DNA OF BROMODEOXYURIDINE–TREATED NEUROBLASTOMA CELLS

Abstract— Cells of an adrenergic clone M1 of the mouse neuroblastoma C 1300 show morphological differentiation when treated with BrdU (10⁻⁵ m ). The involvement in this phenomenon of an action of BrdU at the membrane level has been suggested (see References). Taking into account the alterations observed after addition of BrdU in culture cell lines it was interesting to investigate the incorporation of BrdU into DNA of neuroblastoma cells. Our results showed an active BrdU incorporation into the DNA of treated Ml cells (an average of 25% of all thymidine residues for the 10⁻⁵ m -BrdU concentration present in our experimental conditions). Thymidine substitution by BrdU did not occur randomly, indicating the existence of selective parts in the genome sensitive toward BrdU. In mitomycin–treated cells (at high doses) BrdU incorporation occurred significantly at a very low level when related to the total thymidines (2%). Our findings suggest that since DNA synthesis is not inhibited BrdU may act by being incorporated into DNA and thus altering gene expression in addition to a site of action on the cell membranes.     

Cell synchronization and dynamic G-banding of equine chromosomes by bromodeoxyuridine

Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: initially the BrdU is incorporated during the first part of the S-phase and then it blocks the cells at mid-S-phase. Within the cell cycle, mid-S-phase appears to be the most vulnerable time to various blocking agents. To differentiate the regions of BrdU incorporation from those that have not been substituted, the fluorescence-photolysis-Giemsa (FPG) technique was applied as modified for horse chromosomes. This dynamic technique, which produces many prometaphase and prophase chromosomes showing very sharp G-bands, is certain to enhance the accuracy of cytogenetic analysis and aid in the standardization of equine chromosomes.     

Bone morphogenetic protein (BMP)1-3 enhances bone repair

Small molecules that exhibit biological activity have contributed to the understanding of the molecular mechanisms of various biological phenomena. 5-Bromodeoxyuridine (BrdU) is a thymidine analogue that modulates various biological phenomena such as cellular differentiation and cellular senescence in cultured mammalian cells. Although BrdU is thought to function through changing chromatin structure and gene expression, its precise molecular mechanisms are not understood. To study the molecular mechanism for the action of BrdU, we have employed the yeast Saccharomycescerevisiae as a model system, and screened multi-copy suppressor genes that confer resistance to BrdU. Our genetic screen has revealed that expression of the N-terminal short fragment of TUP1, and also disruption of HDA1 or HOS1, histone deacetylases that interact with TUP1, conferred resistance to BrdU. These results suggest the implication of the chromatin proteins in the function of BrdU, and would provide novel clues to answer the old question of how BrdU modulates various biological phenomena.     

家蚕染色体复制区带的初步显示

该文采用家蚕Bomoyx mori活体注射BrdU结合FPG(fluorochrome photolyusis Giem-sa)显带方法,以生殖腺为材料,成功显示出家蚕有丝分裂中期染色体复制带。由于处于S-期的细胞有早有晚,且同一细胞DNA各片段的复制亦有先后,因此BrdU掺入DNA合成的时间也有所不同,从而可产生出早、中、晚复制带型。BrdU掺入时间早,则会在家蚕部分染色体上出现大面积浅染带纹的早复制带。每一染色体皆有其独特的带纹特征,据此可初步将它与其它染色体相互区分;随着BrdU掺入时间的推后,染色体上会出现深浅交替、丰富的带纹,即中复制带型;至S-期DNA合成晚期掺入BrdU,最终染色体出现以深染带纹为主,浅染带纹仅出现于少数染色体的中部、近中部或端部的晚复制带。     

3-Aminobenzamide-induced sister chromatid exchanges are dependent on incorporated bromodeoxyuridine in DNA

Data are presented establishing a direct correlation between 3-aminobenzamide-induced sister chromatid exchange (SCE) frequency and the level of bromodeoxyuridine (BrdU) incorporated into DNA. In addition, it is shown that most of the SCEs are induced in the second cell cycle, in which BrdU-containing DNA is used as the template for replication.     

Fra(10)(q25): the BrdU effect is substitution-dependent.

Expression in the majority of fra(10)(q25) cases is either induced or enhanced by the presence of bromodeoxyuridine (BrdU) in the culture medium. BrdU is known to exert its effects on cells via two primary mechanisms: substitution-dependent and concentration-dependent. BrdU incorporation into DNA and BrdU concentration in the culture medium can be resolved as independent variables. The results of such experiments indicate that at three fixed levels of BrdU substitution, 100-fold variation of BrdU concentration had little or no effect on fra(10)(q25) expression. At a fixed BrdU concentration, the level of fra(10)(q25) expression rises as a function of increased BrdU substitution, approaching 100% expression at 100% substitution. Thus, BrdU induction or enhancement of fra(10)(q25) expression is clearly substitution-dependent. Iododeoxyuridine, another halogenated pyrimidine, has a similar effect. The critical time of incorporation is between 8 and 9 hrs before mitosis. After this time, removal of BrdU (and fluorodeoxyuridine [FdU]) from the culture medium followed by addition of deoxythymidine does not reverse the BrdU effect on fra(10)(q25) expression.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

We describe a reproducible method for combining tritiated thymidine ([H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [3H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [3H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified. The inhibition of uptake into DNA of [3H]TdR from 0.23 to 1.85 MBq (6.25 to 50 mu Ci) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in mu Ci per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [3H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Search for DNA interchange corresponding to sister chromatid exchanges in Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) grown for one cycle in bromodeoxyuridine (BrdU) contain a small amount (0.5%) of unusually dense double stranded DNA. This dense DNA has been previously interpreted as being bifilarly substituted with BrdU and hence evidence that sister chromatid exchange (SCE) formation proceeds via the Holliday model of recombination. However, the amount of this dense DNA is 100 times greater than that expected based on the SCE frequency in similarly cultured CHO cells, and it is not increased by treating the cells with mitomycin C. Moreover, contrary to expectations for bifilary substituted DNA, the amount of this dense DNA is not reduced by growing BrdU-labeled cells for a second cycle in TdR. Finally, DNA isolated from CHO cells contains a minor band (0.5%) with a density 0.025 gm/cc greater than that of the main band, whether or not BrdU has been incorporated. These results call into question the identification of this unusually dense DNA as bifilarly substituted and hence its previously postulated relationship to SCE formation.     

Measuring drug-related receptor occupancy with positron emission tomography

Several techniques can be used to measure indirectly the effect of drugs (e.g., EEG, fMRI) in healthy volunteers and in patients. Although each technique has its merits, a direct link between drug efficacy and site of action in vivo usually cannot be established. In addition, when the specific mode of action of a drug has been determined from preclinical studies, it is often not known whether the administered dose is optimal for humans. Both industry and academia are becoming more and more interested in determining the dose-related occupancy of specific targets caused by administration of drugs under test. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are noninvasive imaging techniques that can give insight into the relationship between target occupancy and drug efficacy, provided a suitable radioligand is available. Although SPECT has certain advantages (e.g., a long half-life of the radionuclides), the spatial and temporal resolution as well as the labeling possibilities of this technique are limited. This review focuses on PET methodology for conducting drug occupancy studies in humans.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

Abstract We describe a reproducible method for combining tritiated thymidine ([³H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [³H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [³H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [³H]TdR from 0.23 to 1.85 MBq (6.25 to 50 μCi) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in μCi per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [³H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Inhibition of Friend erythroleukemic cell differentiation by bromodeoxyuridine: correlation with the amount of bromodeoxyuridine in DNA

These studies were undertaken to examine the relationship between the inhibition by 5-bromodeoxyuridine (BrdU) of erythroid differentiation in Friend erythroleukemia cells and the incorporation of BrdU into DNA. Experiments were carried out in which the incorporation of BrdU into DNA and the concentration of BrdU to which the cells were exposed were varied independently of each other. In addition, the ability of deoxycytidine (dC) to reverse the effects of BrdU on hemoglobin production and to reduce the amount of BrdU in DNA was analyzed. Under all the conditions tested, the effects of BrdU were correlated with the amount of BrdU incorporated into nuclear DNA. These results differ from those of recent studies on the inhibition of pigmentation and the induction of mutations by BrdU in Syrian hamster melanoma cells. The results suggest that BrdU may be producing its biological effects by a variety of different mechanisms.     

DNA replication during endospore development in Metabacterium polyspora

The guinea pig intestinal symbiont Metabacterium polyspora is an uncultured, endospore-forming member of the Firmicutes. Unlike most endospore-forming bacteria, sporulation is an obligate part of the M. polyspora life cycle when it is associated with a guinea pig. Binary fission is limited to a brief period in its life cycle, if exhibited at all. Instead, M. polyspora relies on the formation of multiple endospores for reproduction. Sporulation is initiated immediately after germination, which leaves little time for the cell to accumulate resources to support spore formation. Using immunolocalization of the nucleotide analogue bromodeoxyuridine (BrdU), we were able to follow replication dynamics in M. polyspora . BrdU was provided to cells within the guinea pig intestinal tract. BrdU was incorporated into DNA located within the forespores throughout development, at all stages prior to spore maturation. Our results suggest that in M. polyspora , DNA replication within the forespore is not suppressed during sporulation as it is in other endospore-forming bacteria. Replication within forespores would allow M. polyspora to maximize its reproductive potential and supply each endospore with at least one complete copy of the genome.     

Deoxycytidine reverses the suppression of nucleolar organizer regions' activity caused by BrdU

Summary In this article we report that 5-bromodeoxyuridine (BrdU) significantly suppresses the nucleolar organizer regions (NORs) activity of Chinese hamster cells (both dipliod cells and cell line Wg3-h) (P<0.001). One of the most obvious characteristics of the suppression is a significant decrease in the total number of the Ag-NORs per cell rather than in a frequency variation of the associated Ag-NORs. The decrease in the Ag-NORs number is mainly because of the decrease in number of chromosomes bearing 2 Ag-NORs. The degree of the suppression increases with increase in BrdU concentration in the culture medium. There is a close relationship between the suppression and the BrdU-treatment time, i.e. for a given concentration of the BrdU, the longer the BrdU-treatment time, the stronger the suppression. When the BrdU-treated cells are transferred into BrdU-free medium and allowed to grow in it for another 30 h, NORs activity can be restored. Therefore, the suppression of NORs activity may be due to BrdU toxicity. When deoxycytidine (dC) is added into medium containing 30 g/ml of BrdU, the total number of both the Ag-NORs and the chromosomes bearing Ag-NORs per cell increases to the level of untreated cells. Our results thus indicate that the addition of dC reverses the suppression of the NORs activity caused by BrdU.     

The cytotoxic and mutagenic effects of 5-bromodeoxyuridine-plus-black-light treatment in cultured mammalian cells

The cytotoxic and mutagenic effects of the incorporation of 5-bromodeoxyuridine (BrdU)_followed by exposure to black light were investigated with Chinese hamster ovary (CHO) cells in cell culture. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus was determined by selection for 6-thioguanine resistant (TG^r) mutants (CHO/HGPRT system). BrdU alone has been shown to be mutagenic only at concentrations of 50 μM or greater. This study was performed in an effort to determine whether BrdU is actually incorporated into the hgprt gene when lower, nonmutagenic concentrations are employed. Neither BrdU (1–20 μM) nor exposure to black light alone was mutagenic, but the combined treatment did result in the induction of TG^r mutants. The mutant frequency increased with increasing light exposure at constant BrdU and with inreasing BrdU at constant light exposure. These results show that BrdU is incorporated into the hgprt gene, but that this does not result in mutation induction in the absence of light exposure. Such a BrdU-plus-light procedure might be applied to studies of DNA repair at this locus, since mutation induction requires both BrdU incorporation and subsequent exposure to black light.     

Effect of thymidine analogs on tyrosinase activity and mRNA accumulation in mouse melanoma cells

The thymidine analog 5-bromodeoxyuridine (BrdU) has been found to inhibit terminal differentiation of a variety of cell types without significantly affecting the growth of these cells. We have compared the effect of BrdU with two other thymidine analogs, 5-iododeoxyuridine and 5-fluorodeoxyuridine, on the growth, tyrosinase activity, and tyrosinase-mRNA accumulation in BL-6 mouse melanoma cells. We show that all three analogs inhibit growth and tyrosinase activity, but only BrdU significantly inhibits the accumulation of tyrosinase mRNA. We consider these results in the light of current understanding of BrdU action.     

The diverse effects of 5'-bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells.

Reuber (H35) hepatoma cells were grown in medium containing 10(-5)M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino-transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU-containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.     

Histogenesis and dependent glutamine synthetase inducibility in embryonic neural retina. Irreversible inhibition of differentiation by 5-bromodeoxyuridine

Young, mitotically active neural retinas from 7-day chick embryos were cultured with 5-bromodeoxyuridine (BrdU) for 8 hr or more. After this treatment, they failed to differentiate beyond the stage at which they were explanted; there was no histogenesis or increase in glutamine synthetase (GS) inducibility in intact tissues or in aggregates of dissociated cells. Normally GS can be induced in the retina with hydrocortisone as the cells cease to be mitotically active and begin showing histological organization after day 7. This inhibition by BrdU was irreversible even in the presence of excess thymidine. Overall incorporation of ¹⁴C-amino acids into protein was only slightly inhibited, and the ability of cells from treated tissue to aggregate and sort out from nonneural cell types was unaffected. Control cultures without BrdU showed considerable histogenesis and a parallel increase in enzyme inducibility. Postmitotic 10-day retinas appeared to be unaffected by BrdU. The incorporation rates of both tritiated BrdU and thymidine (dT) into DNA were 14× higher in 7- than in 10-day retinas. Simultaneous addition of excess unlabeled dT with either of the labeled nucleosides reduced their incorporation and reduced the inhibitory action of BrdU on differentiation.It is concluded that BrdU irreversibly inhibits the differentiation of retina cell surface properties involved in histogenesis and dependent cytodifferentiation without affecting already differentiated properties of the cell surface. The results support the hypothesis that histogenesis is directed by genes affecting specific cell surface properties.     

Survey of bromodeoxyuridine uptake among environmental bacteria and variation in uptake rates in a taxonomically diverse set of bacterial isolates

Incorporation of 5-Bromo-2′-Deoxyuridine (BrdU) into DNA can be used to target replicating bacteria in the environment, but differential uptake capacity is a potential bias. Among 23 bacterial isolates commonly found in soils, most took up BrdU, but at up to 10-fold different cell-specific rates. Combined with results from an in silico analysis of 1000 BrdU-labeled 16S rRNA gene sequences, our results demonstrate a BrdU uptake bias with no apparent relationship between taxa affiliation and ability to incorporate BrdU.     

Mutagens and the tumor promoter

We studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and saccharin on the frequency of induced mutations of resistance to 6-mercaptopurine and ouabain in Chinese hamster and mouse cells. UV-rays, bovine adenovirus-3 (BAV-3) and 5-bromdeoxyuridine (BrdU) were used as mutagens. In the case of BAV-3 and BrdU, we investigated, apart from the mutagenic effect, the tumor-inducing activity of these mutagens in mice, BrdU proved to have no carcinogenic effect. The data about the influence of TPA on the mutagenic effect of the three different mutagens indicate that TPA increases the frequency of the gene mutations induced by UV-rays and BAV-3. The results of the study of BrdU and TPA combined action revealed the fact that TPA does not increase the mutagenic effect of BrdU. We demonstrated that saccharin also possesses the promoter activity; it increases the mutagenic effect of BAV-3. The results described above lead to the assumption that TPA influence on the mutagenic effect only takes place when carcinogenic mutagens are used.