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Study of the effect of roscovitine and its derivatives on the expression of drugs transporters in vitro
Mlčochová, Alice ; Čečková, Martina (advisor) ; Carazo Fernández, Alejandro (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Mgr. Alice Mlčochová Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of rigorous thesis: Study of the effect of roscovitine and its derivatives on the expression of drugs transporters in vitro In this study we aimed to evaluate interactions of selected inhibitors of cyclin-dependent kinases (CDKi) with efflux transporters ABCB1 (P-glycoprotein, P-gp, MDR1) and ABCG2 (breast cancer resistance protein, BCRP) in cell line LS174T derived from human adenocarcinoma of colon. Among the selected compounds belong roskovitin and its derivates designed as BA-12 and BP-14. Previous studies demonstrated that these substances inhibit both mentioned transporters and therefore increase the effect of simultaneously administrated cytotoxic drugs. Since the drug interactions on the level of transporters can arise from induction of their corresponding genes, the aim of this work was to extend the current knowledge on mentioned derivates of roskovitin in terms of possible affection of ABCB1 and ABCG2 expression. Enhanced expression could promote developement of multidrug resistance (MDR). Results of this work obtained with qRT- PCR method show that neither BA-12 nor BP-14 increase expression of...
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Optimization of HEK293 cell line expression system by regulation of cell cycle and apoptosis
Poláchová, Edita ; Vaněk, Ondřej (advisor) ; Pavlíček, Jiří (referee)
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...
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