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
• Cytidine * metabolism   MeSH
• Cytidine Deaminase metabolism   MeSH
• DCMP Deaminase * MeSH
• Deoxycytidine MeSH
• Deoxycytidine Kinase genetics   metabolism   MeSH
• Metabolic Networks and Pathways MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytidine * MeSH
• Cytidine Deaminase MeSH
• DCMP Deaminase * MeSH
• Deoxycytidine MeSH
• Deoxycytidine Kinase 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.

• Keywords
• BrdU, DNA labeling, EdU, cell cycle, labeled nucleosides, markers of cell cycle phases, time lapse microscopy,
• MeSH
• Bromodeoxyuridine * metabolism   MeSH
• Cell Division MeSH
• Cell Cycle MeSH
• Cell Proliferation MeSH
• Reproducibility of Results MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Bromodeoxyuridine * 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.

• Keywords
• click chemistry, indirect immunocytochemistry, isotopes, nucleoside and nucleotide analogues,
• MeSH
• Click Chemistry MeSH
• DNA chemistry   genetics   MeSH
• Halogenation MeSH
• In Situ Hybridization MeSH
• Immunohistochemistry MeSH
• Isotope Labeling MeSH
• Copper chemistry   MeSH
• Nucleosides chemistry   MeSH
• Nucleotides chemistry   MeSH
• Radioisotopes MeSH
• DNA Replication * MeSH
• Research MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• Copper MeSH
• Nucleosides MeSH
• Nucleotides MeSH
• Radioisotopes 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
• Bromodeoxyuridine chemistry   MeSH
• Cell Cycle MeSH
• A549 Cells MeSH
• Microscopy, Fluorescence MeSH
• HeLa Cells MeSH
• Humans MeSH
• Copper chemistry   MeSH
• Antibodies chemistry   MeSH
• Flow Cytometry MeSH
• DNA Replication physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bromodeoxyuridine MeSH
• Copper MeSH
• Antibodies 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.

• Keywords
• 5-ethynyl-2′-deoxycytidine, 5-ethynyl-2′-deoxyuridine, DNA replication, cytidine deaminase, dCMP deaminase,
• MeSH
• Bromodeoxyuridine metabolism   MeSH
• Cell Death MeSH
• Cell Nucleus metabolism   MeSH
• Cytidine Deaminase metabolism   MeSH
• Deoxycytidine analogs & derivatives   metabolism   MeSH
• Deoxyuridine analogs & derivatives   metabolism   MeSH
• DNA metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• Metabolome MeSH
• Cell Line, Tumor MeSH
• Antibodies metabolism   MeSH
• DNA Replication MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-ethynyl-2'-deoxycytidine MeSH Browser
• 5-ethynyl-2'-deoxyuridine MeSH Browser
• Bromodeoxyuridine MeSH
• Cytidine Deaminase MeSH
• Deoxycytidine MeSH
• Deoxyuridine MeSH
• DNA MeSH
• RNA, Small Interfering MeSH
• Antibodies 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
• Bromodeoxyuridine metabolism   MeSH
• Clone Cells MeSH
• DNA metabolism   MeSH
• HCT116 Cells MeSH
• HeLa Cells MeSH
• Idoxuridine analogs & derivatives   metabolism   MeSH
• Humans MeSH
• Antibodies, Monoclonal metabolism   MeSH
• Peptide Mapping * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5'-deoxy-5'-iodouridine MeSH Browser
• Bromodeoxyuridine MeSH
• DNA MeSH
• Idoxuridine MeSH
• Antibodies, Monoclonal MeSH

2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2'-deoxythymidine 5'-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.

• MeSH
• Cell Line MeSH
• Cytotoxins metabolism   toxicity   MeSH
• Deoxyuridine analogs & derivatives   metabolism   toxicity   MeSH
• DNA biosynthesis   genetics   metabolism   MeSH
• Enzyme Inhibitors metabolism   toxicity   MeSH
• Intracellular Space drug effects   metabolism   MeSH
• Humans MeSH
• DNA Damage * MeSH
• Cell Proliferation drug effects   MeSH
• DNA Replication drug effects   MeSH
• S Phase drug effects   MeSH
• Tetrahydrofolates biosynthesis   MeSH
• Thymidine metabolism   pharmacology   MeSH
• Thymidine Monophosphate metabolism   MeSH
• Thymidylate Synthase antagonists & inhibitors   MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-ethynyl-2'-deoxyuridine MeSH Browser
• 5,6,7,8-tetrahydrofolic acid MeSH Browser
• Cytotoxins MeSH
• Deoxyuridine MeSH
• DNA MeSH
• Enzyme Inhibitors MeSH
• Tetrahydrofolates MeSH
• Thymidine MeSH
• Thymidine Monophosphate MeSH
• Thymidylate Synthase MeSH

The study describes the method of a sensitive detection of double-stranded DNA molecules in situ. It is based on the oxidative attack on the deoxyribose moiety by copper(I) in the presence of oxygen. We have shown previously that the oxidative attack leads to the formation of frequent gaps in DNA. Here we have demonstrated that the gaps can be utilized as the origins for an efficient synthesis of complementary labeled strands by DNA polymerase I and that such enzymatic detection of the double-stranded DNA is a sensitive approach enabling in-situ detection of both the nuclear and mitochondrial genomes in formaldehyde-fixed human cells.

• MeSH
• Cell Nucleus genetics   MeSH
• DNA Polymerase I metabolism   MeSH
• DNA genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• DNA, Mitochondrial genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Polymerase I MeSH
• DNA MeSH
• DNA, Mitochondrial MeSH