DNA damage can impair normal cellular functions and result in various pathophysiological processes including cardiovascular diseases and cancer. We compared the genotoxic potential of diverse DNA damaging agents, and focused on their effects on the DNA damage response (DDR) and cell fate in human lung cells BEAS-2B. Polycyclic aromatic hydrocarbons [PAHs; benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP)] induced DNA strand breaks and oxidative damage to DNA; anticancer drugs doxorubicin (DOX) and 5-bromo-2'-deoxyuridine (BrdU) were less effective. DOX triggered the most robust p53 signaling indicating activation of DDR, followed by cell cycle arrest in the G2/M phase, induction of apoptosis and senescence, possibly due to the severe and irreparable DNA lesions. BrdU not only activated p53, but also increased the percentage of G1-phased cells and caused a massive accumulation of senescent cells. In contrast, regardless the activation of p53, both PAHs did not substantially affect the cell cycle distribution or senescence. Finally, a small fraction of cells accumulated only in the G2/M phase and exhibited increased cell death after the prolonged incubation with B[a]P. Overall, we characterized differential responses to diverse DNA damaging agents resulting in specific cell fate and highlighted the key role of DNA lesion type and the p53 signaling persistence.
- MeSH
- Apoptosis MeSH
- Bromodeoxyuridine pharmacology MeSH
- DNA MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 * metabolism MeSH
- Lung metabolism MeSH
- Polycyclic Aromatic Hydrocarbons * toxicity MeSH
- DNA Damage MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article 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.
The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.
- MeSH
- Staining and Labeling methods MeSH
- Bromodeoxyuridine administration & dosage MeSH
- Cell Cycle * MeSH
- DNA analysis MeSH
- Fluorescent Dyes administration & dosage MeSH
- HeLa Cells MeSH
- HL-60 Cells MeSH
- Jurkat Cells MeSH
- Carbocyanines administration & dosage MeSH
- Humans MeSH
- Flow Cytometry methods MeSH
- DNA Replication * MeSH
- Reproducibility of Results MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters.In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.
- MeSH
- Data Analysis MeSH
- Staining and Labeling methods MeSH
- Bromodeoxyuridine metabolism MeSH
- Cell Differentiation MeSH
- Cell Cycle * MeSH
- Bone Marrow Cells metabolism MeSH
- Deoxyuridine analogs & derivatives MeSH
- DNA biosynthesis MeSH
- Immunophenotyping MeSH
- Mitosis MeSH
- Mice MeSH
- Flow Cytometry MeSH
- Rheology MeSH
- S Phase MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
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
The methods of the detection of (1) non-labeled and (2) BrdU-labeled mitochondrial DNA (mtDNA) are described. They are based on the production of singlet oxygen by monovalent copper ions and the subsequent induction of DNA gaps. The ends of interrupted DNA serve as origins for the labeling of mtDNA by DNA polymerase I or they are utilized by exonuclease that degrades DNA strands, unmasking BrdU in BrdU-labeled DNA. Both methods are sensitive approaches without the need of additional enhancement of the signal or the use of highly sensitive optical systems.
- MeSH
- Staining and Labeling methods MeSH
- Biotin chemistry MeSH
- Bromodeoxyuridine chemistry MeSH
- DNA Polymerase I metabolism MeSH
- Indoles chemistry MeSH
- Cells, Cultured MeSH
- Oxygen chemistry MeSH
- Humans MeSH
- Copper chemistry MeSH
- DNA, Mitochondrial genetics MeSH
- Mitochondria genetics MeSH
- DNA Replication MeSH
- Green Fluorescent Proteins chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- 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
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
Labelling of DNA in replicating cells using 5-bromo-2 ́-deoxyuridine (BrdU) is widely used, however the rapid clearance and metabolisation of BrdU in the living organism is a critical issue. Although the pharmacokinetic of BrdU in experimental animals is empirically approximated, the exact time-curve remains unknown. Here we present novel method for estimation of the BrdU content in the blood serum. The application is based on the in vitro cocultivation of tumour cells with the examined serum and the subsequent quantification of the incorporated BrdU in the DNA using flow cytometry analysis. Our results demonstrate that this approach can quantify the BrdU concentration in serum at 1 micromol.dm(-3) and might represent an attractive alternative to conventional chromatographic analysis. The employment of tumour cells as "detectors" of the BrdU content in serum provides an advantage over high pressure liquid chromatography (HPLC), as this approach allows us to approximate not only the concentration of BrdU, but also to determine, whether BrdU is present in the blood serum in effective concentration to reliable label all cells undergoing the S-phase of the cell cycle. The presented application might be a helpful tool for studies on pharmacokinetics of BrdU or other thymidine analogues when testing various administration routes or protocols.
- MeSH
- Antimetabolites blood MeSH
- Bromodeoxyuridine blood MeSH
- Cell Adhesion MeSH
- Cell Line MeSH
- Injections, Intraperitoneal MeSH
- Rats MeSH
- Humans MeSH
- Cell Proliferation drug effects MeSH
- Flow Cytometry MeSH
- Chromatography, High Pressure Liquid MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
