Effect Of 5-bromodeoxyuridine Research Articles

Binding of 5-bromouracil-containing S/MAR DNA to the nuclear matrix.

Substitution of thymine with 5-bromouracil in DNA is known to change interaction between DNA and proteins, thereby inducing various biological phenomena. We hypothesize that A/T-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are involved in the effects of 5-bromodeoxyuridine. We examined an interaction between DNA containing an intronic S/MAR sequence of the immunoglobulin heavy chain gene and nuclear halos prepared from HeLa cells. Upon substitution with 5-bromouracil, the S/MAR DNA bound more tightly to the nuclear halos. The multi-functional nuclear matrix protein YY1 was also found to bind more strongly to 5-bromouracil-substituted DNA containing its recognition motif. These results are consistent with the above hypothesis.

The effect of 5-bromodeoxyuridine on mouse embryos during neurulation in vitro.

Mouse embryos explanted at various stages during neurulation were cultured for 20-28 h in the presence of 25-900 micrograms/ml of 5-bromodeoxyuridine (BUdR). BUdR strongly inhibited closure of the cranial neural tube, which was found to be stage-dependent. When mouse embryos were exposed to BUdR after development of the concave curvature in the neuroepithelium of the midbrain to the upper hindbrain regions, they became insensitive to the drug-induced open cranial neural tube. Histological observations showed that BUdR interfered with interkinetic migration and cytokinesis of the neuroepithelial cells. These cellular abnormalities were not dependent on the morphological development of the cranial neural folds. The 3H-BUdR experiment confirmed that the label was mostly incorporated into the DNA fraction.

Effect of 5-bromodeoxyuridine on the appearance of the liver isoenzyme of pyruvate kinase.

Hepatocyte cultures derived from 15-day foetal rats produce the liver form of pyruvate kinase (EC 2.7.1.40) only after 3 days of culture. The appearance of the liver form of the enzyme can be blocked by the addition of 5-bromodeoxyuridine on day 2 of culture, but not by addition on day 3 of culture. The reversibility of the action of 5-bromodeoxyuridine was shown when the inhibitor was added on day 2 and removed on day 4. By day 6 of culture the liver form of pyruvate kinase was detectable. The specificity of the action of 5-bromodeoxyuridine was monitored by following changes in the closely related embryonic form of the enzyme as a control. This was unaltered by the inhibitor.

Effect of 5-bromodeoxyuridine on pupariation and adult differentiation in the flesh fly, Sarcophaga bullata

Two stages of postfeeding, third-instar larvae of Sarcophaga bullata were injected with 5-bromodeoxyuridine to examine the effect of the analogue on pupariation and adult differentiation. While pupariation was inhibited in prered spiracle-stage larvae, adult differentiation was unaffected in both stages as revealed by in vivo culture of imaginal discs. It is suggested BudR may be more effective in disturbing certain biochemical processes than in inhibiting morphogenesis.

Frequency of sister-chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA

Chinese hamster D-6 cells were grown for two cell cycles. The effect of 5-bromodeoxyuridine (BrdU) on the frequencies of sister-chromatid exchanges (SCEs) in these cells was investigated by the BrdU-labeling method. A low concentration (5 μM) of BrdU was inoculated in the first cell cycle for SCE counting. When excess concentrations (100–1000 μM) of BrdU were added subsequently in the second cell cycle, a 1–2-fold increase of SCE frequencies was observed. When excess thymidine (dT) (100–1000 μM) was supplied instead of BrdU, the incidence of SCE also increased. When cells were exposed to high concentrations (50–200 μM) of BrdU in the first cell cycle, a 1–4-fold increase in SCE frequencies was observed. This incidence of SCE was largely dependent on the concentration of BrdU and dT used in the second cell cycle. These results suggest that efficient SCE induction by BrdU is related to the BrdU residue incorporated into parental DNA strands.

Bromodeoxyuridine-induced molecular species conversion of sialic acids of gangliosides and the alteration of cellular phenotypic expression in B16 mouse melanoma cells.

Gangliosides of B16 mouse melanoma cells were characterized and the effects of 5-bromodeoxyuridine (BrdU) on gangliosides were examined in relation to the alteration of cellular phenotypic expression. In melanotic cells, gangliosides were shown to be composed of both hematosides containing N-acetylneuraminic acid (NeuAc-hematoside) and N-glycolylneuraminic acid (NeuGc-hematoside) as determined by thin-layer chromatography, gas chromatography-mass spectrometry and the use of anti-NeuGc-hematoside antiserum. Gangliosides other than hematosides were not detectable under the conditions of thin-layer chromatography employed. BrdU treatment of the melanotic cells induced about a two-fold increase in the ratio of NeuGc-hematosides to the total hematosides without any significant change in the cellular ganglioside content. The treatment concomitantly induced a change in cellular morphology, an increase in cell-to-substrate adhesiveness, and the suppression of tyrosinase activity. Amelanotic cells, which showed cellular phenotypes similar to those of BrdU-treated melanotic cells, were demonstrated to have over twice as much NeuGc-hematosides as compared with the melanotic cells. These results indicate a possible relationship in melanoma cells between the molecular species conversion of sialic acids of hematosides and the alteration of cellular phenotypic expression by BrdU treatment or spontaneous amelanization.

Effect of 5-bromodeoxyuridine on appearance of cell-surface saccharides in organ cultures of embryonic pancreas

Rat pancreatic rudiments from Day 15 embryos cultured for 11 days with 20 μ M 5-bromodeoxyuridine (BrdU) show selective suppression of acinar cell cytodifferentiation with production of fluid-filled vacuoles lined by undifferentiated cells. Using a battery of lectin-ferritin conjugates (Con A, RCA I, WGA, SBA, and Ulex lectin) we have shown that the majority of these cells express cell-surface glycoconjugate patterns reminiscent of both protodifferentiated cells from Day 15 rudiments and of adult centroacinar (i.e., duct-like) cells. A smaller population of the undifferentiated cells which contain abundant elements of the rough-surfaced endoplasmic reticulum expresses surface saccharide patterns equivalent to those of acinar cells in Day 19 rudiments. These cells, however, lack zymogen granules characteristic of acinar cells in Day 19 rudiments and are similar morphologically to presumptive acinar cells in Day 17 rudiments. Culture of Day 14 pancreatic rudiments with BrdU leads to growth only of undifferentiated cells with duct-like cell-surface saccharide patterns. We interpret these results to indicate that (1) the differentiation program for the acinar cell plasmalemma is established earlier than that for its intracellular organelles; (2) these two developmental programs are independently regulated; and (3) the progenitor of the acinar cell in the protodifferentiated rudiment may be related to the centroacinar or duct-like cell.

The effect of 5-bromodeoxyuridine on closure of the cranial neural tube in cultured mouse embryo

The effect of 5-bromodeoxyuridine on closure of the cranial neural tube in cultured mouse embryo

Effect of 5-bromodeoxyuridine on extracellular matrix during odontogenesis in vitro.

Maxillary and mandibular incisor primorida were dissected from 16-day old rat embryos and maintained in organ culture for up to 120 h. Substantial histological, cytological, and biochemical odontogenic differentiation had occurred during the 5 days in vitro. Whenever incisor primordia were incubated in the presence of 10 μg/ml of bromodeoxyuridine (BrdU) for the first 16 h of organ culture, no comparable evidence for subsequent odontogenic differentiation could be detected during the 120 h in vitro. Because previous work had shown that overall DNA, RNA, and protein biosynthetic rates were relatively unaffected by incorporation of the thymidine analog (Schwartz and Kirsten, 1974), we examined the relative composition and organization of the specialized extracellular matrix characteristic of rat embryonic incisor primordia. The control rudiments utilized increasing amounts of radiolabeled proline, glucosamine, and sulfate into acid-insoluble products during 120 h in vitro. In contrast, BrdU-treated counterparts incorporated far less amounts of the matrix precursors into acid-insoluble radioactive materials. Furthermore, whereas control rudiments elaborated mostly type I collagen, analog-treated primordia retained the more characteristically immature mixture of types I and III after 5 days in vitro. In light of the presumed “instructional” and inductive role of the extracellular matrix in odontogenic epithelial-mesenchymal interactions, these findings suggest that BrdU specifically and differentially terminated differentiation in vitro through compositional and organizational modifications of matrix constituents.

The effects of 5-bromodeoxyuridine on cyclic AMP levels and cytoskeletal organization in malignant melanoma cells

5-Bromodeoxyuridine (BUdR) causes mouse melanoma cells to develop a flattened morphology and simultaneously adhere tenaceously to the substratum on which they are growing. Experiments were done to determine if these events are coupled to increases in cAMP levels and to rearrangements in the cells' cytoskeleton. Cyclic AMP assays revealed that cell flattening and the increase in adhesive properties caused by BUdR is not accompanied by an increase in the cellular concentration of cyclic AMP. However, electron micrographs of cells grown in the presence of BUdR show a striking increase in the number of organized microtubules and microfilaments. Colchicine binding revealed no difference in the amount of tubulin present in untreated or BUdR-treated cells indicating that the increase in the number of microtubules is due to the polymerization of pre-existing tubulin subunits. These results are discussed in light of possible similar mechanisms of action of BUdR and cyclic AMP in regulating the organization of microtubules and microfilaments and the role these structures play in altering cell morphology and adhesive properties.

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.

Effect of 5-bromodeoxyuridine on nuclear RNA size in frog embryos

Effect of 5-bromodeoxyuridine on nuclear RNA size in frog embryos

The Effect of 5-Bromodeoxyuridine on Interferon Production in Human Cells

5-Bromodeoxyuridine (BrdUrd) increased interferon production by the Namalwa line of human lymphoblastoid cells treated with Sendai virus, but inhibited their growth. Thymidine, which also inhibited cell growth had no effect on interferon production, so that growth inhibition per se was not the cause of the stimulation. BrdUrd was incorporated into cellular DNA; 5-chlorodeoxyuridine and 5-iododeoxyuridine (which are also incorporated) increased the interferon yield, but 5-fluorodeoxyuridine (which is not incorporated) did not. Thymidine reduced both the incorporation of BrdUrd and its stimulatory effect on interferon production. Deoxycytidine (which prevents the cytotoxic effects of BrdUrd) had no effect on the stimulation. BrdUrd also stimulated interferon production in response to poly(rI) . poly(rC) in growing human diploid fibroblasts but not in SV40 virus-transformed human cells. Since BrdUrd was incorporated into the DNA of all these cells, we concluded that incorporation is necessary, but not sufficient for the stimulation of interferon formation.

Effect of 5-bromodeoxyuridine on heterogeneous nuclear RNA in rat hepatoma cells.

Heterogeneous nuclear RNA HnRNA) was isolated from untreated and 5-bromodeoxyuridine (BrdUrd) treated hepatoma tissue culture (HTC) cells. analysis of this RNA by either electrophoresis on polyacrylamide-agarose gels or centrifugation in sucrose gradients demonstrated that BrdUrd caused a shift in the labeled HnRNA population toward a smaller size distribution. This effect was produced by concentrations of BrdUrd which specifically lower the level of the differentiated enzyme tyrosine aminotransferase, but do not greatly affect cell growth. Differential binding to oligo(dT) cellulose was used to fractionate HnRNA further into classes containing poly(A) (alpha), oligo(A) (beta) or neither category of A-rich sequences (gamma). BrdUrd did not alter the relative rates of uridine incorporation into the three classes. The shift in the labeled HnRNA population due to BrdUrd was observed in all three subclasses of HnRNA.

Spontaneous 5-Bromodeoxyuridine Tolerance of Nicotiana Genetic Tumours

Summary Hormone independent Nicotiana glauca × N. langsdorffii genetic tumours are insensitive to cytotoxic effects of 5-bromodeoxyuridine up to the concentrations of 10–4 M. N. glauca pith callus, Constitutively cytokinin independent, exhibits significantly lower BUdR tolerance and hormone dependent N. langsdorffii pith callus is BUdR sensitive. Thus the degree of BUdR tolerance parallels the degree of hormonal autonomy. These results further substantiate our previour hypothesis that BUdR tolerance of plant cells may be connected with cytokinin-auxin autonomy.

Effect of 5-bromodeoxyuridine on the induction of adenosine 3′:5′-monophosphate phosphodiesterase in 3T3-L fibroblasts

Cyclic adenosine 3′:5′-monophosphate (cAMP) phosphodiesterase activity is increased threefold in 3T3-L fibroblasts by 1-methyl-3-isobutylxanthine. Actinomycin D (2 μg/ml) and cycloheximide (10 μg/ml) prevent this increase. Cells pretreated with inducer (0.5 m m) in the presence of cycloheximide for 12 h, rinsed, and then treated with actinomycin D ( t = 0) show a nearly twofold increase in enzyme activity in the next 90 min. The results suggest that cAMP phosphodiesterase is an inducible enzyme in 3T3-L cells and that the increase in enzyme activity requires transcription. The induction of cAMP phosphodiesterase was employed as a model to investigate enzyme induction in cells grown in the presence of the thymidine analog, 5-bromodeoxyuridine. Growth of 3T3-L fibroblasts in 1 μ m bromodeoxyuridine results in an inhibition of the induction of cAMP phosphodiesterase by 1-methyl-3-isobutylxanthine (0.5 m m). This effect increases when bromodeoxyuridine is raised from 0.1 to 10 μ m. The basal level of the enzyme is not altered by bromodeoxyuridine. Addition of thymidine (0.1 to 10 μ m) to the culture medium prevents the bromodeoxyridine effect, indicating that the analog must be incorporated into DNA to act. An increase in the inducer concentration from 0.1 to 1.0 m m partially overcame the bromodeoxyuridine effect on enzyme induction. These results suggest that the induction of cAMP phosphodiesterase can be inhibited by the incorporation of bromodeoxyuridine into DNA, and that an inducer of the enzyme can override the inhibition but at higher concentrations than are necessary in control cultures.

Effect of 5-Bromodeoxyuridine on Differentiation: II. The Effect of Early BUdR Treatment on Gastrulation of Sea Urchin Embryos

BUdR treatment of early cleavage stages is more effective on gastrulation the earlier the drug is administered before the 16-cell stage. The distribution of percentages of embryos gastrulating is in good agreement with the theoretical calculated values presented in part I and permit the assumption that BUdR may act by preventing normal transcription at, or shortly after, the 16-cell stage.

Effect of 5-Bromodeoxyuridine on Differentiation: I. Probability Distribution of BUdR-Containing DNA-Strands in Subsequent Divisions

The consequences on transcription of BUdR substitution of the normal thymidine in nuclear DNA are analyzed from a probabilistic point of view. The probability that at any stage subsequent to the administration of BUdR, a randomly chosen cell will be able to make good transcripts is calculated both under the assumption that incorporation of BUdR is complete and under the assumption that incorporation is partial. In the latter case both gradually increasing and decreasing incorporation rates are considered. The presented calculations may be of value for the investigation of any type of differentiating cells. Using the example of the sea urchin embryo it is shown how to calculate the probability that a particular group of cells, which in further development differentiate to a certain organ, will inherit as sufficient number of essential genes unaffected by BUdR in order to ensure a normal development. It is shown that the probability that the four macromeres (or micromeres) in the vegetal half of the 16-cell stage of the sea urchin embryo will contain at least one, two or four plus (or minus) strands of DNA uninfluenced by BUdR gradually decreases the earlier BUdR is administered. The theoretical calculations are in agreement with experimental observations on sea urchin embryos (described in part II).

Biological and biochemical effects of bromodeoxyuridine and deoxycytidine on Syrian hamster melanoma cells.

The addition of deoxycytidine (dCyd) to the growth medium of cultured Syrian hamster melanoma cells causes a reversal of the toxic effects of 5-bromodeoxyuridine (BrdU) and a decrease in the extent of incorporation of BrdU into nuclear DNA. These effects of dCyd can be accounted for, in part, by the intracellular conversion of the exogenously supplied dCyd to thymidine (dThd) nucleotides which can compete with BrdU nucleotides for incorporation into DNA. To some extent, the conversion of dCyd to dThd nucleotides can be inhibited by increasing the concentration of BrdU in the growth medium. The conversion of dCyd to dThd nucleotides is inhibited completely by aminopterin (Apt), and Apt also prevents dCyd from reversing BrdU toxicity and from decreasing the level of BrdU incorporation into nuclear DNA. In a clone of Syrian hamster melanoma cells, increasing the concentration of dCyd in the growth medium from 1 micron to 1000 micron resulted in a progressive increase in the percentage of dThd residues in nuclear DNA being derived from the exogenous dCyd, until more than 90% of the dThd residues came from the exogenous dCyd. However, despite the increasing amount of dThd derived from exogenous dCyd, there was a plateau in the decrease in BrdU incorporation into nuclear DNA at concentrations of dCyd above 8 micron.

Effects of 5-bromodeoxyuridine on development of Mauthner's neuron and neural retina of Xenopus laevis embryos

Bromodeoxyuridine (BrUdR) injected into Xenopus embryos at stages before, during or after the last period of DNA synthesis in Mauthner's neurons had no serious effect on differentiation of Maunthner's neurons. However, the BrUdR caused widespread cell degeneration in the central nervous system. The action of BrUdR on retinal neuronal differentiation depended on dose and stage of injection. At all doses (0.005–0.9 μg per injection), and all stages (22–29), the main effect was retinal cell degeneration. The drug produced complete absence of differentiated retinal neurons only when given at early stages (22–24) and at the highest doses (0.5–0.9 μg) that enable the embryo to survive to stage 44. At stages 22–24 at lower doses (0.005 or 0.05 μg per injection) and at stages 26–29 at all doses, some retinal neurons differentiated, although retinal cell degeneration was the main effect. In these two system BrUdR did not selectively inhibit neuronal differentiation.