Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raška, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

• MeSH
• DNA-ligasa ATP genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA účinky záření   MeSH
• homologní rekombinace MeSH
• kultivované buňky MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   fyziologie   MeSH
• nestabilita genomu * MeSH
• oprava DNA spojením konců fyziologie   účinky záření   MeSH
• oprava DNA fyziologie   účinky záření   MeSH
• proteiny vázající poly-ADP-ribosu genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.

• Publikační typ
• časopisecké články MeSH

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.

• MeSH
• buněčné linie MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA MeSH
• embryonální kmenové buňky enzymologie   metabolismus   MeSH
• imunohistochemie MeSH
• karyotypizace MeSH
• lidé MeSH
• oprava DNA genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

• Publikační typ
• časopisecké články MeSH

Křišťálová studánka v Chicagu?; Fejetony a příhody, v nichž Josef Václav Sládek seznamoval soudobou českou veřejnost se svými zážitky z cesty po Severní Americe, obohacují tradiční portrét básníka o nové rysy, a představují ho tak v nezvyklé roli zpravodaje a publicisty.Soubor je uspořádán chronologicky, aby věrně rekonstruoval Sládkovo putování po Spojených státech, kam z politických, ekonomických nebo osobních důvodů mířily v šedesátých letech 19. století vlny emigrantů ze střední Evropy, aby si splnily svůj „americký sen“. Sládek si s respektem i dojetím všímá individuálních osudů bezejmenných tuláků, groteskních podivínů, romantických dobrodruhů i šťastně usazených farmářů, vypráví o vtipných i pohnutých setkáních s českým, potažmo slovanským živlem, popisuje stereotypy spjaté s irskými nebo čínskými přistěhovalci, skepticky sleduje domorodé komunity černochů a míšenců, a především odsuzuje kruté zacházení s indiány, jejichž údělu a kulturnímu odkazu věnuje nejvíc pozornosti.

• Klíčová slova
• slovníky odborné, slovník věcný, slovník synonymický,
• Publikační typ
• slovníky MeSH

• Konspekt
• Čeština
• NLK Obory
• lingvistika, lékařská terminologie