Concentrations Of BUdR Research Articles

Decrease in TTP pools mediated by 5-bromo-2'-deoxyuridine exposure in a human glioblastoma cell line

The antitumor and radiosensitizing properties of 5-bromo-2'-deoxyuridine (BUdR) appear to be due, in part, to its incorporation into cellular DNA. To optimize conditions for incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP) into DNA, we investigated the metabolism of BUdR to its DNA precursor form, the 5'-triphosphate BrdUTP, in the U251 human glioblastoma cell line. The results demonstrated that BrdUTP accumulated rapidly in this cell line, achieving steadystate values within 2 hr of drug addition. The level of BrdUTP accumulation was proportional to the amount of exogenous BUdR up to a concentration of 100 μM, without apparent saturation. Exposure of glioblastoma cells to BUdR was associated with substantial selective decreases in both the cellular dCTP and TTP pools, the extent of which was dependent on the exogenous BUdR concentration. In the absence of exogenous BUdR, BrdUTP was eliminated rapidly from cells with an initial half-life of approximately 15 min. As the cellular BrdUTP level declined, the dCTP and TTP levels increased to control values. Incorporation of BrdUMP into DNA appeared linear with time as long as the cellular BrdUTP level remained constant. This incorporation was not enhanced by the addition of 5-fluoro-2'-deoxyuridine (FUdR), a potent inhibitor of thymidylate synthetase, which at a concentration of 10 nM had no effect on TTP pools in this cell line. Thus, the decrease in cellular TTP pools mediated by BrdUTP allows the halogenated pyrimidine to enhance its own incorporation into DNA.

Dynamics of bromodeoxyuridine incorporation into DNA of squamous carcinoma cells during mid and late logarithmic growth.

Head and neck squamous carcinoma cell lines, UM-SCC 1, 5, 9, 11B, and 14B, were exposed in vitro to bromodeoxyuridine (BUdR) during logarithmic growth to determine the effects of drug concentration (0.01 to 10 microM) and duration of exposure (3, 7, and 10 days) on cell growth and on incorporation of BUdR into DNA. Concentrations of less than 1.0 microM were not growth inhibitory except with UM-SCC-11B. After 10 days of exposure to 5 microM BUdR, survival fractions for all lines ranged from 2 to 65% of controls. Replacement of thymidine by BUdR in DNA was assessed by gas chromatography/mass spectroscopy. Percentage replacement (% R) was described by the equation % R = 100 (C/t)s/[(C/t)50s + (C/t)s], where C is the concentration of BUdR (microM), t is the time in days, s is a constant, and (C/t)50 is a constant corresponding to % R = 50%. BUdR incorporation reached a time- and concentration-dependent maximum that, after 3 to 7 days of culture in 10 microM BUdR, ranged from 30 to 60% R. Subsequently, % R declined with time even though the cells were fed daily with fresh BUdR-containing medium.

Improved high-performance liquid chromatographic assay for the quantification of 5-bromo-2′-deoxyuridine and 5-bromouracil in plasma

A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2′-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane—methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 μ M, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).

Effects of 5-bromo-2-deoxyuridine concentration and α-naphthoflavone on the association between smoking and the frequency of sister-chromatid exchanges in lymphocytes from maternal and cord blood

The frequency of sister-chromatid exchanges was analyzed in maternal and cord blood lymphocytes obtained at delivery from 23 nonsmokers and 21 smokers. Lymphocytes were cultured under 3 conditions: in the presence of 100 μM 5-bromo-2-deoxyuridine (BUdR), 20 μM BUdR and 20 μM BUdR with 40 μM α-naphthoflavone (ANF). Under all assay conditions, frequencies of SCEs were consistently higher for maternal lymphocytes than for cord lymphocytes. There was no association between SCE values for cultures of the same blood specimen with 100 μM BUdR and 20 μM BUdR. When cultured with 100 μM BUdR, maternal lymphocytes from smokers had a mean SCE frequency of 13.5, which was significantly higher than the value of 11.1 observed for nonsmokers ( p = 0.001 by the Wilcoxon rank sum test). Maternal smoking had no significant effect on overall frequencies of SCEs in maternal blood cultured with 20 μM BUdR either with or without ANF or when the differential between cells cultured with and without ANF was considered. Use of caffeinated beverages was associated with increased SCE values for maternal lymphocytes cultured with 20 μM BUdR (Tau β = 0.36, p = 0.02 for the Kendall's Rank Correlation), but no such association was seen with 100 μM BUdR. For cord blood lymphocytes, however, neither smoking nor caffeine use were associated with SCE values obtained by any of the assay conditions used. The findings suggest that results of human monitoring studies using SCEs could differ depending on the concentration of BUdR used in cultures.

Sister Chromatid Exchange in Garlic (Allium SativumL.) Meristem Root-Tip Cells

SUMMARYDifferential staining of sister chromatids by the fluorescent plus Giemsa (FPG) technique was performed in Allium sativum L. chromosomes unifilarly substituted with BudR. For this purpose optimal concentrations of FudR, BudR and Thd were determined and their influence was studied on cell cycle kinetics. The distribution of sister chromatid exchanges (SCEs) per chromosome was in good agreement with the expected Poisson distribution. There were statistically significant differences in mean SCEs frequencies per cell among respective plants tested. The mean frequency recorded was 99.78 SCEs per cell.

Simultaneous determination of thymine and 5-bromouracil in DNA hydrolysates using gas chromatography—mass spectrometry with selected-ion monitoring

The gas chromatographic—mass spectrometric method using selected-ion monitoring (GC—MS—SIM) described here quantitatively determines the amount of DNA thymine replacement by 5-bromouracil (BU) after exposure to 5-bromo-2′-deoxyuridine (BUDR) in as few as 10 5 cells. DNA is extracted, enzymatically hydrolyzed, the nucleic acid bases (with added internal standards, 5-iodouracil and 5-chlorouracil) are extracted into ethyl acetate, concentrated and derivatized with bis(trimethylsilyl)trifluoroacetamide. Thymine and BU are then quantitated by GC—MS—SIM. Response is linear to thymine over the range of 100–2000 ng per sample and BU of 1.3–52 ng per sample with a coefficient of variation of less than 10% and an accuracy for seeded samples within 8% of theoretical value. With V79 cells in culture, exposure to increasing BUDR concentrations (0.03– 1.0 μ M) results in increasing thymine substitution by BU over a range of 1–28%. Other important applications of this technique are mentioned.

The role of feeding rejection in Drosophila mutation assays

The frequencies of sex-linked recessive lethal mutations recovered in the male post-meiotic germ cells of Drosophila after feeding on solutions containing a mutagen (either 1 mM methyl methanesulfonate, MMS or 2 mM ethyl methanesulfonate, EMS) and 5-bromo-2-deoxyuridine (BUdR) were significantly lower than the frequencies observed after treatment with the mutagen alone. In an attempt to explain the apparent ‘protective’ effect of BUdR, the feeding behavior of the flies was monitored for differences in the uptake of the mutagen-containing solution in the presence and absence of BUdR. This was accomplished by measuring the uptake of [ 14C]sucrose. The results indicated that the uptake of the feeding solution is inhibited by the presence of the selected concentration of BUdR (1.0 or 32.5 mM). Such a reduced uptake of the mutagen could alone account for the reduction in mutational yields noticed in treatments containing mutagen + BudR compared to the ones with the mutagen alone. These results emphasize the need to monitor the feeding behavior of flies in experiments involving adult feeding.

The amplified expression of factors regulating myogenesis in L6 myoblasts.

A strategy for increasing the expression of the factors regulating myogenesis was developed based upon the observation that increased amounts of regulatory factors could overcome the inhibition of differentiation produced by 5-bromodeoxyuridine (BUdR). L6 rat myoblasts were subjected to multiple cycles of cloning in progressively increasing concentrations of BUdR. The first clones to differentiate were picked and replated for the next cycle of selection. After 28 cycles in BUdR, cells were isolated that could differentiate in the presence of 8 microM BUdR. Cell hybrids between myoblasts subjected to 21 cycles of selection (BU21 cells) and differentiation-defective myoblasts exhibited a high probability of differentiation, consistent with the hypothesis that BU21 cells were overproducing factor(s) involved in the decision to differentiate. The selection of cells able to differentiate in the presence of BUdR may provide a general approach for increasing the expression of the regulatory molecules controlling terminal differentiation.

An improved micronuclear assay in lymphocytes

We have found that by increasing the bromodeoxyuridine (BUdR) concentration to 4 × 10 −4 M in cultures and using the conventional harlequin staining procedure we can distinguish the proliferating lymphocytes by their blue color from the non-proliferating cells which have red nuclei. The micronuclei (MN) are also blue and are associated with the proliferating cells. The high BUdR concentration does not appear to affect the yield of MN. By scoring the MN only in the (blue) proliferating cells the MN assay is made less sensitive to perturbations in the lymphocyte cultures caused by radiation or by variations in the culture time on PHA concentration. Moreover, the frequency of cells with MN approximately doubles and thus comes into better agreement with the frequency of cells with chromosome aberrations. A better agreement yet can be achieved by correcting the assay for the effect of cell division.

Radiosensitization of hematopoietic precursor cells (CFUc) in glioblastoma patients receiving intermittent intravenous infusions of bromodeoxyuridine (BUdR)

The potential use of bromodeoxyuridine (BUdR) as a radiosensitizer given by an intermittent intravenous route is being studied in a Phase I/II trial at the National Cancer Institute. In order to assess the extent of radiosensitization, we have studied the radiation response of human bone marrow cells CFUc taken from 6 patients prior to and after a 14-day infusion of BUdR. Varying concentrations (1000–1500 mg) of BUdR were infused for 12 hours every 24 hours for up to 14 consecutive days. Cell survival was determined by colony formation of CFUc in soft agar suspensions. X ray survival curves were generated over a dose range of 0–300 rad and the slopes of the survival curves (D 0) before and after BUdR infusion were compared. Radiation enhancement ratios (ER) (D 0 pre-BUdR/D 0 post-BUdR) ranged from 1.0–2.2 and appeared to be BUdR dose dependent. Above 650 mg/m 2 the radiation ER was ≥ 1.5. Dose dependent systemic toxicity to bone marrow and skin was also observed with intermittent intravenous infusions of BUdR. From our study, it appears that an intravenous dose of <700 mg/m 2/12 hours is well tolerated and may result in radiosensitization of CFUc in man.

Spontaneous sister-chromatid exchanges in Chinese hamster cells in vivo and in vitro

The incidence of sister-chromatid exchanges (SCEs) was studied in Chinese hamster cells. The cells used were an established cell line (Don), an aneuploid secondary culture still exhibiting contact inhibition of growth, a primary culture, bone-marrow cells in vivo, a Don-derived clone having ring chromosomes, and endoreduplicated Don cells. The frequency of SCEs in the presence of bromodeoxyuridine (BUdR) increased in the following order: bone-marrow cells < Don < secondary culture cells < primary culture cells. Marked increases in BUdR concentration induced only slight increases of SCEs. Some ring chromosomes showed Moebius strip and concatenated ring structures, indicating spontaneous occurrence of SCEs in the absence of BUdR. The incidence of spontaneous SCEs observed in rign chromosomes approximates that of SCEs observed in ordinary rod chromosomes in the presence of low dose levels of BUdR. SCEs occurring in the first and second cell cycles were separately counted in endoreduplicated cells. The ratio of single SCEs, which occurred in the second cell cycle, to twin SCEs, which occurred in the first cell cycle, was about 2, when the count of single SCEs was corrected for the induction effect. This implies that uninemy chromatids retain the polarity of DNA when SCEs occur, that bifilarly and trifilarly BUdR-substituted DNA strands give equal numbers of SCEs and that incidence of SCEs is independent of the length of S in the Don cells used here.

Induction of sister chromatid exchanges by BUdR is largely independent of the BUdR content of DNA.

The halogenated thymidine (dT) analogue, 5-bromodeoxy-uridine (BUdR), has a variety of effects on mammalian cells, including toxicity, suppression of differentiation, and mutagenesis. Although it is generally assumed that the effects of BUdR are due primarily to its presence in DNA, results from our laboratory have raised doubts about such assumptions. We have shown, for example, that BUdR mutagenesis in mammalian cells is determined by the concentration of BUdR in the medium rather than in DNA, and that mutagenesis can be suppressed by deoxycytidine (dC) without changing the amount of BUdR in DNA. BUdR has also been shown to induce sister chromatid exchanges (SCEs) in mammalian cells. Initial results suggested that the relationship between BUdR and SCEs might not be explained by a single factor, and various correlations between BUdR and SCEs have been proposed. However, the results to date have been inconclusive, because the experiments did not resolve as independent variables the concentration of BUdR in the medium and the amount of BUdR incorporated into nuclear DNA. We have now carried out experiments to resolve these two factors; the results indicate that the major factor in determining the frequency of SCEs is the concentration of BUdR in the medium.

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml. Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR. To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml. On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.

Reversible inhibition of somatic embryo differentiation by bromodeoxyuridine in cultured cells of Daucus carota L.

Somatic embryogenesis induced in suspension cultures of carrot ( Daucus carota) was inhibited by BUdR at concentrations which do not influence the rate of RNA or protein synthesis, or the growth of the cells. At the time of embryo induction 2 days treatment with 6 μM BUdR was sufficient for the complete supression of differentiation. The degree of inhibition showed a dependence on the applied concentration of BUdR, as well as on the level of BUdR substitution in DNA. The BUdR inhibition proved to be reversible and the embryogenic capacity of the carrot cells was restored after removel of the analogue. Addition of thymidine prevented the inhibitory effect of BUdR by decreasing the incorporation of BUdR into DNA. The experiments reported here indicate that the effect of BUdR on the somatic embryo differentiation was probably associated with BUdR substitution of thymidine in the DNA.

Lack of spontaneous sister chromatid exchanges in somatic cells of Drosophila melanogaster.

Neural ganglia of wild type third-instar larvae of Drosophila melanogaster were incubated for 13 hours at various concentrations of BUdR (1, 3, 9, 27 micrograms/ml). Metaphases were collected with colchicine, stained with Hoechst 33258, and scored under a fluorescence microscope. Metaphases in which the sister chromatids were clearly differentiated were scored for the presence of sister-chromatid exchanges (SCEs). At the lowest concentration of BUdR (1 microgram/ml), no SCEs were observed in either male or female neuroblasts. The SCEs were found at the higher concentrations of BUdR (3, 9, And 27 micrograms/ml) and with a greater frequency in females than in males. Therefore SCEs are not a spontaneous phenomenon in D. melanogaster, but are induced by BUdR incorporated in the DNA. A striking nonrandomness was found in the distribution of SCEs along the chromosomes. More than a third of the SCEs were clustered in the junctions between euchromatin and heterochromatin. The remaining SCEs were preferentially localized within the heterochromatic regions of the X chromosome and the autosomes and primarily on the entirely heterochromatic Y chromosome.--In order to find an alternative way of measuring the frequency of SCEs in the Drosophila neuroblasts, the occurrence of double dicentric rings was studied in two stocks carrying monocentric ring-X chromosomes. One ring chromosome, C(1)TR94--2, shows a rate of dicentric ring formation corresponding to the frequency of SCEs observed in the BUdR-labelled rod chromosomes. The other ring studied, R(1)2, exhibits a frequency of SCEs higher than that observed with both C(1) TR94--2 and rod chromosomes.

Requirements for RNA and protein synthesis in the induction of several differentiation‐markers in a myeloid leukemia cell line

In a myeloid leukemia cell line, the inducibilities of the Fc receptor, phagocytosis and cell motility were compared. Thymidine analogues such as BUdR, BCdR and IUdR blocked the induction of phagocytosis and motility but not induction of the Fc receptor. This BUdR susceptibility in the induction of phagocytosis and motility was lost in a BUdR resistant line which was isolated for its growth capability in a high concentration of BUdR. Actinomycin D and puromycin brought about a marked decrease in the inducibility of phagocytosis but not in that of the Fc receptor. This led us to the following conclusion: There is a genetic control in the inducibility of phagocytosis and motility in this cell line, and the incorporation of BUdR into cellular DNA results in the DNA becoming unresponsive to a differentiation-stimulating factor. In contrast, gene activation does not seem to be necessary for induction of the Fc receptor. The order of induction of several differentiation markers was also discussed.

Effects of BUdR concentration on the incidence of sister chromatid exchanges in cultured lymphocytes from patients with Down&apos;s syndrome

Effects of BUdR concentration on the incidence of sister chromatid exchanges in cultured lymphocytes from patients with Down&apos;s syndrome

Bromodeoxyuridine mutagenesis in mammalian cells is stimulated by thymidine and suppressed by deoxycytidine.

THE most widely accepted explanation for the mutagenicity of 5-bromodeoxyuridine (BUdR) is that mutations occur as the result of occasional mispairing of the analogue during DNA replication1,2, although the correlation between mutagenesis and the amount of bromouracil (BU) incorporated into DNA has been questioned3,4. We have previously shown that BUdR mutagenesis in Syrian hamster cells (measured by the induction of ouabain-resistant (OBr) or 6-thioguanine-resistant (TGr) mutants) is determined by the concentration of BUdR in the medium rather than the amount of BU in DNA5. This suggested that BUdR nucleotides are involved in BUdR mutagenicity in mammalian cells5 (see also ref. 4). BUdR triphosphate (BUdRTP) inhibits the ribonucleotide reductase-catalysed reduction of cytidine diphosphate to deoxycytidine (CdR) diphosphate6, as does thymidine (TdR) triphosphate (dTTP)7,8. Therefore, we have investigated the effects of CdR and TdR on BUdR mutagenesis, and report here that the former suppresses and the latter stimulates this process.

Replication of Mouse Cytomegalovirus in Thymidine Kinase‐Deficient Mouse Cells

AbstractIn an attempt to determine whether mouse cytomegalovirus (MCMV) requires thymidine kinase (TK) for replication and whether it induces TK, TK‐deficient mouse cells were isolated and used as host cells for MCMV. Mutant cells resistant to 200 μg/ml of 5‐Bromodeoxyuridine (BUdR) were selected from SV40‐transformed mouse cells, mks‐A TU‐7, by propagating the cells in the presence of varying concentrations of BUdR graded by serial 2‐fold increments. The mutant cells, designated as TU‐7 BU, showed a very low TK activity (less than 1/20 that of mks‐A TU‐7). Herpes simplex virus type 1 (HSV‐1) replicated in starved as well as in unstarved TU‐7 BU, whereas MCMV could replicate only in growing TU‐7 BU and could not form plaques in monolayers of mks‐A TU‐7 or TU‐7 BU. HSV‐1 infection enhanced TK activity equally in both mks‐A TU‐7 and TU‐7 BU. In contrast, TK activity of MCMV‐infected mks‐A TU‐7 was lower than that of uninfected cells or cells inoculated with UV‐inactivated virus. In addition, TK activity of the MCMV‐infected TU‐7 BU remained minimal without showing any increase. The replication of HSV‐1 was completely inhibited in the presence of BUdR (10 μg/ml), whereas MCMV could replicate even in the presence of 50 μg/ml of BUdR. The results indicate that MCMV neither requires TK nor induces TK activity in the infected cells.

Direct evidence for the role of incorporated BUdR in the induction of sister chromatid exchanges

Abstract The level of bromodeoxyuridine (BUdR) substitution for thymidine (dThd) in nascent DNA of Chinese hamster cells increases rapidly with increasing BUdR concentration at low BUdR/base pair (BU/bp) ratios and then increases much more slowly at higher ratios. The relationship between percentage substitution and BU/bp indicates that BUdR competes with dThd for incorporation into DNA and that BUdR concentration has no effect on the effective dThd/base pair ratio in these cells. Equal amounts of BUdR are incorporated during both the first and second cell cycles (S phases) after provision of the dThd analog. Incorporation of BUdR induces sister chromatid exchanges (SCEs), and induction appears to occur during the cell cycle (S phase) in which incorporation occurs, with incorporated BUdR from previous cycles having little effect. BUdR-induced SCE frequencies are linearly proportional to the percentage substitution of BUdR for dThd. Extrapolation to zero substitution indicates that SCEs occur spontaneously at a frequency of 0.045 per chromosome per cycle, and that many so-called “spontaneous” SCEs are actually induced by BUdR.