ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.
ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o. ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.
cell lines using multiple methodical approaches. Human metastatic cancer cell lines were used as positive controls. The goal of this work was to find an optimal functional assay which could be used for subsequent functional assessment of specific molecules in cell migration and invasion. To study cell migration, we performed chemotactic migration assays using transwell chambers or real-time analysis with xCELLigence system. Alternatively, a “wound healing - scratch assay” or agarose spot invasion assay was tested. Cell invasion was studied in a similar setup to cell migration, after coating the membranes with ECM proteins such as collagen or Matrigel. Activity of ECM degrading enzymes was analyzed by zymography or a fluorescence microscopy-based gelatinase assay, expression of MMP proteins was evaluated by western blotting. Cell migration towards chemoattractant was enhanced by starving the cells in growth factor depleted media. We observed a specific and robust activation of TGF-beta signaling pathway under these conditions. These results were correlated with expression of a set of secreted proteins on a cytokine array. Altogether, we observed that cancer transformation of human benign prostate hyperplasia-derived BPH-1 cells is associated with increased migration potential, but not with significant enhancement of cell invasion associated with ECM protein degradation. This work was supported by grant no. P301/12/P407 of the Czech Science Foundation; by grant no. NT13573-4/2012 of the Ministry of Health of the Czech Republic; by the Academy of Sciences of the Czech Republic, grant no. AV0Z50040702, and by the project FNUSA-ICRC no. CZ.1.05/1.1.00/02.0123 from the European Regional Development Fund. References: Slabakova, E., et al., TGF-beta1-induced EMT of non-transformed prostate hyperplasia cells is characterized by early induction of SNAI2/Slug. Prostate, 2011. P29. NKD1- CREER T2 MOUSE STRAIN: A NEW TOOL FOR SITE-SPECIFIC RECOMBINATION IN WNT RESPONSIVE CELLS OF MOUSE INTESTINE AND LIVER Fafílek Bohumil 1 , Stančíková Jitka 1,2 , Hlavatá Adéla 1,2 , Kořínek Vladimír 1 1 Laboratory of Cell and Developmental Biology, IMG AV CR (jitka.stancikova@img.cas.cz) 2 Faculty of Science, Charles University in Prague Wnt signaling pathway plays a crucial role in ontogenesis and development of all metazoans. In adult mammals, the Wnt signaling pathway is required for the maintenance of the intestinal homeostasis and establishment of proper hepatic zonation. In contrary to that, aberrant activation of the Wnt pathway leads to neoplasia and cancer development, notably in the intestine and liver. To investigate the role of the Wnt pathway in gut epithelium homeostasis and its malignant transformation we employed chromatin immunoprecipitation method (ChIP) in combination with DNA microarrays (so-called ChIP-on-chip) to identify genes regulated by the Wnt signaling. One of the most prominent targets was the NKD1 (Naked Cuticle Analytical Cytometry VII 121
Homolog 1) gene; previously identified as a Wnt-induced intracellular negative regulator of the canonical Wnt signaling. With use of BAC recombineering, we generated mice with CreER T2 recombinase produced corresponding to the gene NKD1 (NKD1-CreER T2 ) expression. Comparing the natural NKD1 expression with transgenic Cre in NKD1-Cre x Rosa-lacZ reporter strain hybrids proved that the transgenic mouse produces Cre in NKD1 + cells only. Two of the most interesting sites of the NKD1-CreER T2 expression in adult mice are perivenous hepatocytes and intestinal crypt compartment, which was confirmed by the expression profiling. New mouse strain NKD1-Cre ER T2 is therefore a unique tool for gene manipulation particularly in hepatocytes localized in the perivenous zone. P30. EFFECTS OF CHOLESTANE BRASSINOSTEROID DERIVATIVES ON HORMONE-IN/ SENSITIVE BREAST AND PROSTATE CANCER CELL LINES Steigerová J. 1,2 , Rárová L. 3 , Křížová K. 2 , Oklešťková J. 4 , Šváchová M. 5 , Levková M. 2 , Kolář Z. 1,2 a Strnad M. 3,4 1 Laboratory of Molecular Pathology, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech Republic 2 Institute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry, Palacký University and Faculty Hospital in Olomouc, Puškinova 6, 775 20 Olomouc, Czech Republic 3 Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Growth Regulators, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic 4 Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic 5 Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacký University and University Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic Brassinosteroids (BRs), polyhydroxylated sterol derivatives with close structural similarity to animal and insect steroid hormones, are plant growth regulators representing a group of newly-discovered agents with relatively wide-ranging effects in plants. Based on a structural domain similarity between steroids and BRs, the brassinosteroid cytotoxic activity could be, at least partially, related to brassinosteroid-steroid receptor interactions. Investigation of the mechanisms of action of BRs in human cancer cells using cellular and molecular techniques indicated the possible involvement of steroid receptors in BR action. Understanding the mechanisms of nuclear receptor action will enhance our knowledge of transcription and hormone influences on disease and facilitate the design of drugs with greater therapeutic value. Molecular and cellular effects of natural BRs and their synthetic cholestane derivatives were examined in human hormone-in/sensitive breast (MCF-7, MDA-MB-68) and 122 Analytical Cytometry VII
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Homolog 1) gene; previously identified as a Wnt-induced intracellular negative regulator<br />
of the canonical Wnt signaling.<br />
With use of BAC recombineering, we generated mice with CreER T2 recombinase produced<br />
corresponding to the gene NKD1 (NKD1-CreER T2 ) expression. Comparing the natural<br />
NKD1 expression with transgenic Cre in NKD1-Cre x Rosa-lacZ reporter strain hybrids<br />
proved that the transgenic mouse produces Cre in NKD1 + cells only. Two of the most<br />
interesting sites of the NKD1-CreER T2 expression in adult mice are perivenous hepatocytes<br />
and intestinal crypt compartment, which was confirmed by the expression profiling. New<br />
mouse strain NKD1-Cre ER T2 is therefore a unique tool for gene manipulation particularly<br />
in hepatocytes localized in the perivenous zone.<br />
P30. EFFECTS OF CHOLESTANE BRASSINOSTEROID DERIVATIVES ON HORMONE-IN/<br />
SENSITIVE BREAST AND PROSTATE CANCER CELL LINES<br />
Steigerová J. 1,2 , Rárová L. 3 , Křížová K. 2 , Oklešťková J. 4 , Šváchová M. 5 , Levková M. 2 , Kolář<br />
Z. 1,2 a Strnad M. 3,4<br />
1<br />
Laboratory of Molecular Pathology, Department of Clinical and Molecular Pathology,<br />
Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc,<br />
Czech Republic<br />
2<br />
Institute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry,<br />
Palacký University and Faculty Hospital in Olomouc, Puškinova 6, 775 20 Olomouc,<br />
Czech Republic<br />
3<br />
Centre of the Region Haná for Biotechnological and Agricultural Research, Department<br />
of Growth Regulators, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71<br />
Olomouc, Czech Republic<br />
4<br />
Laboratory of Growth Regulators, Palacký University & Institute of Experimental<br />
Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic<br />
5<br />
Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry,<br />
Palacký University and University Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc,<br />
Czech Republic<br />
Brassinosteroids (BRs), polyhydroxylated sterol derivatives with close structural similarity<br />
to animal and insect steroid hormones, are plant growth regulators representing a<br />
group of newly-discovered agents with relatively wide-ranging effects in plants. Based<br />
on a structural domain similarity between steroids and BRs, the brassinosteroid<br />
cytotoxic activity could be, at least partially, related to brassinosteroid-steroid receptor<br />
interactions. Investigation of the mechanisms of action of BRs in human cancer cells<br />
using cellular and molecular techniques indicated the possible involvement of steroid<br />
receptors in BR action. Understanding the mechanisms of nuclear receptor action<br />
will enhance our knowledge of transcription and hormone influences on disease and<br />
facilitate the design of drugs with greater therapeutic value.<br />
Molecular and cellular effects of natural BRs and their synthetic cholestane derivatives<br />
were examined in human hormone-in/sensitive breast (MCF-7, MDA-MB-68) and<br />
122 Analytical Cytometry VII
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