At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků MeSH
- bisbenzimidazol chemie MeSH
- buněčná smrt účinky léků MeSH
- buněčné jádro účinky léků metabolismus MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- cisplatina farmakologie MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie metody MeSH
- fragmentace DNA účinky léků MeSH
- kamptothecin farmakologie MeSH
- lidé MeSH
- průtoková cytometrie metody MeSH
- reprodukovatelnost výsledků MeSH
- staurosporin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- aktiny metabolismus MeSH
- buněčné jádro účinky léků metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- glykoly farmakologie MeSH
- lidé MeSH
- myosin typu I metabolismus MeSH
- nádorové buněčné linie MeSH
- proteinové domény MeSH
- RNA-polymerasa II metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- vazba proteinů účinky léků MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nuclear myosin 1 (NM1) has been implicated in key nuclear functions. Together with actin, it has been shown to initiate and regulate transcription, it is part of the chromatin remodeling complex B-WICH, and is responsible for rearrangements of chromosomal territories in response to external stimuli. Here we show that deletion of NM1 in mouse embryonic fibroblasts leads to chromatin and transcription dysregulation affecting the expression of DNA damage and cell cycle genes. NM1 KO cells exhibit increased DNA damage and changes in cell cycle progression, proliferation, and apoptosis, compatible with a phenotype resulting from impaired p53 signaling. We show that upon DNA damage, NM1 forms a complex with p53 and activates the expression of checkpoint regulator p21 (Cdkn1A) by PCAF and Set1 recruitment to its promoter for histone H3 acetylation and methylation. We propose a role for NM1 in the transcriptional response to DNA damage response and maintenance of genome stability.
- MeSH
- apoptóza MeSH
- buněčné jádro účinky léků genetika metabolismus patologie MeSH
- buněčné linie MeSH
- buněčný cyklus MeSH
- epigeneze genetická MeSH
- etoposid toxicita MeSH
- genetická transkripce * MeSH
- histonlysin-N-methyltransferasa genetika metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika metabolismus MeSH
- myosin typu I genetika metabolismus MeSH
- myši MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- poškození DNA * MeSH
- proliferace buněk MeSH
- restrukturace chromatinu * MeSH
- transkripční faktory p300-CBP genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mediator is a multiprotein complex that connects regulation mediated by transcription factors with RNA polymerase II transcriptional machinery and integrates signals from the cell regulatory cascades with gene expression. One of the Mediator subunits, Mediator complex subunit 28 (MED28), has a dual nuclear and cytoplasmic localization and function. In the nucleus, MED28 functions as part of Mediator and in the cytoplasm, it interacts with cytoskeletal proteins and is part of the regulatory cascades including that of Grb2. MED28 thus has the potential to bring cytoplasmic regulatory interactions towards the centre of gene expression regulation. In this study, we identified MDT-28, the nematode orthologue of MED28, as a likely target of lysine acetylation using bioinformatic prediction of posttranslational modifications. Lysine acetylation was experimentally confirmed using anti-acetyl lysine antibody on immunoprecipitated GFP::MDT-28 expressed in synchronized C. elegans. Valproic acid (VPA), a known inhibitor of lysine deacetylases, enhanced the lysine acetylation of GFP::MDT-28. At the subcellular level, VPA decreased the nuclear localization of GFP::MDT-28 detected by fluorescencelifetime imaging microscopy (FLIM). This indicates that the nuclear pool of MDT-28 is regulated by a mechanism sensitive to VPA and provides an indirect support for a variable relative proportion of MED28 orthologues with other Mediator subunits.
- MeSH
- acetylace MeSH
- buněčné jádro účinky léků metabolismus MeSH
- Caenorhabditis elegans účinky léků metabolismus MeSH
- denzitometrie MeSH
- jaderné proteiny chemie metabolismus MeSH
- kyselina valproová farmakologie MeSH
- larva účinky léků MeSH
- lidé MeSH
- lysin metabolismus MeSH
- mediátorový komplex chemie metabolismus MeSH
- proteiny Caenorhabditis elegans chemie metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin * MeSH
- transport proteinů účinky léků MeSH
- výpočetní biologie MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2-LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models. Mol Cancer Res; 16(10); 1556-67. ©2018 AACR.
- MeSH
- adaptorové proteiny signální transdukční genetika MeSH
- buněčné jádro účinky léků MeSH
- cholangiokarcinom farmakoterapie genetika patologie MeSH
- cytoplazma účinky léků MeSH
- dasatinib aplikace a dávkování MeSH
- fosfoproteiny genetika MeSH
- fosforylace účinky léků MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- protein-serin-threoninkinasy genetika MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- signální transdukce účinky léků MeSH
- skupina kinas odvozených od src-genu antagonisté a inhibitory genetika MeSH
- tyrosin genetika MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty genetika MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.
- MeSH
- antisense oligonukleotidy farmakologie MeSH
- buněčné jádro účinky léků metabolismus MeSH
- endoteliální buňky metabolismus MeSH
- expanze trinukleotidových repetic genetika MeSH
- Fuchsova endoteliální dystrofie genetika patologie MeSH
- genetická predispozice k nemoci * MeSH
- kohortové studie MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- myši inbrední C57BL MeSH
- orgánová specificita MeSH
- posttranskripční úpravy RNA MeSH
- prekurzory RNA genetika MeSH
- rizikové faktory MeSH
- rohovkový endotel patologie MeSH
- senioři MeSH
- sestřihové faktory metabolismus MeSH
- transkripční faktor 4 genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Results of a number of studies indicate that electroplaters have increased cancer risks as a consequence of exposure to genotoxic metals such as chromium (VI) and nickel. These effects may be due to induction of damage of the genetic material which plays a key role in the etiology of cancer, and it was found that workers in galvanization factories exhibited increased levels of DNA damage. The aim of the present study was to investigate genetic stability in workers of a bright plating factory who are exposed to chromium (Cr) and cobalt (Co). Exfoliated cells were collected from the buccal and nasal mucosa of workers (n = 42) and matched controls (n = 43) and analyzed for induction of micronuclei (MN) which are formed as a consequence of chromosomal aberrations. In addition, other nuclear anomalies namely nuclear buds (Nbuds) which are formed as a consequence of gene amplification and markers indicating different stages of cell death (condensed chromatin, karyorrhexis, karyolysis, and pyknosis) were also assessed. No evidence was noted for induction of MN, but significantly increased rates of Nbuds in cells from both, buccal and nasal mucosa, were found. Parameters which are indicative for cytotoxic effects were more pronounced in nasal cells and rose with duration of employment period. Overall, our findings indicated that no apparent chromosomal damage occurred in bright electroplaters. However, data demonstrated that acute cytotoxic effects may lead to inflammations and/or lesions in epithelia of the respiratory tract of the workers.
- MeSH
- buněčná smrt účinky léků MeSH
- buněčné jádro účinky léků MeSH
- chrom toxicita MeSH
- dospělí MeSH
- kobalt toxicita MeSH
- lidé MeSH
- lidské chromozomy účinky léků MeSH
- mikrojádra chromozomálně defektní chemicky indukované MeSH
- nosní sliznice účinky léků MeSH
- pokovování galvanické * MeSH
- pracovní expozice škodlivé účinky MeSH
- ústní sliznice účinky léků MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Oxidative stress and persistent activation of DNA damage response (DDR) are causally involved in the development of cellular senescence, a phenomenon implicated in fundamental (patho)physiological processes such as aging, fetal development and tumorigenesis. Here, we report that adenine nucleotide translocase-2 (ANT2) is consistently down-regulated in all three major forms of cellular senescence: replicative, oncogene-induced and drug-induced, in both normal and cancerous human cells. We previously reported formation of novel NF1/Smad transcription repressor complexes in growth-arrested fibroblasts. Here we show that such complexes form in senescent cells. Mechanistically, binding of the NF1/Smad complexes to the NF1-dependent repressor elements in the ANT2 gene promoter repressed ANT2 expression. Etoposide-induced formation of these complexes and repression of ANT2 were relatively late events co-incident with production and secretion of, and dependent on, TGF-β. siRNA-mediated knock-down of ANT2 in proliferating cells resulted in increased levels of reactive oxygen species (ROS) and activation of the DDR. Knock-down of ANT2, together with etoposide treatment, further intensified ROS production and DNA damage signaling, leading to enhanced apoptosis. Together, our data show that TGF-β-mediated suppression of ANT2 through NF1/Smad4 complexes contributes to oxidative stress and DNA damage during induction of cellular senescence.
- MeSH
- buněčné jádro účinky léků metabolismus MeSH
- buněčné linie MeSH
- cytoprotekce účinky léků MeSH
- down regulace účinky léků MeSH
- etoposid farmakologie MeSH
- lidé MeSH
- mutace MeSH
- oxidační stres * účinky léků MeSH
- poškození DNA MeSH
- promotorové oblasti (genetika) MeSH
- protein Smad4 metabolismus MeSH
- represorové proteiny metabolismus MeSH
- stárnutí buněk * účinky léků MeSH
- transformující růstový faktor beta metabolismus MeSH
- transkripční faktory NFI metabolismus MeSH
- translokátor adeninových nukleotidů 2 genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY-2 cells treated with 50 µM CdSO4. The cadmium-induced alterations in cell morphology occurred gradually over a period of 3-4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium-induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis.
- MeSH
- buněčná smrt účinky léků MeSH
- buněčné jádro účinky léků metabolismus ultrastruktura MeSH
- kadmium toxicita MeSH
- kultivované buňky MeSH
- tabák cytologie MeSH
- tvar buňky účinky léků MeSH
- vakuoly účinky léků metabolismus ultrastruktura MeSH
- viabilita buněk účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIM: The optimal balance between histone acetylation and deacetylation is important for proper gene function. Therefore, we addressed how inhibitors of histone-modifying enzymes can modulate nuclear events, including replication, transcription, splicing and DNA repair. MATERIALS & METHODS: Changes in cell signaling pathways upon treatment with histone acetyltransferases and/or histone deacetylase inhibitors were studied by cDNA microarrays and western blots. RESULTS: We analyzed the effects of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and the histone acetylase inhibitor MG149. SAHA altered the expression of factors involved in DNA replication complexes, basal transcription and the spliceosome pathway. DNA repair-related genes, including Rad51, Rad54 and BRCA2, were significantly downregulated by SAHA. However, MG149 had no effect on the investigated nuclear processes, with the exception of the spliceosome network and Sestrins, involved in DNA repair. CONCLUSION: Based on our results, we propose that the studied epigenetic drugs have the distinct potential to affect specific cell signaling pathways depending on their respective molecular targets.
- MeSH
- acetylace účinky léků MeSH
- apoptóza účinky léků MeSH
- buněčné jádro účinky léků metabolismus MeSH
- buňky K562 MeSH
- DNA-helikasy MeSH
- epigeneze genetická MeSH
- histonacetyltransferasy genetika metabolismus MeSH
- histony metabolismus MeSH
- inhibitory histondeacetylas farmakologie MeSH
- jaderné proteiny genetika MeSH
- kyseliny hydroxamové farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika MeSH
- NF-kappa B genetika MeSH
- oprava DNA účinky léků MeSH
- protein BRCA2 genetika MeSH
- regulace genové exprese MeSH
- rekombinasa Rad51 genetika MeSH
- RNA genetika MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- signální transdukce účinky léků MeSH
- spliceozomy genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH