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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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Neurofibromatosis type 1 and NF1 germline mutations in Czech patients
Bendová, Šárka ; Křepelová, Anna (advisor) ; Kleibl, Zdeněk (referee) ; Kadlecová, Jitka (referee)
Neurofibromatosis type 1 (NF1, MIM 162200) is an autosomal dominant disorder affecting about 1 of 3000 live births, involving many cell types and organs, and associated with an increased risk of malignancy, predominantly of the central and peripheral nervous system 1. Tumour development is caused by inactivation of the NF1 tumour suppressor gene and subsequent cell cycle deregulation 2. Mutational analysis of NF1 is a challenge due to the presence of pseudogenes, large size of the gene, lack of mutational hotspots, and occurrence of a very diverse spectrum of mutations. There is no clear-cut genotype-phenotype correlation allowing accurate prediction of severity of the disorder. Only two mutations have been associated with a particular NF1 phenotype 3,4. This PhD thesis is composed of six publications dealing with NF1. Publications 1 and 6 are focused on NF1 mutation analysis in 67 patients from the Czech Republic. Genotypes and spectra of causal mutations are presented together with phenotypes of the patients and comparison of efficiency of various methods. Sporadic or familial cases with known germline mutation were distinguished by mutational analysis of other family members. This led to a hypothesis that the incidence of sporadic cases could had been overestimated in the past because of overlooked...
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Microsatellite instability in hereditary nonpolyposis colorectal cancer patients
Sekowská, Martina ; Křepelová, Anna (advisor) ; Mareš, Jaroslav (referee) ; Kleibl, Zdeněk (referee)
Detection of microsatellite instability (MSI) is the standard part of mutational analysis in hereditary nonpolyposis colorectal cancers (HNPCC). Characteristic phenotypic feature of MSI indicates loss of mismatch repair (MMR) in tumor cells. We studied MSI in 205 tumors from 152 patients with HNPCC. Of these, 37 patients fulfilled Amsterdam criteria, 72 patients were familial and 43 were sporadic cases. We used methods of fragmentation analysis on polyacrylamide gel and/or with fluorescent labelled primers (ABI Prism 310 Genetic Analyzer). Three mononucleotide (BAT-RII, BAT-25, BAT-26) and five dinucleotide (D2S123, D3S1029, D5S346, D17S250, D18S58) repeat loci were analysed. We detected 75 tumors with high degree of MSI (MSI-H), 12 tumors with low degree of MSI (MSI-L) and 118 tumors with stable microsatellites (MSS). In 44 of these, loss of heterozygozity (LOH) was found. In 30 patients with MSI-H tumors a mutation in one of mismatch repair genes was detected. Microsatellite analysis was positively correlated with immunohistochemical detection of MLH1 and MSH2 proteins.
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Structure, function and importace of BRCA 1protein
Hojný, Jan ; Falk, Martin (referee) ; Kleibl, Zdeněk (advisor)
Studies of factors contributed to the development of hereditary breast and ovary cancers lead to the discovery of Breast Cancer 1 gene (BRCA1). The protein product of this tumor suppressor gene is nuclear phosphoprotein that plays a critical role in DNA repair and it is required for genome integrity control. The BRCA1 protein is the key component for correct assembly of reparation complexes formed in sites of DNA double strand breaks. Furthermore, BRCA1 protein is implicated in regulation of cell cycle checkpoints and it is also involved in regulation of gene expression in response to DNA damage. These activities suggest that BRCA1 protein plays a crucial role in orchestration of intracellular response to genotoxic DNA damage. Loss of BRCA1 functions leads to the DNA-damage repair mechanisms failure resulting in genomic instability and a tolerance of genomic alterations in affected cells. The genomic instability is the initial step toward early malignant transformation of cells lacking BRCA1 proteins. The aim of this work is to summarize the information about structure, functions known and the importance of BRCA1 protein with respect to the current discoveries enabling elucidation of versatile BRCA1-containing multiprotein complexes in which BRCA1 protein acts as the multiplatform interacting...
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Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines
Lhota, Filip ; Kleibl, Zdeněk (advisor) ; Souček, Pavel (referee)
Filip Lhota: Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines Abstract: Estrogen receptor α (ER-α) is a transcription factor responsible for mediation of the activities of its natural ligand 17-β-estradiol (E2), the hormone that together with progesterone belongs to the key regulators of mammary epithelial as well as breast cancer cells proliferation. Except to the major gene product consisting of all eight coding exons of ER-α, numerous qualitatively and quantitatively different spliced variants originated from primary transcript by activity of alternative splicing is expressed. Despite that some of these spliced variants have been functionally characterized, their precise role on final ER-α cellular activity remains to be elucidated. The functional characterization of individual alternative forms of ER-α and description of its participation on the overall ER-α activity is important for our understanding of their biogenesis and is also critical for the delineation of molecular bases for ER-α regulation during anti cancer chemotherapy. This work aimed to study the influence of alternatively spliced ER-α variants on the growth characteristics of clones constructed from stable mammary tissue cell lines in regulation to cultivation conditions and cellular...
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