Discrimination of cell nuclei in early S-phase, mid-to-late S-phase, and G(2)/M-phase by sequential administration of 5-bromo-2'-deoxyuridine and 5-chloro-2'-deoxyuridine.

5-Bromo-2'-deoxyuridine (BrdU) and 5-chloro-2'-deoxyuridine (CldU) were sequentially administered intraperitoneally into mice at 1-hr intervals. After one additional hr, the small intestines were resected, fixed, and embedded in paraffin. In histological sections stained with monoclonal antibody Br-3 reactive to both BrdU and CldU, and CldU antibody reactive only to CldU, three types of staining could be identified in the proliferating zone. Cells with nuclei stained only with Br-3 antibody were estimated to have completed DNA replication during the first 1 hr and were fixed in G(2)/M-phase. Those nuclei were frequently found in apical areas of the simple columnar epithelium of the intestine, whereas other nuclei were located basally in the cells. This observation suggested intracellular movement of cell nuclei in G(2)/M-phase. Identification of cells in early S-phase became possible using these antibodies in combination with DAB and fluorescence stainings. Replication sites in early S-phase nuclei were found to be numerous, whereas in late S-phase they were larger in size and much smaller in number.     

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.     

Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2'-deoxyuridine

We report procedures to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast, a thymidine analogue which has some technical advantages over use of bromodeoxyuridine. Low concentrations of EdU (1 μM) are sufficient to allow detection of incorporation in cells expressing thymidine kinase and human equilibrative nucleoside transporter 1 (hENT1). However EdU is toxic and activates the rad3-dependent checkpoint, resulting in cell cycle arrest, potentially limiting its applications for procedures which require labelling over more than one cell cycle. Limited DNA synthesis, when elongation is largely blocked by hydroxyurea, can be readily detected by EdU incorporation using fluorescence microscopy. Thus EdU should be useful for detecting early stages of S phase, or DNA synthesis associated with DNA repair and recombination.     

Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5'-bromo-2'-deoxyuridine

In this study we present a method for the measurement of in vitro mitogenesis in fish leucocytes that is based on the incorporation of the thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) into the DNA of replicating cells, followed by ELISA-based detection. This technique, adapted from methods developed for mammalian cells, operates on a similar biological principle to (3)H-thymidine incorporation, but circumvents the logistical and safety issues inherent with the radioactive label. Because it directly measures DNA proliferation, the assay has advantages over other colorimetric methods that may be strongly influenced by leucocyte metabolic status. Using BrdU incorporation followed by ELISA, we evaluate the responsiveness of rainbow trout (Oncorhynchus mykiss [Walbaum]) leucocytes to the mammalian T-cell mitogen Concanavalin A (Con A) as well as the differential response of white perch (Morone americana [Gmelin]) leucocytes to Con A and pokeweed mitogen. Specific considerations intrinsic to the assay system are discussed, including the implications of utilising enzyme-based detection.     

4-Thio-5-bromo-2'-deoxyuridine: chemical synthesis and therapeutic potential of UVA-induced DNA damage

4-Thio-5-bromo-2'-deoxyuridine (3a) is prepared from 5-bromo-2'-deoxyuridine (BrdU) and its key properties are explored. The thionucleoside (3a) can react readily with monobromobimane and produces high fluorescence. 3a has UV maximum absorption at 340 nm and can be incorporated into cellular DNA. The cells containing 3a become sensitive to UVA light, offering therapeutic potential for UVA-induced cell killing.     

Quantification of cell turnover kinetics using 5-bromo-2'-deoxyuridine

5-Bromo-2'-deoxyuridine (BrdU) is frequently used to measure the turnover of cell populations in vivo. However, due to a lack of detailed mathematical models that describe the uptake and loss of BrdU in dividing cell populations, assessments of cell turnover kinetics have been largely qualitative rather than quantitative. In this study, we develop a mathematical framework for the analysis of BrdU-labeling experiments. We derive analytical expressions for the fraction of labeled cells within cell populations that are growing, declining, or at equilibrium. Fitting the analytical functions to data allows us to quantify the rates of cell proliferation and cell loss, as well as the rate of cell input from a source. We illustrate this for the BrdU labeling of T lymphocytes of uninfected and SIV-infected rhesus macaques.     

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed ‘unscheduled DNA synthesis (UDS)’, is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.     

Anomalous neural differentiation induced by 5-bromo-2'-deoxyuridine during organogenesis in the rat

The influence of 5-bromo-2'-deoxyuridine (BrdU) on rat embryo development and neurogenesis was investigated using a rat conceptus culture system during organogenesis (pregnancy days 10-13). The embryos and visceral yolk sacs of conceptuses cultured with BrdU were examined for overall growth, morphological anomalies, incorporation of radiolabeled BrdU into DNA, and neurotransmitter enzyme activities in embryos. In addition, neural tubes from cultured whole embryos were isolated and mechanically dissociated into fragments and cultured again to assess neural cell differentiation into neuron-like cells. BrdU was found to incorporate differentially into embryonic and visceral yolk sac DNA with simultaneous stage-specific retardation and anomalous organogenesis in proportion to the increasing concentrations used. Neural tube differentiation of cultured embryos was markedly altered, and there were morphologically distinct neural anomalies. The neurite outgrowth from neuroblast cells (type 1) of explanted spinal neural tube fragments from BrdU-treated embryos was markedly reduced in length and number compared to those from similar areas of embryos grown without BrdU. In contrast, BrdU at the same doses did not affect differentiation of a number of neural tissue-related enzymes. These results indicate that BrdU incorporation into DNA of primordial embryonic cells significantly affects neurogenesis and differentiation of neurites from neuroblasts, which is a specific neural cytodifferentiation characteristic of neuronal cells.     

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.     

5-Ethynyl-2'-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells

The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [³H]thymidine or 5-bromo-2′-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via “click” chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2′-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells.     

Dual labeling with 5‐bromo‐2'‐deoxyuridine and 5‐ethynyl‐2'‐deoxyuridine for estimation of cell migration rate in the small intestinal epithelium

Small intestinal epithelium is a self‐renewing system in which the entire sequence of cell proliferation, differentiation, and removal is coupled to cell migration along the crypt‐villus axis. We examined whether dual labeling with different thymidine analogues, 5‐bromo‐2'‐deoxyuridine (BrdU) and 5‐ethynyl‐2'‐deoxyuridine (EdU), can be used to estimate cell migration rates on the villi of small intestines in rats. Rats received a single intraperitoneal injection of BrdU and EdU within a time interval, and signals in tissue sections were examined by immunohistochemistry and the “click” reaction, respectively. We successfully observed BrdU‐ and EdU‐positive cells on the epithelium with no cross‐reaction. In addition, we observed an almost complete overlapping of BrdU‐ and EdU‐positive cells in rats administered simultaneously with BrdU and EdU. By calculating the cell migration rate by dividing the distance between the median cell positions of the distribution of BrdU‐ and EdU‐positive cells by the time between the injection of BrdU and EdU, we estimated approximately 9 and 5 μm/h for the cell migration rates on the villi in the jejunum and ileum, respectively. We propose that dual labeling with BrdU and EdU within a time interval, followed by detecting with immunohistochemistry and the click reaction, respectively, is useful to estimate accurately the cell migration rate in the intestinal epithelium in a single animal.     

Suppression by 5-Bromo-2'-deoxyuridine of Transdifferentiation into Tracheary Elements of Isolated Mesophyll Cells of Zinnia elegans

5-Bromo-2'-deoxyuridine (BrdU), a thymidine analog, suppressedthe transdifferentiation into tracheary elements (TE) of isolatedmesophyll cells of Zinnia elegans without affecting cell division.Tracer experiments with [³H]BrdU indicated that 76% and 24%of the incorporated radioactivity was located in the DNA andthe RNA, respectively. Both thymidine and uridine counteractedthe inhibitory effect of BrdU on transdifferentiation but thymidinewas much more effective than uridine. These results suggestthat BrdU might interfere with transdifferentiation via itsincorporation into DNA. The timing of effective suppressionby BrdU was examined by monitoring the effects of the sequentialaddition of BrdU and thymidine with an interval of 12 h at varioustimes in culture. Transdifferentiation was most sensitive toBrdU between the 24th and the 36th hour of culture. This resultsuggests that this window of time is critical for DNA synthesisduring the transdifferentiation of isolated mesophyll cellsof Zinnia elegans into TE. ³Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, Mejiro, Tokyo,112 Japan     

Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.     

Comparison of cell proliferation index in equine and caprine embryos using a modified BrdU incorporation assay

The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min at 39 degrees C to permeabilise the capsule, and then embryos were rinsed in PBS containing 10% of foetal calf serum. After washing, embryos were immediately fixed in 2.5% paraformaldehyde with 0.3M NaOH during 15 min at ambient temperature. The S-phase was detected by immunocytochemistry technique. In caprine embryos, BrdU was visualised by the same technique but without the trypsin treatment. The percentage of cells (+/-S.E.M.) with BrdU incorporated into newly synthesised DNA strands was significantly higher in equine embryos (74+/-1) than in caprine (38+/-2). Our results demonstrated that BrdU incorporation assay can be used in equine embryos. This assay allows the determination of the proliferation index of live cells and could be used as an additional tool for evaluating the viability of embryos. The high percentage of cells incorporating BrdU during 1h of incubation with BrdU suggests that in comparison with the caprine embryos the cellular activity of proliferation is more intense in equine embryos and suggests that the cellular cycle is shorter in equine embryos.     

UVB-Induced formation of intrastrand cross-link products of DNA in MCF-7 cells treated with 5-bromo-2'-deoxyuridine

Nucleoside 5-bromo-2'-deoxyuridine (BrdU), after being incorporated into cellular DNA, is well-known to sensitize cells to ionizing radiation and UV irradiation. We reported here, for the first time, the sequence-dependent formation of intrastrand cross-link products from the UVB irradiation of BrdU-treated MCF-7 human breast cancer cells. Our results showed that BrdU replaced more than 30% dT in genomic DNA after the cells were treated with 10 microM BrdU for 48 h. LC-MS/MS data revealed that more than 50% of the incorporated BrdU was consumed during UVB irradiation, of which more than half was dehalogenated to yield dU. Low-dose (5.0 kJ/m2) UVB irradiation of BrdU-treated cells yielded four intrastrand cross-link products, where the C5 of uracil is covalently bonded to the C8 of its neighboring 5' or 3' guanine to give G[8-5]U and U[5-8]G, respectively, and the C5 of uracil could couple with the C2 or C8 of its vicinal 5' adenine to give A[2-5]U and A[8-5]U, respectively. All the above cross-link products except A[2-5]U could also be induced in BrdU-treated cells upon UVB irradiation at a dose of 39 kJ/m2. We further demonstrated, by using LC-MS/MS, that the yield of G[8-5]U was much greater than the total yields of A[2-5]U and A[8-5]U. In addition, our results revealed that BrdU treatment stimulated considerably the UVB-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in vivo. The formation of these intrastrand cross-link products and 8-oxo-dG in vivo underscores the importance of these products in the photosensitizing effect of BrdU.     

Most Anti-BrdU Antibodies React with 2′-Deoxy-5-Ethynyluridine — The Method for the Effective Suppression of This Cross-Reactivity

5-Bromo-2′-deoxyuridine (BrdU) and 2′-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.     

5-chloro-2'-deoxyuridine cytotoxicity results from base excision repair of uracil subsequent to thymidylate synthase inhibition

The lack of a phenotypic alteration of 5-hydroxymethyluracil (hmUra) DNA glycosylase (hmUDG) deficient Chinese hamster V79mut1 cells exposed to DNA-damaging agents known to produce hmUra has raised the question whether there might be DNA substrates other than hmUra for hmUDG. Based on the structural similarity between 5-chlorouracil (ClUra) and hmUra and the observations that 5-chloro-2'-deoxyuridine (CldUrd) induces base excision repair (BER) events, we asked whether hmUDG or some other DNA BER enzyme is responsible for the removal of ClUra from DNA. An in vivo flow cytometry assay with FITC-anti-BrdUrd (which cross-reacts with CldUrd) showed that exogenous CldUrd is incorporated into DNA. However, both in vivo and in vitro experiments indicated that ClUra is not excised from DNA by hmUDG or other DNA glycosylase activities. The absence of removal of ClUra by hmUDG raised the question whether DNA strand breaks occurred subsequent to thymidylate synthase inhibition, leading to deoxyuridine incorporation, followed by cleavage of uracil from DNA by uracil DNA glycosylase (UDG). An in vivo thymidylate synthase activity assay in V79 cells demonstrated that CldUrd treatment inhibits thymidylate synthase as effectively as 5-fluoro-2'-deoxyuridine (FdUrd) treatment. Uracil, a known UDG inhibitor, partially reverses the cytotoxic effects of CldUrd on V79 cells, thus confirming that CldUrd induced cytotoxicity is a result of UDG activity. Our results demonstrated that while CldUrd is not directly repaired from DNA, its cytotoxicity is directly due to the UDG removing uracil subsequent to inhibition of thymidylate synthase by CldUMP.     

Measurement of 5-fluoro-2'-deoxyuridine serum levels by gas chromatography chemical ionization mass spectrometry

Serum levels of 5-fluoro-2'-deoxyuridine in cancer treated patients were measured by gas chromatography mass spectrometry under chemical ionization conditions; 1-(2-deoxy-beta-D-lyxofuranosyl)-5-fluorouracil (3'-epi-5-fluoro-2'-deoxyuridine) was used as an internal standard. The drug and internal standard were quantitatively isolated from the serum sample by a mini-column anion exchange method and the extract permethylated using potassium-tert-butoxide in dimethylsulphoxide and methyl iodide. The derivatized nucleosides were then re-extracted from the reaction mixture and analysed on a glass capillary column coated with Superox-4. The column was coupled directly to the chemical ionization source of the mass spectrometer; NH3 was used as the reagent gas. The gas chromatographic effluent was monitored at m/z 289, the [MH]+ ion of the N,O-permethyl derivatives of 5-fluoro-2'-deoxyuridine and the internal standard. Recovery of 5-fluoro-2'-deoxyuridine from serum in the 0-1 microgram ml-1 concentration range averaged 93 +/- 2% (SD); a linear detector response was observed up to 50 ng 5-fluoro-2'-deoxyuridine ml-1. At the 10 ng ml-1 level, a within-run assay precision of 10% (CV) (n = 5) was found, while a detection limit of about 1 ng 5-fluoro-2'-deoxyuridine ml-1 of serum was attained. The method was applied to the measurement of disappearance curves of 5-fluoro-2'-deoxyuridine in the serum of treated patients.     

Subnuclear localization,rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield,confocal and super-resolution microscopy

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2′-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.     

A broad replication origin of Drosophila melanogaster, oriD α, consists of AT-rich multiple discrete initiation sites

This work shows that the replication origin of Drosophila melanogaster, oriDalpha, consists of multiple discrete initiation sites. We attempted to map at high resolution the initiation sites in oriDalpha with a quantitative nascent DNA abundance assay using a competitive polymerase chain reaction (PCR) method. Nascent DNA was prepared from either cells blocked in very early S-phase and then labeled with 5-bromo-2'-deoxyuridine (BrdU), or asynchronously growing cells labeled briefly with BrdU. Denatured DNA was size-fractionated in alkaline sucrose gradients. BrdU-labeled nascent DNA was immuno-affinity purified using anti-BrdU antibodies. DNA was quantified with a competitive PCR method before and after immuno-purification. The results indicated that oriDalpha, whose size was presumed to be about 10 kb from two-dimensional gel electrophoretic analysis, contained four major initiation sites in its central 2.8 kb region, and six to approximately eight sites in 8.4 kb. All initiation sites corresponded with AT-rich sequences. Detailed analysis of one major initiation site indicated that its range was restricted to 700 bp.