Latest Publications

@article{paculova_brca1_2017,

title = {BRCA1 or CDK12 loss sensitizes cells to CHK1 inhibitors.},

author = {Hana Paculová and Juraj Kramara and Šárka Šimečková and Radek Fedr and Karel Souček and Ondřej Hylse and Kamil Paruch and Marek Svoboda and Martin Mistrík and Jiří Kohoutek},

doi = {10.1177/1010428317727479},

issn = {1423-0380 1010-4283},

year  = {2017},

date = {2017-10-01},

journal = {Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine},

volume = {39},

number = {10},

pages = {1010428317727479},

abstract = {A broad spectrum of tumors develop resistance to classic chemotherapy, necessitating the discovery of new therapies. One successful strategy exploits the synthetic lethality between poly(ADP-ribose) polymerase 1/2 proteins and DNA damage response genes, including BRCA1, a factor involved in homologous recombination-mediated DNA repair, and CDK12, a transcriptional kinase known to regulate the expression of DDR genes. CHK1 inhibitors have been shown to enhance the anti-cancer effect of DNA-damaging compounds. Since loss of BRCA1 increases replication stress and leads to DNA damage, we tested a hypothesis that CDK12- or BRCA1-depleted cells rely extensively on S-phase-related CHK1 functions for survival. The silencing of BRCA1 or CDK12 sensitized tumor cells to CHK1 inhibitors in vitro and in vivo. BRCA1 downregulation combined with CHK1 inhibition induced excessive amounts of DNA damage, resulting in an inability to complete the S-phase. Therefore, we suggest CHK1 inhibition as a strategy for targeting BRCA1- or CDK12-deficient tumors.},

note = {Place: Netherlands},

keywords = {Animals, BRCA1, BRCA1 Protein/antagonists & inhibitors/*genetics, CDK12, Checkpoint Kinase 1/*genetics, CHK1 inhibitor, Colorectal Neoplasms/drug therapy/*genetics/pathology, Cyclin-Dependent Kinases/antagonists & inhibitors/*genetics, DNA damage response, DNA Damage/drug effects, Drug resistance, Gene Expression Regulation, Gene Silencing, HCT116 Cells, Humans, Mice, Neoplasm/genetics, Neoplastic/drug effects, Poly (ADP-Ribose) Polymerase-1/genetics, Pyrazoles/administration & dosage, Pyrimidines/administration & dosage, Transcription, Xenograft Model Antitumor Assays},

pubstate = {published},

tppubtype = {article}

}

@article{libalova_analysis_2014,

title = {Analysis of gene expression changes in A549 cells induced by organic compounds from respirable air particles.},

author = {Helena Líbalová and Simona Krčková and Kateřina Uhlířová and Jiří Kléma and Miroslav Ciganek and Pavel Jr Rössner and Radim J. Šrám and Jan Vondráček and Miroslav Machala and Jan Topinka},

doi = {10.1016/j.mrfmmm.2014.10.002},

issn = {1873-135X 0027-5107},

year  = {2014},

date = {2014-12-01},

journal = {Mutation research},

volume = {770},

pages = {94–105},

abstract = {A number of toxic effects of respirable ambient air particles (genotoxic effects, inflammation, oxidative damage) have been attributed to organic compounds bound onto the particle surface. In this study, we analyzed global gene expression changes caused by the extractable organic matters (EOMs) from respirable airborne particles <2.5μm (PM2.5), collected at 3 localities from heavily polluted areas of the Czech Republic and a control locality with low pollution levels, in human lung epithelial A549 cells. Although the sampled localities differed in both extent and sources of air pollution, EOMs did not induce substantially different gene expression profiles. The number of transcripts deregulated in A549 cells treated with the lowest EOM concentration (10μg/ml) ranged from 65 to 85 in 4 sampling localities compared to the number of transcripts deregulated after 30μg/ml and 60μg/ml of EOMs, which ranged from 90 to 109, and from 149 to 452, respectively. We found numerous commonly deregulated genes and pathways related to activation of the aryl hydrocarbon receptor (AhR) and metabolism of xenobiotics and endogenous compounds. We further identified deregulation of expression of the genes involved in pro-inflammatory processes, oxidative stress response and in cancer and developmental pathways, such as TGF-β and Wnt signaling pathways. No cell cycle arrest, DNA repair or pro-apoptotic responses were identified at the transcriptional level after the treatment of A549 cells with EOMs. In conclusion, numerous processes and pathways deregulated in response to EOMs suggest a significant role of activated AhR. Interestingly, we did not observe substantial gene expression changes related to DNA damage response, possibly due to the antagonistic effect of non-genotoxic EOM components. Moreover, a comparison of EOM effects with other available data on modulation of global gene expression suggests possible overlap among the effects of PM2.5, EOMs and various types of AhR agonists.},

note = {Place: Netherlands},

keywords = {A549 Cells, Adenocarcinoma of Lung, Adenocarcinoma/genetics/pathology, Ah receptor, Cultured, gene expression profile, Gene Expression Profiling, Gene Expression Regulation, Gene Expression/*drug effects, Humans, Lung Neoplasms/genetics/pathology, Microarray Analysis, Neoplastic/drug effects, Organic Chemicals/*pharmacology, PAHs, Particulate Matter/*pharmacology, PM2.5, Respiratory Mucosa/drug effects/metabolism, Signal Transduction/drug effects/genetics, Tumor Cells},

pubstate = {published},

tppubtype = {article}

}

@article{smerdova_upregulation_2014,

title = {Upregulation of CYP1B1 expression by inflammatory cytokines is mediated by the p38 MAP kinase signal transduction pathway.},

author = {Lenka Smerdová and Jana Šmerdová and Markéta Kabátková and Jiří Kohoutek and Dalibor Blažek and Miroslav Machala and Jan Vondráček},

doi = {10.1093/carcin/bgu190},

issn = {1460-2180 0143-3334},

year  = {2014},

date = {2014-11-01},

journal = {Carcinogenesis},

volume = {35},

number = {11},

pages = {2534–2543},

abstract = {Cytochrome P450 1B1 (CYP1B1) is an enzyme that has a unique tumor-specific pattern of expression and is capable of bioactivating a wide range of carcinogenic compounds. We have reported previously that coordinated upregulation of CYP1B1 by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and the aryl hydrocarbon receptor ligands, may increase bioactivation of promutagens, such as benzo[a]pyrene (BaP) in epithelial cells. Here, we extend those studies by describing a novel mechanism participating in the regulation of CYP1B1 expression, which involves activation of the p38 mitogen-activated protein kinase (p38) and mitogen- and stress-activated protein kinase 1 (MSK1). Using inhibitors of p38 and MSKs, as well as mouse embryonic cells derived from p38α-deficient and MSK1/2 double knockout mice, we show here that TNF-α potentiates CYP1B1 upregulation via the p38/MSK1 kinase cascade. Effects of this inflammatory cytokine on CYP1B1 expression further involve the positive transcription elongation factor b (P-TEFb). The inhibition of the P-TEFb subunit, cyclin-dependent kinase 9 (CDK9), which phosphorylates RNA polymerase II (RNAPII), prevented the enhanced CYP1B1 induction by a combination of BaP and inflammatory cytokine. Furthermore, using chromatin immunoprecipitation assays, we found that cotreatment of epithelial cells with TNF-α and BaP resulted in enhanced recruitment of both CDK9 and RNAPII to the Cyp1b1 gene promoter. Overall, these results have implications concerning the contribution of inflammatory factors to carcinogenesis, since enhanced CYP1B1 induction during inflammation may alter metabolism of exogenous carcinogens, as well as endogenous CYP1B1 substrates playing role in tumor development.},

note = {Place: England},

keywords = {Animals, Carcinogenesis/drug effects/*genetics, Carcinogens/toxicity, Cyclin-Dependent Kinase 9/genetics, Cytochrome P-450 CYP1B1/*biosynthesis/genetics, Cytokines/metabolism, Gene Expression Regulation, Humans, Mice, Neoplasms/chemically induced/*genetics/pathology, Neoplastic/drug effects, p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*genetics/metabolism, Positive Transcriptional Elongation Factor B/genetics, RNA Polymerase II/genetics, Signal Transduction/drug effects, Tumor Necrosis Factor-alpha/metabolism},

pubstate = {published},

tppubtype = {article}

}

@article{hruba_genotoxic_2010,

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

author = {Eva Hrubá and Lenka Trilecová and Sona Marvanová and Pavel Krcmár and Lenka Vykopalová and Alena Milcová and Helena Líbalová and Jan Topinka and Andrea Starsíchová and Karel Soucek and Jan Vondrácek and Miroslav Machala},

doi = {10.1016/j.toxlet.2010.06.004},

issn = {1879-3169 0378-4274},

year  = {2010},

date = {2010-09-01},

journal = {Toxicology letters},

volume = {197},

number = {3},

pages = {227–235},

abstract = {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.},

note = {Place: Netherlands},

keywords = {Animals, Apoptosis/*drug effects, Carcinoma/*metabolism, Cell Line, DNA Damage/*drug effects, Environmental Pollutants/toxicity, Gene Expression Regulation, Male, Neoplastic/drug effects, Polycyclic Aromatic Hydrocarbons/*toxicity, Prostatic Neoplasms/*metabolism, Tumor, Tumor Suppressor Protein p53/*metabolism},

pubstate = {published},

tppubtype = {article}

}

@article{ondrouskova_heavy_2009,

title = {Heavy metals induce phosphorylation of the Bcl-2 protein by Jun N-terminal kinase.},

author = {Eva Ondrousková and Jana Slovácková and Vendula Pelková and Jirina Procházková and Karel Soucek and Petr Benes and Jan Smarda},

doi = {10.1515/BC.2009.007},

issn = {1431-6730},

year  = {2009},

date = {2009-01-01},

journal = {Biological chemistry},

volume = {390},

number = {1},

pages = {49–58},

abstract = {The Bcl-2 protein is one of the key components of biochemical pathways controlling programmed cell death. The function of this protein can be regulated by posttranslational modifications. Phosphorylation of Bcl-2 has been considered to be significantly associated with cell cycle arrest in the G2/M phase of the cell cycle, and with cell death caused by defects of microtubule dynamics. This study shows that phosphorylation of Bcl-2 can be induced by heavy metals due to activation of the Jun N-terminal kinase pathway that is not linked to the G2/M cell cycle arrest. Furthermore, we demonstrate that hyperphosphorylated Bcl-2 protein is a more potent inhibitor of zinc-induced cell death than its hypophosphorylated mutant form. These data suggest that regulation of Bcl-2 protein function by phosphorylation is an important part of cell responses to stress.},

note = {Place: Germany},

keywords = {Animals, Apoptosis/drug effects, Cell Line, Electrophoresis, Gene Expression Regulation, Heavy/*pharmacology, Humans, JNK Mitogen-Activated Protein Kinases/*metabolism, Metals, Neoplastic/drug effects, Phosphorylation/drug effects, Physiological/drug effects, Post-Translational/drug effects, Protein Processing, Proto-Oncogene Proteins c-bcl-2/*metabolism, Signal Transduction/drug effects, Stress, Tumor, Zinc/pharmacology},

pubstate = {published},

tppubtype = {article}

}

@article{gavelova_reduction_2008,

title = {Reduction of doxorubicin and oracin and induction of carbonyl reductase in human breast carcinoma MCF-7 cells.},

author = {Martina Gavelová and Jana Hladíková and Lenka Vildová and Romana Novotná and Jan Vondrácek and Pavel Krcmár and Miroslav Machala and Lenka Skálová},

doi = {10.1016/j.cbi.2008.07.011},

issn = {0009-2797},

year  = {2008},

date = {2008-10-01},

journal = {Chemico-biological interactions},

volume = {176},

number = {1},

pages = {9–18},

abstract = {In cancer cells, the drug-metabolizing enzymes may deactivate cytostatics, thus contributing to their survival. Moreover, the induction of these enzymes may also contribute to development of drug-resistance through acceleration of cytostatics deactivation. However, the principal metabolic pathways contributing to deactivation of many cytostatics still remain poorly defined. The main aims of the present study were: (i) to compare the reductive deactivation of cytostatic drugs doxorubicin (DOX) and oracin (ORC) in human breast cancer MCF-7 cells; (ii) to identify major enzyme(s) involved in the carbonyl reduction; and iii) to evaluate the activities and expression of selected carbonyl reducing enzymes in MCF-7 cells upon a short-term (48 h) exposure to either DOX or ORC. We found that MCF-7 cells were able to effectively metabolize both DOX and ORC through reduction of their carbonyl groups. The reduction of ORC was stereospecific, with a preferential formation of + enantiomer of dihydrooracin (DHO). The cytosolic carbonyl reductase CBR1 seemed to be a principal enzyme reducing both drugs, while cytosolic aldo-keto reductase AKR1C3 or microsomal reductases probably did not play important role in metabolism of either DOX or ORC. The exposure of MCF-7 cells to low (nanomolar) concentrations of DOX or ORC caused a significant elevation of reduction rates of both cytostatics, accompanied with an increase of CBR1 protein levels. Taken together, the present results seem to suggest that the accelerated metabolic deactivation of ORC or DOX might contribute to the survival of breast cancer cells during exposure to these cytostatics.},

note = {Place: Ireland},

keywords = {3-Hydroxysteroid Dehydrogenases/antagonists & inhibitors/genetics/metabolism, Alcohol Oxidoreductases/antagonists & inhibitors/*biosynthesis/genetics/metabolism, Aldehyde Reductase, Aldo-Keto Reductase Family 1 Member C3, Aldo-Keto Reductases, Biotransformation/drug effects, Blotting, Breast Neoplasms/*enzymology/genetics, Cell Line, Dose-Response Relationship, Doxorubicin/analogs & derivatives/chemistry/*metabolism/pharmacology, Drug, Enzyme Induction/drug effects, Enzyme Inhibitors/pharmacology, Ethanolamines/chemistry/*metabolism/pharmacology, Gene Expression Regulation, Humans, Hydroxyprostaglandin Dehydrogenases/antagonists & inhibitors/genetics/metabolism, Isoquinolines/chemistry/*metabolism/pharmacology, Kinetics, Messenger/genetics/metabolism, Methacrylates/pharmacology, Neoplastic/drug effects, Oxidation-Reduction/drug effects, Phenylpropionates/pharmacology, Quercetin/analogs & derivatives/pharmacology, RNA, Subcellular Fractions/drug effects/metabolism, Tumor, Western},

pubstate = {published},

tppubtype = {article}

}