Laboratory of Subcellular Structures Analyses

 


 

Head:
Eliza Wyszko Associate Prof, 

 

 


Staff:

Małgorzata Giel-Pietraszuk

Associate Prof.;

Agnieszka Fedoruk-Wyszomirska,

Ph D;

Dorota Gurda

Ph D


PhD Students:
Marta Orlicka-Płocka, MSc;

Kinga Gwóźdź-Bąk, MSc

 

 

 


 

 

 

Key words:

confocal microscopy, fluorescent microscopy, flow cytometry, thermocycler LightCycler 480, xCELLigence system, CO2 incubators, laminar flow cabinet, HPLC

low molecular compounds, nucleosides, nucleic acids modifications, DNA – and RNA-adducts, catalytic nucleic acids

 

Scientific profile:

The laboratory possesses equipment to analysis of intracellular structures and monitoring cellular processes as apoptosis, necrosis, proliferation and influence of reactive oxygen species (ROS) on regulation of physiological conditions.

Our equipment allow searching for new therapeutics (low molecular compounds) through cytotoxicity analysis, changes in gene expression (LightCycler 480 Real-Time PCR),transfer of genetic information and signaling, initiation or cell death programming (flow cytometry), intravital confocal analysis of fluorescently labeled cells in monolayer or 3D using Leica TCS SP5 microscope. The Laboratory is concerned with analysis of cellular parameters such as proliferation, cytotoxicity and cell cycle through impedance measurement using xCELLigence system or using flow cytometer.

 

Current research activity

  • Implementation of the NCS research project OPUS: ” Kinetin riboside and its derivatives-analysis of apoptotic properties and mechanism of action in brain tumor cells (2015-2018). We estimated IC50 value for kinetin riboside (KR) on tumorigenic cell lines: glioblastoma (U87, A172), ovary (A2780) and lungs (A-549) and non-tumorigenic as lung fibroblasts (MRC-5). Using flow cytometry we estimated influence of RK on cell viability. We showed that RK decreases mitochondrial membrane potential. It was shown that RK is able to induce oxidative stress. We also tested in vitro phosphorylation of RK by adenosine kinase.
  • Analysis of intracellular processes (apoptosis, necrosis, DNA content, cell proliferation, cell membrane oxidative potential) in the presence of low molecular compounds and their derivatives with potential anti-cancer (e.g. 6-N-furfuryladenosine, Cu- and Ag-complexes), anti-aging (4-N-furfurylcytosine) and anti-neurodegenerative properties (e.g. β-carbolines) using confocal microscopy, flow cytometry and real-time PCR.
  • Participation to NCS project OPUS:” RNA hybryd duplex-quadruplex structures with dual functionality as a new tool for silencing of gene expression”. Analysis of biological activity different variants of RNA quadruplex in cell lines transfecte with pEGFP vector coding target sequence for analyzed RNA molecules and subsequent evaluation of EGFR expression silencing with mutation in exon 21.

 

Most important research achievements:

  • We examined three structural variants of hammerhead ribozymes (RzHH): minimal, truncated and extended, that hydrolyzes target sequence within GUC trinucleotides GUC, AUC and GAA. Structural differences between analyzed RzHH defined their catalytic activity. On the basis of the in vitro results as well as in eukaryotic cells we selected the best variant of hammerhead ribozyme.
  • The subject of the recent study were L- and D- optic enantiomers of hammerhead ribozymes and DNAzymes where D-ribose or D-deoxyribose is replaced with its D-enantiomer. We showed that these L-ribozymes nad L-DNAzymes are highly stable in human serum, similarly to Spiegelmers – non-toxic and non-immunogenic aptamers.
  • Our current research issue concerns analysis of molecular compounds e.g. 4-N-furfurylcytosine (FC), which is cytosine analog N4-substituted with furfural group. Our experiments showed that FC has beneficial influence on human normal cell with its anti-oxidative properties. FC is especially effective in reducing oxidative damage in age-differentiated cells. Its mechanism of action may be considered on many levels from changes in gene expression to impact on cellular structures.

 

Actual research projects:

  • NSC project, OPUS 7, Kinetin riboside and its derivatives-analysis of apoptotic properties and mechanism of action in brain tumor cells ( 2015-2018)
  • NSC project, OPUS 7 RNA hybryd duplex-quadruplex structures with dual functionality as a new tool for silencing of gene expression  (2014-2017)
  • NSC project, OPUS 5, Ribozymes as a new tool for mitochondrial genome research (2014 -2017)
  • NSC project, OPUS 10, Regulation of dietary neuroactive β-carbolines level based on coffee substitutes (2016-2019)

 

Selected publications:

  • A. Fedoruk-Wyszomirska, M. Szymański, P. Głodowicz, M.M. Gabryelska, E. Wyszko, W.J. Estrin, J. Barciszewski, Inhibition of HIV-1 gp41 expression with hammerhead ribozymes. Biochem J., 471, 53-66 (2015)
  • A.M. Barciszewska, D. Gurda, P. Głodowicz, S. Nowak, M.Z. Naskręt-Barciszewska, A New Epigenetic Mechanism of Temozolomide Action in Glioma Cells, PLoS One, 10, e0136669 (2015)
  • D. Gurda, L. Handschuh, W. Kotkowiak, H. Jakubowski, Homocysteine thiolactone and N-homocysteinylated protein induce pro-atherogenic changes in gene expression in human vascular endothelial cells. Amino Acids, 47, 1319-1339 (2015)
  • D. Mileshina, A.K. Niazi, E. Wyszko, M. Szymanski, R. Val, C. Valentin, J. Barciszewski, A. Dietrich,Mitochondrial targeting of catalytic RNAs. Methods Mol. Biol., 1265, 227-254 (2015)
  • M. Kucinska, P. Skupin-Mrugalska, W. Szczolko, L. Sobotta, M. Sciepura, E. Tykarska, M. Wierzchowski, A. Teubert, A. Fedoruk-Wyszomirska, E. Wyszko, M. Gdaniec, M. Kaczmarek, T. Goslinski, J. Mielcarek, M. Murias, Phthalocyanine derivatives possessing 2-(morpholin-4-yl)ethoxy groups as potential agents for photodynamic therapy. J. Med. Chem., 58, 2240-2255 (2015)
  • M.A. Fik, A. Gorczyński, M. Kubicki, Z. Hnatejko, A. Fedoruk-Wyszomirska, E. Wyszko, M. Giel-Pietraszuk, V. Patroniak, 6,6″-Dimethyl-2,2':6',2″-terpyridine revisited: new fluorescent silver(I) helicates with in vitro antiproliferative activity via selective nucleoli targeting. Eur. J. Med. Chem., 86, 456-468 (2014)
  • M. Małgowska, D. Gudanis, R. Kierzek, E. Wyszko, V. Gabelica, Z. Gdaniec, Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats. Nucleic Acids Res., 42, 10196-207 (2014)
  • E. Wyszko, F. Mueller, M Gabryelska, A. Bondzio, M. Popenda, J. Barciszewski, V.A. Erdmann, Spiegelzymes® mirror-image hammerhead ribozymes and mirror-image DNAzymes, an alternative to siRNAs and microRNAs to cleave mRNAs in vivo? PLoS One, 9, e86673 (2014)
  • M.M. Gabryelska, E. Wyszko, M. Szymański, M. Popenda, J. Barciszewski, Prediction of hammerhead ribozyme intracellular activity with the catalytic core fingerprint. Biochem J., 451, 439-451 (2013)
  • E. Wyszko, M. Szymański, H. Zeichhardt, F. Müller, J. Barciszewski, V.A. Erdmann, Spiegelzymes: Sequence Specific Hydrolysis of L-RNA with Mirror Image Hammerhead Ribozymes and DNAzymes. PLoS One, 8, e54741. (2013)