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Molecular mechanisms of tumor pathogenesis of Hedgehog signaling pathway in selected tumor types
Kreisingerová, Kateřina ; Vachtenheim, Jiří (advisor) ; Macůrek, Libor (referee) ; Uldrijan, Stjepan (referee)
The presented doctoral thesis is focused on the role of the Hedgehog (HH) signaling pathway in cancer pathogenesis. HH signaling pathway is an evolutionarily conserved signaling pathway that plays an essential role in embryonic development. Its activity is strictly limited to stem and progenitor cells for example in brain, lung, skin or prostate. HH pathway also plays a key role in tissue homeostasis and regeneration. Aberrantly activated HH pathway is essential in cancer progression. The aim of the presented thesis was to elucidate new details about the HH signaling pathway. We identified a new target gene of the HH pathway - the anti-apoptotic protein survivin. Survivin is considered to be an important tumor marker associated with a poor prognosis of patients. We showed that the inhibitor of HH pathway effectors GLI1 and GLI2 GANT61 reduced the survivin level in cancer cells. Subsequently, we used GANT61 and the inhibitor of the anti-apoptotic BCL2 protein family obatoclax to inhibit melanoma cells growth. We showed that the combination of these inhibitors was very effective in the eradication of melanoma cells in vitro. We also proved that GANT61 triggers the process of apoptosis in melanoma cells. We found out that the HH signaling pathway is canonically activated in many cell lines of various...
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Role of the MRN complex in the nucleolar DNA damage response
Palková, Natálie ; Macůrek, Libor (advisor) ; Hanzlíková, Hana (referee)
Genome integrity maintenance is crucial for proper functioning and survival of all organisms, especially if the cell is constantly exposed to various genotoxic agents. For that reason, there are specific mechanisms that detect DNA damage, facilitate signalling and promote repair of the damaged region. These processes are referred to as DNA damage response (DDR). Necessary part of the DDR is also the MRE11-RAD50-NBS1 complex (MRN), comprised of the nuclease MRE11, ATPase RAD50 and regulatory docking protein NBS1. The MRN complex has an indispensable role in the detection and immediate resection of double-strand breaks (DSBs), signal transduction and activation of ataxia telangiectasia mutated (ATM) kinase with its downstream effectors necessary for the DDR. The compounds of the MRN complex are involved in processes crucial for efficient DNA repair, cell survival and maintenance of genomic stability. The main aim of this work is to elucidate less known functions of the MRN complex in the nucleoli, nuclear membrane-less organelles formed around the copies of genes coding rRNA. This work discusses how the MRN complex is involved in the repair of rDNA double-strand breaks, transient inhibition of rRNA transcription or nucleolar segregation. Thereafter, this work puts into context the latest knowledge in...
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The functional in vitro analysis of the BRCA1alternative splicing variants
Ševčík, Jan ; Kleibl, Zdeněk (advisor) ; Stopka, Tomáš (referee) ; Macůrek, Libor (referee)
BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...
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Genomic instability in patient tumors due to excesive AID activity
Vaníčková, Karolína ; Drbal, Karel (advisor) ; Macůrek, Libor (referee)
AID is a member of APOBEC family of mutational enzymes. AID generates U:G mismatches in ssDNA by deaminating cytosine to uracil. In B cells error-prone repair of these mismatches induces a mutational burden in the process of somatic hypermutation of Ig locus during affinity maturation of immunoglobulins (Ig). AID also induces double-strand breaks during Ig class switch recombination or primary Ig diversification through templated gene conversion in some vertebrate species. AID might gain tumorigenic potential in case of insufficient regulation of induction and repair processes, causing genomic instability and possibly leading to tumorigenesis. AID is induced in epithelial tissues by proinflammatory cytokines via canonical NF-B pathway. Both exogenous factors (pathogens Helicobacter pylori or HCV), endogenous factors (bile acid) or even physiological state such as ovulation are the initiating factors. Thus, AID might be the link between inflammation and carcinogenesis. AID is expressed in different stages of carcinomas, mostly during the initial oncogenic transformation. Mice with ectopic AID expression develop lung, gastric, oral and hepatic carcinomas as well as melanomas. AID also regulates epithelial-mesenchymal transition in other tumors. AID is responsible for treatment resistance in both CML...
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Role of Rad18 in genome stability
Palek, Matouš ; Macůrek, Libor (advisor) ; Šolc, Petr (referee)
Rad18 is an E3 ubiquitin ligase well-known for its function in DNA damage tolerance (DDT). Especially, its role in translesion DNA synthesis, one of two DDT branches, was extensively studied in the past. Recently, Rad18 was shown to be involved in the repair of DNA double- strand breaks (DSBs) in mammalian cells. The role of Rad18 in human cells seems to be important since DSB repair as well as DDT pathway are essential for maintenance of genome stability. In this work, I introduce the function of Rad18 in both DDT pathways, translesion DNA synthesis (TLS) and template switching (TS). Then I summarize current knowledge about the role of human Rad18 in DSB repair. Finally, I describe potential involvement of Rad18 dysregulation in human cancer, since loss of genome integrity is an important driving force for tumorigenesis. Keywords: Rad18, genome stability, DNA double-strand break repair, tumorigenesis, DNA damage tolerance, translesion DNA synthesis, template switching.
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Molecular characterization of γ -tubulin interactions with signalling molecules
Macůrek, Libor ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Svoboda, Augustin (referee)
52 V. CONCLUSIONS The results of presented PhD thesis can be summarized as follows: For the first time it has been demonstrated that γ-tubulin forms complexes with αβ-tubulin dimers in brain tissue as well as in other models of neuronal differentiation. Two forms of γ- tubulin have been identified in complexes of various sizes. It has been shown that γ-tubulin is posttranslationally modified. One of the identified posttranslational modifications of γ-tubulin is phosphorylation that appears to depend on Src family kinase activity. It has been proposed that posttranslational modifications of γ-tubulin may regulate interactions of γ-tubulin with αβ-tubulin heterodimers or other associated proteins during neurogenesis. It has been shown that γ-tubulin associates with protein tyrosine kinases involved in signal transduction events. γ-Tubulin interaction with Src family kinases significantly increased after long-term RA-activation embryonal carcinoma P19 cells. A similar increase has been observed after rapid activation of mast cells, indicating that this regulatory mechanism is not restricted to a particular model system. In both models, Src family kinases bound to γ-tubulin are active and phosphorylate proteins present in γ-tubulin complexes. Fyn kinase interacts with γ-tubulin through its SH2 domain in a...
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Investigating critical mechanisms of oncogenesis using cell model systems
Hušková, Hana ; Stopka, Tomáš (advisor) ; Macůrek, Libor (referee) ; Vojtěšek, Bořivoj (referee)
(EN) Humans and cells in their bodies are exposed to various mutagens in their lifetime that cause DNA damage and mutations, which affect the biology and physiology of the target cell, and can lead to the expansion of an immortalized cell clone. Genome-wide massively parallel sequencing allows the identification of DNA mutations in the coding sequences (whole exome sequencing, WES), or even the entire genome of a tumour. Mutational signatures of individual mutagenic processes can be extracted from these data, as well as mutations in genes potentially important for cancer development ('cancer drivers', as opposed to 'passengers', which do not confer a comparative growth advantage to a cell clone). Many known mutational signatures do not yet have an attributed cause; and many known mutagens do not have an attributed signature. Similarly, it is estimated that many cancer driver genes remain to be identified. This Thesis proposes a system based on immortalization of mouse embryonic fibroblasts (MEF) upon mutagen treatment for modelling of mutational signatures and identification and testing of cancer driver genes and mutations. The signatures extracted from WES data of 25 immortalized MEF cell lines, which arose upon treatment with a variety of mutagens, showed that the assay recapitulates the...
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Role of genetic factors in the prognosis and prediction of efficacy of chemotherapy in breast carcinoma patients
Brynychová, Veronika ; Souček, Pavel (advisor) ; Macůrek, Libor (referee) ; Stružinská, Ivana (referee)
Changes in the regulation of apoptosis and cell cycle are involved in tumor development, progression, and resistance to antitumor therapy. The aim of this work was to evaluate the importance of apoptotic caspases and regulators of cytokineses as possible prognostic and predictive markers in breast carcinoma patients. In addition to determining the transcript levels of selected genes in tumor and control tissues obtained from breast carcinoma patients, we have also focused on the importance of alternative splice variants of caspases and their potential genetially determined regulation. We analysed the obtained data in relation to the clinical-pathological characteristics of the tumors, the progression-free survival of patients and to the response of the patients to the neoadjuvant chemotherapeutic treatment. Part of the work was determination of protein expression levels and verification of the importance of selected candidates for the effect of chemotherapy by functional study. The transcript levels of caspase 2, 3, 7, 8, 9, 10, the specifically detected splice variants caspase 2S, 2L, 3A and B, 3S, 9A, 9B, 8L, and the transcript levels of KIF14 and CIT in breast carcinomas were unrelated to the progression-free survival of patients, or to the response of patients to neoadjuvant treatment. The...
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Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
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Regulation of cell cycle and DNA damage response in mouse oocytes.
Mayer, Alexandra ; Šolc, Petr (advisor) ; Macůrek, Libor (referee) ; Macek, Milan (referee)
A specific feature of mammalian oocytes is a long prophase I arrest, which can be maintained for many years in humans. The oocytes must ensure robust mechanisms, which can keep them in prophase I, but effectively trigger meiotic resumption when required. Consequently, throughout the maturation of an oocyte, non-erroneous chromosome segregation is a prerequisite for the generation of healthy offspring. In this study we aimed to investigate the new roles of Aurora A (AURKA) and polo-like kinase 1 (PLK1) in the regulation of the cell cycle progression. For this purpose, we used transgenic mice that specifically overexpress wild type (WT-) or kinase-dead (KD-) AURKA in oocytes only, and to study PLK1 we treated oocytes with BI2536, a small molecule inhibitor known to specifically inhibit PLK1 in somatic cells. Our data show, that both AURKA and PLK1 are not essential for meiotic resumption, however they participate in this process. Active AURKA regualtes the increase in microtubule organizing centers (MTOC) in prophase I, which is the first visible marker of resumption of meiosis in oocytes. AURKA activation is biphasic, and the initial increase in MTOC is transient, while full AURKA activation needed for the stability of MTOC requires the activity of Cyclin-dependent kinase 1 (CDK1). We show that PLK1...
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