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.     

Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine

The replacement of thymidine with 5-bromo-2′-deoxyuridine (^BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a ^BrdU or its closely related 5-bromo-2′-deoxycytidine (^BrdC). Our results showed that two types of crosslink products could be induced from d(^BrCG), d(^BrUG), d(G^BrU), or d(A^BrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5′ flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5′ neighboring nucleoside was a dG than while it was a dA, and ^BrdC induced the formation of crosslink products much more efficiently than ^BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.     

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.     

Continuous measurement and analysis of staining kinetics by flow cytometry

The measurement of color development with time in cells following the start of a staining reaction is of interest in a number of biological systems. These include the subsets of peripheral white blood cells after acridine orange staining, the uptake by cells and nuclei of fluorescent agents, especially antitumor drugs, and measurement of intracellular enzyme kinetics using fluorogenic or absorbing substrates. The present work describes a simple computer program for analyzing flow cytometric (FCM) data versus time, including both the population kinetics of color development and the variability of staining speed within one population of cells. A single-channel absorption measurement in flow (Technicon Hemalog D) was used to record peroxidase kinetics in peripheral blood cells. Every 5 s, a 64-channel absorption histogram was recorded, up to a maximum of 64 histograms. The data were then analyzed by a computer program which searched for the peak channel of each histogram. A least-squares fit was computed for these maxima. The asymmetries of the 64 absorption histograms were compared to see if there was more than one population present with different time constants. Although developed for enzyme kinetic measurements, this program may have wider usefulness in any measurements of time-dependent phenomena by FCM.     

Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis.

The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases. CF3dUrd was incorporated into the DNA of mammalian cells in culture. We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis. Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide. After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol. alpha). When CF3dUrd residue was located at an internucleotide site in the template, however, pol. alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue. These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.     

Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.     

Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining

Abstract. The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.     

Measurement of DNA damage in mammalian cells using flow cytometry

A technique for the detection of DNA damage induced by radiation insult has been developed. Cells were lysed with a buffer containing 2 M sodium chloride to release the DNA in a supercoiled form, the nucleoid. These were stained with the DNA intercalating dye, ethidium bromide, and exposed to laser light within a flow cytometer. Scattered and fluorescent light was analyzed from the laser/nucleoid interaction following irradiation of viable cells with gamma rays. The addition of ethidium bromide to prepared nucleoids caused a reduction in scattered light due to condensation of the nucleoid. Irradiation of cells prior to nucleoid production and ethidium bromide treatment restricted this condensation and produced a dose-dependent increase in laser scatter. Nucleoids derived from human lymphocytes showed enhanced light scatter from 5 Gy, compared to Chinese hamster ovary (CHO) fibroblasts where doses above 10 Gy were required. Up to 30 Gy CHO nucleoids showed a dose-dependent reduction in the ethidium bromide fluorescence. This technique allows detection of altered light scattering and fluorescent behavior of nucleoids after cellular irradiation; these may be related to structural changes within the nucleus induced by the radiation. The use of flow cytometry compared to other methods allows a rapid analysis of nuclear damage within individual cells.     

Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining

The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.     

Cytogenetic effect of the radiosensitizing action of 5-bromo-2'-deoxyuridine on eukaryotic cells at various periods of the DNA synthesis phase

The effect of 5-bromo-2'-deoxyuridine (BUDR) radiosensitizing action on meristematic cells of wheat seedlings and human peripheral blood lymphocytes synchronized with 5-fluoro-2'-deoxyuridine at the G1-S boundary was revealed. This effect was only found in the beginning of the S phase. We show that it was conditioned by incorporation of BUDR into special early replicating DNA sequences which are specific for chromosomal mutagenesis. Using the molecular biology methods non-random distribution of 3H-BUDR in the early replicating wheat DNA sequences with different degree of repeating was found.     

Changes in the synthesis of minor cartilage collagens after growth of chick chondrocytes in 5-bromo-2'-deoxyuridine or to senescence

Analyses were made of the minor collagens synthesized by cultures of chondrocytes derived from 14-day chick embryo sterna. Comparisons were made between control cultures, cultures grown for 9 days in 5-bromo-2'-deoxyuridine (BrdU) and clones of chondrocytes grown to senescence. Separation of minor collagens from interstitial collagens was achieved by differential salt precipitation in the presence of carrier collagens in acid conditions. The precipitate at 0.9 M NaCl 0.5 M acetic acid from control cultures was shown by CNBr peptide analysis to contain only the alpha 1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only alpha 1(I) and alpha 2(I) chains occurred. The synthesis of type III collagen was not detected. Analysis of the precipitate at 2.0 M NaCl, 0.5 M HAc from control cultures demonstrated the synthesis of 1 alpha, 2 alpha and 3 alpha chains together with the synthesis of short chain (SC) collagen of Mr 43000 after pepsin digestion. After BrdU treatment or growth to senescence alpha chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the alpha 1(V) and alpha 2(V) chains of type V collagen. In addition, for BrdU-treated but not for control cultures, intracellular immunofluorescent staining was observed with a monoclonal antibody which specifically recognizes an epitope present in the triple helix of type V collagen. Synthesis of short chain (SC) collagen was not detected after BrdU treatment or growth to senescence. These results suggest that chick chondrocytes grown in conditions known to cause switching of collagen synthesis from type II to type I collagen also undergo a switch from the synthesis of 1 alpha, 2 alpha and 3 alpha chains to the synthesis of the alpha 1(V) and alpha 2(V) chains of type V collagen. It appears that there are several cartilage-specific collagens which together undergo a regulatory control to the synthesis of collagens typical of other connective tissues.     

Rate of DNA synthesis determined by flow cytometry using the BrdUrd/Hoechst technique in combination with propidium-iodide staining

Concanavalin A (conA) and phytohemagglutinin (PHA), at relatively high concentrations, induce spreading of human T lymphocytes on adhesive surfaces. After 24–48 h of mitogen stimulation of such lymphocytes in suspension, approx. 50% of the cells had acquired the capacity to develop prominent substrate-attached actin-containing projections with a length of 1–7 μm when subsequently induced to spread on a surface. In addition, cells stimulated with mitogen when in continuous contact with a surface developed similar projections after the same stimulation period. The spreading of lymphocytes was accompanied by a disappearance of the microvilli with a length of 0.2–0.9 μm present in large numbers on activated cells in suspension. Thus, on the basis both of their size as well as on the presence in relation to substrate contact, these microvilli and the substrate-attached projections are separate structures. Acquisition of the capacity to form projections after substrate contact was dependent on protein synthesis during the stimulation period and not detectable until 10–18 h after starting the stimulation. Control experiments indicated that the inhibiting of projection formation by inhibitors of protein synthesis was not due to a toxic effect, since the presence of these inhibitors did not prevent the formation of actin-containing projections in cells that had acquired the capacity to form such projections. T-enriched lymphocytes did not develop substrate-attached projections during continuous adhesion to a surface mediated by the non-mitogenic ligands poly-l-lysine and wheat germ lectin. Nor did cells cultured under these conditions develop prominent projections when subsequently transferred to another substrate and induced to spread in the presence of conA.     

The use of antibodies to 5-bromo-2'-deoxyuridine for the isolation of DNA sequences containing excision-repair sites

We have developed an immunological method for isolation and identification of DNA sequences containing 5-bromo-2'-deoxyuridine (BrdUrd) incorporated during UV-induced excision-repair synthesis. DNA fragments containing BrdUrd incorporated during repair synthesis were incubated with goat anti-BrdUrd and rabbit anti-goat IgG, and the antibody-DNA complexes were separated from bulk DNA by nitrocellulose filter binding. With this method, 80% of DNA sequences containing BrdUrd-labeled excision-repair sites were recovered, contaminated with less than 1% of DNA fragments devoid of excision-repair sites. Recovery of DNA fragments containing repair sites was independent of size from 2 to 20 kilobases. We have used this method in conjunction with blot hybridization to demonstrate that repair synthesis occurs in human ribosomal gene sequences in cells treated with UV.     

Alteration of the Cloudman melanoma cell cycle by prostaglandins E1 and E2 determined by using a 5-bromo-2'-deoxyuridine method of DNA analysis

Prostaglandins (PGs) E1 and E2 stimulate tyrosinase activity and suppress the proliferation of Cloudman S91 melanoma cells by altering their progression through the cell cycle. Prostaglandin E1 and PGE2 have prolonged or residual effects on melanoma cells. Cells treated for 5 or 24 hours with 10 micrograms/ml PGE1 or cells treated for 8 or 24 hours with 10 micrograms/ml PGE2 demonstrated decreased proliferation and increased tyrosinase activity for 48 hours after removal of the PGs. The effects of PGs on the cell cycle were investigated by determining total DNA content in cells stained with propidium iodide (PI) and analyzed by a fluorescence activated cell sorter (FACS). Prostaglandin E1 blocked cells in G2 phase after 5 hours of treatment, corresponding to when inhibition of proliferation was first evident. Similarly, after 9 hours of treatment with PGE2, more cells were in late S, early G2 phase and less in G1 than their control counterparts. Also, melanoma cells were pulse-labeled with 5-bromo-2'-deoxyuridine (BrdUrd) prior to or at the end of PG treatment and then stained with a fluoresceinated monoclonal antibody to BrdUrd, and with PI. This allows one to observe how BrdUrd-labeled S-phase cells cycle with time. Both PGE1 and PGE2 inhibit proliferation by blocking cells in G2 phase of the cell cycle. The PG-induced block in G2 may be required by melanoma cells to synthesize mRNA and proteins that are essential for stimulation of tyrosinase activity. Ultrastructurally, only a subpopulation of the cells treated with PGE1 or PGE2 contained more mature melanosomes than control cells.     

The demonstration of proliferating cells by using monoclonal antibodies to 5-bromo-2'-deoxyuridine in the peroxidase-antiperoxidase method

Monoclonal antibodies (MAb) to the thymidine analog 5-bromo-2'-deoxyuridine (BrdUrd) and to horseradish peroxidase (HRPO) have been produced and characterized. On the basis of Mab to HRPO, complexes of the antibodies and HRPO (PAP-complexes) for immunochemical investigations are prepared. The possibility to identify proliferating cells in cultures of mouse myeloma Sp2/0 and mouse fibroblasts NIH 3T3 using Mab to BrdURd by the PAP-method is shown. The conditions of performing the analysis were optimized. The effect of various techniques ot cell fixation and cell DNA denaturation on cell morphology and on specific staining of nuclei of BrdUrd-containing cells in investigated.     

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.