functional assay
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- MeSH
- biologický transport MeSH
- buněčné linie MeSH
- finanční podpora výzkumu jako téma MeSH
- fluorescenční barviva MeSH
- lidé MeSH
- mnohočetná léková rezistence MeSH
- P-glykoprotein antagonisté a inhibitory MeSH
- protinádorové látky MeSH
- průtoková cytometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- srovnávací studie MeSH
Nádorové kmenové buňky (cancer stem cells – CSCs) jsou považovány za populaci buněk, která odpovídá za iniciaci a progresi nádoru, účastní se procesu metastazování a je možnou příčinou získané lékové rezistence a rekurence nádorů. CSCs disponují schopností sebeobnovy a mají tumorigenní potenciál. Funkční testy, které umožňují detekovat zmiňované vlastnosti, jsou hlavním nástrojem pro identifikaci nádorových kmenových buněk. Tento článek přináší ucelený přehled in vivo a in vitro metod využívaných pro průkaz CSCs s důrazem na recentně zaváděné techniky detekce CSCs. Mezi nejčastěji prováděné funkční testy patří test tumorigenicity in vivo, testy tvorby sfér (sphere formation assay) a kolonií (colony‑forming unit assay) a rovněž detekce tzv. vedlejší populace (side population). Dále jsou popsány metody zadržování detekční značky (label‑retention assay) a test aktivity aldehyddehydrogenázy.
Cancer stem cells (CSCs) are considered to be a population of tumor cells, which are responsible for tumor initiation and progression. They are also involved in metastasizing and may be a possible cause of multidrug resistance and tumor recurrence. CSCs possess the ability to self‑renew and show a tumorigenic potential. Functional assays, which enable the detection of these properties, represent the main tool for identification of CSCs. This article summarizes both in vitro and in vivo methods used to identify the CSCs with emphasis on recently employed techniques of CSCs detection. In vivo tumorigenicity assay, sphere formation assay and colony‑forming unit assay belong to the most commonly used functional assays. Further, label‑retention assay and aldehyde dehydrogenase activity assay are described in this article. Key words: cancer stem cells – functional assays – tumorigenicity – tumor spheres – colony‑forming unit assay – side population cells – aldehyde dehydrogenase The study was supported by grant of Internal Grant Agency of the Czech Ministry of Health No. NT13443-4 and by the European Regional Development Fund and the State Budget of the Czech Republic – RECAMO, CZ.1.05//2.1.00/03.0101 and by the project CEB, OP VK CZ.1.07/2.3.00/20.0183. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 16. 1. 2014 Accepted: 4. 4. 2014
- Klíčová slova
- tumorigenicita, tvorba kolonií, nádorové sféry, myši NSG, test zadržování detekční značky, label-retention assay,
- MeSH
- aldehyddehydrogenasa analýza MeSH
- analýza kolonii tvořících jednotek * metody MeSH
- buněčné kultury MeSH
- buněčné sféroidy MeSH
- fluoresceiny metabolismus MeSH
- kultivační média MeSH
- myši inbrední NOD MeSH
- myši SCID MeSH
- myši MeSH
- nádorové buňky kultivované * MeSH
- nádorové kmenové buňky MeSH
- testy karcinogenity * metody MeSH
- testy nádorových kmenových buněk * metody MeSH
- transplantace nádorů * metody MeSH
- vedlejší populace buněk MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Methods in enzymology ; Vol. 200
763 s. : obr., tab., přeruš.bibliogr.
Methods in molecular biology ; Vol. 272
[1st ed.] xix, 385 s. : il.
Cytokinins, like other phytohormones, act in plants as signaling molecules at very low concentrations. The system that mediates between their chemical recognition and the responses that they induce requires a hormone receptor that, together with down-stream located elements, forms a signaling network, converting the signal into a specific response. Identification of the cytokinin-binding histidine kinases CRE1/AHK4, AHK3, and AHK2 as cytokinin receptors in Arabidopsis was an important milestone in the elucidation of cytokinin signal transduction pathways. Their molecular characterization through the use of transgenic E. coli strains revealed that a variety of cytokinin compounds may have signaling functions, but only with specific receptors. This indicates that differential ligand specificities of the receptors may be a mechanism to fine-tune the various cytokinin responses. This chapter describes the detailed protocol of a method employing transgenic E. coli which substantially contributes to our understanding of cytokinin perception, a crucial step in the cytokinin regulation of diverse plant growth and development processes.
- MeSH
- Arabidopsis enzymologie MeSH
- beta-galaktosidasa genetika metabolismus MeSH
- biotest metody MeSH
- cytokininy metabolismus farmakologie MeSH
- Escherichia coli genetika metabolismus MeSH
- proteinkinasy genetika metabolismus MeSH
- regulace genové exprese u bakterií účinky léků MeSH
- regulátory růstu rostlin metabolismus farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.
- MeSH
- acetylace MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- biotest metody MeSH
- buněčné jádro genetika metabolismus MeSH
- fosforylace MeSH
- interakční proteinové domény a motivy MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- nádory kostí genetika metabolismus patologie MeSH
- oprava DNA MeSH
- osteosarkom genetika metabolismus patologie MeSH
- poškození DNA MeSH
- proteinfosfatasa 2C genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Evaluating nanoparticle (NP) toxicity in human cell systems is a fundamental requirement for future NP biomedical applications. In this study, we have designed a screening assay for assessing different types of cell death induced by NPs in human umbilical vein endothelial cell (HUVEC) culture. This assay consists of WST-8, LDH and Hoechst 33342 staining, all performed in one well, which enables an evaluation of cell viability, necrosis and apoptosis, respectively, in the same cell sample. The 96-well format and automated processing of fluorescent images enhances the assay rapidity and reproducibility. After testing the assay functionality with agents that induced different types of cell death, we investigated the endothelial toxicity of superparamagnetic iron oxide nanoparticles (SPIONs, 8 nm), silica nanoparticles (SiNPs, 7-14 nm) and carboxylated multiwall carbon nanotubes (CNTCOOHs, 60 nm). Our results indicated that all the tested NP types induced decreases in cell viability after 24 hours at a concentration of 100 μg/ml. SPIONs caused the lowest toxicity in HUVECs. By contrast, SiNPs induced pronounced necrosis and apoptosis. A time course experiment showed the gradual toxic effect of all the tested NPs. CNTCOOHs inhibited tetrazolium derivatives at 100 μg/ml, causing false negative results from the WST-8 and LDH assay. In summary, our data demonstrate that the presented "three-in-one" screening assay is capable of evaluating NP toxicity effectively and reliably. Due to its simultaneous utilization of two different methods to assess cell viability, this assay is also capable of revealing, if NPs interfere with tetrazolium salts.
- MeSH
- apoptóza účinky léků MeSH
- biotest MeSH
- endoteliální buňky pupečníkové žíly (lidské) MeSH
- endoteliální buňky účinky léků MeSH
- lidé MeSH
- nanočástice aplikace a dávkování MeSH
- preklinické hodnocení léčiv metody MeSH
- velikost částic MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The last decade witnessed an increase in the use of comet assay for DNA damage monitoring in cancer patients and controls. Apart from case-control studies, reports described the determination of DNA damage prior to (baseline value) and after chemo-/radiotherapy, the treatment resulted in significantly elevated DNA damage. However, studies on DNA damage as a factor reflecting cancer prognosis and therapy prediction are scarce. In most cases, DNA damage was analysed in surrogate tissues. The data on DNA damage are available for 17 types of cancer. The reviewed data unambiguously pinpoint the usefulness of the comet assay in human cancer research due to its sensitivity and cost-effectiveness in evaluating DNA damage associated with the disease and with the treatment. DNA repair capacity (DRC) represents a complex marker for functional evaluation of multigene DNA repair processes in cancer onset with future prospects in personalized prevention and/or cancer treatment. A comparison between studies and more general conclusions are precluded by a variable design of the studies and a lack of standard protocol for both DNA damage and DRC determination. Since cancer is a heterogeneous complex disease, numerous points have to be considered: a) DNA damage and DRC measured in surrogate/target tissues, b) changes in the levels of DNA damage and DRC may be a cause or a consequence of the disease, c) changes in DRC alter sensitivity of tumour cells to antineoplastic drugs, d) one time point-sampling of patients provides insufficient information on the role of DNA damage and its repair in carcinogenesis. Finally, systemic cancer therapy is targeted at DNA damage and its repair. A proper understanding of these processes is a key precondition for the optimisation of therapy regimens, prediction of therapeutic response and prognosis in cancer patients.
- MeSH
- analýza nákladů a výnosů MeSH
- DNA krev MeSH
- epitelové buňky chemie MeSH
- karcinom genetika metabolismus MeSH
- kometový test * ekonomika metody MeSH
- koncentrace vodíkových iontů MeSH
- krevní buňky chemie MeSH
- lidé MeSH
- melanom genetika metabolismus MeSH
- monitorování léčiv metody MeSH
- nádory genetika metabolismus terapie MeSH
- oprava DNA * MeSH
- orgánová specificita MeSH
- poškození DNA * MeSH
- senzitivita a specificita MeSH
- spermie chemie MeSH
- zlomy DNA MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
