Zuzana Pernicová

Publications (8)27.96 Total impact

• Article: Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

  Radek Fedr, Zuzana Pernicová, Eva Slabáková, Nicol Straková, Jan Bouchal, Michal Grepl, Alois Kozubík, Karel Souček
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  ABSTRACT: The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. © 2013 International Society for Advancement of Cytometry.
  Cytometry Part A 02/2013; · 3.73 Impact Factor
• Article: TGF-β1 signaling plays a dominant role in the crosstalk between TGF-β1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells.

  Andrea Staršíchová, Eva Hrubá, Eva Slabáková, Zuzana Pernicová, Jiřina Procházková, Kateřina Pěnčíková, Václav Seda, Markéta Kabátková, Jan Vondráček, Alois Kozubík, Miroslav Machala, Karel Souček
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  ABSTRACT: Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-β1 (TGF-β1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-β1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-β1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-β1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-β1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context.
  Cellular signalling 04/2012; 24(8):1665-76. · 4.09 Impact Factor
• Article: Androgen depletion induces senescence in prostate cancer cells through down-regulation of Skp2.

  Zuzana Pernicová, Eva Slabáková, Gvantsa Kharaishvili, Jan Bouchal, Milan Král, Zuzana Kunická, Miroslav Machala, Alois Kozubík, Karel Souček
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  ABSTRACT: Although the induction of senescence in cancer cells is a potent mechanism of tumor suppression, senescent cells remain metabolically active and may secrete a broad spectrum of factors that promote tumorigenicity in neighboring malignant cells. Here we show that androgen deprivation therapy (ADT), a widely used treatment for advanced prostate cancer, induces a senescence-associated secretory phenotype in prostate cancer epithelial cells, indicated by increases in senescence-associated β-galactosidase activity, heterochromatin protein 1β foci, and expression of cathepsin B and insulin-like growth factor binding protein 3. Interestingly, ADT also induced high levels of vimentin expression in prostate cancer cell lines in vitro and in human prostate tumors in vivo. The induction of the senescence-associated secretory phenotype by androgen depletion was mediated, at least in part, by down-regulation of S-phase kinase-associated protein 2, whereas the neuroendocrine differentiation of prostate cancer cells was under separate control. These data demonstrate a previously unrecognized link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. We propose that ADT may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells.
  Neoplasia (New York, N.Y.) 06/2011; 13(6):526-36. · 5.48 Impact Factor
• Article: TGF-β1-induced EMT of non-transformed prostate hyperplasia cells is characterized by early induction of SNAI2/Slug.

  Eva Slabáková, Zuzana Pernicová, Eva Slavíčková, Andrea Staršíchová, Alois Kozubík, Karel Souček
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  ABSTRACT: Epithelial-mesenchymal transition (EMT) underlying cancer cell invasion and metastasis has been thoroughly studied in prostate cancer. Although EMT markers have been clinically observed in benign prostate hyperplasia, molecular events underlying the onset and progression of EMT in benign prostate cells have not been described. EMT in BPH-1 cells was induced by TGF-β1 treatment and the kinetics of expression of EMT markers, regulators, and selected miRNAs was assessed by western blotting and quantitative RT-PCR. EMT in BPH-1 cells was accompanied by rapid up-regulation of SNAI2/Slug and ZEB1 transcription factors, while changes in expression levels of ZEB2 and miR-200 family members were observed after extended time intervals. Invasive phenotype with EMT hallmarks, characterizing tumorigenic clones derived from BPH-1 cells, was associated with increased mRNA levels of SNAI2, ZEB1, and ZEB2, but was not associated with significant changes in basal levels of miR-200 family members. RNA interference revealed that SNAI2/Slug is crucial for TGF-β1-induced vimentin up-regulation and migration of BPH-1 cells. This study suggests that in BPH-1 cells the transcription factor SNAI2/Slug is important for EMT initiation, while the ZEB family of transcription factors in cooperation with the miR-200 family may oppose the reversal of the EMT phenotype.
  The Prostate 02/2011; 71(12):1332-43. · 3.48 Impact Factor
• Article: TGF-beta1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells.

  Andrea Starsíchová, Eva Lincová, Zuzana Pernicová, Alois Kozubík, Karel Soucek
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  ABSTRACT: Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-beta1 (TGF-beta1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-beta1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-beta1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-beta1 and IL-6 signalling and suggested another mechanism of how defects in TGF-beta signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.
  Cellular signalling 11/2010; 22(11):1734-44. · 4.09 Impact Factor
• Source

  Available from: Karel Soucek

  Article: Fetal colon cell line FHC exhibits tumorigenic phenotype, complex karyotype, and TP53 gene mutation.

  Karel Soucek, Pavla Gajdusková, Marie Brázdová, Martina Hýzd'alová, Lenka Kocí, David Vydra, Radek Trojanec, Zuzana Pernicová, Lenka Lentvorská, Marián Hajdúch, Jirina Hofmanová, Alois Kozubík
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  ABSTRACT: Stable cell lines obtained by spontaneous immortalization might represent early stages of malignant transformation and be useful experimental models for studies of mechanisms of cancer development. The FHC (fetal human cells) cell line has been established from normal fetal colonic mucosa. Detailed characterization of this cell line and mechanism of spontaneously acquired immortality have not been described yet. Therefore, we characterized the FHC cell line in terms of its tumorigenicity, cytogenetics, and TP53 gene mutation analysis. FHC cells displayed capability for anchorage-independent growth in semisolid media in vitro and formed solid tumors after transplantation into SCID (severe combined immunodeficiency) mice. This tumorigenic phenotype was associated with hypotriploidy and chromosome number ranging from 66 to 69. Results of comparative genetic hybridization arrays showed that most chromosomes included regions of copy number gains or losses. Region 8q23 approximately 8q24.3 (containing, e.g., MYC proto-oncogene) was present in more than 20 copies per nucleus. Moreover, we identified mutation of TP53 gene in codon 273; triplet CGT coding Arg was changed to CAG coding His. Expression of Pro codon 72 polymorphic variant of p53 was also detected. Mutation of TP53 gene was associated with abolished induction of p21(Waf1/Cip1) and MDM-2 proteins and resistance to apoptosis after genotoxic treatment. Because of their origin from normal fetal colon and their relative resistance to the induction of apoptosis, FHC cells can be considered a valuable experimental model for various studies.
  Cancer genetics and cytogenetics 03/2010; 197(2):107-16. · 1.54 Impact Factor
• Source

  Available from: Karel Soucek

  Article: Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1.

  Andrea Staršíchová, Lukáš Kubala, Eva Lincová, Zuzana Pernicová, Alois Kozubík, Karel Souček
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  ABSTRACT: The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial-mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.
  Biological Procedures Online 09/2009; 11:316-24. · 1.29 Impact Factor
• Source

  Available from: Karel Soucek

  Article: Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs.

  Eva Lincová, Ales Hampl, Zuzana Pernicová, Andrea Starsíchová, Pavel Krcmár, Miroslav Machala, Alois Kozubík, Karel Soucek
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  ABSTRACT: Antitumorigenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. However, the mechanisms driving these processes are not understood in all details. In our study, we observed significant differences in sensitivity of cancer epithelial cell lines to COX-independent antiproliferative effects of NSAIDs. The prostate cancer cell line LNCaP, lacking both critical enzymes in the negative control of PKB/Akt activation, PTEN and SHIP2, was the most sensitive to these effects, as assessed by analysing the cell cycle profile and expression of cell cycle regulating proteins. We found that p53 protein and its signalling pathway is not involved in early antiproliferative action of the selected NSAID-indomethacin. RNAi provided evidence for the involvement of p21(Cip1/Waf1), but not GDF-15, in antiproliferative effects of indomethacin in LNCaP cells. Interestingly, we also found that indomethacin activated PKB/Akt and induced nuclear localisation of p21(Cip1/Waf1) and Akt2 isoform. Our results are in agreement with other studies and suggest that maintaining of the p21(Cip1/Waf1) level and its intracellular localisation might be influenced by Akt2. Knock-down of SHIP2 by RNAi in PTEN negative prostate and colon cancer cell lines resulted in higher sensitivity to antiproliferative effects of indomethacin. Our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.
  Biochemical pharmacology 06/2009; 78(6):561-72. · 4.25 Impact Factor