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.     

Approximation of baseline and BrdU-induced SCE frequencies

In the present paper we have developed a new rationale and an experimental schedule to approximate the frequency of SCEs which occur independently of BrdU incorporation, namely, the baseline frequency of SCEs. The method used includes the analysis of SCE yields in second and third division chromosomes after BrdU-substitution for 1, 2, and/or 3 successive replication rounds in the presence of this thymidine analogue, leading to a set of ten different experimental results. As a result of formulating various mathematical equations and applying them to the data, an accurate estimation of the frequency of baseline (BrdU-independent) and BrdU-induced SCEs, can be made, thus avoiding the difficulties inherent in the current extrapolation methods. The conclusions are that 1) SCEs seem to be formed after DNA synthesis (by exchanging post-replicative DNA portions), but, obviously, very near to the replication fork and 2) that under our experimental conditions about 0.065 SCEs per picogram of DNA per cell cycle occur as a consequence of chromosome replication, this frequency being increased by BrdU-substitution. The methodology seems to be reliable enough to be used in other species and systems in order to compare baseline SCE frequencies.Abbreviations SCEs sister-chromatid exchanges - BrdU(BrdUrd) 5-bromodeoxyuridine - dTh(dThd) thymidine - ³H-dTh(³H-dThd) tritiated thymidine - FdU(FdUrd) 5-fluorodeoxyuridine - Urd uridine - FPG fluorescent plus Giemsa     

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.     

Evidence That BRCA1- or BRCA2-Associated Cancers Are Not Inevitable

Inheriting a BRCA1 or BRCA2 gene mutation can cause a deficiency in repairing complex DNA damage. This step leads to genomic instability and probably contributes to an inherited predisposition to breast and ovarian cancer. Complex DNA damage has been viewed as an integral part of DNA replication before cell division. It causes temporary replication blocks, replication fork collapse, chromosome breaks and sister chromatid exchanges (SCEs). Chemical modification of DNA may also occur spontaneously as a byproduct of normal processes. Pathways containing BRCA1 and BRCA2 gene products are essential to repair spontaneous complex DNA damage or to carry out SCEs if repair is not possible. This scenario creates a theoretical limit that effectively means there are spontaneous BRCA1/2-associated cancers that cannot be prevented or delayed. However, much evidence for high rates of spontaneous DNA mutation is based on measuring SCEs by using bromodeoxyuridine (BrdU). Here we find that the routine use of BrdU has probably led to overestimating spontaneous DNA damage and SCEs because BrdU is itself a mutagen. Evidence based on spontaneous chromosome abnormalities and epidemiologic data indicates strong effects from exogenous mutagens and does not support the inevitability of cancer in all BRCA1/2 mutation carriers. We therefore remove a theoretical argument that has limited efforts to develop chemoprevention strategies to delay or prevent cancers in BRCA1/2 mutation carriers.     

The asymmetric methylation of CG palindromic dinucleotides increases sister-chromatid exchanges

Treatment with the base analogue, 5azaC, increases SCEs in CHO but not in mosquito cells. On the other hand, both types of cells show equivalent increases in exchanges when treated with other compounds, such as mitomycin C. Vertebrate DNA is heavily methylated while diptera DNA is heavily demethylated. The sequence of events leading to an increase in SCEs in CHO cells is as follows: first of all, Cs are replaced by 5azaC; in the next cell cycle, CG palindromic dinucleotides exhibit an asymmetric configuration, the Cs in the parental DNA strand being methylated and the Cs in the daughter DNA strand demethylated; after one more cycle, half of the chromosomes show symmetric methylation and the other half symmetric demethylation of both Cs in CG palindromes. The increase of SCEs occurs in the second cell cycle when the hemimethylated DNA enters replication. DNA hemimethylation is believed to be an intermediate stage in the process of demethylation that accompanies gene expression. If so, gene demethylation would be a cause of SCE increase in normal vertebrate cells.     

Aphidicolin-Resistant Mutants of Mouse Lymphoma L5178y Cells with a High Incidence of Spontaneous Sister Chromatid Exchanges

Two aphidicolin-resistant cell mutants (AC 12 and AC 41) with a fourfold increase in spontaneous frequency of sister chromatid exchanges (SCEs) were obtained out of over 400 aphidicolin-resistant mutants isolated from mouse lymphoma L5178Y cells. They also exhibited three- to fourfold increases in spontaneous frequency of chromosome aberrations (CAs). To determine whether the high level of SCE frequency in AC 12 is caused by 5-bromodeoxyuridine (BrdUrd) used for visualizing SCEs, the effect of BrdUrd incorporated into DNA on SCE induction was analyzed. The SCE frequencies in AC 12 remained constant at BrdUrd incorporation levels corresponding to 2-90% substitution for thymidine in DNA. In addition, the small amount of BrdUrd incorporated into both daughter and parenteral DNA strands in AC 12 had minimal effect on SCE induction. Furthermore, AC 12 and AC 41 were slightly resistant to BrdUrd with respect to the induction of CAs, the inhibition of cell-cycle progression and the decrease in mitotic activity. These findings suggest that the high incidence of SCEs in AC 12 and AC 41 is formed by their intrinsic defects, not by the effects of BrdUrd used. The analysis of SCE frequencies in hybrid cells between these mutants and the parental L5178Y revealed that the genetic defects in AC 12 and AC 41 appear to be recessive, and that these two mutants belong to the same complementation group. Furthermore, AC 12 belonged to a different complementation group from ES 4, which was isolated previously from L5178Y as an SCE mutant with a twofold higher frequency of spontaneous SCEs. This finding indicates that at least two different genetic defects participate in the formation of the high incidence of spontaneous SCEs in mouse cells. These SCE mutants would provide valuable cell materials for studying the molecular mechanism of SCE formation.     

A replication model for sister-chromatid exchange

A model for the production of sister-chromatid exchanges is presented, based on the idea that double-strand breaks are generated at junctions between a completely duplicated replicon cluster and a partially duplicated replicon cluster. Agents that induce absolute blocks to DNA fork displacement will cause this condition to persist longer than normal, whereas agents that inhibit initiation of whole clusters will rarely cause it at all. During the blunt-end repair of the double-strand breaks, sister-chromatid exchange would be initiated when daughter strands of a duplicated cluster recombine with the parental strands of the partially replicated cluster. When the latter finishes replication, sister-chromatid exchange would be completed.     

The diplochromosome of endoreduplicated cells: A new approach to highlight the mechanism of sister chromatid exchange

Chinese hamster lung embryonic cells (CL1) were treated with colchicine in order to induce endoreduplication and subsequently with mitomycin-C (MMC) to induce exchanges within the diplochromosome. The use of chromosomal differential staining through incorporation of 5-bromodeoxyuridine, resulting in only one stained chromatid, has allowed the analysis of all classes of exchanges among the four chromatids of the diplochromosome. Three classes of exchanges may occur: intradiplochromatid exchanges (ICEs) between the two inner chromatids, cousin chromatid exchanges (CCEs) between one inner and one outer chromatid, and sister chromatid exchanges (SCEs) between the two sister chromatids of the diplochromosome. The results show that MMC treatment, in the last cell cycle of endoreduplication, as expected, significantly increases only the frequency of SCEs, whereas the frequency of ICEs and CCEs remains unchanged. This result supports replication models of formation of SCEs. Furthermore the fact that the number of ICEs does not increase means that the molecular mechanism of somatic crossing over is not related to that of SCE formation, or very rarely. The results also indicate a statistically significant lower induction of SCEs in endoreduplicated metaphases as compared with diploid ones both in control and MMC-treated cells. Such a result may be due to structural restrictions within the diplochromosome. Received: 29 December 1995; in revised form; 4 March 1996 / Accepted: 24 March 1996     

Antagonization by cycloheximide of ethyl methanesulfonate-induced 6-thioguanine-resistant mutation and sister-chromatid exchanges

We studied the effect of cycloheximide (CH) on the induction of mutation and sister-chromatid exchanges (SCEs) in ethyl methanesulfonate (EMS)-treated Chinese hamster cells. At 10^?6 M, CH strongly antagonized the induction of mutation and SCEs and cell survival increased. This suggests that protein synthesis is essential for the induction of mutation as well as SCEs. Results of experiments in which CH treatment preceded or followed exposure to mutagens were similar with respect to the response curves obtained for mutation and SCEs.     

Influence of sodium butyrate on the induction of radiation-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells.

CHO cells were pre-treated with sodium butyrate (SB) for 24 h and then X-irradiated in G1. Metaphases were scored for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs). The data were compared with those obtained after irradiation of cells not pre-treated with SB and showed that SB has different effects on the endpoints examined. The frequencies of dicentric chromosomes were elevated and of small acentric rings (double minutes, DMs) reduced. These results are discussed to be a consequence of conformational changes in hyperacetylated chromatin which could lead to more interchromosomal and to less intrachromosomal exchanges. SB itself induces a few SCEs but suppresses the induction of SCEs by X-rays. We assume that a minor part of radiation induced SCEs are 'false' resulting from structural chromosomal aberrations, such as inversions, induced in G1. Inversions are the symmetrical counterparts of DMs. If inversions are suppressed by SB treatment to a similar extent as DMs a small reduction of SCEs by SB can be expected.     

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.     

The Bloom's syndrome helicase can promote the regression of a model replication fork

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called "chicken foot" structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges.     

Insights into the mechanisms of sister chromatid exchange formation

The DNA lesions responsible for the formation of sister chromatid exchanges (SCEs) have been the object of research for a long time. SCEs can be visualized by growing cells for either two rounds of replication in the presence of 5-bromo-2'-deoxyuridine (BrdU) or for one round with BrdU and the next without. If BrdU is added after cells were treated with a DNA-damaging agent, the effect on SCEs can only be analyzed in the second post-treatment mitosis. If one wishes to analyze the first post-treatment mitosis, cells unifilarily labeled with BrdU must be treated. Due to the highly reactive bromine atom, BrdU interacts with such agents like ionizing and UV radiation enhancing the frequency of SCEs. However, its precise role in this process was difficult to assess for a long time, because no alternative technique existed that allowed differential staining of chromatids. We have recently developed a method to differentially label sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) circumventing the disadvantage of BrdU. This technique was applied to study the SCEs induced by ionizing and UV radiation as well as by mitomycin C, DNaseI and AluI. This article is a review of the results and conclusions of our previous studies.     

Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 265-313 nm monochromatic ultraviolet wavelengths and photoreactivating light

Exposure of ICR 2A frog cells to 265 nm, 289 nm, 302 nm or 313 nm monochromatic ultraviolet (UV) wavelengths induced the formation of sister-chromatid exchanges (SCEs). However, treatment of cells with photoreactivating light (PRL) following the UV irradiations resulted in a lower level of SCEs compared with cells incubated in the dark. Hence, it can be concluded that pyrimidine dimers are the principal photoproducts responsible for the induction of SCEs in cells exposed to 265-313 nm UV due to the specificity of DNA photolyase for the light-dependent monomerization of dimers in DNA. It was also found that the maximum yield of induced SCEs in 313 nm-irradiated cells was only about 7 SCEs per cell whereas the plateau values for the shorter wavelengths were approximately 15-20 SCEs per cell. In addition, treatment of cells with 313 nm plus 265 nm light resulted in a lower level of SCEs than in cells exposed to 265 nm UV alone. These results can be interpreted in the context of a replication model for SCE, in which the high level of non-dimer damages produced in the DNA of 313 nm-irradiated cells inhibits the induction of SCEs by the pyrimidine dimers that are also produced by this wavelength.     

Simultaneous staining of sister chromatid exchanges and Q-bands in human chromosomes after treatment with methyl methane sulphonate,quinacrine mustard,and quinacrine

Summary Human peripheral lymphocyte chromosomes were stained simultaneously for sister chromatid exchanges (SCEs) and Q-banding. No effect of treatment with MMS, QM, and Q on the distribution of SCEs in chromosomes was found compared with controls. The SCEs were distributed between chromosomes roughly according to metaphase length, with the shorter chromosomes underrepresented. The majority of SCEs were located to pale bands, while a few occurred in bright bands and at interfaces between pale and bright bands. A greater frequency than expected of SCEs had occurred at identical sites in homologous chromosomes. This frequency was significantly increased after treatment with MMS.     

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.     

Persistence of DNA lesions and the cytological cancellation of sister chromatid exchanges

The ability of UV light, mitomycin C and ionizing radiation to induce the formation of sister chromatid exchanges (SCEs) at the same locus in successive cell generations was investigated in human lymphocytes. Cells were exposed to the DNA damaging agents after they had completed their first round of DNA replication, and SCEs were examined at the third division in chromosomes that had been differentially stained three ways. Although some of these treatments induced long-lived lesions that increased the frequency of SCEs in successive cell generations, none of the lesions led to the formation of consecutive SCEs at the same locus in successive cell generations. This observation seriously challenges the hypothesis that SCE cancellation results as a consequence of persistence of the lesions induced by these agents.     

In vivo sister chromatid differentiation and base line sister chromatid exchanges in Channa punctatus.

An in vivo system for differentially stained sister chromatids by incorporating 5' Bromo 2' deoxyuridine at two consecutive round of DNA replication has been developed in C. punctatus. The base line developed frequency of sister chromatid exchanges (SCEs) was found to be 0.038 SCE/chromosome. This low baseline frequency of SCEs could be useful in detecting genotoxicity of pollutants in aquatic medium.     

Induction of chromosome damage by benzo[a]pyrene, 2-aminofluorene and cyclophosphamide in the root cells of Vicia faba

The induction of sister-chromatid exchanges (SCEs) and chromosome aberrations (CAs) by benzo[a]pyrene (BP), 2-aminofluorene (2-AF) and cyclophosphamide (CP) in the root cells of Vicia faba was examined. BP and 2-AF induced CAs, but not SCEs. CP induced both SCEs and CAs.     

Sister chromatid exchanges in Drosophila melanogaster cell lines in vitro

The frequency of sister chromatid exchanges (SCEs) in two cell lines of Drosophila melanogaster with different karyotypes (XX and XY) was determined, considering (1) the distribution of SCEs within each chromosome, with reference to eu- and heterochromatin and (2) the distribution of SCEs in different chromosomes. A comparison was made between chromosome pairs within each karyotype and between the two different karyotypes. The following results were obtained. The SCEs are not randomly distributed along chromosomes, since exchanges were never observed in heterochromatin. SCEs are more frequent in XY than in XX cells; moreover, in both cell types there exists a significantly higher frequency of SCEs in the X chromosome than in the autosomes. These findings are discussed in relation to chromosome aberrations and mitotic recombination.