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
• 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

Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.

• MeSH
• cytidin * metabolismus   MeSH
• cytidindeaminasa metabolismus   MeSH
• dCMP-deaminasa * MeSH
• deoxycytidin MeSH
• deoxycytidinkinasa genetika   metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytidin * MeSH
• cytidindeaminasa MeSH
• dCMP-deaminasa * MeSH
• deoxycytidin MeSH
• deoxycytidinkinasa MeSH

Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.

• Klíčová slova
• BrdU, DNA labeling, EdU, cell cycle, labeled nucleosides, markers of cell cycle phases, time lapse microscopy,
• MeSH
• bromodeoxyuridin * metabolismus   MeSH
• buněčné dělení MeSH
• buněčný cyklus MeSH
• proliferace buněk MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• bromodeoxyuridin * MeSH

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

• Klíčová slova
• cytocentrifugation, microscopy, sample processing, staining,
• MeSH
• barvení a značení metody   MeSH
• centrifugace přístrojové vybavení   metody   MeSH
• cytologické techniky přístrojové vybavení   metody   MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• odběr biologického vzorku metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.

• MeSH
• barvení a značení metody   MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• cytofotometrie metody   MeSH
• diploidie * MeSH
• DNA analýza   chemie   MeSH
• dodecylsíran sodný chemie   MeSH
• fluorescenční barviva chemie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• počet buněk přístrojové vybavení   metody   MeSH
• replikace DNA * MeSH
• reprodukovatelnost výsledků MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• dodecylsíran sodný MeSH
• fluorescenční barviva MeSH

The replication of nuclear and mitochondrial DNA are basic processes assuring the doubling of the genetic information of eukaryotic cells. In research of the basic principles of DNA replication, and also in the studies focused on the cell cycle, an important role is played by artificially-prepared nucleoside and nucleotide analogues that serve as markers of newly synthesized DNA. These analogues are incorporated into the DNA during DNA replication, and are subsequently visualized. Several methods are used for their detection, including the highly popular click chemistry. This review aims to provide the readers with basic information about the various possibilities of the detection of replication activity using nucleoside and nucleotide analogues, and to show the strengths and weaknesses of those different detection systems, including click chemistry for microscopic studies.

• Klíčová slova
• click chemistry, indirect immunocytochemistry, isotopes, nucleoside and nucleotide analogues,
• MeSH
• click chemie MeSH
• DNA chemie   genetika   MeSH
• halogenace MeSH
• hybridizace in situ MeSH
• imunohistochemie MeSH
• izotopové značení MeSH
• měď chemie   MeSH
• nukleosidy chemie   MeSH
• nukleotidy chemie   MeSH
• radionuklidy MeSH
• replikace DNA * MeSH
• výzkum MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH
• měď MeSH
• nukleosidy MeSH
• nukleotidy MeSH
• radionuklidy MeSH

The approach for the detection of replicational activity in cells using 5-bromo-2'-deoxyuridine, a low concentration of hydrochloric acid and exonuclease III is presented in the study. The described method was optimised with the aim to provide a fast and robust tool for the detection of DNA synthesis with minimal impact on the cellular structures using image and flow cytometry. The approach is based on the introduction of breaks into the DNA by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III. Our data showed that the method has only a minimal effect on the tested protein localisations and is applicable both for formaldehyde- and ethanol-fixed cells. The approach partially also preserves the fluorescence of the fluorescent proteins in the HeLa cells expressing Fluorescent Ubiquitin Cell Cycle Indicator. In the case of the short labelling pulses that disabled the use of 5-ethynyl-2'-deoxyuridine because of the low specific signal, the described method provided a bright signal enabling reliable recognition of replicating cells. The optimized protocol was also successfully tested for the detection of trifluridine, the nucleoside used as an antiviral drug and in combination with tipiracil also for the treatment of some types of cancer.

• MeSH
• bromodeoxyuridin metabolismus   MeSH
• buněčný cyklus * MeSH
• buňky A549 MeSH
• exodeoxyribonukleasy metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• HeLa buňky MeSH
• kyselina chlorovodíková farmakologie   MeSH
• lidé MeSH
• průtoková cytometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bromodeoxyuridin MeSH
• exodeoxyribonuclease III MeSH Prohlížeč
• exodeoxyribonukleasy MeSH
• kyselina chlorovodíková MeSH

5-Bromo-2'-deoxyuridine (BrdU) labelling and immunostaining is commonly used for the detection of DNA replication using specific antibodies. Previously, we found that these antibodies significantly differ in their affinity to BrdU. Our present data showed that one of the reasons for the differences in the replication signal is the speed of antibody dissociation. Whereas highly efficient antibodies created stable complexes with BrdU, the low efficiency antibodies were unstable. A substantial loss of the signal occurred within several minutes. The increase of the complex stability can be achieved by i) formaldehyde fixation or ii) a quick reaction with a secondary antibody. These steps allowed the same or even higher signal/background ratio to be reached as in the highly efficient antibodies. Based on our findings, we optimised an approach for the fully enzymatic detection of BrdU enabling the fast detection of replicational activity without a significant effect on the tested proteins or the fluorescence of the fluorescent proteins. The method was successfully applied for image and flow cytometry. The speed of the method is comparable to the approach based on 5-ethynyl-2'-deoxyuridine. Moreover, in the case of short labelling pulses, the optimised method is even more sensitive. The approach is also applicable for the detection of 5-trifluoromethyl-2'-deoxyuridine.

• MeSH
• bromodeoxyuridin chemie   MeSH
• buněčný cyklus MeSH
• buňky A549 MeSH
• fluorescenční mikroskopie MeSH
• HeLa buňky MeSH
• lidé MeSH
• měď chemie   MeSH
• protilátky chemie   MeSH
• průtoková cytometrie MeSH
• replikace DNA fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bromodeoxyuridin MeSH
• měď MeSH
• protilátky MeSH

5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.

• Klíčová slova
• 5-ethynyl-2′-deoxycytidine, 5-ethynyl-2′-deoxyuridine, DNA replication, cytidine deaminase, dCMP deaminase,
• MeSH
• bromodeoxyuridin metabolismus   MeSH
• buněčná smrt MeSH
• buněčné jádro metabolismus   MeSH
• cytidindeaminasa metabolismus   MeSH
• deoxycytidin analogy a deriváty   metabolismus   MeSH
• deoxyuridin analogy a deriváty   metabolismus   MeSH
• DNA metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• metabolom MeSH
• nádorové buněčné linie MeSH
• protilátky metabolismus   MeSH
• replikace DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5-ethynyl-2'-deoxycytidine MeSH Prohlížeč
• 5-ethynyl-2'-deoxyuridine MeSH Prohlížeč
• bromodeoxyuridin MeSH
• cytidindeaminasa MeSH
• deoxycytidin MeSH
• deoxyuridin MeSH
• DNA MeSH
• malá interferující RNA MeSH
• protilátky MeSH

We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.

• MeSH
• bromodeoxyuridin metabolismus   MeSH
• buněčné klony MeSH
• DNA metabolismus   MeSH
• HCT116 buňky MeSH
• HeLa buňky MeSH
• idoxuridin analogy a deriváty   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky metabolismus   MeSH
• peptidové mapování * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5'-deoxy-5'-iodouridine MeSH Prohlížeč
• bromodeoxyuridin MeSH
• DNA MeSH
• idoxuridin MeSH
• monoklonální protilátky MeSH