Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), a Wnt pathway member, has been previously recognised as a stem cell marker in numerous epithelial tissues. In this study, we used Lgr5-EGFP-CreERT2 mice to analyse the distribution of LGR5-positive cells during craniofacial development. LGR5 expressing cells were primarily located in the mesenchyme adjacent to the craniofacial epithelial structures undergoing folding, such as the nasopharyngeal duct, lingual groove, and vomeronasal organ. To follow the fate of LGR5-positive cells, we performed lineage tracing using an inducible Cre knock-in allele in combination with Rosa26-tdTomato reporter mice. The slight expansion of LGR5-positive cells was found around the vomeronasal organ, in the nasal cavity, and around the epithelium in the lingual groove. However, most LGR5 expressing cells remained in their original location, possibly supporting their signalling function for adjacent epithelium rather than exerting their role as progenitor cells for the craniofacial structures. Moreover, Lgr5 knockout mice displayed distinct defects in LGR5-positive areas, especially in the reduction of the nasopharyngeal duct, the alteration of the palatal shelves shape, abnormal epithelial folding in the lingual groove area, and the disruption of salivary gland development. The latter defect manifested as an atypical number and localisation of the glandular ducts. The gene expression of several Wnt pathway members (Rspo1-3, Axin2) was altered in Lgr5-deficient animals. However, the difference was not found in sorted EGFP-positive cells obtained from Lgr5+/+ and Lgr5-/- animals. Expression profiling of LGR5-positive cells revealed the expression of several markers of mesenchymal cells, antagonists, as well as agonists, of Wnt signalling, and molecules associated with the basal membrane. Therefore, LGR5-positive cells in the craniofacial area represent a very specific population of mesenchymal cells adjacent to the epithelium undergoing folding or groove formation. Our results indicate a possible novel role of LGR5 in the regulation of morphogenetic processes during the formation of complex epithelial structures in the craniofacial areas, a role which is not related to the stem cell properties of LGR5-positive cells as was previously defined for various epithelial tissues.
- Publikační typ
- časopisecké články MeSH
Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.
Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs.
- MeSH
- buněčná diferenciace * MeSH
- buněčná sebeobnova * MeSH
- centrozom metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- intracelulární signální peptidy a proteiny antagonisté a inhibitory metabolismus MeSH
- lidé MeSH
- mitóza MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- proliferace buněk MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the past decade, automated microscopy has become an important tool for the drug discovery and development process. The establishment of imaging modalities as screening tools depended on technological breakthroughs in the domain of automated microscopy and automated image analysis. These types of assays are often referred to as high content screening or high content analysis (HCS/HCA). The driving force to adopt imaging for drug development is the quantity and quality of cellular information that can be collected and the enhanced physiological relevance of cellular screening compared to biochemical screening. Most imaging in drug development is performed on fixed cells as this allows uncoupling the preparation of the cells from the acquisition of the images. Live-cell imaging is technically challenging, but is very useful for many aspects of the drug development pipeline such as kinetic studies of compound mode of action or to analyze the motion of cellular components. Most vendors of HCS microscopy systems offer the option of environmental chambers and onboard pipetting on their platforms. This reflects the wish and desire of many customers to have the ability to perform live-cell assays on their HCS automated microscopes. This book chapter summarizes the challenges and advantages of live-cell imaging in drug discovery. Examples of applications are presented and the motivation to perform these assays in kinetic mode is discussed.
- MeSH
- buněčné kultury MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikroskopie MeSH
- molekulární zobrazování metody MeSH
- objevování léků * metody MeSH
- počítačové zpracování obrazu MeSH
- rychlé screeningové testy * MeSH
- software MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
HMGB1 and HMGB2 proteins have been implicated in numerous cellular processes, including proliferation, differentiation, apoptosis, and tumor growth. It is unknown whether they are involved in regulating the typical functions of pluripotent human embryonic stem cells (hESCs) and/or those of the differentiated derivatives of hESCs. Using inducible, stably transfected hESCs capable of shRNA-mediated knockdown of HMGB1 and HMGB2, we provide evidence that downregulation of HMGB1 and/or HMGB2 in undifferentiated hESCs does not affect the stemness of cells and induces only minor changes to the proliferation rate, cell-cycle profile, and apoptosis. After differentiation is induced, however, the downregulation of those proteins has important effects on proliferation, apoptosis, telomerase activity, and the efficiency of differentiation toward the neuroectodermal lineage. Furthermore, those processes are affected only when one, but not both, of the two proteins is downregulated; the knockdown of both HMGB1 and HMGB2 results in a normal phenotype. Those results advance our knowledge of regulation of hESC and human neuroectodermal cell differentiation and illustrate the distinct roles of HMGB1 and HMGB2 during early human development.
- MeSH
- apoptóza genetika MeSH
- buněčná diferenciace * MeSH
- buněčná sebeobnova genetika MeSH
- buněčné linie MeSH
- buněčný cyklus genetika MeSH
- buněčný rodokmen genetika MeSH
- down regulace genetika MeSH
- histony metabolismus MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus MeSH
- neurální ploténka cytologie MeSH
- proliferace buněk genetika MeSH
- protein HMGB1 metabolismus MeSH
- protein HMGB2 metabolismus MeSH
- telomerasa metabolismus MeSH
- transfekce MeSH
- tvar buňky genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A significant part of current research studies utilizes various cellular models which imply specific antibiotics-containing media as well as antibiotics used for clonal selection or promoter de/activation. With the great success of developing such tools, mitochondria, once originated from bacteria, can be effectively targeted by antibiotics. For that reason, some studies propose antibiotics-targeting of mitochondria as part of anticancer therapy. Here, we have focused on the effects of various classes of antibiotics on mitochondria in cancer and non-cancer cells and demonlow mitochondrial membrane potential, reduced ATP production, altered morphology and lowered respiration rate which altogether suggested mitochondrial dysfunction (MDF). This was in parallel with increased level of reactive oxygen species (ROS) and decreased activity of mitochondrial respiration complexes. However, both survival and repopulation capacity of cancer cells was not significantly affected by the antibiotics, perhaps due to a glycolytic shift or activated autophagy. In turn, simultaneous inhibition of autophagy and treatment with antibiotics largely reduced tumorigenic properties of cancer cells suggesting potential strategy for anticancer therapy.
- MeSH
- adenin analogy a deriváty farmakologie MeSH
- adenosintrifosfát metabolismus MeSH
- antibakteriální látky farmakologie MeSH
- autofagie účinky léků MeSH
- časové faktory MeSH
- elektronový transportní řetězec metabolismus MeSH
- energetický metabolismus účinky léků MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondrie účinky léků metabolismus patologie MeSH
- mitofagie účinky léků MeSH
- nádorové buněčné linie MeSH
- nádory prsu farmakoterapie genetika metabolismus patologie MeSH
- proliferace buněk účinky léků MeSH
- proteiny asociované s mikrotubuly genetika metabolismus MeSH
- protokoly protinádorové kombinované chemoterapie farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- signální transdukce účinky léků MeSH
- synergismus léků MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Checkpoint-mediated dependency of tumor cells can be deployed to selectively kill them without substantial toxicity to normal cells. Specifically, loss of CHK1, a serine threonine kinase involved in the surveillance of the G2-M checkpoint in the presence of replication stress inflicted by DNA-damaging drugs, has been reported to dramatically influence the viability of tumor cells. CHK1's pivotal role in maintaining genomic stability offers attractive opportunity for increasing the selectivity, effectivity, and reduced toxicity of chemotherapy. Some recently identified CHK1 inhibitors entered clinical trials in combination with DNA antimetabolites. Herein, we report synthesis and profiling of MU380, a nontrivial analogue of clinically profiled compound SCH900776 possessing the highly unusual N-trifluoromethylpyrazole motif, which was envisioned not to undergo metabolic oxidative dealkylation and thereby provide greater robustness to the compound. MU380 is a selective and potent inhibitor of CHK1 which sensitizes a variety of tumor cell lines to hydroxyurea or gemcitabine up to 10 times. MU380 shows extended inhibitory effects in cells, and unlike SCH900776, does not undergo in vivo N-dealkylation to the significantly less selective metabolite. Compared with SCH900776, MU380 in combination with GEM causes higher accumulation of DNA damage in tumor cells and subsequent enhanced cell death, and is more efficacious in the A2780 xenograft mouse model. Overall, MU380 represents a novel state-of-the-art CHK1 inhibitor with high potency, selectivity, and improved metabolic robustness to oxidative N-dealkylation. Mol Cancer Ther; 16(9); 1831-42. ©2017 AACR.
- MeSH
- apoptóza účinky léků MeSH
- biologické markery MeSH
- buněčný cyklus účinky léků MeSH
- checkpoint kinasa 1 antagonisté a inhibitory MeSH
- chemorezistence účinky léků MeSH
- dealkylace účinky léků MeSH
- inhibitory proteinkinas chemická syntéza farmakologie MeSH
- kontrolní body buněčného cyklu účinky léků MeSH
- lidé MeSH
- metylace MeSH
- modely nemocí na zvířatech MeSH
- molekulární struktura MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- protinádorové látky chemická syntéza farmakologie MeSH
- pyrazoly farmakologie MeSH
- pyrimidiny farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.
- MeSH
- autofagie MeSH
- buněčné linie MeSH
- Fanconiho anemie patofyziologie MeSH
- fluorescenční mikroskopie MeSH
- lidé MeSH
- mitochondriální dynamika * MeSH
- mitochondrie patologie ultrastruktura MeSH
- mitofagie * MeSH
- oxidační stres MeSH
- reaktivní formy kyslíku metabolismus MeSH
- transmisní elektronová mikroskopie MeSH
- vzácné nemoci patofyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Ovarian cancer is one of the most common malignancies in women and contributes greatly to cancer-related deaths. Tumor suppressor candidate 3 (TUSC3) is a putative tumor suppressor gene located at chromosomal region 8p22, which is often lost in epithelial cancers. Epigenetic silencing of TUSC3 has been associated with poor prognosis, and hypermethylation of its promoter provides an independent biomarker of overall and disease-free survival in ovarian cancer patients. TUSC3 is localized to the endoplasmic reticulum in an oligosaccharyl tranferase complex responsible for the N-glycosylation of proteins. However, the precise molecular role of TUSC3 in ovarian cancer remains unclear. In this study, we establish TUSC3 as a novel ovarian cancer tumor suppressor using a xenograft mouse model and demonstrate that loss of TUSC3 alters the molecular response to endoplasmic reticulum stress and induces hallmarks of the epithelial-to-mesenchymal transition in ovarian cancer cells. In summary, we have confirmed the tumor-suppressive function of TUSC3 and identified the possible mechanism driving TUSC3-deficient ovarian cancer cells toward a malignant phenotype.
- MeSH
- epitelo-mezenchymální tranzice genetika MeSH
- heterografty MeSH
- lidé MeSH
- membránové proteiny genetika MeSH
- myši inbrední NOD MeSH
- myši SCID MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádorové supresorové proteiny genetika MeSH
- nádory vaječníků genetika MeSH
- stres endoplazmatického retikula genetika MeSH
- tumor supresorové geny fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.
- MeSH
- ADP-ribosylační faktor 1 genetika metabolismus MeSH
- buněčná membrána enzymologie MeSH
- endozomy enzymologie MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- genové regulační sítě MeSH
- HeLa buňky MeSH
- lidé MeSH
- mapy interakcí proteinů MeSH
- rac1 protein vázající GTP genetika metabolismus MeSH
- receptor IGF typ 2 genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- RNA interference * MeSH
- shluková analýza MeSH
- signální transdukce MeSH
- skupina kinas odvozených od src-genu genetika metabolismus MeSH
- trans-Golgiho síť enzymologie MeSH
- transfekce MeSH
- transport proteinů genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
