|
|
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...
|
|
|
Molecular mechanisms of G2/M checkpoint regulation
Kořínková, Klára ; Macůrek, Libor (advisor) ; Forman, Martin (referee)
Cell division is necessary for maintaining tissue homoeostasis, but at the same time its defects are closely related to the development of many diseases including cancer and premature ageing. Activation of oncogenes leads to replication stress and directly threatens genome stability. The right control of transition between interphase and mitosis is an important mechanism for the protection of genome integrity. Nuclear division is only possible with those cells in which flawless duplication of genetic information occurred. By contrast, cells with damaged DNA structure remain temporarily or permanently stopped at G2 phase of the cell cycle. The topic of this thesis is a detailed literature overview with the subject of molecular mechanisms of the G2/M transition regulation under unperturbed conditions and in the presence of damaged DNA.
|
|
|
Characterisation of the mechanisms regulating 53BP1 nuclear transport
Liďák, Tomáš ; Macůrek, Libor (advisor) ; Brábek, Jan (referee)
Tumor suppressor p53-binding protein 1 (53BP1) is an integral part of a sophisticated network of cellular pathways termed as the DNA damage response (DDR). These pathways are specialized in the maintenance of genome integrity. Recently, it was reported that nuclear import of 53BP1 depends on importin ß. Here, I used fluorescence microscopy and co-immunoprecipitation experiments to identify its nuclear localization signal (NLS). Clusters of basic amino acids 1667-KRK-1669 and 1681-KRGRK- 1685 were required for 53BP1 interaction with importin ß and for its nuclear localization. Short peptide containing these two clusters was sufficient for interaction with importin ß and targeting EGFP to the nucleus. Additionally, the effect of 53BP1 phosphorylation at S1678 on its nuclear import was examined. Mimicking the phosphorylation in the 53BP1-S1678D mutant decreased the binding to importin ß and resulted in a mild defect in 53BP1 nuclear import. However, 53BP1 entered the nucleus continuously during the cell cycle, suggesting that CDK-dependent phosphorylation of S1678 probably does not significantly contribute to the regulation of 53BP1 nuclear transport. Taken together, 53BP1 NLS meets the attributes of a classical bipartite NLS. Although no cell cycle-dependent regulation of its import was observed, the...
|
|
|
Role of Polo-like kinases in the cell cycle and DNA damage response
Kudláčková, Radmila ; Macůrek, Libor (advisor) ; Šolc, Petr (referee)
Within the process of cell division, genetic material must be equally distributed between the two daughter cells. In the next phase, the missing portion of the genome must be synthesized. The entire cycle is regulated by cyclin-dependent kinases (Cdks) in cooperation with cyclins. If the DNA is damaged during the cell cycle, signaling pathways of checkpoints supress cycle progression and enforce the cell cycle arrest until the damage is repaired. Malfunction of the checkpoints results in tumorigenesis. Polo-like kinases (Plks) are, much like Cdks, important regulators of the cell cycle. Plks play significant role mainly in the mitosis and also in a response to the DNA damage. This thesis is focused on human homologues, nevertheless conservation of homologues among organisms is considerable, thus presented findings are of general relevance. Human cells express five proteins from the family of Polo-like kinases, from which Plk1 corresponds to Polo-like kinases of lower eukaryotes. Knowledge on the remaining four kinases is still on the rise.
|
|
|
Wnt signaling in intestinal homeostasis and tumorigenesis
Janečková, Lucie ; Kořínek, Vladimír (advisor) ; Macůrek, Libor (referee) ; Truksa, Jaroslav (referee)
The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....
|
|
|
The role of 53BP1 in the cellular response to double-strand DNA breaks
Liďák, Tomáš ; Macůrek, Libor (advisor) ; Rösel, Daniel (referee)
DNA damage may result in various pathological conditions and contributes to aging and development of cancer. Evolutionarily conserved DNA damage response prevents the acumulation of mutations and protects against genomic instability. Tumor suppressor p53-binding protein 1 (53BP1) is an important regulator of the cellular response to DNA double-strand breaks (DSB) and is a canonical component of ionizing radiation-induced foci which are formed at DNA DSB following radiation exposure. Recently, new insights have been gained into its functions in the DNA damage response. Apart from its subtle role in the DNA damage checkpoints signaling, 53BP1 is a well established player in the DNA DSB repair pathway choice. The outcome of DNA repair is influenced by 53BP1 in several contexts. 53BP1 controls 5' end resection at DNA ends, improves DSB repair in heterochromatin, promotes the mobility of uncapped telomeres and mediates synapsis of DNA ends during V(D)J and class switch recombination. 53BP1 contributes to repair defect in BRCA1 (breast cancer type 1 susceptibility protein)-deficient cells, which may have an impact on the treatment of some types of breast cancer. The aim of this bachelor's thesis is to summarize new findings about the role of 53BP1 in the cellular response to DNA DSB. Powered by TCPDF (www.tcpdf.org)
|
|
|
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...
|
|
|
Molecular mechanisms of checkpoint signalling and termination
Benada, Jan ; Macůrek, Libor (advisor) ; Brábek, Jan (referee) ; Truksa, Jaroslav (referee)
Cells employ an extensive signalling network to protect their genome integrity, termed DNA damage response (DDR). The DDR can trigger cell cycle checkpoints which prevent cell cycle progression and allow repair of DNA damage. The failures in these safeguarding mechanism are represented by serious human malignancies, most predominantly by cancer development. This work aims to contribute to the understanding of how do the cells negatively regulate DDR and cell cycle checkpoint signalling. We focused mainly on Wip1 (PPM1D) phosphatase, which is a major negative regulator of DDR and is indispensable for checkpoint recovery. Firstly, we have shown that Wip1 is degraded during mitosis in APC-Cdc20 dependent manner. Moreover, Wip1 is phosphorylated at multiple residues during mitosis, resulting in inhibition of its enzymatic activity. We suggest that the abrogation of Wip1 activity enables cells to react adequately even to low levels of DNA damage encountered during unperturbed mitosis. In the following publication, we have investigated why the mitotic cells trigger only early events of DDR and do not proceed to the recruitment of DNA repair factors such as 53BP1. We showed that 53BP1 is phosphorylated within its ubiquitination-dependent recruitment domain by CDK1 and Plk1. These phosphorylations prevents...
|
|
| |
|
| |
guest ::
