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Targeted therapy of AML1-ETO positive acute myeloid leukemia with histone deacetylase inhibitors
Zápotocký, Michal ; Trka, Jan (advisor) ; Stopka, Tomáš (referee) ; Trbušek, Martin (referee)
In t(8;21) acute myeloid leukaemia (AML), the leukemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1-ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. We aimed to characterize the differentiation effect of VPA on AML1-ETO-positive leukemic cells and to determine the expression pattern of AML1 target genes. Kasumi-1 (M2 AML1-ETO-positive), Kasumi-6 (M2 AML1-ETO- negative), MV4-11 (MLL-AF4-positive) and K562 cells were treated with VPA and 12-0-tetra- decanoylphorbol-13-acetate (TPA) and examined by flow cytometry and qRT-PCR. Two AML1-ETO- positive and two negative patients' bone marrow diagnostic samples were treated with VPA and TPA to confirm in vitro findings. Valproic acid induced apoptosis in AML1-ETO-positive and MLL- AF4-positive cells in dose dependent manner. But changes of immunophenotype proving the differentiation were observed purely in AML1-ETO-positive cell line (decreased CD33/34/117 and increased CD11a/11b expression). However, differentiated cells exhibited positivity of AnnexinV; hence the relationship between cell death and differentiation had to be evaluated. Apoptosis was blocked by...
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Role onkogenní mikroRNA-155 a proto-onkogenu MYB u chronické lymfatické leukémie
Vargová, Karina ; Stopka, Tomáš (advisor) ; Móciková, Heidi (referee) ; Trka, Jan (referee)
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B- CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Molecular detection of invasive fungal disease in immunocompromised patients
Bašková, Lenka ; Živný, Pavel (advisor) ; Bílková, Zuzana (referee) ; Trka, Jan (referee)
5 3. Summary Molecular detection of invasive fungal disease in immunocompromised patients In my work I have been able to establish three different PCR-based assays for the quantitative detection and identification of fungal DNA. Two DNA-based detection assays termed PanAC PCR and panfungal PCR based on the real-time quantitative (RQ-PCR) technology were designed to detect and quantify the most important fungal genera currently associated with IFD including a large number of pathogenic moulds and yeasts. Upon standardization of both RQ-PCR techniques, the applicability in the clinical setting was assessed by investigating a series of clinical specimens from patients with documented fungal infection, and by prospectively studying patient cohorts at high risk of IFD. In view of the importance of precise identification of the causative fungal pathogen, a semi-nested PCR method coupled with fluorescent capillary electrophoresis detection was established. It facilitates rapid identification of fungal species in clinical materials that test positive for IFD using one of the broad-range screening assays. This method was also tested in a population of patients with documented fungal infections to assess its clinical potential.
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The role of WT1 and its isoforms in normal haematopoiesis and leukaemogenesis
Kramarzová, Karolina ; Trka, Jan (advisor) ; Pospíšilová, Dagmar (referee) ; Živný, Jan (referee)
61 Summary Wilms' tumor gene 1 (WT1) is highly expressed in acute leukemia and other hematological malignancies. It has been therefore suggested as a potential universal marker of minimal residual disease (MRD), particularly in patients with acute myeloid leukemia (AML). Due to controversial results of some of the studies, the role of WT1 in MRD follow-up and WT1 prognostic significance remain unclear. WT1 protein is produced in more than 36 different isoforms. These variants have distinct, partially overlapping functions and their ratio is supposed to influence the final effect of WT1. However, despite the increasing number of studies, the clinical impact of WT1 and its isoforms in acute leukemia have not yet been elucidated. We established a unique qPCR method to assess the expression pattern of the main 4 WT1 isoforms. Using this method, we determined the ratio of WT1 variants in the samples of patients with AML, myelodysplastic syndrome (MDS) and healthy controls. Our data showed that this pattern can distinguish among particular hematological malignancies, but lacks a prognostic significance. Within our international study group we determined the prognostic significance of total WT1 expression in childhood AML. Based on our results of a large cohort of patients we can conclude that WT1 expression at...
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Molecular mechanisms of Diamond-Blackfan anemia
Handrková, Helena ; Petrák, Jiří (advisor) ; Šebela, Marek (referee) ; Trka, Jan (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital syndrome that presents with ane- mia and selective deficiency of erythroid precursors, while other blood lineages are usu- ally unaffected. Approximately half of the patients display additional somatic anoma- lies and growth retardation. The therapy is mostly symptomatic and is dominated by corticosteroids, other modalities include regular blood transfusions or hematopoietic stem cell transplantation. At the beginning of this work, only two DBA causal genes were known, RPS19 and RPS24, being mutated in approximately 1/4 of all DBA patients. The goals of this work were to study the consequences of the known DBA causal mutations on cellular level and to find novel DBA causal genes. To date, over a half of DBA patients have been reported to carry a mutation in one of nine known DBA causal genes, including RPS17, RPL11 and RPL5, that are reported in this dissertation. All confirmed DBA causal genes encode for ribosomal proteins (RPs) that were essential for ribosome assembly. We further hypothesized a non- ribosomal protein participating in this process might be involved in DBA pathogenesis, too. In one DBA patient, we identified a rare sequence variant in one such candidate, a protein arginine methyltransferase 3 (PRMT3). We reported that the patient PRMT3...
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Regulatory mechanisms of WNT signalling
Pospíchalová, Vendula ; Kořínek, Vladimír (advisor) ; Trka, Jan (referee) ; Bryja, Josef (referee)
AB T mu hom dise β sign mu liga stab tran sign T the focu disc cate of t cell targ the inte con sec I sign BSTRACT The Wnt si lticellular o meostasis. A eases, most β-catenin is nalling). In ltiprotein c ands when t bilized and nscription f nalling is tig This thesis knowledge uses on seq cusses the enin signall the Wnt pa ls of intest geted mous thesis des eraction wit nditional Hi retory cell t In conclusi nalling path T ignalling pa organisms Accordingly notably can s a central m n unstimula complex and they engage d transloca factors and ghtly regula is based on e of the reg quential po positive ro ling outcom athway whic tinal epithe e strains th scribes unp th members ic1 deletion types and en ion, our fin hway in dev athway is o ensuring s y, mutations ncer. mediator of ated cells d degraded e their recep tes to the to drive th ated at vario n four origin gulation of sttranslation ole of nucle me. The third ch reduces lium. Final at enable st ublished da s of the Wn n in the inte nhanced tum ndings contr velopment an one of the m successful s in the pat f canonical W β-catenin d in the pro ptors, degrad nucleus t he transcrip ous levels b nal articles f the Wnt s nal process ear protein d study repo the levels o lly, the las tudying the ata on the nt pathway, estinal epith mourigenesi ributed to t...
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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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Analysis of TRAIL-induced apoptosis in acute myeloid leukemia cells
Klener, Pavel ; Nečas, Emanuel (advisor) ; Herget, Jan (referee) ; Trka, Jan (referee)
Advanced tumors, including leukemia, represent heterogeneous cell populations evolved from original malignant clones. Chemotherapy of leukemia is often associated with selection of drug-resistant cells followed by progression/relapse of the disease. Implementation of molecules that specifically target leukemia cells with minimal toxicity to normal tissues might significantly improve outcome of leukemia treatment. TRAIL belongs to the tumor necrosis factor (TNF) ligand family of cytokines. TRAIL triggers apoptosis in target cells via the receptor-mediated apoptotic pathway. Receptors for TRAIL can be divided into death receptors, TRAILR1/ DR4 , TRAIL-R2/DR5, and decoy receptors, TRAIL-R3/DcR1, TRAIL-R4/DcR2, osteoprotegerin/OPG/TRAIL-R5, based on their ability to transduce apoptotic signal. While normal tissues, including hematopoietic progenitor cells, are resistant to TRAIL-induced apoptosis, TRAIL induces programmed death in many tumor cell lines and primary cells. Various malignant cell lines and primary tumor cells, however, show resistance to TRAIL-induced apoptosis. TRAIL-resistance could represent important limitation for the potential TRAIL anti-tumor therapy. Combined in vitro application of TRAIL with other anti-cancer agents often increased sensitivity or overcame resistance of the tumor cells to...
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