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The role of DNA repair pathways in ovarian cancer therapy response
Vallušová, Dominika ; Opattová, Alena (advisor) ; Rössner, Pavel (referee)
Ovarian cancer is serious and one of the most common gynecologic cancers. Carboplatin is the therapeutic agent of the first choice in the ovarian cancer therapy. However, after the primary therapeutic response to carboplatin, the relapse of the disease may occur with developed resistance to carboplatin. Chemoresistance and insufficient therapy response are considered to be the reason of the high mortality rate of ovarian cancer. The DNA damage response pathways play an important role in the therapeutic response and chemoresistance development. Restoration of homologous recombination function in cancers is the key mechanism of resistance development to platinum agents. Based on this knowledge, we formed our hypothesis, that the inhibition of homologous recombination could increase the sensibility to carboplatin. The main goal of this thesis was to define the role of double-strand breaks repair in response to chemotherapy of ovarian cancer. Protein MRE11 is part of the MRN complex, that participates in double-strand breaks repair. Using mirin as a pharmaceutic inhibitor of MRE11 we were aiming to determine the impact of homologous recombination on the effect of carboplatin and its role in resistant development to carboplatin. In the practical part of the thesis, we described the association between...
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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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The role of DNA repair mechanisms in the pathogenesis of myelodysplastic syndrome.
Válka, Jan ; Čermák, Jaroslav (advisor) ; Pospíšilová, Dagmar (referee) ; Penka, Miroslav (referee)
Background: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests the involvement of DNA repair mechanism defects in the pathogenesis of this disorder. The first part of this work was focused on monitoring of gene expression of DNA repair genes in MDS patients and on their alterations during disease progression. In the second part, next generation sequencing was used to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with MDS development was evaluated. Methods: Expression profiling of 84 DNA repair genes was performed on bone marrow CD34+ cells of patients with MDS. Screening cohort consisted of 28 patients and expression of selected genes was further validated on larger cohort of 122 patients with all subtypes of MDS. Paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 recombinase protein was done on samples acquired by trephine-biopsy. Targeted enrichment resequencing of exonic parts of 84 DNA repair genes was performed on the screening cohort of MDS patients. Real-time PCR was used for genotyping of selected SNPs in the population study. Results: RAD51 and XRCC2 genes showed...
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BopN function in the Bordetella type III secretion system
Kincová, Veronika ; Kamanová, Jana (advisor) ; Večerek, Branislav (referee)
Species of the Bordetella genus cause the highly contagious whooping cough disease in humans (B. pertussis, B. parapertussis) and related respiratory diseases in other mammals (B. bronchiseptica, B. parapertussis). One of the virulence systems of Bordetellae is the type III secretion system (T3SS) employed for translocation of effector proteins directly from bacterial cytosol into the cytosol of host cells. The T3SS protein BopN protein has been categorized as a Bordetella effector protein. Nevertheless, the homologous proteins in other gram-negative bacteria function in establishing the secretion hierarchy through T3SS and some of them block T3SS secretion in high calcium environments before bacteria-host cell contact has been established. In this thesis I examined the function of the BopN protein and the role of calcium ions in T3SS activity of B. bronchiseptica. Two independent methods have been used for determination of T3SS secretion activity. Addition of 2 mM calcium ions into bacterial media decreased secretion of the T3SS reporter, while no such effect was observed in a B. bronchiseptica strain lacking the bopN gene. Mass spectrometry data confirmed the inhibition of T3SS activity in the presence of calcium ions. Enhanced calcium levels resulted in decreased mobilization and secretion of...
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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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DNA damage response in mammalian oocytes
Vachová, Veronika ; Šolc, Petr (advisor) ; Nevoral, Jan (referee)
During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...
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Mechanisms of DNA repair in the moss Physcomitrella patens
Holá, Marcela ; Angelis, Karel (advisor) ; Bříza, Jindřich (referee) ; Fajkus, Jiří (referee)
Over the course of an organism's life, its genome is exposed to endogenous and exogenous chemical, physical and biological agents - genotoxins. These genotoxins alter its basic structural components - sugar residues, phosphodiester bonds, and nitrogenous bases. Organisms have therefore evolved a plethora of different strategies to both repair DNA lesions and maintain genomic stability. These DNA repair pathways are linked with several other cell pathways, including chromatin remodelling, DNA replication, transcription, cell cycle control, apoptosis - programmed cell death (PCD), thereby providing a coordinated cellular response to DNA damage. Biochemical mechanisms of DNA repair are relatively well understood in yeast and mammals, however, far less so in plants. While these repair mechanisms are evolutionary conserved, significant differences still remain. Therefore, further investigation is required. This thesis summarises the introduction of a novel plant model - the moss, Physcomitrella patens (Physcomitrella). As a haploid gametophyte with unique characteristics of high frequency of homologous recombination (HR), and apical growth of filaments, it is an ideal organism to study DNA repair in plants. Previous research on Physcomitrella regarding mechanisms of DNA lesion repair induced by...
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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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A DNA double-strand break repair and it's disorders with a relationship to the cancerogenesis
Komžák, Josef ; Ševčík, Jan (advisor) ; Vopálenský, Václav (referee)
The DNA-double strand break (DSB) repair has an essential importance for the genomic integrity maintenance. The main DSB repair pathways are homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). The most important protein factors contributing to the maintenance of genomic integrity by direct participation in DSB repair are MRN, ATM, Rad51, BRCA1/2 and PALB2 in the case of HR; Ku70/80 DNA-PKcs, XRCC4 and DNA ligase IV in the case of NHEJ and Msh2-Msh3 and Rad1- Rad10 in the case of SSA. If mutated, these proteins can cause the inability to repair DNA lesions leading to a malignant transformation. The predominant phenotype manifestation of BRCA1/2 inactivation is the hereditary breast and/or ovarian cancer (HBOC). Mutations in ATM have been described as a cause of ataxia telangiectasia and inactivation of NBN gene (Nbs1 protein) causes the Nijmegen breakage syndrome. Other syndromes connected with defects in a DSB repair pathways are Fanconi anemia and Werner syndrome. Detail knowledge of DSB repair process is a mandatory for diagnostics and effective therapy of a number of malignances. An example of practical and clinically relevant utilization of current knowledge about the DSB repair process is the concept of a synthetic lethality as a specific therapy. This...
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Genes of early meiotic prophase I of spermatogenesis in house mouse
Škaloudová, Eliška ; Trachtulec, Zdeněk (advisor) ; Forman, Martin (referee)
Meiosis is an essential cellular process that is necessary for gamete formation in all sexually reproducing organisms. This work is focused on the description of the genes of early stages of meiotic division in males of a mammalian model, the house mouse. The first part summarizes meiosis focusing on prophase I, which is longer than prophase II. Prophase I is divided into five stages, namely leptotene, zygotene, pachytene, diplotene, and diakinesis. Mouse spermatogenesis and its differences from oogenesis are also briefly described. The second part provides a list of genes encoding proteins required for initiation of meiotic division, pairing and synapses of chromosomes, and initiation of the catalysis of double-strand breaks. Double-strand breaks are repaired by homologous recombination, which may result in so-called crossing-over, the major source of genetic variability. The work deals with the early stage of homologous recombination and components required for this process. Localization of meiotic double-strand breaks in the genome is not random and is under the control of the Prdm9 gene, which seems to take multiple roles, such as the formation of new subspecies of the house mouse. Knowledge of the genes controlling the early stages of meiotic division is a prerequisite to understanding some of...
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