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Functional analysis of the population-specific checkpoint kinase gene CHEK2 sequence variants
Stolařová, Lenka ; Kleibl, Zdeněk (advisor) ; Živný, Jan (referee)
CHEK2 gene codes for serin/threonine kinase Chk2 (Checkpoint kinase 2). In response to genomic DNA damage, Chk2 phosphorylates its substrates (proteins Cdc25C, BRCA1 or p53), whose activation leads either to cell cycle arrest, DNA damage repair or induction of apoptosis. Germline mutations in CHEK2 gene increase risk of cancer development. Analysis of high risk breast cancer patients in Czech Republic reveals rare CHEK2 mutations (mainly missense) with yet unknown clinical significance. This work focuses on functional impact of these variants and analysis of kinase activity of variant isoforms of Chk2 kinase. For this purpose, recombinant constructs were expressed in bacterial cells of E. coli. Enzymatic activity of Chk2 kinase isoforms in crude cell lysates was measured by the phosphorylation of Chk2 arteficial substrate spectrophotometrically. Results of in vitro kinase assay were correlated to the results of in silico prediction software. The results show that from 15 analyzed mutations (together with one in frame deletion), kinase activity was abrogated in all variants affecting the kinase domain of Chk2, in concordance with in silico predictions. The same result has been found for a FHA domain variant p.R145Q. No significant changes in kinase activity were observed in case of two FHA domain variants...
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Current methods of genome analysis and their use in identification of genetic determinants of human diseases
Stránecký, Viktor ; Kmoch, Stanislav (advisor) ; Kleibl, Zdeněk (referee) ; Pačes, Jan (referee)
The study of rare genetic diseases presents unique opportunity to uncover the genetic and molecular basis of human traits and greatly helped to the identification of genes, to the elucidation of their function and to the characterization of metabolic pathways and cellular processes. Over the past decades, linkage analysis has been appropriate approach to search for the genes causing Mendelian diseases and contributed to the identification of many genes, but the genetic cause of many diseases remains unknown. New methods of studying the human genome, microarray technology and massively parallel sequencing (next generation sequencing), represent a way to efficiently identify the cause of genetically determined diseases, based on direct observation of mutations in the genome of affected individuals. These techniques replaced the traditional method of disease gene identification represented by linkage analysis and sequencing of candidate genes and have become the standard approach to elucidate the molecular basis of diseases. In this work, i describe the the results achieved by using these methods - identification of the genes underlying mucopolysacharidosis type IIIC, isolated defect of ATP synthase, Rotor syndrome, autosomal dominat ANCL and GAPO syndrome.
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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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Intracellular signalling of Chk2 kinase and impact of its defects in oncogenesis
Stolařová, Lenka ; Kleibl, Zdeněk (advisor) ; Brábek, Jan (referee)
Chk2 (checkpoint kinase 2), a regulatory protein of the cell cycle checkpoints, is coded by CHEK2 gene. Chk2 belongs to serine/threonine kinase family and its dominant activity is in regulation and signal distribution of intracellular response to DNA damage. The upstream regulator of Chk2 protein is the ATM kinase that activates Chk2 by its phosphorylation on Thr68 localized in FHA domain. This in turn leads to the conformation change inducing homodimerization of Chk2 protomers and their activating phosphorylation within their kinase domains. Upon phosphorylation, catalytically active Chk2 protomers dissociate and phosphorylate various intracellular proteins (incl. p53, E2F-1, BRCA1, Cdc25A a C, BRCA2 a PLK3). By regulation of these proteins, Chk2 contributes to the cell cycle arrest, regulation of DNA repair and apoptosis. Germline mutations in CHEK2 gene were identified with the increased frequency in many human cancers, including breast and colorectal cancer. Hence, the failure of Chk2 intracellular activity contributes to the process of malignant transformation.
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Genetic factors of resistance to chemotherapy and clinical characteristics of breast tumors.
Kunická, Tereza ; Souček, Pavel (advisor) ; Kleibl, Zdeněk (referee)
Breast cancer is the most frequent malignancy in women population both in the Czech Republic and worldwide. Treatment of this disease involves surgical removal of the tumor, radiotherapy, chemotherapy and hormonal therapy. Recently, targeted biological treatment is also approached. Each patient reacts to the treatment individually and thus high variability in response is common. Multidrug resistance (MDR) presents one of the most important obstacles to successful chemotherapy. MDR is often associated with a decreased intracellular accumulation of anticancer drugs and an increased expression of ABC transporters such asABCC1 of our interest. The ABC family of membrane transport proteins includes the well-known mediators of resistance to anticancer drugs. In particular, ABCB1, ABCC1 and ABCG2 actively perform efflux of various types of drugs from cancer cells, thereby conferring resistance to those agents. The main aim of this study was to asses the genetic variability of the ABCC1 gene in 191 patients with breast cancer and to determinate the expression profile of ABCC1 in 30 patients from this cohort who were treated preoperatively. We evaluated relations between ABCC1 genotype, or phenotype and prognostic factors including the result of chemotherapy. Gene expression was measured in preamplificated...
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Analysis and characterization of BRCA1 splicing variants.
Hojný, Jan ; Kleibl, Zdeněk (advisor) ; Souček, Pavel (referee)
The Breast cancer gene 1 (BRCA1) codes for nuclear phosphoprotein with a key function in the regulation of DNA damage response. The BRCA1 protein contributes to the formation and regulation of protein supercomplexes that participates on the DNA double-strand break repair. These protein supercomplexes are formed by the protein-protein interactions between highly conservative protein motives in BRCA1 and its binding partners. Except to the wild type form of BRCA1 mRNA containing entire set of 22 exons coding for the 220 kD protein, numerous alternative splicing variants (ASVs) BRCA1 mRNA has been described. These ASVs code for BRCA1 isoforms lacking several critical functional domains. It has been proposed, that formation of BRCA1's ASVs represent a tool for regulation of BRCA1 function. Only poorly has been characterized a complex catalogue of in various human tissues and their expression. This study aims to address these questions. We optimized the identification of BRCA1's ASVs including those covering the entire transcripts of the wt BRCA1 mRNA with length exceeding 5.5 kb. In further analysis, we characterized 13 BRCA1's ASVs in RNA samples isolated from peripheral blood mononuclear cells (PBMNC) obtained from patients with breast cancer (BC) and control subjects. The majority of the identified...
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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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