Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA uracil glycosylases. The data presented in this study strongly indicate that the nuclear uracil-N-glycosylase activity and nuclear protein content in human cell lines is highest in the S phase of the cell cycle and that its distribution kinetics partially reflect the DNA replication activity in replication foci. In this respect, the data demonstrate structural changes of the replication focus related to the uracil-N-glycosylase distribution several dozens of minutes before end of its replication. The analysis also showed that very popular synchronisation protocols based on the double thymidine block can result in changes in the UNG2 content and uracil excision rate. In response, we propose a new method for the description of the changes of the content and the activity of different cell components during cell cycle without the necessity to use synchronisation protocols.
- MeSH
- buněčné jádro metabolismus MeSH
- buněčný cyklus MeSH
- kinetika MeSH
- lidé MeSH
- oprava DNA MeSH
- replikace DNA * MeSH
- S fáze MeSH
- uracil-DNA-glykosidasa * metabolismus MeSH
- uracil metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- uracil-DNA-glykosidasa * MeSH
- uracil MeSH
Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.
- Klíčová slova
- NM1, PI(4,5)P2, cell nucleus, lamin A/C, nuclear lamina, nuclear myosin 1, nucleoplasm, phosphoinositides, phosphorylation,
- MeSH
- buněčné jádro * metabolismus MeSH
- interfáze MeSH
- intermediární filamenta metabolismus MeSH
- lamin typ A * metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- lamin typ A * MeSH
Several cancer core regulatory circuitries (CRCs) depend on the sustained generation of DNA accessibility by SWI/SNF chromatin remodelers. However, the window when SWI/SNF is acutely essential in these settings has not been identified. Here we used neuroblastoma (NB) cells to model and dissect the relationship between cell-cycle progression and SWI/SNF ATPase activity. We find that SWI/SNF inactivation impairs coordinated occupancy of non-pioneer CRC members at enhancers within 1 hour, rapidly breaking their autoregulation. By precisely timing inhibitor treatment following synchronization, we show that SWI/SNF is dispensable for survival in S and G2/M, but becomes acutely essential only during G1 phase. We furthermore developed a new approach to analyze the oscillating patterns of genome-wide DNA accessibility across the cell cycle, which revealed that SWI/SNF-dependent CRC binding sites are enriched at enhancers with peak accessibility during G1 phase, where they activate genes involved in cell-cycle progression. SWI/SNF inhibition strongly impairs G1-S transition and potentiates the ability of retinoids used clinically to induce cell-cycle exit. Similar cell-cycle effects in diverse SWI/SNF-addicted settings highlight G1-S transition as a common cause of SWI/SNF dependency. Our results illustrate that deeper knowledge of the temporal patterns of enhancer-related dependencies may aid the rational targeting of addicted cancers.
Cancer cells driven by runaway transcription factor networks frequently depend on the cellular machinery that promotes DNA accessibility. For this reason, recently developed small molecules that impair SWI/SNF (or BAF) chromatin remodeling activity have been under active evaluation as anti-cancer agents. However, exactly when SWI/SNF activity is essential in dependent cancers has remained unknown. By combining live-cell imaging and genome-wide profiling in neuroblastoma cells, Cermakova et al. discover that SWI/SNF activity is needed for survival only during G1 phase of the cell cycle. The authors reveal that in several cancer settings, dependency on SWI/SNF arises from the need to reactivate factors involved in G1-S transition. Because of this role, authors find that SWI/SNF inhibition potentiates cell-cycle exit by retinoic acid.
- MeSH
- buněčný cyklus MeSH
- chromatin genetika MeSH
- DNA MeSH
- G1 fáze * MeSH
- lidé MeSH
- nádory * MeSH
- regulační oblasti nukleových kyselin MeSH
- restrukturace chromatinu MeSH
- transkripční faktory * metabolismus MeSH
- zesilovače transkripce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chromatin MeSH
- DNA MeSH
- SWI-SNF-B chromatin-remodeling complex MeSH Prohlížeč
- transkripční faktory * MeSH
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.
- MeSH
- DNA vazebné proteiny * metabolismus MeSH
- DNA MeSH
- hydroxymočovina farmakologie MeSH
- lidé MeSH
- reaktivní formy kyslíku MeSH
- replikace DNA * MeSH
- S fáze genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA vazebné proteiny * MeSH
- DNA MeSH
- hydroxymočovina MeSH
- reaktivní formy kyslíku MeSH
The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.
Microtubule dynamic is exceptionally sensitive to modulation by small-molecule ligands. Our previous work presented the preparation of microtubule-targeting estradiol dimer (ED) with anticancer activity. In the present study, we explore the effect of selected linkers on the biological activity of the dimer. The linkers were designed as five-atom chains with carbon, nitrogen or oxygen in their centre. In addition, the central nitrogen was modified by a benzyl group with hydroxy or methoxy substituents and one derivative possessed an extended linker length. Thirteen new dimers were subjected to cytotoxicity assay and cell cycle profiling. Dimers containing linker with benzyl moiety substituted with one or more methoxy groups and longer branched ones were found inactive, whereas other structures had comparable efficacy as the original ED (e.g. D1 with IC50 = 1.53 µM). Cell cycle analysis and immunofluorescence proved the interference of dimers with microtubule assembly and mitosis. The proposed in silico model and calculated binding free energy by the MM-PBSA method were closely correlated with in vitro tubulin assembly assay.
- Klíčová slova
- Antimitotic activity, Cell cycle, Chemistry, CuAAC reaction, Cytotoxicity, Estradiol, G2/M arrest, In silico simulations, Microtubules, Steroid dimer, Tubulin assembly,
- MeSH
- apoptóza MeSH
- ethinylestradiol * chemie farmakologie MeSH
- kontrolní body fáze G2 buněčného cyklu účinky léků MeSH
- mikrotubuly MeSH
- modulátory tubulinu * chemie farmakologie MeSH
- nádorové buněčné linie MeSH
- protinádorové látky * chemie farmakologie MeSH
- triazoly * chemie farmakologie MeSH
- tubulin * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ethinylestradiol * MeSH
- modulátory tubulinu * MeSH
- protinádorové látky * MeSH
- triazoly * MeSH
- tubulin * MeSH
The meadowfoam family (Limnanthaceae) is one of the smallest and genomically underexplored families of the Brassicales. The Limnanthaceae harbor about seven species in the genus Limnanthes (meadowfoam) and Floerkea proserpinacoides (false mermaidweed), all native to North America. Because all Limnanthes and Floerkea species have only five chromosome pairs, i.e., a chromosome number rare in Brassicales and shared with Arabidopsis thaliana (Arabidopsis), we examined the Limnanthaceae genomes as a potential model system. Using low-coverage whole-genome sequencing data, we reexamined phylogenetic relationships and characterized the repeatomes of Limnanthaceae genomes. Phylogenies based on complete chloroplast and 35S rDNA sequences corroborated the sister relationship between Floerkea and Limnanthes and two major clades in the latter genus. The genome size of Limnanthaceae species ranges from 1.5 to 2.1 Gb, apparently due to the large increase in DNA repeats, which constitute 60-70% of their genomes. Repeatomes are dominated by long terminal repeat retrotransposons, while tandem repeats represent only less than 0.5% of the genomes. The average chromosome size in Limnanthaceae species (340-420 Mb) is more than 10 times larger than in Arabidopsis (32 Mb). A three-dimensional fluorescence in situ hybridization analysis demonstrated that the five chromosome pairs in interphase nuclei of Limnanthes species adopt the Rabl-like configuration.
- Klíčová slova
- Brassicales, DNA repeats, Limnanthes, Rabl, chromosomes, interphase, meadowfoam, repeatome,
- MeSH
- Arabidopsis * genetika MeSH
- chromozomy MeSH
- fylogeneze MeSH
- hybridizace in situ fluorescenční MeSH
- interfáze MeSH
- Magnoliopsida * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Fluorescent carbon dots (CDs) are potential tools for the labeling of cells with many advantages such as photostability, multicolor emission, small size, rapid uptake, biocompatibility, and easy preparation. Affinity towards organelles can be influenced by the surface properties of CDs which affect the interaction with the cell and cytoplasmic distribution. Organelle targeting by carbon dots is promising for anticancer treatment; thus, intracellular trafficking and cytotoxicity of cationic CDs was investigated. Based on our previous study, we used quaternized carbon dots (QCDs) for treatment and monitoring the behavior of two human cancer cell MCF-7 and HeLa lines. We found similarities between human cancer cells and mouse fibroblasts in the case of QCDs uptake. Time lapse microscopy of QCDs-labeled MCF-7 cells showed that cells are dying during the first two hours, faster at lower doses than at higher ones. QCDs at a concentration of 100 µg/mL entered into the nucleus before cellular death; however, at a dose of 200 µg/mL, blebbing of the cellular membrane occurred, with a subsequent penetration of QCDs into the nuclear area. In the case of HeLa cells, the dose-depended effect did not happen; however, the labeled cells were also dying in mitosis and genotoxicity occurred nearly at all doses. Moreover, contrasted intracellular compartments, probably mitochondria, were obvious after 24 h incubation with 100 µg/mL of QCDs. The levels of reactive oxygen species (ROS) slightly increased after 24 h, depending on the concentration, thus the genotoxicity was likely evoked by the nanomaterial. A decrease in viability did not reach IC 50 as the DNA damage was probably partly repaired in the prolonged G0/G1 phase of the cell cycle. Thus, the defects in the G2/M phase may have allowed a damaged cell to enter mitosis and undergo apoptosis. The anticancer effect in both cell lines was manifested mainly through genotoxicity.
- Klíčová slova
- HeLa, MCF-7, cancer cells, cationic carbon dots, cytotoxicity, fluorescence microspectroscopy, genotoxicity, nucleus,
- MeSH
- biologický transport MeSH
- buněčné linie MeSH
- časosběrné zobrazování metody MeSH
- fibroblasty cytologie účinky léků metabolismus MeSH
- HeLa buňky MeSH
- kontrolní body fáze G2 buněčného cyklu účinky léků MeSH
- kvantové tečky chemie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- myši MeSH
- nádory farmakoterapie genetika metabolismus MeSH
- optické zobrazování MeSH
- poškození DNA MeSH
- proliferace buněk MeSH
- reaktivní formy kyslíku metabolismus MeSH
- uhlík chemie farmakokinetika farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- reaktivní formy kyslíku MeSH
- uhlík MeSH
Arabidopsis thaliana has become a major plant research model, where interphase nuclear organization exhibits unique features, including nucleolus-associated telomere clustering. The chromocenter (CC)-loop model, or rosette-like configuration, describes intranuclear chromatin organization in Arabidopsis as megabase-long loops anchored in, and emanating from, peripherally positioned CCs, with those containing telomeres associating with the nucleolus. To investigate whether the CC-loop organization is universal across the mustard family (crucifers), the nuclear distributions of centromeres, telomeres and nucleoli were analyzed by fluorescence in situ hybridization in seven diploid species (2n = 10-16) representing major crucifer clades with an up to 26-fold variation in genome size (160-4260 Mb). Nucleolus-associated telomere clustering was confirmed in Arabidopsis (157 Mb) and was newly identified as the major nuclear phenotype in other species with a small genome (215-381 Mb). In large-genome species (2611-4264 Mb), centromeres and telomeres adopted a Rabl-like configuration or dispersed distribution in the nuclear interior; telomeres only rarely associated with the nucleolus. In Arabis cypria (381 Mb) and Bunias orientalis (2611 Mb), tissue-specific patterns deviating from the major nuclear phenotypes were observed in anther and stem tissues, respectively. The rosette-like configuration, including nucleolus-associated telomere clustering in small-genome species from different infrafamiliar clades, suggests that genomic properties rather than phylogenetic position determine the interphase nuclear organization. Our data suggest that nuclear genome size, average chromosome size and degree of longitudinal chromosome compartmentalization affect interphase chromosome organization in crucifer genomes.
- Klíčová slova
- Arabidopsis thaliana, Crucifereae, Rabl, centromere, chromocenter-loop model, interphase, nucleolus, telomere,
- MeSH
- Arabidopsis genetika MeSH
- Arabis genetika MeSH
- Brassicaceae genetika MeSH
- buněčné jadérko genetika MeSH
- centromera genetika MeSH
- chromatin genetika MeSH
- délka genomu MeSH
- fylogeneze MeSH
- genom rostlinný * MeSH
- heterochromatin genetika MeSH
- hybridizace in situ fluorescenční MeSH
- interfáze MeSH
- ribozomální DNA genetika MeSH
- telomery genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chromatin MeSH
- heterochromatin MeSH
- ribozomální DNA MeSH
Condensins are best known for their role in shaping chromosomes. Other functions such as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals, but little is known about their function in plants. To elucidate the specific composition of condensin complexes and the expression of CAP-D2 (condensin I) and CAP-D3 (condensin II), we performed biochemical analyses in Arabidopsis. The role of CAP-D3 in interphase chromatin organization and function was evaluated using cytogenetic and transcriptome analysis in cap-d3 T-DNA insertion mutants. CAP-D2 and CAP-D3 are highly expressed in mitotically active tissues. In silico and pull-down experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. In cap-d3 mutants, an association of heterochromatic sequences occurs, but the nuclear size and the general histone and DNA methylation patterns remain unchanged. Also, CAP-D3 influences the expression of genes affecting the response to water, chemicals, and stress. The expression and composition of the condensin complexes in Arabidopsis are similar to those in other higher eukaryotes. We propose a model for the CAP-D3 function during interphase in which CAP-D3 localizes in euchromatin loops to stiffen them and consequently separates centromeric regions and 45S rDNA repeats.
- Klíčová slova
- Arabidopsis thaliana, SMC proteins, chromatin organization, chromosomes, condensin, interphase nuclei,
- MeSH
- adenosintrifosfatasy genetika MeSH
- Arabidopsis * genetika MeSH
- chromatin * MeSH
- DNA vazebné proteiny MeSH
- interfáze MeSH
- multiproteinové komplexy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenosintrifosfatasy MeSH
- chromatin * MeSH
- condensin complexes MeSH Prohlížeč
- DNA vazebné proteiny MeSH
- multiproteinové komplexy MeSH
