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Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The TEAD family of transcription factors regulates expression of genes affecting cell proliferation, differentiation and apoptosis. The activity of a particular transcription factor called TEAD1 is regulated by the Hippo signalling pathway. The Hippo pathway has been implicated to play a role in cancer suppression, however its precise mechanism remains unclear. MYC and GLUT1, genes which are coding two key regulators of glycolysis, were recently described as potential targets of the Hippo signalling pathway in human leukemia cells. In this diploma thesis, I tried to confirm the proposed interaction of the transcription factor TEAD1 with regulatory sequences of MYC and GLUT1 genes using chromatin immunoprecipitation (ChIP) analysis in human leukemic cells. However, I failed to successfully isolate TEAD1 complexes using ChIP. So, I discuss in my diploma thesis also possible reasons for this outcome, including biological and methodological issues. (In Czech) Key words: Transcriptional regulation, TEAD transcription factors, chromatin immunoprecipitation, leukemia
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Molecular mechanism of anti-cancer activity of selected natural compounds.
Smolová, Dagmar ; Vališ, Karel (advisor) ; Flieger, Miroslav (referee)
Molecular mechanism of anti-cancer activity of selected natural compounds Přírodovědecká fakulta Univerzity Karlovi v Praze Smolová Dagmar Nowadays, cancer is affecting more then a third of the world's population and it's the cau- se of more than 20% of all deaths. A better understanding of cancer at the epigenic, gene- tic, molecular and cellular levels is opening up lots of opportunities to intervene with the goal of preventing end-stage of the invasive disease. Recently, much attention is paid to the natural substances because of its ability to interact specifically in the tumorgenesis. This compounds are preferred before the standard treatment because of its availability and safe- ty. In this work, there were observed the effects of 6-shogaol, 6-gingerol and galangin in the leukemic cells Jurkat. The greatest efficiency was showed by 6-shogaol wich caused apoptosis in cells by activating the antiproliferative signalling of Hippo pathway and it also decreased the expression of C-MYC oncogene. There was also a decline of C-MYC onco- protein wich has a key role in the tumor cells metabolism through it's ability to provide enought of energy for the growth and active proliferation. (In Czech)
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Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The family of transcription factors TEAD regulates the expression of genes that affect cell proliferation, differentiation and apoptosis. Activity of TEAD1 is regulated via the Hippo signaling pathway. General mechanism of tumor cell suppression by the Hippo signaling pathway remains unclear. C-MYC and GLUT1, the two key regulators of glycolysis, were recently described as targets of the Hippo signaling pathway in human leukemia cells. In this diploma thesis, the interaction of TEAD1 with M-CAT binding motifs was experimentally confirmed in the first exon of C-MYC gene. In addition, a new interaction of TEAD1 with M-CAT binding motifs has been found in the enhancer of C-MYC promoter and enhancer of GLUT1 promoter by ChIP analysis. Regulation of glucose metabolism by the Hippo signaling pathway may represent a new mechanism of tumor cell suppression. Key words: Gene regulation, transcription factors, chromatin immunoprecipitation, bioinformatics
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Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The TEAD family of transcription factors regulates expression of genes affecting cell proliferation, differentiation and apoptosis. The activity of a particular transcription factor called TEAD1 is regulated by the Hippo signalling pathway. The Hippo pathway has been implicated to play a role in cancer suppression, however its precise mechanism remains unclear. MYC and GLUT1, genes which are coding two key regulators of glycolysis, were recently described as potential targets of the Hippo signalling pathway in human leukemia cells. In this diploma thesis, I tried to confirm the proposed interaction of the transcription factor TEAD1 with regulatory sequences of MYC and GLUT1 genes using chromatin immunoprecipitation (ChIP) analysis in human leukemic cells. However, I failed to successfully isolate TEAD1 complexes using ChIP. So, I discuss in my diploma thesis also possible reasons for this outcome, including biological and methodological issues. (In Czech) Key words: Transcriptional regulation, TEAD transcription factors, chromatin immunoprecipitation, leukemia
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The effect of curcumin on carcinogenesis and metastasis development
Kašík, Petr ; Kábelová, Adéla (advisor) ; Vališ, Karel (referee)
Curcumin is a polyphenol from the roots of plant Curcuma longa. Highly pleiotropic effect of curcumin has presently application not only in prevention, but also in treatment of various diseases including cancer. Therefore, this bachelor thesis aims to summarize the current knowledge concerning the effect of curcumin on carcinogenesis and metastasis development. Considering the inhibition of cancer cells proliferation, curcumin stops the progression of cell cycle, primarily via regulating the activity of cyclins and cyclin-dependent kinases, their inhibitory proteins or tumor suppressor protein p53. Additionally, p53 also seems to have an important role in the initiation of intrinsic apoptotic pathway, which was observed in many cancer cell lines after curcumin administration. Apoptotic proteins involved in apoptosis induction after curcumin application also comprise Bcl-2 family proteins and, caspases. Other mechanisms exerted by which curcumin prevent cancer development include suppression of angiogenesis, increase of oxidative stress or modulation of inflammatory response. Finally, curcumin also suppress cancer cells invasion and migration by enhancing the transcription of E- cadherin and decreasing of the transcription of matrix metalloproteinases. Because the application of curcumin in clinical...
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Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The family of transcription factors TEAD regulates the expression of genes that affect cell proliferation, differentiation and apoptosis. Activity of TEAD1 is regulated via the Hippo signaling pathway. General mechanism of tumor cell suppression by the Hippo signaling pathway remains unclear. C-MYC and GLUT1, the two key regulators of glycolysis, were recently described as targets of the Hippo signaling pathway in human leukemia cells. In this diploma thesis, the interaction of TEAD1 with M-CAT binding motifs was experimentally confirmed in the first exon of C-MYC gene. In addition, a new interaction of TEAD1 with M-CAT binding motifs has been found in the enhancer of C-MYC promoter and enhancer of GLUT1 promoter by ChIP analysis. Regulation of glucose metabolism by the Hippo signaling pathway may represent a new mechanism of tumor cell suppression. Key words: Gene regulation, transcription factors, chromatin immunoprecipitation, bioinformatics
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Interplay between cellular signaling and metabolism in cancer cells.
Záhumenská, Romana ; Vališ, Karel (advisor) ; Starková, Júlia (referee)
Hippo signaling pathway represents organ size control mechanism constrained between all metazoans. Individual components of the Hippo signaling pathway were identified as key tumor-suppressors which phosphorylate and inhibit activity of several oncogenic factors and signaling pathways (such as YAP/TAZ, PI3K and mTOR). MST1 kinase is a part of central protein complex of the Hippo signaling pathway and its activation is involved in anti-cancer activity of several drugs. We have demonstrated activation of the MST1 kinase by natural compounds in leukemic cells followed by inhibition of proliferation and induction of apoptosis. Shikonin represents natural naphthoquinonic compound isolated from Lithospermum erythrorhizon which acts as inhibitor of glycolysis and mitochondrial respiratory chain in human cells. Shikonin induces fast activation of the MST1 protein in leukemic cells however mechanism of this activation remains unknown. Therefore, we tried to characterize posttranslational modifications of the MST1 kinase during shikonin treatment of leukemic cells. Firstly, we isolated MST1 kinase from control and shikonin-treated cells using immunoprecipitation. Then we characterized posttranslational modifications of the MST1 protein employing mass spectrometry. Using this approach we found out...
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Molecular mechanism of MST1 kinase activation in cancer cells
Smetanová, Jitka ; Vališ, Karel (advisor) ; Grobárová, Valéria (referee)
MST1 kinase is an internal part of the Hippo signal pathway. The Hippo pathway is an evolutionary conserved regulator of tissue and organ growth and affects proliferation and apoptosis. Active MST1 kinase phosphorylates YAP and TAZ oncoproteins, which regulate the activity of transcription factors in their unphosphorylated state, including TEAD and SMAD. Furthermore active MST1 kinase phosphorylates FOXO transcription factors and induces their translocation into the cell nucleus. Finally the activation of MST1 kinase leads to cell apoptosis or halt cell cycle in G1 phase. Activation of MST1 protein depends on its auto-phosphorylation and cleavage. Recently, there are several articles which take interest in the issue of activation of MST1. Some of them describe the activation of MST1 by the effector caspase-3 and -7, on the other hand the latest articles argue that MST1 kinase itself is responsible for the activation of caspases. The molecular mechanism of MST1 kinase activation was studied in this bachelor thesis. We used the biologically active compounds GDC-0941 for the activation of MST1 protein. The activity of caspase was inhibited by specific inhibitor Z-DEVD. Using electrophoresis and Western blot it was demonstrated that MST1 is active in the case when caspases are inhibited. This fact...
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Molecular mechanism of anti-cancer activity of selected natural compounds.
Smolová, Dagmar ; Vališ, Karel (advisor) ; Flieger, Miroslav (referee)
Molecular mechanism of anti-cancer activity of selected natural compounds Přírodovědecká fakulta Univerzity Karlovi v Praze Smolová Dagmar Nowadays, cancer is affecting more then a third of the world's population and it's the cau- se of more than 20% of all deaths. A better understanding of cancer at the epigenic, gene- tic, molecular and cellular levels is opening up lots of opportunities to intervene with the goal of preventing end-stage of the invasive disease. Recently, much attention is paid to the natural substances because of its ability to interact specifically in the tumorgenesis. This compounds are preferred before the standard treatment because of its availability and safe- ty. In this work, there were observed the effects of 6-shogaol, 6-gingerol and galangin in the leukemic cells Jurkat. The greatest efficiency was showed by 6-shogaol wich caused apoptosis in cells by activating the antiproliferative signalling of Hippo pathway and it also decreased the expression of C-MYC oncogene. There was also a decline of C-MYC onco- protein wich has a key role in the tumor cells metabolism through it's ability to provide enought of energy for the growth and active proliferation. (In Czech)
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Mechanisms of anticancer drug action in neuroblastomas
Groh, Tomáš ; Stiborová, Marie (advisor) ; Levová, Kateřina (referee) ; Vališ, Karel (referee)
Cancer cells are able to adapt to different stress factors such as hypoxia, which is caused by insufficient tumor vascularization. An increased acetylation status of histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines was found to be a mechanism of adaptation of these cells to hypoxia. An increase in acetylation of histones H3 and H4 is suggested to cause changes in the structure of chromatin that lead to activation of gene transcription. In addition, cultivation of tested neuroblastoma cells under hypoxic conditions changes expression of proteins of a transcription factor N-myc, which is essential for development of neuroblastomas. This transcription factor is also responsible for a metabolic adaptation of neuroblastoma cells, increases their aggressiveness and its expression leads to a worse prognosis of the disease. Inhibitors of histone deacetylases (HDAC) are suggested to be the promising agents exhibiting various anticancer effects. They can induce cell cycle arrest, differentiation or programmed cell death in sensitive tumors. In this study, the effect of one of inhibitors of HDACs, valproate, on expression of proteins of transcription factors N-myc and hypoxia inducible factor 1α (HIF-1α) was investigated. Valproate decreases protein levels of both transcription factors in...
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