Publications by authors named "Eva Hruba"

21 Publications

  • Page 1 of 1

An RNA aptamer restores defective bone growth in FGFR3-related skeletal dysplasia in mice.

Sci Transl Med 2021 May;13(592)

Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic.

Achondroplasia is the most prevalent genetic form of dwarfism in humans and is caused by activating mutations in FGFR3 tyrosine kinase. The clinical need for a safe and effective inhibitor of FGFR3 is unmet, leaving achondroplasia currently incurable. Here, we evaluated RBM-007, an RNA aptamer previously developed to neutralize the FGFR3 ligand FGF2, for its activity against FGFR3. In cultured rat chondrocytes or mouse embryonal tibia organ culture, RBM-007 rescued the proliferation arrest, degradation of cartilaginous extracellular matrix, premature senescence, and impaired hypertrophic differentiation induced by FGFR3 signaling. In cartilage xenografts derived from induced pluripotent stem cells from individuals with achondroplasia, RBM-007 rescued impaired chondrocyte differentiation and maturation. When delivered by subcutaneous injection, RBM-007 restored defective skeletal growth in a mouse model of achondroplasia. We thus demonstrate a ligand-trap concept of targeting the cartilage FGFR3 and delineate a potential therapeutic approach for achondroplasia and other FGFR3-related skeletal dysplasias.
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http://dx.doi.org/10.1126/scitranslmed.aba4226DOI Listing
May 2021

Adherence to Oral Nutritional Supplements After Being Discharged from the Hospital is Low but Improves Outcome in Patients with Advanced Chronic Liver Disease.

Patient Prefer Adherence 2020 5;14:2559-2572. Epub 2021 Jan 5.

Gastroenterology and Hepatology Subdiv. of the 5th Department of Medicine, Comenius University Faculty of Medicine, University Hospital Bratislava, Bratislava, Slovakia.

Purpose: Patients with advanced chronic liver disease (ACLD) often have a poor nutritional status. In the management, current guidelines recommend dietary counseling and oral nutritional supplements (ONS). Nutritional goals and adherence to ONS are difficult to achieve while studies addressing adherence are scarce. We aimed to evaluate adherence to ONS, the associated factors, and its impact on outcome among ALCD patients who are discharged from the hospital.

Patients And Methods: We identified consecutive hospitalized patients with ACLD from the cirrhosis registry and ONS prescription at discharge. Baseline demographics, anthropometrics, hand-grip strength (HGS), nutritional, and laboratory parameters were recorded. Adherence was assessed at 30, 90, and 180 days, but not in patients who did not survive or in those who underwent liver transplantation (LT) before the time-point.

Results: From the registry containing 1004 patients, we included 450 cases, the median age was 56.3 (IQR 47-62), 60% were males, 63.8% had alcoholic etiology, and the median model for end-stage liver disease score (MELD) was 16 (11-21). During follow-up, 13.6%, 23.6%, and 31.1% of patients have died within 30, 90, and 180 days, respectively, and 21 underwent LT. Adherence to ONS in surviving patients was observed in 46%, 26.1%, and 16.9% within 30, 90, and 180 days, respectively. Baseline refractory ascites (HR=0.43, 0.24-0.76), HGS (HR=1.03, 1.01-1.06), and mid-arm circumference (HR=0.93, 0.88-0.99) were independently associated with 30-day adherence. Among patients who survived beyond 30 days, adherents for >30 days had improved synthetic liver function, HGS, a higher probability of LT (HR=1.7, 1.03-2.8) and lower risk of death (HR=0.65, 0.45-0.89), particularly those with MELD>16 (OR=0.55, 0.36-0.85) and low HGS (OR=0.61, 0.39-0.93).

Conclusion: In ACLD patients after discharge, adherence to ONS steeply declined and was associated with baseline refractory ascites and low muscle strength. Adherence to ONS also improved liver function, muscle strength, and survival.
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http://dx.doi.org/10.2147/PPA.S283034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802017PMC
January 2021

A prolonged exposure of human lung carcinoma epithelial cells to benzo[a]pyrene induces p21-dependent epithelial-to-mesenchymal transition (EMT)-like phenotype.

Chemosphere 2021 Jan 10;263:128126. Epub 2020 Sep 10.

Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic. Electronic address:

Deciphering the role of the aryl hydrocarbon receptor (AhR) in lung cancer cells may help us to better understand the role of toxic AhR ligands in lung carcinogenesis, including cancer progression. We employed human lung carcinoma A549 cells to investigate their fate after continuous two-week exposure to model AhR agonists, genotoxic benzo[a]pyrene (BaP; 1 μM) and non-genotoxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM). While TCDD increased proliferative rate of A549 cells, exposure to BaP decreased cell proliferation and induced epithelial-to-mesenchymal transition (EMT)-like phenotype, which was associated with enhanced cell migration, invasion, and altered cell morphology. Although TCDD also suppressed expression of E-cadherin and activated some genes linked to EMT, it did not induce the EMT-like phenotype. The results of transcriptomic analysis, and the opposite effects of BaP and TCDD on cell proliferation, indicated that a delay in cell cycle progression, together with a slight increase of senescence (when coupled with AhR activation), favors the induction of EMT-like phenotype. The shift towards EMT-like phenotype observed after simultaneous treatment with TCDD and mitomycin C (an inhibitor of cell proliferation) confirmed the hypothesis. Since BaP decreased cell proliferative rate via induction of p21 expression, we generated the A549 cell model with reduced p21 expression and exposed it to BaP for two weeks. The p21 knockdown suppressed the BaP-mediated EMT-like phenotype in A549 cells, thus confirming that a delayed cell cycle progression, together with p21-dependent induction of senescence-related chemokine CCL2, may contribute to induction of EMT-like cell phenotype in lung cells exposed to genotoxic AhR ligands.
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http://dx.doi.org/10.1016/j.chemosphere.2020.128126DOI Listing
January 2021

Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling.

EMBO Mol Med 2020 11 14;12(11):e11739. Epub 2020 Oct 14.

Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

Mutations in genes affecting primary cilia cause ciliopathies, a diverse group of disorders often affecting skeletal development. This includes Jeune syndrome or asphyxiating thoracic dystrophy (ATD), an autosomal recessive skeletal disorder. Unraveling the responsible molecular pathology helps illuminate mechanisms responsible for functional primary cilia. We identified two families with ATD caused by loss-of-function mutations in the gene encoding adrenergic receptor kinase 1 (ADRBK1 or GRK2). GRK2 cells from an affected individual homozygous for the p.R158* mutation resulted in loss of GRK2, and disrupted chondrocyte growth and differentiation in the cartilage growth plate. GRK2 null cells displayed normal cilia morphology, yet loss of GRK2 compromised cilia-based signaling of Hedgehog (Hh) pathway. Canonical Wnt signaling was also impaired, manifested as a failure to respond to Wnt ligand due to impaired phosphorylation of the Wnt co-receptor LRP6. We have identified GRK2 as an essential regulator of skeletogenesis and demonstrate how both Hh and Wnt signaling mechanistically contribute to skeletal ciliopathies.
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http://dx.doi.org/10.15252/emmm.201911739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645380PMC
November 2020

NOTCH2NLC CGG Repeats Are Not Expanded and Skin Biopsy Was Negative in an Infantile Patient With Neuronal Intranuclear Inclusion Disease.

J Neuropathol Exp Neurol 2020 10;79(10):1065-1071

Department of Pediatrics and Adolescent Medicine, Research Unit for Rare Diseases.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disorder categorized into 3 phenotypic variants: infantile, juvenile, and adult. Four recent reports have linked NIID to CGG expansions in the NOTCH2NLC gene in adult NIID (aNIID) and several juvenile patients. Infantile NIID (iNIID) is an extremely rare neuropediatric condition. We present a 7-year-old male patient with severe progressive neurodegenerative disease that included cerebellar symptoms with cerebellar atrophy on brain MRI, psychomotor developmental regression, pseudobulbar syndrome, and polyneuropathy. The diagnosis of iNIID was established through a postmortem neuropathology work-up. We performed long-read sequencing of the critical NOTCH2NLC repeat motif and found no expansion in the patient. We also re-evaluated an antemortem skin biopsy that was collected when the patient was 2 years and 8 months old and did not identify the intranuclear inclusions. In our report, we highlight that the 2 methods (skin biopsy and CGG expansion testing in NOTCH2NLC) used to identify aNIID patients may provide negative results in iNIID patients.
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http://dx.doi.org/10.1093/jnen/nlaa070DOI Listing
October 2020

In Vitro Transformation of Human Bronchial Epithelial Cells by Diesel Exhaust Particles: Gene Expression Profiling and Early Toxic Responses.

Toxicol Sci 2018 11;166(1):51-64

Section for Toxicology and Biological Work Environment, Department of Chemical and Biological Work Environment, National Institute of Occupational Health, N-0304 Oslo, Norway.

Occupational exposure to diesel exhaust may cause lung cancer in humans. Mechanisms include DNA-damage and inflammatory responses. Here, the potential of NIST SRM2975 diesel exhaust particles (DEP) to transform human bronchial epithelial cells (HBEC3) in vitro was investigated. Long-term exposure of HBEC3 to DEP led to increased colony growth in soft agar. Several DEP-transformed cell lines were established and based on the expression of epithelial-to-mesenchymal-transition (EMT) marker genes, one of them (T2-HBEC3) was further characterized. T2-HBEC3 showed a mesenchymal/fibroblast-like morphology, reduced expression of CDH1, and induction of CDH2 and VIM. T2-HBEC3 had reduced migration potential compared with HBEC3 and little invasion capacity. Gene expression profiling showed baseline differences between HBEC3 and T2-HBEC3 linked to lung carcinogenesis. Next, to assess differences in sensitivity to DEP between parental HBEC3 and T2-HBEC3, gene expression profiling was carried out after DEP short-term exposure. Results revealed changes in genes involved in metabolism of xenobiotics and lipids, as well as inflammation. HBEC3 displayed a higher steady state of IL1B gene expression and release of IL-1β compared with T2-HBEC3. HBEC3 and T2-HBEC3 showed similar susceptibility towards DEP-induced genotoxic effects. Liquid-chromatography-tandem-mass-spectrometry was used to measure secretion of eicosanoids. Generally, major prostaglandin species were released in higher concentrations from T2-HBEC3 than from HBEC3 and several analytes were altered after DEP-exposure. In conclusion, long-term exposure to DEP-transformed human bronchial epithelial cells in vitro. Differences between HBEC3 and T2-HBEC3 regarding baseline levels and DEP-induced changes of particularly CYP1A1, IL-1β, PGE2, and PGF2α may have implications for acute inflammation and carcinogenesis.
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http://dx.doi.org/10.1093/toxsci/kfy183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204768PMC
November 2018

Adaptive changes in global gene expression profile of lung carcinoma A549 cells acutely exposed to distinct types of AhR ligands.

Toxicol Lett 2018 Aug 25;292:162-174. Epub 2018 Apr 25.

Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic. Electronic address:

Exposure to persistent ligands of aryl hydrocarbon receptor (AhR) has been found to cause lung cancer in experimental animals, and lung adenocarcinomas are often associated with enhanced AhR expression and aberrant AhR activation. In order to better understand the action of toxic AhR ligands in lung epithelial cells, we performed global gene expression profiling and analyze TCDD-induced changes in A549 transcriptome, both sensitive and non-sensitive to CH223191 co-treatment. Comparison of our data with results from previously reported microarray and ChIP-seq experiments enabled us to identify candidate genes, which expression status reflects exposure of lung cancer cells to TCDD, and to predict processes, pathways (e.g. ER stress, Wnt/β-cat, IFNɣ, EGFR/Erbb1), putative TFs (e.g. STAT, AP1, E2F1, TCF4), which may be implicated in adaptive response of lung cells to TCDD-induced AhR activation. Importantly, TCDD-like expression fingerprint of selected genes was observed also in A549 cells exposed acutely to both toxic (benzo[a]pyrene, benzo[k]fluoranthene) and endogenous AhR ligands (2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester and 6-formylindolo[3,2-b]carbazole). Overall, our results suggest novel cellular candidates, which could help to improve monitoring of AhR-dependent transcriptional activity during acute exposure of lung cells to distinct types of environmental pollutants.
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http://dx.doi.org/10.1016/j.toxlet.2018.04.024DOI Listing
August 2018

Atropisomers of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) exhibit stereoselective effects on activation of nuclear receptors in vitro.

Environ Sci Pollut Res Int 2018 Jun 9;25(17):16411-16419. Epub 2017 Nov 9.

Department of Chemistry and Toxicology, Veterinary Research Institute, 62100, Brno, Czech Republic.

PCB 136 is an environmentally relevant chiral PCB congener, which has been found in vivo to be present in form of rotational isomers (atropisomers). Its atropselective biotransformation or neurotoxic effects linked with sensitization of ryanodine receptor suggest that it might interact also with other intracellular receptors in a stereospecific manner. However, possible atropselective effects of PCB 136 on nuclear receptor transactivation remain unknown. Therefore, in this study, atropselective effects of PCB 136 on nuclear receptors controlling endocrine signaling and/or expression of xenobiotic and steroid hormone catabolism were investigated. PCB136 atropisomers were found to exert differential effects on estrogen receptor (ER) activation; (+)-PCB 136 was estrogenic, while (-)-PCB 136 was antiestrogenic. In contrast, inhibition of androgen receptor (AR) activity was not stereospecific. Both PCB136 stereoisomers induced the constitutive androgen receptor (CAR)-dependent gene expression; however, no significant stereospecificity of PCB 136 atropisomers was observed. PCB136 was a partial inducer of the pregnane X receptor (PXR)-dependent gene expression. Here, (-)-PCB 136 was a significantly more potent inducer of PXR activity than (+)-PCB 136. Taken together, the present results indicate that at least two nuclear receptors participating in endocrine regulation or metabolism, ER and PXR, could be regulated in an atropselective manner by chiral PCB 136. The enantioselective enrichment of PCB atropisomers in animal and human tissues may thus have significant consequences for endocrine-disrupting effects of chiral ortho-substituted PCB congeners.
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http://dx.doi.org/10.1007/s11356-017-0683-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943194PMC
June 2018

One reporter for in-cell activity profiling of majority of protein kinase oncogenes.

Elife 2017 02 15;6. Epub 2017 Feb 15.

Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The promoter was engineered to enhance -activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies.
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http://dx.doi.org/10.7554/eLife.21536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310841PMC
February 2017

7-MEOTA-donepezil like compounds as cholinesterase inhibitors: Synthesis, pharmacological evaluation, molecular modeling and QSAR studies.

Eur J Med Chem 2014 Jul 27;82:426-38. Epub 2014 May 27.

Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic; Department of Toxicology, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic; Centre for Advanced Studies, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic; Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, 500 03 Hradec Kralove, Czech Republic. Electronic address:

A novel series of 7-methoxytacrine (7-MEOTA)-donepezil like compounds was synthesized and tested for their ability to inhibit electric eel acetylcholinesterase (EeAChE), human recombinant AChE (hAChE), equine serum butyrylcholinesterase (eqBChE) and human plasmatic BChE (hBChE). New hybrids consist of a 7-MEOTA unit, representing less toxic tacrine (THA) derivative, connected with analogues of N-benzylpiperazine moieties mimicking N-benzylpiperidine fragment from donepezil. 7-MEOTA-donepezil like compounds exerted mostly non-selective profile in inhibiting cholinesterases of different origin with IC50 ranging from micromolar to sub-micromolar concentration scale. Kinetic analysis confirmed mixed-type inhibition presuming that these inhibitors are capable to simultaneously bind peripheral anionic site (PAS) as well as catalytic anionic site (CAS) of AChE. Molecular modeling studies and QSAR studies were performed to rationalize studies from in vitro. Overall, 7-MEOTA-donepezil like derivatives can be considered as interesting candidates for Alzheimer's disease treatment.
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http://dx.doi.org/10.1016/j.ejmech.2014.05.066DOI Listing
July 2014

Transcriptional and epigenetic mechanisms underlying enhanced in vitro adipocyte differentiation by the brominated flame retardant BDE-47.

Environ Sci Technol 2014 Apr 11;48(7):4110-9. Epub 2014 Mar 11.

Institute for Environmental Studies, VU University Amsterdam , Amsterdam, 1081HV, The Netherlands.

Recent studies suggest that exposure to endocrine-disrupting compounds (EDCs) may play a role in the development of obesity. EDCs such as the flame retardant 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) have been shown to enhance adipocyte differentiation in the murine 3T3-L1 model. The mechanisms by which EDCs direct preadipocytes to form adipocytes are poorly understood. Here, we examined transcriptional and epigenetic mechanisms underlying the induction of in vitro adipocyte differentiation by BDE-47. Quantitative high content microscopy revealed concentration-dependent enhanced adipocyte differentiation following exposure to BDE-47 or the antidiabetic drug troglitazone (TROG). BDE-47 modestly activated the key adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) in COS7 cells, transiently transfected with a GAL4 reporter construct. Increased gene expression was observed for Pparγ2, leptin (Lep), and glucose-6-phophatase catalytic subunit (G6pc) in differentiated 3T3-L1 cells after BDE-47 exposure compared to TROG. Methylation-sensitive high resolution melting (MS-HRM) revealed significant demethylation of three CpG sites in the Pparγ2 promoter after exposure to both BDE-47 and TROG in differentiated 3T3-L1 cells. This study shows the potential of BDE-47 to induce adipocyte differentiation through various mechanisms that include Pparγ2 gene induction and promoter demethylation accompanied by activation of PPARγ, and possible disruption of glucose homeostasis and IGF1 signaling.
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http://dx.doi.org/10.1021/es405524bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983330PMC
April 2014

Aryl hydrocarbon receptor negatively regulates expression of the plakoglobin gene (jup).

Toxicol Sci 2013 Aug 20;134(2):258-70. Epub 2013 May 20.

Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, 61265 Brno, Czech Republic.

Plakoglobin is an important component of intercellular junctions, including both desmosomes and adherens junctions, which is known as a tumor suppressor. Although mutations in the plakoglobin gene (Jup) and/or changes in its protein levels have been observed in various disease states, including cancer progression or cardiovascular defects, the information about endogenous or exogenous stimuli orchestrating Jup expression is limited. Here we show that the aryl hydrocarbon receptor (AhR) may regulate Jup expression in a cell-specific manner. We observed a significant suppressive effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model toxic exogenous activator of the AhR signaling, on Jup expression in a variety of experimental models derived from rodent tissues, including contact-inhibited rat liver progenitor cells (where TCDD induces cell proliferation), rat and mouse hepatoma cell models (where TCDD inhibits cell cycle progression), cardiac cells derived from the mouse embryonic stem cells, or cardiomyocytes isolated from neonatal rat hearts. The small interfering RNA (siRNA)-mediated knockdown of AhR confirmed its role in both basal and TCDD-deregulated Jup expression. The analysis of genomic DNA located ~2.5kb upstream of rat Jup gene revealed a presence of evolutionarily conserved AhR binding motifs, which were confirmed upon their cloning into luciferase reporter construct. The siRNA-mediated knockdown of Jup expression affected both proliferation and attachment of liver progenitor cells. The present data indicate that the AhR may contribute to negative regulation of Jup gene expression in rodent cellular models, which may affect cell adherence and proliferation.
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http://dx.doi.org/10.1093/toxsci/kft110DOI Listing
August 2013

AhR-mediated changes in global gene expression in rat liver progenitor cells.

Arch Toxicol 2013 Apr 30;87(4):681-98. Epub 2012 Nov 30.

Institute of Toxicology, University Medical Center of the Johannes Gutenberg-University Mainz, Germany.

Although the tumor-promoting effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar polychlorinated biphenyls (PCBs), and related compounds in liver tissue are primarily attributed to the activation of the aryl hydrocarbon receptor (AhR), the underlying molecular mechanisms are still unclear. Liver progenitor (oval) cells have been suggested to constitute a potential target for hepatocarcinogenic chemicals. To better understand AhR-driven pathways, we analyzed the transcriptional program in response to coplanar PCB 126 in contact-inhibited rat liver progenitor WB-F344 cells using high-density microarrays. After 6-h treatment, we identified 145 significantly deregulated genes considered to be direct AhR-dependent target genes. The number of differentially regulated genes increased to 658 and 968 genes after 24 and 72 h, respectively. Gene ontology analysis revealed that these genes were primarily involved in drug and lipid metabolism, cell cycle and growth control, cancer developmental processes, cell-cell communication, and adhesion. Interestingly, the Wnt and TGF-β signaling pathways, both being involved in developmental and tumorigenic processes, belonged to the most affected pathways. AhR- and ARNT-dependent regulation of selected target genes of interest was then confirmed using TCDD as a model AhR agonist, together with pharmacological inhibition of the AhR and by RNA-interference techniques. We demonstrated AhR-dependent regulation of emerging and novel AhR target genes, such as Fst, Areg, Hbegf, Ctgf, Btg2, and Foxq1. Among them, the transcription factor Foxq1, recently suggested to contribute to tumor promotion and/or progression, was found to be regulated at both mRNA and protein levels by AhR/ARNT activation.
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http://dx.doi.org/10.1007/s00204-012-0979-zDOI Listing
April 2013

TGF-β1 signaling plays a dominant role in the crosstalk between TGF-β1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells.

Cell Signal 2012 Aug 25;24(8):1665-76. Epub 2012 Apr 25.

Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.

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.
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http://dx.doi.org/10.1016/j.cellsig.2012.04.008DOI Listing
August 2012

[Intended pharmacotherapeutical approaches of Alzheimer's disease therapy].

Ceska Slov Farm 2012 Feb;61(1-2):4-10

Univerzita Karlova v Praze, Farmaceutická fakulta v Hradci Králové, Katedra farmaceutické chemie a kontroly léciv.

Alzheimer's disease is a progressive neurodegenerative disorder mainly manifested by memory loss, personality changes, and cognitive dysfunction. Despite the fact that tireless research is being conducted, up-to-date pharmacotherapy of AD is presented only by two groups diverging in the mechanism of action. The larger one uses acetylcholinesterase inhibitors, and the second group is represented by the N-methyl-D-aspartate antagonist memantine. Even though the etiology of Alzheimer's disease is unknown, several different therapeutic approaches are being investigated. The aim of this paper is to provide an overview of the present state of intended therapeutics for AD, describing their mechanism of action if known, displaying chemical structures, and the state of clinical trials if any.
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February 2012

Genotoxicity of 7H-dibenzo[c,g]carbazole and its methyl derivatives in human keratinocytes.

Mutat Res 2012 Mar 28;743(1-2):91-8. Epub 2012 Jan 28.

Laboratory of Mutagenesis and Carcinogenesis, Cancer Research Institute, SAS, 833 91 Bratislava, Slovakia.

Differences between tissues in the expression of drug-metabolizing enzymes may substantially contribute to tissue-specificity of chemical carcinogens. To verify this hypothesis, the spontaneously immortalized human keratinocytes HaCaT were used, in order to evaluate the genotoxic potential of 7H-dibenzo[c,g]carbazole (DBC), a known hepatocarcinogen and sarcomagen, and its synthetic tissue-specific derivatives, 5,9-dimethyl-DBC (DiMeDBC) and N-methyl-DBC (N-MeDBC), which manifest specific tropism to the liver and skin, respectively. HaCaT cells mainly express cytochrome P4501A1 (CYP1A1), which is involved in metabolism of DBC and N-MeDBC, but not DiMeDBC [10]. Both DBC and the sarcomagen N-MeDBC induced significant levels of DNA strand-breaks, micronuclei, and DNA adducts followed by the phosphorylation of the p53 protein and histone H2AX in HaCaT cells. In contrast, the specific hepatocarcinogen DiMeDBC was devoid of any significant genotoxic activity in this cell line. Our study demonstrates that the absence of drug-metabolizing enzyme(s) involved in DiMeDBC metabolism may contribute substantially to the tissue-specific genotoxicity of this hepatocarcinogen.
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http://dx.doi.org/10.1016/j.mrgentox.2011.12.030DOI Listing
March 2012

Genotoxicity of 7H-dibenzo[c,g]carbazole and its tissue-specific derivatives in human hepatoma HepG2 cells is related to CYP1A1/1A2 expression.

Environ Mol Mutagen 2011 Oct 1;52(8):636-45. Epub 2011 Aug 1.

Laboratory of Mutagenesis and Carcinogenesis, Cancer Research Institute, Bratislava, Slovakia.

The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression.
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http://dx.doi.org/10.1002/em.20664DOI Listing
October 2011

Gene expression changes in human prostate carcinoma cells exposed to genotoxic and nongenotoxic aryl hydrocarbon receptor ligands.

Toxicol Lett 2011 Oct 23;206(2):178-88. Epub 2011 Jul 23.

Department of Toxicology, Pharmacology and Immunotherapy, Veterinary Research Institute, Hudcova 70, Brno, Czech Republic.

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) are known as efficient mutagens and ligands of the aryl hydrocarbon receptor (AhR), which has been suggested to play an important role in prostate carcinogenesis. In order to evaluate the complex relationship between the genotoxicity and the AhR-mediated activity of PAHs in prostate cells, we selected benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as model genotoxic and nongenotoxic AhR ligands, respectively, to explore global changes in gene expression in LNCaP cells by microarray analysis. We identified 112 genes that were differentially expressed in cells treated for 24h with BaP, TCDD or both compounds. Our data indicated that the impacts of BaP and TCDD on transcriptome of LNCaP cells significantly overlap, since over 64% of significantly up-regulated genes and 47% of down-regulated genes were similarly affected by both AhR ligands. This suggested that the activation of AhR played a prominent role in the nongenotoxic effects of BaP in the prostate carcinoma cell model LNCaP. Both AhR ligands suppressed expression of genes associated with cell cycle progression, DNA replication, spindle assembly checkpoint or DNA repair, which probably occurred secondary to inhibition of cell cycle progression. In contrast, we identified Wnt5a, an important regulator of prostate cancer progression, to be induced as early as 6h after exposure to both AhR ligands. The AhR ligand-induced Wnt5a upregulation, together with other observed alterations of gene expression, may further contribute to enhanced cell plasticity of prostate carcinoma cells.
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http://dx.doi.org/10.1016/j.toxlet.2011.07.011DOI Listing
October 2011

Genotoxic polycyclic aromatic hydrocarbons fail to induce the p53-dependent DNA damage response, apoptosis or cell-cycle arrest in human prostate carcinoma LNCaP cells.

Toxicol Lett 2010 Sep 11;197(3):227-35. Epub 2010 Jun 11.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been positively associated with prostate cancer, but knowledge of the formation of PAH-DNA adducts and related genotoxic events in prostatic cells is limited. In the present study, benzo[a]pyrene (BaP), a potent mutagenic PAH, formed significant levels of DNA adducts in cell lines derived from human prostate carcinoma. When analyzing the effect of BaP on the induction of CYP1 enzymes participating in the metabolic activation of PAHs in LNCaP cells, we found that BaP induced expression of CYP1A1 and CYP1A2, but not CYP1B1 enzyme. Despite a significant amount of DNA adducts being formed by BaP and, to a lesser extent also by another strong genotoxin, dibenzo[a,l]pyrene, neither apoptosis nor cell-cycle arrest were induced in LNCaP cells. LNCaP cells were not sensitized to the induction of apoptosis by PAHs even through inhibition of the phosphoinositide-3-kinase/Akt pro-survival pathway. The lack of apoptosis was not due a disruption of expression of pro-apoptotic and pro-survival members of the Bcl-2 family of apoptosis regulators. In contrast to other genotoxic stimuli, genotoxic PAHs failed to induce DNA double-strand breaks, as illustrated by the lack of phosphorylation of histone H2AX or checkpoint kinase-2. BaP did not activate p53, as evidenced by the lack of p53 accumulation, phosphorylation at Ser15, or induction of p53 transcriptional targets. Taken together, although genotoxic PAHs produced significant levels of DNA adducts in a model of human prostate carcinoma cells, they did not activate the mechanisms leading to elimination of cells with significant damage to DNA, presumably due to their failure to activate the p53-dependent DNA damage response.
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http://dx.doi.org/10.1016/j.toxlet.2010.06.004DOI Listing
September 2010

Clinical presentation and metabolic consequences in 40 breastfed infants with nutritional vitamin B12 deficiency--what have we learned?

Eur J Paediatr Neurol 2010 Nov 20;14(6):488-95. Epub 2010 Jan 20.

Department of Paediatrics, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic.

Background: Maternal vitamin B(12) (Cbl) deficiency causes nutritional Cbl deficiency in breastfed infants.

Aims: To analyse clinical presentation and metabolic consequences in 40 breastfed infants with Cbl deficiency.

Methods: Cbl levels in serum and breast milk were determined by an electrochemiluminescence immunoassay, methylmalonic acid level by GC/MS, plasma homocysteine by HPLC and propionylcarnitine by MS/MS. Profound Cbl deficiency was found in 17 children (69 ± 17 ng/l, controls 200-900), and milder Cbl deficiency in 23 children (167 ± 40 ng/l). Maternal Cbl deficiency was mostly caused by insufficient Cbl absorption. Only six mothers were vegetarian.

Results: The average age at diagnosis was 4.4 ± 2.5 months. Clinical symptoms included failure to thrive (48% of children), hypotonia (40%), developmental delay (38%) and microcephaly (23%). 63% of children had anaemia (megaloblastic in 28% of all children). All but one patient had methylmalonic aciduria, 80% of patients had hyperhomocysteinemia and 87% had increased aminotransferases. Propionylcarnitine was elevated in two out of 25 infants. Comparing groups with severe and mild Cbl deficiency, a marked difference was found in severity of clinical and laboratory changes.

Conclusion: Maternal Cbl status and diagnostic delay are the major factors influencing severity and progression of Cbl deficiency in breastfed infants. In our cohort, propionylcarnitine was not sufficiently sensitive marker of Cbl deficiency. Although symptoms are reversible on Cbl substitution, permanent neurological damage can result. Selective screening for Cbl deficiency is indicated in all breastfed infants with failure to thrive, hypotonia, developmental delay, microcephaly or megaloblastic anaemia. The best prevention in future could be the screening of all pregnant women.
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http://dx.doi.org/10.1016/j.ejpn.2009.12.003DOI Listing
November 2010

New polymorphic sites within ornithine transcarbamylase gene: population genetics studies and implications for diagnosis.

Mol Genet Metab 2003 Feb;78(2):152-7

IPATIMUP-Institute of Molecular Pathology and Immunology, University of Porto, Portugal.

Ornithine transcarbamylase (OTC) deficiency, transmitted as an X-linked trait, is the most common disorder of the urea cycle. At least 3.5% out of more than 230 mutations consist of large gene deletions, involving one or more exons. Only in 78% of OTC patients the diagnosis was confirmed on DNA level. We analysed OTC intragenic polymorphisms and haplotypes, in an attempt to contribute to the clarification of unresolved cases, in three populations (Czech, Portuguese, and Mozambican) and identified six novel nucleotide changes, all of them occurring with frequency higher than 12.5% in Europeans. Five of these polymorphisms occur with a significant frequency also in Africans. The number and frequency of haplotypes defined with the newly reported markers differ in individual populations.
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http://dx.doi.org/10.1016/s1096-7192(03)00019-2DOI Listing
February 2003