other hematologic malignancies. Myelodysplastic syndrome (MDS) is hematopoietic stem cell disorder with extremely heterogeneous features and outcome. It is characterized by improper differentiation of blood cells resulting in loss of function, dysplasia and blasts accumulation in bone marrow. About one third of MDS cases transforms into AML. MDS is also characterized by silencing of gene expression caused by aberrant DNA hypermethylation. Using DNA Methyltransferase inhibitors (DNMTi) such as 5-azacitidine (AZA) has good clinical results for the MDS patients with higher risk of disease. Indeed, AZA became standard therapy of high risk MDS in recent years. Nonetheless, our understanding of molecular mechanisms of AZA remains incomplete. This PhD thesis reports about the role of transcription factor PU.1 in MDS. We found that significant subset of high risk MDS patients express low level of PU.1 due to DNA hypermethylation of PU.1 upstream regulatory element (URE). We also found significant...
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Molecular mechanisms of mutagenesis and resistence in CML cell lineages
Karasová, Dominika ; Čuřík, Nikola (advisor) ; Savvulidi Vargová, Karina (referee)
Chronic myeloid leukemia is a clonal haematopoietic disease, with characteristic BCR-ABL1 fusion gene. Despite the significant improvement in patient treated with tyrosine kinase inhibitors (TKI), 20-30 % of patients develop resistance. One of the main causes of treatment failure are mutations in the BCR-ABL1 kinase domain (KD). The aim of this work was to elucidate the molecular mechanisms of resistance and mutagenesis development in CML using an in vitro CML model KCL-22. The main part of this work was focused on the identification of genes involved in DNA damage response and repair, that could play a role in the process of mutagenesis of BCR-ABL1. We used the RT2 Profiler PCR Arrays method for the group of selected genes regulating DNA damage response and repair. We identified the genes XRCC6 and PARP1 whose gene expression was significantly and specifically decreased during KD BCR-ABL1 mutagenesis. Products of these genes are involved in repairing DNA double-strand breaks through non-homologous end joining (NHEJ). During study of the KD BCR-ABL1 mutagenesis we also found that clones, which developed mutations, did not show the increased BCR-ABL1 expression in the beginning of the culture compared to the clones in which mutations have not evolved. Key words: myeloid leukemia, mutation,...
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Study of dysregulation of DLX1 protein in myeloid leukemia cells in in vitro and in vivo models
Jelínková, Alena ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
The heterogeneous nature of acute myeloid leukemia (AML) worsens the results of patients treated with standard therapy. Understanding the processes of leukemogenesis can contribute to identification of more appropriate treatment. Family of DLX genes (Distal-less homeobox), belonging to the homeobox genes, are associated with haematological malignancies and solid tumors. In the analysis of expression data, the low level of the DLX1 gene was associated with a worse prognosis of patients with AML. In this work we studied phenotypic changes of cell lines with different expression of the DLX1 gene. We silenced the DLX1 gene in AML cell line (sh cells) and compared it to the parental line with higher expression of DLX1 (NSC cells). By cell cycle analysis and apoptosis assays in vitro and in vivo, we have observed the arrest of sh cells in the G0 phase and a lower number of apoptotic cells. Differences were found when measuring the absolute number of cells in time. In in vitro conditions there were less sh cells, in in vivo environment there was significantly higher number of sh cells engrafted in comparison to NSC cells. Further results have shown that sh cells have lower levels of pro-apoptotic proteins and exhibit a higher level of TGF-β targeting PAI-1 gene that activates replicative senescence. We...
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Epigenetic control of PU.1 gene transcription during development of 5-Azacytidine resistance in acute myeloid leukemia
Křtěnová, Petra ; Burda, Pavel (advisor) ; Čuřík, Nikola (referee)
Hematopoiesis is a highly orchestrated process, in which a single hematopoietic stem cell (HSC) gives a rise to all blood cellular components. For myeloid and lymphoid development precise controlled expression of the PU.1 transcription factor is needed. Deletion of PU.1 gene in mouse is lethal and its dysregulation during hematopoietic differentiation is associated with blood malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). MDS and AML are serious blood disorders characterized by expansion of immature blood cells and lack of differentiated functional cells. Not only genetic but also epigenetic aberrations represent a very important field for studying pathophysiology of leukemia genesis and dysregulation of the PU.1 gene represents intensively studied candidate mechanism. Modern therapy of selected MDS and subset of AML patients is based on treatment with DNA hypomethylating agent Azacytidine (AZA) interfering in PU.1 gene regulatory mechanism. However, poor response or resistance to this therapy often occurs. In this thesis we present data obtained from AZA-resistant clones of MDS/AML cell line OCI-M2. We analysed DNA methylation and DNA hydroxymethylation at the key regulatory element of the PU.1 gene (URE). We found that these epigenetic modifications at URE...
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Study of leukemic cells' metabolism in association with response to the therapy
Šimčíková, Markéta ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
Acute lymphoblastic leukemia (ALL) is the most common malignant dise- ase in children. Despite great advancements in treatment of this disease, around 15-20 % of patients suffer a relapse. One of the possible reasons for relapse is developed resistance to cytostatic drugs. L-asparaginase is an im- portant chemotherapy component for childhood ALL and resistance to this drug often complicates treatment. To date, causes of developing resistance have not been sufficiently described. This thesis is a part of a greater research project focusing on mechanisms of L-asparaginase's activity and reasons for developing resistance to this chemotherapeutic agent. Differential metabolic requirements of cancerous cells have been described as early as 1924 by O. H. Warburg and they have been subject to scientific inquiry since. This study aimed to describe the relationship between basal metabolic determinants of leukemia cells and their sensitivity to L-asparaginase. For this reason, two metabolic pathways, glycolysis and oxidative phosphorylati- on, were studied in detail using a Seahorse Bioanalyzer. Further, expression of specific genes involved in glycolysis was detected. Content of mitochon- drial reticulum in cells, expression of the asparagine synthetase gene, and cell size were also studied. Experiments were...
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Transcription factor PU.1 is a target of 5-azacitidine during differentiation therapy of myelodysplastic syndrome
Čuřík, Nikola
PU.1 is a key hematopoietic transcription factor. Knock-out of PU.1 in mouse is embryonic lethal due to complete depletion or several disruption of differentiation of multiple blood cell lineages. Low level of PU.1 and the disruption of its regulation are associated in vivo with acute myeloid leukemia and other hematologic malignancies. Myelodysplastic syndrome (MDS) is hematopoietic stem cell disorder with extremely heterogeneous features and outcome. It is characterized by improper differentiation of blood cells resulting in loss of function, dysplasia and blasts accumulation in bone marrow. About one third of MDS cases transforms into AML. MDS is also characterized by silencing of gene expression caused by aberrant DNA hypermethylation. Using DNA Methyltransferase inhibitors (DNMTi) such as 5-azacitidine (AZA) has good clinical results for the MDS patients with higher risk of disease. Indeed, AZA became standard therapy of high risk MDS in recent years. Nonetheless, our understanding of molecular mechanisms of AZA remains incomplete. This PhD thesis reports about the role of transcription factor PU.1 in MDS. We found that significant subset of high risk MDS patients express low level of PU.1 due to DNA hypermethylation of PU.1 upstream regulatory element (URE). We also found significant...
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