Hemoglobin synthesis in murine virus-induced leukemic cells in vitro. 3. Effects of 5-bromo-2'-deoxyuridine, dimethylformamide and dimethylsulfoxide

Erythroid differentiation of Friend leukemia cells is enhanced when the cells are grown for four days in the presence of dimethylsulfoxide (DMSO). Dimethylformamide (DMF) has a similar though less marked effect. 5-Bromo-2′-deoxyuridine (BUdR) (10^?5M) inhibits both DMF- and DMSO-stimulated differentiation. For maximum inhibition, BUdR must be present during the first two days of growth, during which time DNA synthesis is maximal. The addition of BUdR after the third day has no effect. Since BUdR is incorporated into DNA and thymidine prevents BUdR inhibition of DMSO-stimulated differentiation, it is likely that BUdR acts by virtue of its incorporation into DNA. Although BUdR alone had little effect upon cell multiplication, in combination with DMSO, cell growth was inhibited up to 40%. Since the BUdR-inhibition of the DMSO effect was approximately 70%, it is unlikely that its effect on differentiation is due to selective killing of those cells which are stimulated to differentiate.     

Flow cytometric cell cycle analysis using the quenching of 33258 Hoechst fluorescence by bromodeoxyuridine incorporation.

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, The cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G1, S and G2 + M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.     

FLOW CYTOMETRIC CELL CYCLE ANALYSIS USING THE QUENCHING OF 33258 HOECHST FLUORESCENCE BY BROMODEOXYURIDINE INCORPORATION

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, the cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G₁, S and G₂+ M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.     

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.     

INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

Morphogenic Effects of Halogenated Thymidine Analogues on Drosophila

Variations in the treatment conditions which affect the frequency of BUdR-induced morphogenic lesions in Drosophila melanogaster have been studied. By varying the concentrations of BUdR and FU, or by changing the duration of treatment, it has been demonstrated that increased incorporation of BUdR into DNA results in a concomitant increase in morphogenic lesions.
A quantitative analysis of the data on BUdR-induced lesions as a function of the age of the larvae at the time of treatment indicates that an analog pulse during early larval life induces fewer, but larger lesions than a similar treatment given during later stages of development. These observations support the hypothesis that the developmental modifications are the result of genetic changes which in subsequent replication cycles of DNA are transmitted to descendant cells, and are not necessarily due to the presence of BUdR in DNA per se of cells in the developing organism.     

An Enrichment for Temperature-Sensitive Mutants in TETRAHYMENA THERMOPHILA

The parameters for the killing of Tetrahymena by 5-bromodeoxyuridine(BUdR) and near-ultraviolet light have been determined. Significant preferential killing by UV of cells that have incorporated BUdR was obtained when the cells were irradiated in a nonnutrient buffer. UV alone was found to be toxic to cells irradiated in growth medium. Mutants defective in division at a restrictive temperature were isolated from mutagenized cultures that had been treated with BUdR and UV and from mutagenized cultures that had no such treatment. Results indicate that the number of temperature sensitive (ts) growth mutants can be increase five to six times using the BUdR/UV treatment. Data are presented that indicate differences in the frequency of occurrence of various types of ts mutants, with and without enrichment. A mutant that immediately stopped macromolecular synthesis and cell division upon being placed at the restrictive temperature was more resistant to BUdR/UV treatment than wild type by 1000-fold. Using the above techniques, BUdR-resistant mutants altered in the phosphorylation of thymidine have been isolated.     

Defective transport of thymidine by cultured cells resistant to 5-bromodeoxyuridine

A line of HeLa cells resistant to 5-bromo-2′-deoxyuridine (BUdR) was established by continuous culture in growth medium containing BUdR; during the selection period, BUdR concentrations, initially 15 μM, were gradually increased to 100 μM. Cells of a clone (HeLa/B5) established from this line were also resistant to 5-fluoro-2′-deoxyuridine (FUdR), but not to the free base, 5-fluorouracil. Although extracts of HeLa/B5 cells exhibited levels of thymidine kinase activity comparable to those of parental cells, rates of uptake of BUdR, FUdR, and thymidine into intact cells were much reduced. The kinetics of uptake of uridine and adenosine, nucleosides which appear to be transported independently of thymidine in HeLa cells, were similar for HeLa/B5 and the parental line (HeLa/0). Relative to thymidine uptake by HeLa/0 cells, that by HeLa/B5 cells was distinctly less sensitive to nitrobenzlthionosine (NBMPR), a specific inhibitor of nucleoside transport in various types of animal cells. Despite this difference in NBMPR sensitivity, both cell lines possessed the same number of high affinity NBMPR binding sites per mg cell protein. The altered kinetics of thymidine uptake and the NBMPR insensitivity of that function in HeLa/B5 cells suggest that resistance to BUdR is due to an altered thymidine transport mechanism.     

Flow cytometry and cell proliferation kinetics

Flow cytometric techniques are presented which allow to determine parameters of cell proliferation kinetics by means of histogram sequences after special manipulations of the cell culture under investigation: (a) In the stathmokinetic method metaphase blocking agents are applied which allow the cells of the population to continue progression through interphase and accumulate at 4C DNA content. The development of DNA specific histograms during this process is analysed as to the G1 phase duration and the fraction of nonproliferating cells. (b) In the BUdR/Hoechst method the suppression of Hoechst fluorescence after BUdR incorporation during S phase is taken as a means for inducing a temporal change of histogram shapes without perturbing the cell cycle progression of the population. This temporal development of histogram shapes is analysed as to phase duration, whole cycle time and fraction of nonproliferating cells. (c) By combining the BUdR/Hoechst technique with a simultanous DNA specific stain and analysing with a two-parametrical flow cytometer, more information is obtained from each histogram after BUdR incorporation: The location of cells in the cycle at the beginning of the experiment, the cycle stage at cell harvest, and from this the distance and velocity of progression through the cycle during drug incubation. By introduction of these dynamic methods flow cytometry has become a powerful tool for the study of cell proliferation kinetics in culture.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

Differential sensitivity to 5-bromodeoxyuridine during the S phase of synchronized myogenic cells

Synchronized myogenic cell cultures have been used to demonstrate differential sensitivity to BUdR during segments of the S period. Synchronization of the cells was achieved by two methods. First, cells were initiated in medium containing FUdR, an inhibitor of DNA synthesis. Following FUdR blockade reversal with TdR after 19 hr in vitro, the synchronized cells were allowed to replicate their DNA with BUdR for periods corresponding to early and late S. Determinations of percentage labeled cells during synchronization with FUdR indicate that about 90% of the cycling population of cells accumulates at the G1/S interface of the cell-cycle and that the duration of the S period following blockade reversal with TdR is not altered. Since BUdR is pulsed to these cultures immediately after the point of synchronization, a high degree of synchrony is obtained. In the second method of synchrony, cohorts of cells which had been in G2, late S, or early S during a BUdR pulse were collected in metaphase arrest with Colcemid and selectively removed from the cultures. With the mitotic selection method the point of synchronization occurred several hours after the BUdR pulse. In both methods the cells were allowed to resume myogenesis and scored for percentage fused nuclei after approx 50 hr in vitro. With both methods of synchrony, BUdR incorporation into early replicating DNA results in a striking decline in myoblast fusion, whereas incorporation into late replicating DNA is without effect. The results cannot be attributed to a disproportionate uptake of nucleotide during early S. Further fractionation of the 4-hr S phase into 1-hr periods indicates that the BUdR sensitive target is replicated during the second hr of DNA synthesis.     

Morphogenic effects of halogenated thymidine analogues on Drosophila. I. Quantitative analysis of lesions induced by 5-bromodeoxyuridine and 5-fluorouracil

When 5-fluorouracil (FU) is offered simultaneously with 5-bromodeoxyuridine (BUdR) to Drosophila larvae, a variety of bristle modifications and hyperplastic growths are found on the wings of the adult flies. Administration of FU alone will not stimulate growth in Drosophila, while high concentrations of BUdR offered alone will induce a lower frequency of growth modifications than induced by BUdR plus FU. Comparison of the morphological response induced by sequential treatment with the two analogues and that by simultaneous treatment with the analogues at the same concentrations indicates that maximum response is achieved by the presence of both analogues at the same time. These observations suggest that BUdR may be the primary agent in upsetting morphogenesis in Drosophila, while FU plays a subsidiary role leading to intensification of the morphogenic effects when it is present during the treatment period. The incorporation of BUdR-H³ and FU-H³ in Drosophila tissues was demonstrated by autoradiography. BUdR-H³ was incorporated in nuclei of both larval and imaginal disc cells, and the isotopic label was removable by deoxyribonuclease. Following dietary administration of FU-H³, tritium was found in RNA, primarily in cytoplasmic regions. Since BUdR is a known mutagen, consideration was given to the hypothesis that the altered growth patterns in Drosophila wings are the result of somatic cell mutational events induced by BUdR. Validity of the argument that recessive mutations on the X chromosomes can be readily expressed in the somatic cells of the male with one X chromosome as opposed to the female with two X chromosomes was tested by comparing the frequency of the induced somatic cell lesions in male and female zygotes. The males showed a higher frequency of induced supernumeraries, while the icidence of bristle effects and total wings affected was the same in both sexes.This research was supported by grants to T. M. R. from the National Science Foundation (GB-11745) and by an Institutional Research Grant (No. IN-40H) to the University of Michigan from the American Cancer Society.     

For the reproductive death of a cell, damage to a single chromosome is sufficient

The Chinese hamster cells V-79 were treated with BUdR during one cell cycle; after that the cells were grown in the medium without BUdR and were irradiated by longwave-UV-light at different time. The cell survival after photolysis was compared with the percentage of metaphase plates with different number of chromosomes containing BUdR. It is concluded that for cell inactivation the presence of only one destroyed chromosome (or its part) is enough.     

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.     

Mechanism of Oncogenic Transformation by Rous Sarcoma Virus: I. Intracellular Inactivation of Cell-Transforming Ability of Rous Sarcoma Virus by 5-Bromodeoxyuridine and Light

Chick embryo fibroblasts brought into stationary phase of growth by maintenance in serum-free Eagle's MEM medium were infected with the Bryan strain of Rous sarcoma virus (B-RSV) and incubated for 18 hr in the presence of 5-bromo-deoxyuridine (BUdR). The cells were then allowed to resume growth and deoxyribonucleic acid (DNA) synthesis by addition of an enriched F12 medium containing serum and RSV antibody to prevent spread of viral infection. After 48 hr, the cultures were exposed for various periods to visible light, overlaid with solid culture medium, and observed for the appearance of foci of transformed cells. In cultures treated with BUdR at the time of infection, exposure to light resulted in a suppression of focus formation of from 50 to 90% in various experiments. Treatment with BUdR for 18 hr before infection or on the day after infection, followed by exposure to light, had no effect on focus formation. In cultures in which almost all cells were infected, treatment with BUdR followed by exposure to light did not result in cell death. This suggests that suppression of transformation is not due to selective killing of infected cells by this treatment but rather to the intracellular inactivation of the transforming ability of Rous sarcoma proviral DNA.     

Some effects of 5-bromodeoxyuridine on polyoma-transformed mouse cells

The tumor-forming potential of polyoma-transformed mouse cells was found to be diminished but not eliminated after growth in the presence of 5-bromodeoxyuridine (BUdR). Several in vitro characteristics of the cells were also investigated and those which best correlated with the tumor studies were the ability to form colonies either in agar suspension or on monolayers of non-transformed cells. Transformed cells treated with BUdR grew well in culture when plated above a certain critical number of cells per unit area and poorly when plated at lower densities. Non-transformed cells exposed to BUdR for two or three passages were unable to divide.     

Studies on the Effects of Bromodeoxyuridine (BUdR) on Differentiation

The effects of BUdR on morphogenesis in Acetabularia mediterranea and on embryonic development (sea urchin, tunicate and amphibian embryos) have been studied.
In the presence of BUdR, cap development is affected in both the anucleate and nucleate fragment of the unicellular alga Acetabularia mediterranea . In the nucleate fragment, the initiation of cap formation can be inhibited. BUdR exerts various inhibitory effects on embryonic development. Modifications of sensitivity to BUdR during development are also observed: in Paracentrotus , a change in sensitivity towards the drug occurs at the 16 cell stage, while in amphibian eggs, a modification can be shown after the gastrula stage.
The significance of these observations is discussed.     

Mutagenic mechanism of 5-bromodeoxyuridine in Chinese hamster cells

Resistance to 6-thioguanine was induced by 5-bromodeoxyuridine (BUdR) in synchronous Chinese hamster cells. The yield of mutant colonies was not proportional to the amount of BUdR incorporated into DNA; thus mutants were not due to mispairing of BUdR with guanine during replication. Few mutants were induced until BUdR concentrations exceeded that of the intracellular thymidine triphosphate pool and mutant yield was depressed by addition of thymidine to the medium. These data suggest that BUdR exerts an allosteric effect on the DNA synthesizing system which renders it more error prone.