Bloom's syndrome and EM9 cells in BrdU-containing medium exhibit similarly elevated frequencies of sister chromatid exchange but dissimilar amounts of cellular proliferation and chromosome disruption

Bloom's syndrome (BS) and EM9 cells both display elevated frequencies of sister chromatid exchange (SCE) following growth for two rounds of DNA replication in bromodeoxyuridine (BrdU)-containing medium. To learn whether hyperresponsiveness to BrdU itself might play a role in causing the SCE elevation, the effects of BrdU on two other parameters, cellular proliferation and chromosome disruption, were examined, comparing the responses of BS and normal lymphoblastoid cells and of EM9 and CHO cells. BS and normal cells responded similarly with respect to growth for 4 days in BrdU-containing medium (0, 1, 3, and 5 g/ml). Chromosome aberrrations were increased only slightly in the BS and normal cells after 2 days in BrdU. CHO cells responded to growth in BrdU-containing medium like BS and normal cells; however, little growth of EM9 was detected at any of the BrdU concentrations employed. CHO and EM9 cells also exhibited strikingly different amounts of chromosome damage following growth in BrdU. After 2 days in 1, 3, and 5 g/ml BrdU 21%, 46%, and 50%, respectively, of the CHO cells had chromosome aberrations in contrast to 92%, 96%, and 98% of the EM9 cells. Most of the aberrations in the BrdU-treated CHO cells consisted of what appeared to be polycentric and ring chromosomes or chromosomes exhibiting telomere association. Acentric fragments were absent from most cells with polycentric and ring chromosomes, indicating either that the abnormal chromosomes were formed during an earlier cell cycle or that the abnormal chromosomes represent a form of association in which the telomeres are apposed so tightly that the juncture between chromosomes cannot be identified microscopically. EM9 cells treated with BrdU exhibited many chromatid and isochromatid gaps and breaks as well as numerous quadriradial, triradial, and complex interchange configurations. In addition, the types of aberrations present in CHO cells also were increased greatly in number. The different responses of BS and EM9 cells to growth in BrdU suggest that the molecular defects in the two cell types are different.     

Implications of an elevated sister-chromatid exchange frequency in rat lymphocytes cultured in the absence of erythrocytes

One important variable in complex culture systems such as whole blood is the interaction of the cell types present. To investigate the effects of erythrocytes (RBCs) and monocytes on the sister-chromatid exchange (SCE) frequency, Ficoll-Hypaque-separated Fischer-344 rat leukocytes were added to 1.9 ml of culture medium containing either 4 micrograms phytohemagglutinin or 4-8 micrograms concanavalin A/ml. Bromodeoxyuridine (BrdU;2 microM) was added at 24 h, and the cultures were harvested at 54 or 72 h. SCE frequencies in the mononuclear leukocyte cultures were consistently about 1.5- to 2-fold higher than in the whole-blood cultures. The titration of rat or human RBCs (0.05-2.5 X 10(9)) into purified rat leukocyte cultures reduced the SCE frequency to that of whole-blood cultures. Monocyte depletion decreased the elevated SCE frequency by approximately 50%. Scintillation counting of [14C]BrdU uptake in isolated RBCs revealed that less than 8% of the total amount of BrdU was sequestered. Also, BrdU induced a concentration-dependent increase in SCE in purified leukocytes, but the absolute increase was no greater than in whole-blood lymphocytes. Thus, BrdU had a minor role in the elevated SCE frequency in purified lymphocytes. Neither anti-oxidant enzymes such as catalase and superoxide dismutase nor the hydroxyl radical scavenger, dimethyl sulfoxide, decreased the SCE frequency. Although purified human lymphocytes had a small, but significant increase in SCE compared to whole blood, the magnitude of the dichotomous response between man and rat may represent a fundamental species difference.     

Construction of human XRCC1 minigenes that fully correct the CHO DNA repair mutant EM9.

The human gene that corrects the DNA repair defect of the CHO cell mutant EM9 is designated XRCC1 and is the first human gene to be cloned that has an established role in DNA strand-break repair. In this study, either an XRCC1 cosmid genomic fragment or synthetic oligonucleotides were ligated to an incomplete XRCC1 cDNA to generate two full-length XRCC1 cDNA constructs. The ability of these minigene constructs to restore normal levels of sister chromatid exchange (SCE) to EM9 upon transfection was demonstrated, and the transfectants grew at normal rates in selective medium that is fully toxic to EM9 cells. Constructs in which the XRCC1 open reading frame (ORF) was transcribed from the SV40 early promoter or the genomic XRCC1 native promoter were compared in their efficiency of correction. EM9 transfectants derived from the SV40 promoter displayed fewer SCEs and lower sensitivity to CldUrd than either AA8 wild-type cells or transfectants containing the ORF transcribed from the native promoter.     

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19

The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinese-hamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repair-deficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.     

5-Bromodeoxyuridine-dependent increase in sister chromatid exchange formation in Bloom's syndrome is associated with reduction in topoisomerase II activity

Bloom's syndrome is characterized by a high sister chromatid exchange (SCE) frequency, the basis for which is not yet understood. Immunofluorescent detection of SCE formation in dermal fibroblasts was employed over a wide range of 5-bromodeoxyuridine (BrdU) substitution into template DNA to show that this SCE elevation reflects both an increased baseline SCE frequency and an exaggerated increment in SCE formation as BrdU substitution increases. The impact of BrdU on SCE formation in Bloom's syndrome is paralleled by its ability to reduce the activity in nuclear extracts of topoisomerase II, an enzyme important for DNA replication and interchange. The extractable topoisomerase II activity of Bloom's syndrome fibroblasts, as measured by unknotting of page P4 DNA, is much more strongly inhibited by cell growth in medium containing BrdU than is that of normal fibroblasts. The results are consistent with the hypothesis that much of the BrdU-dependent component of SCE formation in Bloom's syndrome may be mediated by an effect of BrdU substitution of template DNA on topoisomerase II activity.     

Strand breaks arising from the repair of the 5-bromodeoxyuridine-substituted template and methyl methanesulphonate-induced lesions can explain the formation of sister chromatid exchanges

5-Bromodeoxyuridine (BrdU)-induced sister chromatid exchanges (SCEs) are mainly determined during replication on a BrdU-substituted template. The BrdU, once incorporated, is rapidly excised as uracil (U), and the gap is repaired with the incorporation of BrdU from the medium, which leads to further repair. During the second S period in BrdU medium, this process continues as the strand acts as template. Experiments suggest that 3-amino-benzamide (3AB) delays the ligation of the gaps formed after U excision, resulting in enhanced SCE levels during the second cycle of BrdU incorporation. When normal templates of G1 cells are treated before BrdU introduction with methyl methanesulphonate (MMS), 3AB in the first cycle doubles the MMS-induced SCEs but has no effect on them during the second cycle. When the BrdU-substituted template is treated with MMS in G1 of the second cycle, 3AB again doubles the SCEs due to MMS and also enhances the SCEs resulting from delays in ligation of the gaps following U excision in the BrdU-substituted template. The repair processes of MMS lesions that are sensitive to 3AB and lead to SCEs take place rapidly, while the repair process of late repairing lesions that lead to SCEs appear to be insensitive to 3AB. A model for SCE induction is proposed involving a single-strand break or gap as the initial requirement for SCE initiation at the replicating fork. Subsequent events represent natural stages in the repair process of a lesion, ensuring replication without loss of genetic information. G1 cells treated with methylnitrosourea (MNU) and grown immediately in BrdU medium rapidly lose the O⁶-methylguanine from their DNA and the rate of loss is BrdU-dose dependent. The rapid excision of the U lesions can explain the effect of BrdU concentration on SCE reduction following both MNU or MMS treatment.     

Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients

The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.     

Cytotoxic and genotoxic effects of bromodeoxyuridine during in vitro labelling for sister-chromatid differentiation in preimplantation mouse embryos

Exposure of preimplantation mouse embryos in culture to bromodeoxyuridine (BrdU) in the concentration range of 10(-9) to 2 x 10(-6) M allows sister-chromatid differentiation at the morula and blastocyst stage. The same BrdU concentrations induced no chromosomal aberrations, but a prolongation of the cell cycle and an increase of the SCE frequency. Even at the lowest BrdU concentration for sister-chromatid differentiation (10(-9) M the background level for SCE was found to be significantly higher in early embryos than in fetal or adult tissues of the mouse. Therefore, the high SCE frequency seems to be characteristic of undifferentiated embryonic cells. Methodological recommendations are also given for SCE assay in preimplantation mouse embryos.     

The interaction of Hoechst 33258 and BrdU substituted DNA in the formation of sister chromatid exchanges

Sister chromatid exchanges (SCEs) are induced in cultured Chinese hamster cells by treatment with 5-bromodeoxyuridine (BrdU) or with Hoechst 33258 (H33258) plus BrdU. The SCE frequencies depend upon the number of H33258 molecules available per cell (or per base pair) and the number of brdU molecules available per cell, and not solely upon molarity. In addition, H 33258 and BrdU act synergistically to induce SCEs. At low BrdU concentrations H33258 induces very few SCEs. At high BrdU concentrations and similar concentrations of H33258, however, SCE frequencies are significantly increased. SCE frequencies decrease with time in successively harvested cells because of the depletion of H33258 from the medium due to DNA binding.     

Factors influencing the frequency of mitomycin C-induced sister-chromatid exchanges in 5-bromodeoxyuridine-substituted human lymphocytes in culture.

Newly developed techniques for the detection of sister-chromatid exchanges (SCE) require the substitution of 5-bromodeoxyuridine (BrdU) for thymidine in DNA. We investigated the possibility of interactions between BrdU and one mutagen--carcinogen, mitomycin C (MMC) for the induction of both chromosomal aberrations and SCE in human peripheral lymphocytes in culture. No effect on aberration yield was found. Neither comparisons between the yields of SCE by BrdU substitution and differential staining and those detected by tritiated thymidine incorporation and autoradiography nor between the yields of SCE for different levels of BrdU incorporation provided any evidence of synergism. It was found, however, that MMC persists in cultures and continues to increase SCE frequencies for about 30 h. It was also observed that some MMC-induced DNA lesions persist long enough so that some of those present prior to S phase of the first cell cycle cause additional SCE in the third cycle.     

Effect of free 5-bromodeoxyuridine on induction of SCE in human lymphocytes gamma-irradiated at the presynthetic stage of primary and secondary mitotic cycles

Formation of SCE was studied in lymphocytes irradiated by 60Co gamma-rays at the G1 stage of the first or second mitotic cycles. The yield of SCEs induced by irradiation in the presence of 5-bromodeoxyuridine (BrdU) proved to be significantly higher than that obtained in the absence of BrdU. The enhancing influence of BrdU on SCE induction depends on neither replication cycle nor the molecular constitution of chromosomes under irradiation. Direct modification of chromosomal radiosensitivity by BrdU is excluded. The results obtained suggest the interference of free BrdU present in culture medium with processes of DNA reparation at the G1 stage.     

Increased sister-chromatid exchanges (SCEs) and chromosomal fragilities by BrdU in a human mutant B-lymphoblastoid cell line

From an X-irradiated human B-lymphoblastoid cell line (CCRF-SB), we have isolated a unique mutant clone (CCRF-SB-T1) which reveals high frequencies of sister-chromatid exchanges (SCEs) and chromosomal fragilities in the C-band regions of chromosomes Nos. 1, 9 and 16, when exposed to high concentrations of bromodeoxyuridine (BrdU). A clear BrdU dose-dependent increase of SCEs (9.6 SCEs/cell at 0.05 mM, 40 SCEs/cell at 0.37 mM on average) in this mutant was observed. Relative contributions of nucleoside and a thymidine (dT) analog of BrdU to high SCEs were studied, since an unusual SCE response to BrdU led us to suspect the significance of BrdU incorporation into DNA and dT pool disturbances. Addition of deoxycytidine (dC), dT or both dC and dT causes an increase of SCEs. On the other hand, deoxyadenosine (dA) and deoxyguanosine (dG) did not have significant effects on SCEs in SB-T1 cells. These results suggest that disturbances of pyrimidine-nucleotide synthesis, including gross imbalance of nucleotide pools, play a pivotal role in the high SCE induction of SB-T1 cells by BrdU.     

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.     

Sister chromatid exchanges in bromine incorporation into DNA cytosine nucleotides. III. 5-bromodeoxycytidine as a DNA thymine precursor. The characteristics of the exchanges]

Cell movement through the mitotic cycle and sister chromatid exchanges (SCE) were studied in human blood lymphocytes cultured in the presence of 5-bromodeoxycytidine (BrdC, 0.05 mM) plus thymidine (dT 0.4, 0.8, and 1.0 mM). In controls, lymphocytes were cultivated in the presence of 5-bromodeoxyuridine (BrdU, 0.05 mM) and deoxycytidine (0.1 mM), or BrdC alone. All nucleosides were added to the cultures 28 hours prior to fixation and were maintained in the medium for 16 hours. As determined from percentage of metaphases of 1st to 3rd divisions, BrdC did not release from thymidine block. This fact leads us to conclude that BrdC in contrast to deoxycytidine does not serve as a cytosine precursor. No significant differences in the frequency of SCE and their distribution among chromosomes were found between cultures treated with BrdC and with BrdU.     

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.     

Spontaneous and mitomycin-C induced sister-chromatid exchanges : Comparison of in vivo and in vitro systems

Frequencies of sister-chromatid exchanges (SCE) were measured in vitro in mouse fibroblasts and in vivo in mouse bone-marrow cells. SCE levels in these cell systems were measured in response to varying concentrations of bromodeoxyuridine (BrdU) and mitomycin-C (MMC). Although BrdU was found to induce SCE in both cellular systems, baseline SCE levels were 2- to 3-fold higher in vitro than in vivo. SCE induction was found to be a linear function of MMC concentration in vivo and in vitro; however the slope of the in vivo curve was 5-fold higher. The interaction of BrdU substituted DNA and MMC was examined by administering a fixed dose of MMC with increasing concentrations of BrdU. The induced SCE frequencies appeared to be additive. In addition to measuring drug-induced SCE, the BrdU differential staining technique allows concomitant measurement of the inhibition of cellular replication by the test drugs.     

Effects of amino acids on sister-chromatid exchanges.

The effects of 10 amino acids on sister-chromatid exchange (SCE) frequency in human peripheral blood lymphocytes (PBL) and six amino acids on the SCE frequency in root tip cells of Hordeum vulgare were studied. Alanine (Ala), glycine (Gly), phenylalanine (Phe), valine (Val), histidine (His) and serine (Ser) induced a significant increase in SCE in PBL but threonine (Thr), isoleucine (Ile), lysine (Lys) and arginine (Arg) did not. Ala, Gly, Thr, Ile and Val induced a significant increase in SCE in root tip cells of Hordeum vulgare but Lys did not. The effect of Lys and bromodeoxyuridine (BrdU) on SCE levels in PBL and the interaction between them were also studied. The results show that Lys can inhibit the SCE induced by BrdU.     

Sister chromatid exchange in the centromere and centromeric area

Central and peripheral sister chromatid exchanges (SCE) were evaluated separately in human phytohemagglutinin (PHA)-stimulated lymphocytes after culture for 72 h in 5-bromodeoxyuridine (BrdU) containing medium. At the same time, the length of chromosome No. 1 was measured in 10 metaphases per case and the mean value taken as a representative parameter for the contraction of chromosomes. The statistical analysis of regression revealed a close relationship between the percentage of SCE observed in the centromere and the contraction state of chromosomes (P less than or equal to 0.01). A statistically significant increase of central exchanges was seen in more condensed chromosomes, due to the difficulty in differentiating clearly between centric and pericentric exchanges. Consequently, if exchanges in the centromere are omitted from evaluation, this would lead to spuriously low SCE rates in more contracted chromosomes. In order to exclude the variable factor of chromosome contraction in SCE studies, we highly recommend inclusion of counts of central exchanges. Results obtained on chromosomes with twisted chromatids, a situation which tends to stimulate SCE, should be omitted.     

Spontaneous sister-chromatid exchange frequencies in human B and T lymphocytes at BrdU borderline concentrations for sister-chromatid differentiation

Human peripheral blood B and T lymphocytes, highly purified by immunologic methods, were supplemented with gamma-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohemagglutinin, respectively. Spontaneous sister-chromatid exchange (SCE) frequencies were investigated in proliferating B and T lymphocyte cultures labeled with the cell-type-specific borderline concentrations of 5-bromodeoxyuridine (BrdU) for sister-chromatid differentiation (SCD). B lymphocytes from 6 different donors showed mean values of 3.28-3.72 SCE events/cell. In T lymphocytes, mean values of 6.30-7.28 SCEs/cell were observed. The differences between the SCE distributions of the cell populations are highly significant. The results show that the differences in the spontaneous SCE frequencies between human B and T lymphocytes were not due to a difference in the uptake of BrdU.     

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.