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The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatin
Černá, Tereza ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
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Metallothioneins and other biomarkers in the treatment decision in pediatric cancer patients
Kruseová, Jarmila ; Eckschlager, Tomáš (advisor) ; Prausová, Jana (referee) ; Raudenská, Martina (referee)
Metallothioneins (MT) are low molecular weight, cysteine-rich proteins maintaining metal ions homeostasis. They play a role in carcinogenesis and may also cause chemoresistance. The aim: was to explore the relationship between MT serum levels in children suffering from malignant tumours and correlate with laboratory findings during anticancer treatment. Material and methods: This prospective study involves 865 samples from 172 patients (71 girls and 101 boys) with malignant tumours treated from 2008 to 2011. Patient median age: 9.9 years (0.1-19.5). Metastatic diseases at diagnosis: 93 patients (54.1%), reccurence: 12(7%), died: 32(18.6%). MT serum levels were determined using differential pulse voltammetry-Brdicka reaction. Results: Mean MT level 2.67 uM/l was five times higher that of control group (0.05). No significant difference between MT levels in different tumours. No correlation between MT levels in active disease and disease in remission (p =0.33). However, we found a positive correlation between MT levels and age (p=0.009), negative correlation between MT and creatinine (p=0.003). Patients who had disease recurrence had lower MT levels during the treatment (remission 2.67 vs. recurrence 2.34, p=0.001) Conclusion: Patients who had early disease recurrence had lower MT levels during treatment...
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H19 methylation determination and KCNQ1OT1 at nephroblasts , pheochromocytomas and paragangliomas using MLPA technique
Jenčová, Pavla ; Vícha, Aleš (advisor) ; Eckschlager, Tomáš (referee)
DNA methylation is an epigenetic mechanism that affects the level of gene expression. Methylation is a physiological for imprinted genes, when is required to express a gene derived only from a particular parent. If a fault occurs in the DNA methylation of these genes, various diseases may develop. The object of this work was to determine the level of methylation of H19 and KCNQ1OT1 genes, located on the short arm of chromosome 11 in 15.5 region. The aim of this study was to investigate the changes in methylation of these genes in nephroblastomas, pheochromocytomas and paragangliomas, determine what changes of target genes are present in the samples, examined and divided samples into groups according to the detected changes. File of nephroblastomas is divided into 3 groups according to the methylation changes in genes and compared with a similar study by Scott et al. 2012. File of pheochromocytomas and paragangliomas are found according to changes in the methylation of genes divided into 4 groups entirety. Found changes are aligned with the research Margetts et al. 2005.
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Monitoring of children's cancer chemoresistance molecular cytogenetic methods
Procházka, Pavel ; Eckschlager, Tomáš (advisor) ; Kočárek, Eduard (referee) ; Kuglík, Petr (referee)
Souhrn Disertacni prace se zabyva chemorezistenci detskych nadorovych onemocneni adetekci cytogenetickych zmen, ktere s ni souvisi. Ovlivneni rizikovych forem detskych nadoru vyzaduje pouziti vysokych davek chemoterapie, ktera muze vest ke vzniku chemorezistence. Prukaz chromozomalnich aberaci muze prispet k urceni prognozy apredikce efektu lecbychemorezistentnichnadoru. Vetsinu vysledku jsme ziskalistudiem neuroblastomu (NBL). Ostatni prezentovane vysledky pak predstavuji geneticke zmeny u Ewingova sarkomu a u detskych feochromocytomu. Ke studiu chromozomalnichaberaci bylypouzitymetodykomparativni genomova hybridizace (CGH)a array CGH doplnene o vysetreni mnohobarevnou nebo int fzni fuor c brdi nst. Zmenyv expr naur mRNA byl s rera l escen ni hy i zaci i iu esi ovni yvyeteny expresni cipovou analyzou doplnenou o kvantitativni polymerazovou retezovou reakci, zmeny v expresiproteinu bylyvysetrenypomoci westernblottingu nebo prutokovou cytometrii. Predkladana disertace je komentovany soubor sesti publikaci. Studium chromozomalnich aberaci Ewingovych sarkomu predstavuje literarni resersi doplnenou o vlastni vysledky. Prace tykajici se feochromocytomu je jednim z nejvetsich souboru podrobnegeneticky vysetrenych detskych feochromocytomu. Nejobsahlejsi castse zabyva cytogenetickymizmenamiu NBL. Dlouhodobou...
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Effects of reovirus on cancer cells
Figová, Katarína ; Eckschlager, Tomáš (advisor) ; Mikyšková, Romana (referee) ; Němečková, Šárka (referee)
Oncolytic viruses are examined to serve as anticancer therapeutics. It is expected that in addition to direct oncolytic effect their action will also help eliciting a solid antitumor immunity. In presented series of experiments we have employed two HPV16- transformed mouse cell lines, TC-1 and MK16, and reovirus type 3, strain Dearing (RV). Both cell lines are highly susceptible to RV and produce large amounts of infectious virus in vitro. Still, some differences were encountered. When inoculating high doses of infected cells into syngeneic animals their oncogenic activity was strongly suppressed, nearly completely in the case of MK16 cells but less efficiently in the case of more oncogenic TC-1 cells. When immunized animals were challenged with TC-1 cells, the irradiated cells proved to be a much better immunogen that the infected cells. However, when challenged with MK16 cells the opposite was true. In another study we demonstrate that RV replicates preferentially in tumor cells and that the virus is able to overcome cellular adaptation to hypoxia (1% O2) and infect and kill hypoxic tumor cells. RV can both replicate in a hypoxic tumor microenvironment and can cause cytophatic effect and subsequently induce cell death. We found that a large proportion of cells are in hypoxia killed by caspase independent...
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Histone deacetylase inhibitors induced caspase-independent cell death
Groh, Tomáš ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
Neuroblastoma is the most common extracranial solid tumor that occurs during infancy. Despite the great progress has been made in contemporary clinic medicine some forms of neuroblastoma disease are still found very difficult to treat . This work focuses on the effects of histone deacetylase inhibitors (HDAC) in the neuroblastoma cell lines. It is known that HDAC inhibitors may contribute to recurrence of the tumor cells by affecting the chromatin structure and thus increase the expression of critical tumor suppressor genes. These genes activate apoptotic pathways that may even be independent of caspases. We observed the efficiency of used HDAC inhibitors as under standard conditions an in hypoxia (1 % O2). Inadequate amount of oxygen supply is one of the characteristic features of tumors and it also may contribute to chemoresistance. With the hypoxia-induced chemoresistance of tumor cells, the influence of HIF-1α is expected. Some HDAC inhibitors reduce the amount of HIF-1α in hypoxia and thus HIF transcription factor activity. Thus, the first part of this study is concerned with the acquisition of suitable experimental arrangement for the monitoring of induction of cellular death in human neuroblastoma cell lines SK-N-AS and UKF-NB-3. Secondly, this paper provides the evaluation of the influence...
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Genetic changes in neuroectodermal tumors detected by molecular biological methods.
Vosecká, Tatiana ; Vícha, Aleš (advisor) ; Eckschlager, Tomáš (referee)
9 ABSTRACT Tatiana Labudová: Genetic changes in neuroectodermal tumours detected by molecular biological methods. Charles University in Prague, Faculty of Science, Department of Anthropology and Human Genetics Thesis, 75 pages,8 supplements, 2012 This thesis is concerned to neuroectodermal tumours that make a major group of infant tumour diseases. Genetic material gained from patients with neuroectodermal tumours was examined using comparative genomic hybridization (CGH) and interphasic fluorescence in situ hybridization (I-FISH). The aim of this Thesis is to prove chromosomal changes and to create the whole genetic profile. According to these profiles can be determined tumourgenetic cascade or specific genetic changes that lead to malignant tumours. In some cases (f.e. neuroblastoms) the genetic profile helps us to determine a subtype of disease and it's biological behaviour. Keywords: tumour diseases, neuroblastoma, CNS tumours, pheochromocytoma, Ewing's sarcoma, neuroectodermal tumour, comparative genomic hybridization, interphase fluorescence in situ hybridization
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From the search for new oncogenes to the effort of redefining the cancerogenesis phenomenon
Pajer, Petr ; Dvořák, Michal (advisor) ; Vodička, Pavel (referee) ; Eckschlager, Tomáš (referee)
The described experimental model of clonal tumors induced through the insertional mutagenesis with MAV-2 proved to be a valid and rich source of information describing the process of transformation of normal into tumor cell. We have mapped more than 2000 individual clonal VISs from several hundreds of tumor tissue samples. We have analyzed five tumor types of different histology and tissue of origin along with their derivative tissue cultures. Furthermore, we have discovered the industasis phenomenon and described it during the course of the study. The goal of my study was to uncover common reasons for neoplastic transformation of the cell. The results of my study led me to the paradoxical conclusion that the significance of genetic changes as the primary cause of induction of neoplastic transformation is being overestimated. Although studying the functions of individual genes and search for new tumor markers and therapeutical targets are still beneficial, I believe that the traditional perception of tumor formation as a function/result of mutation accumulation and selection is becoming a serious drawback in further investigations. These conclusions are further discussed in the last section of the presented Ph.D. thesis.
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Differences in histone acetylation in normoxia and hypoxia
Čepek, Pavel ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
Histones and their N and C terminal tails undergo different covalent modifications that regulate gene transcription. Among these histone modifications are methylation, ubiquitinilation, SUMOylation, ADP- ribosylation, phosphorylation, proline izomerization, deimination and acetylation. Histone acetylation is regulated by histonacetyltransferases (HATs) and histondeacetylases (HDACs). The balance between acetylation/deacetylation influences chromatin condensation and thus regulates gene transcription. Acetylation balance is disrupted in many human cancers and this fact can contribute to the development of malignant diseases. Histondeacetylase inhibitors (HDACi) can restore this acetylation imbalance. One of these HDACi is valproic acid (VPA) which has been used in treatment of epilepsy for decades. VPA shows antitumour effect in many studies. Decreased expression of n-myc oncoprotein, inhibition of tumour growth and angiogenesis are one of these anticancer effects observed in neuroblastoma cell lines after treatment with VPA. Despite the fact that exact mechanism of antitumour effect of VPA remains unclear, one of the most important mechanisms is hyperacetylation of histone H3 and H4. It is shown in this work that VPA increases acetylation of histones H3 and H4 in human neuroblastoma cell lines...
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Affecting the growth of a tumor on an experimental animal model
Kučera, Alexandr ; Škába, Richard (advisor) ; Eckschlager, Tomáš (referee) ; Havránek, Petr (referee) ; Kalous, Martin (referee)
In this study we present the models of preventive and therapeutic vaccination of sarcoma-bearing rats with dendritic cells that present tumor antigens from killed tumor cells. We present the characteristics of DCs-based vaccine and its capacity to induce antitumor immune response both in v vitro and in vivo. We show that preventive vaccination efficiently prevents the growth of tumors. On the other hand vaccination of rats with established tumors did not led to the eradication of tumors. Despite the induction of a vigorous immune response after administration of DCs-based vaccine and transient decrease in tumor progression, tumors eventually resumed their growth and animals vaccinated with DCs succumbed to cancer. In both settings, preventive and therapeutic, DCs-based vaccination induced a vigorous tumor specific T cells response. These results argue for the timing of cancer immunotherapy to the stages of low tumor load. Immunotherapy initiated at the stage of minimal residual disease, after reduction of tumor load by other modalities, will have much better chance to offer a clinical benefit to cancer patients than the immunotherapy at the stage of metastatic disease. Powered by TCPDF (www.tcpdf.org)
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