Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...

Transient transfection of mammalian cell lines is an effective approach for recombinant protein production, which can provide milligrams to grams of proteins in two weeks from cloning of the corresponding cDNA. Native glycosylated proteins prepared via this approach can be used for various purposes in molecular biology, immunology or pharmaceutical industry, i.e. initial phase of pre-clinical therapeutic protein research. One of the most used mammalian host cell lines is the human embryonic kidney cell line, that can be easily cultivated and chemically transfected. The amount of proteins produced by transiently transfected human embryonic kidney cells can be enhanced by a whole range of factors, i.e. co-expression or direct addition of acidic fibroblast growth factor to the culture medium, co-expression of cell cycle regulating proteins or anti-apoptotic proteins. Expression plasmid pTW5 was prepared and further modified by gene insertion of aFGF, cell cycle regulator p18, p21 or p27 (cyclin-dependent kinase inhibitors) or apoptosis inhibitor bcl-2 or bcl-x. These plasmids were then used for optimization of HEK293T cell line expression system. The impact of every single regulator and their combinations, including hitherto undescribed effect of combination of cell cycle regulator and anti-apoptotic...

Mammalian oocytes undergoing meiotic maturation are transcriptionally silent and gene expression is therefore regulated at the level of translation. One of the well established mechanisms employed in translational regulation of maternal mRNAs in oocytes is cytoplasmic polyadenylation. This process is generally controlled by phosphorylation and activation of cytoplasmic polyadenylation element binding protein (CPEB). The aim of this thesis is to determine the role of mitogen-activated protein kinase (MAPK) in regulation of CPEB-mediated cytoplasmic polyadenylation in maturing mouse and porcine oocytes. For this purpose, MAPK activity was inhibited using its specific inhibitor, GDC-0994 and the effect of MAPK inhibition on cyclin B1 mRNA polyadenylation was monitored. In mouse oocytes, MAPK inhibition impaired neither cyclin B1 mRNA polyadenylation nor its translation and MAPK is thus unlikely to be involved in regulation of cytoplasmic polyadenylation in this species. Based on the results of experiments performed using porcine oocytes, the possible role of MAPK in CPEB-mediated cytoplasmic polyadenylation can neither be confirmed nor ruled out. Keywords: cytoplasmic polyadenylation, mouse oocyte, porcine oocyte, mitogen-activated protein kinase (MAPK), cyclin B1, GDC-0994 inhibitor

Despite significant progress in the development of anti-cancer drugs, there is still a need for novel therapeutic strategies that would improve the outcome of cancer patients. Using proteomic technologies and cell lines with different phenotype of p53 tumour suppressor, we monitored cancer cell response to anti-cancer treatment with focus on the development of drug resistance. The different levels of metabolic proteins were identified in our study which may help to explain different anti-cancer activity of drugs with only a subtle difference in structure. More importantly, proteins associated with the development of drug resistance were identified and such expression changes have become a focus of interest. Our findings demonstrate a higher protein level of serine hydroxymethyltransferase, serpin B5 and calretinin in cancer cells resistant to Aurora kinase inhibitors. Such proteins promote the tumour growth with no apparent impact of p53 phenotype whilst voltage-dependent anion-selective channel protein 2 contributes to the development of resistance only in cells with functional p53 which is accompanied by the decreased level of elongation factor 2. On the other hand, cancer cells with loss of p53 appear to amplify alternative mechanisms such as protection against oxidative stress. The results...