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Proteomika CDK inhibice v nádorových buňkách
Kovářová, Hana ; Skalníková, Helena ; Halada, Petr ; Strnad, M. ; Hajdúch, M.
In order to improve our understanding of the biochemical basis of the anti-cancer activity of olomoucine-derived synthetic cyclin-dependent kinase inhibitors (CDKIs) and to search for novel proteins associated with these biological effects we applied complex proteomic approaches. To analyse cellular responses to the CDKI we used two cancer models: the CEM T-lymphoblastic leukemia cell line representing hematological malignancy, and the A549 lung adenocarcinoma cell line as a solid tumor model. Cancer cells of these lines were cultured in both the presence and absence (controls) of the CDKI, bohemine (BOH). Cellular proteins of both of these lines were then extracted and fractionated using conventional two-dimensional gel electrophoresis (2-DE), and for the CEM T-lymphoblastic leukemia cell line we also used a 2-D liquid phase fractionation system ProteomeLab PF 2D ( Beckman Coulter). Computer-assisted data analysis of the resulting 2-D protein expression maps was applied to determine the similarity/dissimilarity of the maps and to select characteristic protein spots or bands based on the quantitative differences between BOH-treated and control cells. Many of these differentially expressed proteins were identified by mass spectrometry, since they represent candidate biomarkers of cancer cell responses to CDK inhibition and cellular pathways that are relevant to the anti-cancer activity of the CDKIs. Subsequently, we focused directly on these proteins in confirmatory studies using various techniques (including quantitative immunoblotting, immunocytochemistry and functional activity analyses) to demonstrate the validity of the proteomic results and extend our knowledge of the CDKI effects.
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CDC25A je schopna indukovat znovuzahájení meiosy, ale inhibuje metafase I – metafase II přechod
Šolc, Petr ; Šašková, Adéla ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein is present at metaphase I (MI) and metaphase II (MII) stages. CDC25A mRNA is stable during entire meiotic maturation. Exogenous CDC25A was sufficient to overcome cAMP-mediated GV-stage block. Using microinjection of GFP-CDC25A and GFP-CDC25B mRNAs constructs we have revealed that CDC25A is exclusively nuclear protein until nuclear envelope break down (NEBD). In contrast CDC25B localizes to cytoplasm at GV-stage oocytes and translocates to nucleus shortly before NEBD. Overexpression of GFP-CDC25A, to interfere with CDC25A degradation during meiotic maturation, resulted in MI block characterized with problems in chromosome congression and spindle formation. This MI block was accompanied with the transient reduction of both CDK1 and MAPK activities. RNAi mediated CDC25A knock-down resulted in a reduced ability to resume meiosis and to reach MII. These data demonstrate that behavior of CDC25A during female meiosis differs significantly from mitosis and CDC25A is involved in both, resumption of meiosis and also in metaphase I spindle formation as a prerequisite for correct MI-MII transition. It is evident that CDC25B is not only important CDC25 phosphatase for meiotic maturation but also CDC25A has its meiotic specific role.
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Aurora-A je zhrnuta v znovuzahájení meiosy a formaci metafáze I spindlu
Šašková, Adéla ; Šolc, Petr ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized entirely to the centrosome (MTOC) shorly before germinal vesicle breakdown (GVBD). Compared to somatic cells, where active Aurora-A is at the centrosomes and the spindle poles, active Aurora-A is strictly localized on MTOCs at metaphase I in oocytes. We show that activation of centrosomal Aurora-A is independent on PI3K-PKB and CDK1 signaling pathways. This was proved by cultivation of oocytes in presence of roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor). Treated oocytes show high phosphorylation of Aurora-A on T288 and centrosome amplification despite the presence of intact nuclear envelope. Silencing of Aurora-A by RNA interference induces incorrect spindle assembly. Oocytes are arrested in prometaphase I and unable to reach metaphase II. After microinjection of eGFP-Aurora-A mRNA into GV-stage oocytes, overexpression of Aurora-A leads to distortion of MI spindle organization as well. Our results indicate that Aurora-A is the key centrosomal player in meiotic maturation, essential for proper spindle formation and metaphase I - metaphase II transition.
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Aurora-A je zhrnuta v znovuzahájení meiosy a formaci metafáze I spindlu
Šašková, Adéla ; Šolc, Petr ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized entirely to the centrosome (MTOC) shorly before germinal vesicle breakdown (GVBD). Compared to somatic cells, where active Aurora-A is at the centrosomes and the spindle poles, active Aurora-A is strictly localized on MTOCs at metaphase I in oocytes. We show that activation of centrosomal Aurora-A is independent on PI3K-PKB and CDK1 signaling pathways. This was proved by cultivation of oocytes in presence of roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor). Treated oocytes show high phosphorylation of Aurora-A on T288 and centrosome amplification despite the presence of intact nuclear envelope. Silencing of Aurora-A by RNA interference induces incorrect spindle assembly. Oocytes are arrested in prometaphase I and unable to reach metaphase II. After microinjection of eGFP-Aurora-A mRNA into GV-stage oocytes, overexpression of Aurora-A leads to distortion of MI spindle organization as well. Our results indicate that Aurora-A is the key centrosomal player in meiotic maturation, essential for proper spindle formation and metaphase I - metaphase II transition.
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CDC25A je schopna indukovat znovuzahájení meiosy, ale inhibuje metafase I – metafase II přechod
Šolc, Petr ; Šašková, Adéla ; Baran, V. ; Kubelka, Michal ; Motlík, Jan
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein is present at metaphase I (MI) and metaphase II (MII) stages. CDC25A mRNA is stable during entire meiotic maturation. Exogenous CDC25A was sufficient to overcome cAMP-mediated GV-stage block. Using microinjection of GFP-CDC25A and GFP-CDC25B mRNAs constructs we have revealed that CDC25A is exclusively nuclear protein until nuclear envelope break down (NEBD). In contrast CDC25B localizes to cytoplasm at GV-stage oocytes and translocates to nucleus shortly before NEBD. Overexpression of GFP-CDC25A, to interfere with CDC25A degradation during meiotic maturation, resulted in MI block characterized with problems in chromosome congression and spindle formation. This MI block was accompanied with the transient reduction of both CDK1 and MAPK activities. RNAi mediated CDC25A knock-down resulted in a reduced ability to resume meiosis and to reach MII. These data demonstrate that behavior of CDC25A during female meiosis differs significantly from mitosis and CDC25A is involved in both, resumption of meiosis and also in metaphase I spindle formation as a prerequisite for correct MI-MII transition. It is evident that CDC25B is not only important CDC25 phosphatase for meiotic maturation but also CDC25A has its meiotic specific role.
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Modulace intracelulární kaspázové mašinérie s využitím explantátových kultur
Matalová, Eva ; Fleischmannová, Jana ; Norek, Adam ; Míšek, Ivan
Organ explant cultures eliminate animal suffering during experiments but simultaneously allow investigations of intact organ, tissue and cell systems with preserved interactions corresponding to the situation in vivo. Moreover, the explants can be manipulated in several ways using modern micromanipulation techniques (e.g. electroporation, inhibition). We show possible usage of explant culture approaches in embryonal apoptosis research in three models – mouse limb digitalization, tooth germ morphogenesis, separation of middle ear ossicles. Data from specific pharmaceutical inhibitions focused on key apoptotic molecules – caspases - are demonstrated.
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Molekulární aspekty hypodoncie
Fleischmannová, Jana ; Krejčí, P. ; Matalová, Eva ; Míšek, Ivan
Numeric dental anomalies are the most common craniofacial congenital malformations in humans. More than 20 % of human population miss one or more third molars, approximately 5 % of population display agenesis of another tooth. In this contribution molecular events underlying tooth formation (more than 200 genes have been identified so far) and defects in tooth germ formation are correlated. Both syndromic forms of hypodonia (e.g. Rieger syndrome, anhidrotic ectodermal dysplasia) and non-syndromic forms (related to pax9, msx1, axin2) were investigated.
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Charakterizace míšních progenitorových buněk in vitro
Vitásková, Martina ; Klíma, Jiří ; Vodička, Petr ; Motlík, Jan
We performed in vitro studies with the spinal cord of mice and miniature pig. Employing papain dissociation system we isolated neural progenitor cells and cultured them in DMEM/F12 HEPES, Ala-Glu, gentamicin, heparin, B27 supplement without vitamin A, N2 supplement, EGF (Epidermal Growth Factor) and bFGF (Basic Fibroblast Growth Factor). We added retinoic acid and 10% fetal bovine serum into cultivation media to induce cell differentiation. Using immunocytochemistry we confirmed cell capability to differentiate into neurons (βIII-tubulin, Map 2A - Microtubule Associated Protein), astroglia (GFAP – Glial Fibrillary Acidic Protein), and oligodendroglia (CNPase - 2',3'-cyclic nucleotide 3'-phosphodiesterase). Cells can keep non-differentiated status for a longer period of time when cultured in low adhesion culturing flask or by addition of LIF (Leukemia Inhibitory Factor) to the cultivation media.
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