Resistance to systemic therapy is a major problem in metastatic breast cancer (MBC) that can be explained by initial tumor heterogeneity as well as by evolutionary changes during therapy and tumor progression. Circulating tumor cells (CTCs) detected in a liquid biopsy can be sampled and characterized repeatedly during therapy in order to monitor treatment response and disease progression.Our aim was to investigate how CTC derived gene expression of treatment predictive markers (ESR1/HER2) and other cancer associated markers changed in patient blood samples during six months of first-line systemic treatment for MBC. CTCs from 36 patients were enriched using CellSearch (Janssen Diagnostics) and AdnaTest (QIAGEN) before gene expression analysis was performed with a customized gene panel (TATAA Biocenter).Our results show that antibodies against HER2 and EGFR were valuable to isolate CTCs unidentified by CellSearch and possibly lacking EpCAM expression. Evaluation of patients with clinically different breast cancer subgroups demonstrated that gene expression of treatment predictive markers changed over time. This change was especially prominent for HER2 expression.In conclusion, we found that changed gene expression during first-line systemic therapy for MBC could be a possible explanation for treatment resistance. Characterization of CTCs at several time-points during therapy could be informative for treatment selection.

• MeSH
• chemorezistence MeSH
• dospělí MeSH
• Kaplanův-Meierův odhad MeSH
• lidé středního věku MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorové biomarkery * MeSH
• nádorové cirkulující buňky metabolismus   patologie   MeSH
• nádory prsu genetika   patologie   terapie   MeSH
• progrese nemoci MeSH
• receptor erbB-2 genetika   MeSH
• receptory pro estrogeny genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state.

• MeSH
• analýza hlavních komponent MeSH
• buněčné klony MeSH
• buněčné linie MeSH
• buněčný rodokmen genetika   MeSH
• časosběrné zobrazování MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• genetická transkripce MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• kinetika MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• stanovení celkové genové exprese MeSH
• stochastické procesy MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3'-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3- and androgen-mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.

• MeSH
• androgenní receptory genetika   metabolismus   MeSH
• buňky PC-3 MeSH
• cholin aplikace a dávkování   analogy a deriváty   MeSH
• čipová analýza tkání MeSH
• down regulace * MeSH
• kohortové studie MeSH
• krystaliny genetika   metabolismus   MeSH
• lidé MeSH
• metabolomika MeSH
• nádorové buněčné linie MeSH
• nádory prostaty diagnostické zobrazování   genetika   metabolismus   patologie   MeSH
• PET/CT MeSH
• prognóza MeSH
• receptory thyreoidních hormonů genetika   MeSH
• regulace genové exprese u nádorů MeSH
• sekvenční analýza RNA MeSH
• signální transdukce * MeSH
• staging nádorů MeSH
• stanovení celkové genové exprese MeSH
• trijodthyronin antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

While numerous genomes of Leishmania spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, Endotrypanum and Porcisia spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from Leishmaniamajor genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the Endotrypanum/Porcisia branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the Endotrypanum/Porcisia species when compared to amastigote-dwelling Leishmania. This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of Leishmania amastigotes inside vertebrate macrophages and, apparently, dispensable for Endotrypanum/Porcisia, which do not infect such cells.

• MeSH
• fylogeneze MeSH
• Leishmania major klasifikace   genetika   MeSH
• Leishmania klasifikace   genetika   MeSH
• membránové proteiny genetika   MeSH
• molekulární evoluce MeSH
• protozoální proteiny genetika   MeSH
• regulace genové exprese MeSH
• sekvenování celého genomu metody   MeSH
• stanovení celkové genové exprese MeSH
• Trypanosomatina klasifikace   genetika   MeSH
• virulence MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

• MeSH
• biologie buňky MeSH
• molekulární biologie MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• cytologie, klinická cytologie

• MeSH
• buňky * MeSH
• molekulární biologie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• molekulární biologie, molekulární medicína
• NLK Publikační typ
• učebnice vysokých škol

Fusion oncogenes are among the most common types of oncogene in human cancers. The gene rearrangements result in new combinations of regulatory elements and functional protein domains. Here we studied a subgroup of sarcomas and leukaemias characterized by the FET (FUS, EWSR1, TAF15) family of fusion oncogenes, including FUS-DDIT3 in myxoid liposarcoma (MLS). We investigated the regulatory mechanisms, expression levels and effects of FUS-DDIT3 in detail. FUS-DDIT3 showed a lower expression than normal FUS at both the mRNA and protein levels, and single-cell analysis revealed a lack of correlation between FUS-DDIT3 and FUS expression. FUS-DDIT3 transcription was regulated by the FUS promotor, while its mRNA stability depended on the DDIT3 sequence. FUS-DDIT3 protein stability was regulated by protein interactions through the FUS part, rather than the leucine zipper containing DDIT3 part. In addition, in vitro as well as in vivo FUS-DDIT3 protein expression data displayed highly variable expression levels between individual MLS cells. Combined mRNA and protein analyses at the single-cell level showed that FUS-DDIT3 protein expression was inversely correlated to the expression of cell proliferation-associated genes. We concluded that FUS-DDIT3 is uniquely regulated at the transcriptional as well as the post-translational level and that its expression level is important for MLS tumour development. The FET fusion oncogenes are potentially powerful drug targets and detailed knowledge about their regulation and functions may help in the development of novel treatments.

• MeSH
• časové faktory MeSH
• fúzní onkogenní proteiny genetika   metabolismus   MeSH
• genetická transkripce MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• myxoidní liposarkom genetika   metabolismus   patologie   MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• poločas MeSH
• posttranslační úpravy proteinů MeSH
• proliferace buněk * MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce MeSH
• stabilita proteinů MeSH
• stabilita RNA MeSH
• transfekce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bordetella pertussis is the causative agent of whooping cough, a respiratory disease still considered as a major public health threat and for which recent re-emergence has been observed. Constant reshuffling of Bordetella pertussis genome organization was observed during evolution. These rearrangements are essentially mediated by Insertion Sequences (IS), a mobile genetic elements present in more than 230 copies in the genome, which are supposed to be one of the driving forces enabling the pathogen to escape from vaccine-induced immunity. Here we use high-throughput sequencing approaches (RNA-seq and differential RNA-seq), to decipher Bordetella pertussis transcriptome characteristics and to evaluate the impact of IS elements on transcriptome architecture. Transcriptional organization was determined by identification of transcription start sites and revealed also a large variety of non-coding RNAs including sRNAs, leaderless mRNAs or long 3' and 5'UTR including seven riboswitches. Unusual topological organizations, such as overlapping 5'- or 3'-extremities between oppositely orientated mRNA were also unveiled. The pivotal role of IS elements in the transcriptome architecture and their effect on the transcription of neighboring genes was examined. This effect is mediated by the introduction of IS harbored promoters or by emergence of hybrid promoters. This study revealed that in addition to their impact on genome rearrangements, most of the IS also impact on the expression of their flanking genes. Furthermore, the transcripts produced by IS are strain-specific due to the strain to strain variation in IS copy number and genomic context.

• MeSH
• 3' nepřekládaná oblast MeSH
• 5' nepřekládaná oblast MeSH
• bakteriální RNA genetika   MeSH
• Bordetella pertussis genetika   MeSH
• genetická transkripce * MeSH
• genom bakteriální genetika   MeSH
• messenger RNA genetika   MeSH
• nekódující RNA genetika   MeSH
• počátek transkripce MeSH
• stanovení celkové genové exprese * MeSH
• transpozibilní elementy DNA genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Analyzing rare DNA and RNA molecules in limited sample sizes, such as liquid biopsies and single cells, often requires preamplification, which makes downstream analyses particularly sensitive to polymerase chain reaction (PCR) generated contamination. Herein, we assessed the feasibility of performing Cod uracil-DNA N-glycosylase (Cod UNG) treatment in combination with targeted preamplification, using deoxyuridine triphosphate (dUTP) to eliminate carry-over DNA. Cod UNG can be completely and irreversibly heat inactivated, a prerequisite in preamplification methods, where any loss of amplicons is detrimental to subsequent quantification. Using 96 target assays and quantitative real-time PCR, we show that replacement of deoxythymidine triphosphate (dTTP) with dUTP in the preamplification reaction mix results in comparable dynamic range, reproducibility, and sensitivity. Moreover, Cod UNG essentially removes all uracil-containing template of most assays, regardless of initial concentration, without affecting downstream analyses. Finally, we demonstrate that the use of Cod UNG and dUTP in targeted preamplification can easily be included in the workflow for single-cell gene expression profiling. In summary, Cod UNG treatment in combination with targeted preamplification using dUTP provides a simple and efficient solution to eliminate carry-over contamination and the generation of false positives and inaccurate quantification.

• MeSH
• analýza jednotlivých buněk MeSH
• deoxyuracilnukleotidy metabolismus   MeSH
• Gadus morhua metabolismus   MeSH
• kontaminace DNA * MeSH
• reprodukovatelnost výsledků MeSH
• stanovení celkové genové exprese MeSH
• uracil-DNA-glykosidasa metabolismus   MeSH
• uracil metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase-signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.

• MeSH
• buněčný cyklus genetika   MeSH
• chemorezistence * MeSH
• chromozomální aberace MeSH
• cílená molekulární terapie MeSH
• exprese genu MeSH
• fenotyp MeSH
• inhibitory proteinkinas farmakologie   MeSH
• Janus kinasy metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• nádorové biomarkery * MeSH
• nádorové buněčné linie MeSH
• oxazoly farmakologie   MeSH
• protinádorové látky farmakologie   terapeutické užití   MeSH
• rychlé screeningové testy * MeSH
• screeningové testy protinádorových léčiv * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• T-buněčná prolymfocytární leukemie farmakoterapie   genetika   metabolismus   MeSH
• thiazoly farmakologie   MeSH
• transkripční faktory STAT metabolismus   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH