Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyuridine

Differential fluorescence of sister chromatids (SCD) and sister chromatid exchanges (SCE) were visualized in chromosomes obtained directly from growing chicken embryos. SCD was obtained by exposing 3-day embryos to BrdU (12.5-50 mug) in ovo for 26 hours and staining air dried chromosome preparations with 33258 Hoechst. Bright, stable fluorescence and continued SCD were achieved if slides were mounted in McIlvaine's pH 4.4 buffer. Embryo growth, mitotic activity and gross chromosome morphology were not adversely altered by the BrdU treatments. The SCE rate was estimated to be 0.07 SCEs per macrochromosome and 0.75 SCEs per metaphase for two cell cycles.     

Three-way differentiation of sister chromatids in endoreduplicated (M3) chromosomes of Bloom syndrome B-lymphoid cell line

Summary The three-way differentiation of sister chromatids (3-way SCD) in M₃ endoreduplicated chromosomes in a Bloom syndrome (BS) B-lymphoid cell line, suggested that in addition to exchanges between sister chromatids (intra-exchanges), non-sister chromatid exchanges (inter-exchanges) also occur, especially in BS high SCE cells. In BS diploid chromosomes such inter-exchanges probably get confused with intra-exchanges when total SCEs are accounted for. Bloom syndrome high SCE cells probably do not follow the same bromodeoxyuridine (BrdU) uptake pattern over three cell cycles as normal cells. The 3-way SCD in M₃ endoreduplicated chromosomes can be explained on the basis of Schvartzman's second model (1979) as well as Miller's model (1976), depending on the pattern of uptake of BrdU over three cell cycles. An interference in the previous events of exchanges in the following cell cycle (i.e., cancellation of SCEs) in BS chromosomes was observed in some regions, though not in high numbers.     

Sister chromatid differentiation and chromosomal in situ suppression hybridization: a combined methodology for analyzing cell proliferation and SCEs in individual chromosomes.

A technique combining sister chromatid differentiation (SCD) with chromosomal in situ suppression (CISS) hybridization is described. This combined methodology allows simultaneous analysis of cell-proliferation kinetics and sister chromatid exchanges (SCEs) in chromosomes identified by probes. To demonstrate the usefulness of this approach, cultured fibroblasts from a patient with Pallister-Killian syndrome, mos46/47,+i(12p), a chromosome mosaicism disorder, were studied. The fibroblasts were cultured in the presence of 5-bromodeoxyuridine (BrdU) for 72 h. Chromosome preparations were stained by a modified fluorescence-plus-Giemsa method to obtain SCD. For identification of the normal chromosome 12 and the i(12p), CISS hybridization with a biotin-labeled chromosome 12-specific library probe (LA 12NS01) was carried out after SCD. The hybridization was detected by an indirect immunofluorescence technique. For the analysis of cell kinetics and SCEs, the technique allows rapid, reliable identification of abnormal and normal cell populations. It also allows analysis of SCEs in individual chromosomes.     

Demonstration of sister chromatid differentiation in human amniotic fluid cells after partial synchronization with BrdU or dT surplus

Summary Protocols are compared demonstrating sister chromatid differentiation (SCD) in human amniotic fluid (AF) cells with and without partial synchronization. Partial synchronization both with an excess of 5-bromodeoxyuridine (BrdU) and an excess of thymidine leads to an increase of metaphases with SCD. Compared with unsynchronized cells, the rate of sister chromatid exchanges (SCE) is not increased. Studies on the late replicating X chromosome of female cells showed that the addition of mitomycin C (MMC) after releasing the thymidine block preferentially induces SCEs in late replicating regions. The partial synchronization with thymidine surplus provides a good basis for SCE experiments with AF cells and facilitates the prenatal diagnosis of diseases characterized by changes in the SCE rate.     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: monoclonal anti-BrdU antibody

A stable staining procedure of sister-chromatid differentiation (SCD) using a monoclonal antibromodeoxyuridine (BrdU) antibody was newly established by combining it with the immunoperoxidase reaction (3,3'-diaminobenzidine, DAB reaction). This procedure permitted detection of SCD and SCE at very low BrdU concentrations. SCD was not usually observed below 2.0 micrograms/ml BrdU with flame-dried chromosome slides. When chromosome slides were prepared by air-drying over 37 degrees C warm water, SCD was detected at 10.0, 5.0, 1.0, 0.5, 0.3 and 0.2 micrograms/ml BrdU with FPG and even at 0.1 microgram/ml BrdU with the antibody technique. SCE levels were evaluated using the antibody technique and endomitotic analysis with FPG at low BrdU concentrations (1.0, 0.5, 0.3, 0.2 microgram/ml) in two BS B-lymphoblastoid cell lines (LCLs). Even though the BS SCE level was approximately 70 per cell at 10 micrograms/ml, the value decreased to the level of 20-30 SCE per cell at 0.1 microgram/ml with the antibody technique. In BrdU-labelled BS endomitoses, single SCEs highly decreased with BrdU concentrations (130-140 level at 10 micrograms/ml: 38-60 level at 0.2 microgram/ml), when compared to the rare twin SCE values (3-6 SCE level) at all BrdU concentrations. These findings conclusively indicate that the spontaneous baseline SCE in BS B-lymphoblastoid cells is low and most BS SCEs are caused by BrdU.     

Three-Way Differentiation of Chinese Hamster Ovary Chromosomes by Immunoperoxidase Technique Using a Monoclonal Anti-Bromodeoxyuridine Antibody

A new permanent staining procedure for sister chromatid differentiation (SCD) in cultured Chinese hamster ovary cells has been established by combining the three-way differentiation in third mitosis (M3) chromosomes and the immunoperoxidase reaction developed with 3,3'-diaminobenzidine using a monoclonal anti-bromodeoxyuridine (BrdU) antibody. This procedure allows SCD at very low BrdU concentrations, and the evaluation of the sister chromatid exchange frequencies on a per cell-cycle basis.     

Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine antibody

A commercially available bromodeoxyuridine (BrdUrd) antibody was used to demonstrate sister chromatid differentiation (SCD) and to evaluate sister chromatid exchanges (SCEs) in V79 Chinese hamster cells. V79 cells were cultivated for one cell cycle in the presence of BrdUrd, followed by a second cell cycle in the absence of BrdUrd. Chromosome preparations were stained by a common immunologic staining technique. The staining pattern observed is similar to that after FPG (fluorescent plus Giemsa) staining, though with reverse staining specificity. The sensitivity of BrdUrd detection is enhanced by a factor of 20 compared to the FPG technique and thus allows the evaluation of SCEs at very low BrdUrd concentrations. The application of the antibody technique gives information about the origin and localization of SCEs and produces further evidence for the spontaneous occurrence of SCEs.     

Influence of low doses of BrdU and estimation of spontaneous SCE in CHO chromosomes: three-way differential staining and an immunoperoxidase method

The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1+SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 M no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.by S. Wolff     

Sister chromatid exchanges induced by sarcolysine in human lymphocytes in vitro

The effect of three concentrations of sarcolysine (0.5 micrograms/ml, 1 microgram/ml and 2 micrograms/ml) on the sister chromatid exchanges (SCE) was investigated in human lymphocytes in vitro. A dose related increase in SCEs frequencies was observed after sarcolysine administration and also a delayed development of cell cycle has been induced by the two last concentrations. The variation range of SCEs per cell was dose-dependent and it was considered to represent the acquired genetic instability induced by the drug.     

Cytotoxic effect and induction of sister chromatid exchange in exponentially growing rat 9L gliosarcoma cells after brief exposure to BrdU

The magnitude of DNA modulation in rat 9L gliosarcoma cells after a brief exposure to bromodeoxyuridine (BrdU) was studied by assaying colony-forming efficiency (CFE) and the number of sister chromatid exchanges (SCEs) per metaphase. The CFE assay showed that a 1-hr exposure to BrdU, at concentrations ranging from 10 to 1000 microM, produced a maximum cell kill of 5%. After a 2-hr exposure to 20 microM BrdU, the surviving fraction was 0.99, and even at a BrdU concentration of 1000 microM, 77% of the 9L cells survived. Compared with control cultures, the relative number of SCEs per metaphase in treated cultures was increased after a 1-hr exposure to BrdU at concentrations of 100 microM or more and after a 2-hr exposure to concentrations of 20 microM or more; no increase was observed in cells treated for 30 min with BrdU at concentrations up to 1000 microM. When the treated cells were allowed to grow in BrdU-free growth medium, the number of SCEs per metaphase returned to the control level within 24 hr, even after exposure to BrdU at concentrations as high as 1000 microM. These results demonstrate that exposure to BrdU at concentrations of up to 1000 microM for 30 min, 100 microM for 1 hr, and 20 microM for 2 hr causes little modulation of DNA.     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.     

Distribution of sister chromatid exchanges in chromosomes of normal Chinese hamster and its cell lines exposed to BrdU and MMC

Sister chromatid exchanges (SCEs) were investigated in chromosomes from normal male Chinese hamster (CH) and its cell lines (CHW, 1102 and 1103). The fibroblasts were grown for two replication cycles in medium containing BrdU and mitomycin C (MMC) at concentrations of 0.01, 0.02 and 0.03 micrograms/ml of medium. The difference in SCEs/cell between male CH and CHW was negligible, but the difference between CHW and 1102 was about 2.6-fold. It is suggested from karyotypic differences between CHW and 1102, that the control of SCEs might be due partly or completely to chromosome 5 in Chinese hamster. The lines CHW and 1102 were less responsive than normal Chinese hamster cells when exposed to different MMC concentrations. It is suggested that the lines CHW and 1102 might be slightly resistant to MMC. The frequency of SCEs decreased with the decrease of chromosome size. SCEs are not preferentially distributed on any autosomal chromosomes. No SCEs were found in normal X-chromosomes. The majority of exchanges appear to be either interband regions or very near band-interband junctions.     

Cytotoxic effect and induction of sister chromatid exchange in exponentially growing rat 9L gliosarcoma cells after brief exposure to BrdU

Abstract. The magnitude of DNA modulation in rat 9L gliosarcoma cells after a brief exposure to bromodeoxyuridine (BrdU) was studied by assaying colony-forming efficiency (CFE) and the number of sister chromatid exchanges (SCEs) per metaphase. The CFE assay showed that a 1-hr exposure to BrdU, at concentrations ranging from 10 to 1000 μ M, produced a maximum cell kill of 5%. After a 2-hr exposure to 20 μ M BrdU, the surviving fraction was 0.99, and even at a BrdU concentration of 1000 μ M, 77% of the 9L cells survived. Compared with control cultures, the relative number of SCEs per metaphase in treated cultures was increased after a 1-hr exposure to BrdU at concentrations of 100 μ M or more and after a 2-hr exposure to concentrations of 20 μ M or more; no increase was observed in cells treated for 30 min with BrdU at concentrations up to 1000 μ M. When the treated cells were allowed to grow in BrdU-free growth medium, the number of SCEs per metaphase returned to the control level within 24 hr, even after exposure to BrdU at concentrations as high as 1000 μ M. These results demonstrate that exposure to BrdU at concentrations of up to 1000 μ M for 30 min, 100 μ M for 1 hr, and 20 μ M for 2 hr causes little modulation of DNA.     

Do the frequencies of sister chromatid exchanges in endoreduplieated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.     

Increased sister chromatid exchange events in the human late replicating X

Summary Human female blood cultures were labeled with BrdU for detecting sister chromatid exchanges (SCEs) by the Hoechst 33258 fluorescence technique. Late labeling with ³H-thymidine and autoradiography allowed the identification of the late replicating X. The mean number of SCEs in the cells was 13. The isopycnotic X showed an exchange frequency according to its relative length in the karyotype; in the late replicating X a doubled number of SCE events was observed.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

Determination of the baseline sister chromatid exchange frequency in human and mouse peripheral lymphocytes using monoclonal antibodies and very low doses of bromodeoxyuridine

We measured the frequency of sister chromatid exchanges (SCEs) in human and mouse peripheral lymphocytes using doses of bromodeoxyuridine (BrdU) ranging from 30 nM to 100 microM (human) and from 10 nM to 10 microM (mouse). Heparinized peripheral blood was obtained from five healthy nonsmokers and from six C57B1/6 male mice. The blood was stimulated with PHA (human) or lipopolysaccharide (LPS, mouse) and grown for the first of two cell cycles in BrdU. Metaphase chromosomes were denatured and exposed to a monoclonal antibody reactive to single-stranded DNA containing BrdU. A second antibody was used to label the first antibody with fluorescein, and propidium iodide was used as a counterstain. Second-division metaphases were thus differentially stained red to indicate DNA content and yellow-green to indicate the presence of BrdU. The results indicate that the baseline SCE frequency in human and mouse peripheral lymphocytes is 3.6 and 2.4 SCEs per cell per generation, and that in the human these frequencies are invariant at the lowest BrdU levels. This suggests that SCEs are an integral part of DNA replication, even in the absence of agents known to induce SCEs. The distribution of SCEs per chromosome was analyzed and found to be Poisson-distributed in all 24 murine cultures and in 25 of 36 human cultures. The distribution of SCEs per chromosome may be due to either species-specific chromosome packaging or to karyotypic differences between the species.     

In vivo sister chromatid differentiation and baseline sister chromatid exchanges in a mouse ascites tumour model.

Induction of differentially stained sister chromatids at G2/M and determination of baseline sister chromatid exchanges (SCEs) in ascites form of mouse sarcoma 180 cell line have been done by in vivo incorporation of 5-bromodeoxyuridine (BrdU) for two consecutive DNA replication cycles. The baseline SCE frequency is 6.24 at log phase of tumour growth.     

The effects of incorporated tritium and bromodeoxyuridine on the frequency of sister chromatid exchanges

Cells in third mitosis treated during the first cell cycle with ³H-TdR and during the next two cycles with BrdU (without ³H-TdR) show a typical pattern of chromosome differentiation which allows identification of sister chromatid exchanges occurring during the first (SCE₁, second (SCE₂) and third cycles (SCE₃). Chromosomes labeled only with ³H-TdR had the most SCEs; those labeled only with BrdU, the second highest number; and those labeled with ³H-TdR plus BrdU, the fewest. Since BrdU and ³H-TdR are well known inducers of SCEs, the relatively low frequency of exchanges produced by the combined action of these two compounds is paradoxical. — It is assumed that SCEs are generated by the abnormal recombination of double-strand DNA breaks occurring at the junctions between completely and partially duplicated replicon clusters. Thus, agents that induce absolute blocks to DNA fork displacement will favor the appearance of SCEs because double-strand breaks have more time to occur at junctions. Conversely, agents that inhibit the initiation of replication will decrease the probability of SCEs. Ionizing radiation delays the onset of cluster replication. Therefore, in ³H-TdR plus BrdU-substituted chromosomes the radiation from tritium may inhibit the appearance of BrdU-induced SCEs. Since the inhibition does not exist in chromosomes substituted only with BrdU, the frequency of SCEs in these elements is higher than in double-substituted chromosomes. During the first cell cycle the onset of cluster replication is normal. However, the incorporation of ³H-TdR in the replication fork may enhance the appearance of double-strand breaks, thus inducing a high frequency of SCEs.