Sister Chromatid Exchange Formation Research Articles

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.

Frequent occurrence of UVB-induced sister chromatid exchanges in telomere regions and its implication to telomere maintenance

Sister chromatid exchanges (SCEs) are symmetrical exchanges between newly replicated chromatids and their sisters. While homologous recombination may be one of the principal mechanisms responsible for SCEs, the full details of their molecular basis and biological significance remain to be elucidated. Following exposure to ultraviolet light B (UVB), mitomycin C (MMC) and cisplatin, we analyzed the location of SCEs on metaphase chromosomes in Chinese hamster CHO cells. The frequency of SCEs increased over the spontaneous level in proportion to the agent’s dose. UVB-induced SCEs occurred frequently in telomere regions, as cisplatin-induced SCEs did, differing from MMC-induced ones. The remarkable difference of intrachromosomal distribution among the three mutagens may be attributed to the specificity of induced DNA lesions and structures of different chromosome regions. Telomeric DNA at the end of chromosomes is composed of multiple copies of a repeated motif, 5′-TTAGGG-3′ in mammalian cells. Telomeric repeats may be potential targets for UVB and cisplatin, which mainly form pyrimidine dimers and intrastrand d(GpG) cross-links, respectively, resulting in SCE formation. UVB irradiation shortened telomeres and augmented the telomerase activity. The possible implications of the frequent occurrence of SCEs in telomere regions are discussed in connection with the maintenance of telomere integrity.

Ablation of PARP-1 does not interfere with the repair of DNA double-strand breaks, but compromises the reactivation of stalled replication forks.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant DNA end-sensing and binding molecule. Inactivation of PARP-1 by chemicals and genetic mutations slows cell proliferation, increases sister chromatid exchange (SCE), micronuclei formation and chromosome instability, and shortens telomeres. Given its affinity to DNA breaks and temporal occupation on DNA strand break sites, PARP-1 is proposed to prevent inappropriate DNA recombination. We investigated the potential role of PARP-1 in repair of DNA double-strand breaks (DSBs) and stalled replication forks. PARP-1-/- embryonic stem cells and embryonic fibroblast cells exhibited normal repair of DNA DSBs by either homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways. Inactivation of PARP-1 did not interfere with gene-targeting efficiency in ES cells. However, PARP-1-/- cells were hypersensitive to the replication damage agent hydroxyurea (HU)-induced cell death and exhibited enhanced SCE formation. Ablation of PARP-1 delayed reactivation of stalled replication forks imposed by HU and re-entry into the G2-M phase after HU release. These data indicate that PARP-1 is dispensable in HR induced by DSBs, but is involved in the repair and reactivation of stalled replication forks.

Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition

We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy- trans-stilbene, 3-hydroxy- trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy- trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy- trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.

Lead acetate genotoxicity on human melanoma cells in vitro.

In this study, chromosomal damage induced in vitro by lead acetate in human melanoma cells (B-Mel) was evaluated using the cytokinesis-blocked micronucleus assay and sister chromatid exchange (SCE) analysis. Lead acetate (10-6, 10-5 and 10-3 mM) induced micronuclei and SCE formation in a dose-dependent manner. Treated cells showed a decrease in cell viability but not concomitant cell death by apoptosis (lead acetate failed to induce internucleosomal DNA fragmentation at any of the doses tested). One important observation emerging from this study was that low-level lead exposure in vitro is able to induce significant cytogenetic damage in human melanoma cells, indicating an increased sensitivity of B-Mel cells to lead acetate.

Effects of Carvacrol on Sister Chromatid Exchanges in Human Lymphocyte Cultures

Carvacrol is a predominant aromatic compound in oil of oregano. It has naturally remarkable antibacterial, antiviral, antifungal and antiparasital effects. In this study, genotoxic and antigenotoxic activities of carvacrol were investigated by the in vitro sister chromatid exchange (SCE) assay on human peripheral blood lymphocytes. The genotoxicity test was performed with carvacrol in two donors. On the other hand, inhibitory effect of carvacrol was tested in the presence of mitomycin C (MMC) in the same assay. According to data, all doses of carvacrol did not increase the formation of SCE, whereas it inhibited the rate of SCE induced by MMC. In conclusion, carvacrol exhibited a significant antigenotoxic activity in mammalian cells, indicating its potential for use as an antigenotoxic agent.

Propofol anesthesia in children does not induce sister chromatid exchanges in lymphocytes

Background: Propofol is frequently used for general anesthesia in children although little is known about possible genotoxic effects in humans. We investigated the formation of sister chromatid exchanges (SCE) in metaphase chromosomes of T-lymphocytes of children as a marker for possible genotoxocity following total intravenous anesthesia with propofol for minor surgical procedures. Methods: 40 children ASA classification I–III were included (ASA I n=34, ASA II n=5, ASA III n=1) in the study. Anesthesia was induced by propofol (3 mg/kg) and alfentanil. Succinylcholine or rocuronium were administered for muscle relaxation. After tracheal intubation anesthesia was maintained by continuos propofol infusion at 12 mg/(kg h). Blood samples were drawn before induction and after termination of anesthesia. Following a 72 h cell culture period, 25 T-lymphocyte metaphases per blood sample for all children were analyzed for SCE frequencies. Results: Total intravenous anesthesia with propofol on children did not influence SCE rates in metaphase chromosomes of T-lymphocytes. No SCE differences could be detected between blood samples before initiation and after termination of anesthesia (Wilcoxon signed rank test). Slightly elevated SCE rates were obtained in T-lymphocytes of girls compared to boys, but these differences did not reach statistical significance. Conclusions: Propofol anesthesia under the chosen conditions did not induce the formation of SCE in children in vivo. No genotoxic effect of a short term exposure to propofol during pediatric anesthesia had been observed.

Functional relation among RecQ family helicases RecQL1, RecQL5, and BLM in cell growth and sister chromatid exchange formation.

Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1(-/-), RECQL5(-/-), RECQL1(-/-)/RECQL5(-/-), RECQL1(-/-)/BLM(-/-), and RECQL5(-/-)/BLM(-/-) cells from chicken B-lymphocyte line DT40 cells. Although BLM(-/-) DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an approximately 10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1(-/-), RECQL5(-/-), and RECQL1(-/-)/RECQL5(-/-) cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1(-/-)/BLM(-/-) and RECQL5(-/-)/BLM(-/-) cells grew more slowly than BLM(-/-) cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5(-/-)/BLM(-/-) cells showed a higher frequency of SCE than BLM(-/-) cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.

Application of the biotin–dUTP chromosome labelling technique to study the role of 5-bromo-2′-deoxyuridine in the formation of UV-induced sister chromatid exchanges in CHO cells

The role of 5-bromo-2′-deoxyuridine (BrdU) in the formation of sister chromatid exchanges (SCEs) in cells exposed to UV radiation was studied. Cells were unifilarily labelled (labelling of one strand of chromosomal DNA) with BrdU or biotin-16-2′-deoxyuridine (biotin-dU) and irradiated in G 1 phase of the cell cycle either with 254 nm, which is absorbed by all nucleobases including bromouracil (BrU) or with 313 nm radiation, which is predominantly absorbed by the BrU moiety. Elevated SCE frequencies were observed in cells irradiated at 254 nm (1.2 and 3.0 J m −2) which were pre-labelled with BrdU or biotin-dU. Following irradiation at 313 nm (38 and 96 J m −2) a statistically elevated SCE frequency was observed in cells pre-labelled with BrdU but not with biotin-dU. In cells pre-labelled with BrdU, UV-radiation at 254 nm was 50–80 times more effective in inducing SCEs than that at 313 nm. This result can be accounted for by the fact that in BrdU-DNA the cross-section for uracilyl radical and bromine atom formation is ∼100-fold higher at 254 nm than that at 313 nm. Upon irradiation at 254 nm, BrdU had a strong sensitising effect on SCE induction: the SCE frequencies observed in cells pre-labelled with BrdU are ∼6 times higher than in cells pre-labelled with biotin-dU. From this it follows that BrdU-induced damage is responsible for more than 80% of the SCEs formed in UV irradiated cells unifilarily labelled with BrdU. Based on photochemical considerations and the fact that chemical agents which form DNA interstrand cross-links are among the most potent inducers of SCEs, we propose that an interstrand cross-link may be the major lesion leading to SCEs in BrdU-labelled cells.

Sevoflurane anaesthesia does not induce the formation of sister chromatid exchanges in peripheral blood lymphocytes of children

Compound A, a degradation product of sevoflurane, has been demonstrated to induce sister chromatid exchanges (SCE) in Chinese hamster ovary cells in vitro as a marker for possible genotoxicity. We investigated the formation of SCE in mitogen-stimulated T-lymphocytes of 40 children undergoing sevoflurane anaesthesia for minor surgical procedures. Anaesthesia was induced by inhalation of up to 8% sevoflurane and maintained at 2.5-3% in oxygen/nitrous oxide (65/35%) at a fresh gas flow of 3 litre min(-1). Soda lime (humidity 12-15%) was used as a carbon dioxide absorbent. Blood was drawn directly before induction and after termination of anaesthesia. Twenty-five second division metaphases of mitogen-stimulated T-lymphocytes per blood sample were screened for SCE rates using standard techniques. Average duration of anaesthesia was 49.6 (SD 24.0) min. Before anaesthesia induction, 7.93 (1.23) SCE per metaphase were determined. After sevoflurane anaesthesia [1.40 (0.77) MAC h] 7.92 (1.19) SCE per metaphase were observed. Additionally, no differences were evident between male or female children. Short-term administration of sevoflurane anaesthesia did not induce SCE in T-lymphocytes of children. No indication for a possible genotoxic effect has been observed.

Accretion of Unstable Heterochromatin as the Origin of Double Minute Chromosomes: Evidence from Bloom Syndrome.

Recent investigations in this laboratory have lead to the formulation of the hypothesis that the mechanism of sister chromatid exchange formation does not involve breakage and rejoining of segments of chromosomes. According to the hypothesis, heterochromatin exists on the chromosome surface exclusively. Sister chromatid exchanges occur when this heterochromatin exists in an unstable state and sections are transposed or translocated between sister chromatids. In order to study this phenomenon further, cells from a high sister chromatid exchange frequency disorder, Bloom syndrome, were analyzed for the presence of unstable heterochromatin. In addition to confirming the presence of unstable heterochromatin, Bloom syndrome cells were often found to contain double minute chromosomes, and these were observed to form by accretion of unstable heterochromatin from the chromosome surface, possibly solving the enigma of their origin and formation.

SCE formation after exposure of CHO cells prelabelled with BrdU or biotin-dUTP to various DNA-damaging agents.

Formation of sister chromatid exchanges (SCE) is a mechanism of repair or bypass of DNA damage during S phase. Although SCE have been studied for a long time, the types of DNA lesions involved and the role of 5-bromodeoxyuridine (BrdU) in SCE formation are a matter of debate. We have developed a novel method of differential labelling of sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) and could show that a substantial proportion of radiation-induced SCE arise via damage to BrdU-moieties. The present investigations were performed to examine the role of BrdU in the formation of SCE by the endonucleases AluI and DNase I, as well as the alkylating agent mitomycin C (MMC). CHO cells unifilarily prelabelled with biotin-dUTP or BrdU were treated and the frequencies of SCE analysed in the first post-treatment mitoses. AluI induced similar frequencies of SCE in cells prelabelled with BrdU or biotin-dUTP. DNase I induced significantly more SCE in cells prelabelled with BrdU than with biotin-dUTP. MMC induced slightly more SCE in cells labelled with biotin-dUTP than BrdU, but the difference was not significant. The possible mechanisms responsible for the enhanced SCE frequency following DNase I treatment of cells prelabelled with BrdU are discussed.

Effect of genotoxic exposure to chromium among electroplating workers in Taiwan

The objective of this study was to investigate the association between chromium (Cr) concentrations and sister chromatid exchanges (SCE) formation in Cr workers and to assess the effects of susceptible genes (glutathione S-transferase M1 and T1) on the frequency of deletion and SCE/cell. Urinary Cr concentration was significantly elevated in Cr workers (3.67±3.89 μg/g creatinine) compared to control group (1.21±1.16 μg/g creatinine, P<0.01). There was also a significant difference of superoxide dismutase (SOD) levels between Cr workers (6.86±0.80 U/mg Hb) and controls (7.16±0.53 U/mg Hb, P<0.01). The frequencies of SCE and high frequency cells (HFC) were significantly correlated with smoking habits and with duration of exposure to Cr. A significantly higher percentage (50%) of Cr workers had both the null GSTM1 and GSTT1 genotype as compared to 10% of the controls ( P<0.01). However, the chromosomal DNA damage effect of GSTM1 and GSTT1 genotypes, individually or in combination, was not revealed in Cr exposed workers.

Role of RAD51 in sister-chromatid exchanges in mammalian cells.

To measure the impact of the RAD51 pathway on Sister-Chromatid Exchanges (SCE), we used hamster cells expressing either the wild-type MmRAD51, which stimulates, or the dominant negative SMRAD51, which inhibits, gene conversion without affecting cell viability of untreated as well as gamma-rays irradiated cells. We show that MmRAD51 did not affect the rate of spontaneous SCE while it strongly stimulated spontaneous recombination between tandem repeats. No spontaneous recombinant was detected when expressing SMRAD51 while spontaneous SCE were only slightly diminished. After treatment by an alkylating agent (MNU), MmRAD51 stimulated MNU-induced recombination whereas no recombinant was detected when expressing SMRAD51. MNU induced SCE in all cell lines, even in the SMRAD51 expressing lines, but the induction of SCE was slightly more efficient in lines expressing MmRAD51 and less efficient in lines expressing SMRAD51. Thus, in mammalian cells, the RAD51-dependent gene conversion pathway drastically affects recombination between intrachromosomal tandem repeats, whereas it only partially participates in SCE formation, measured at a chromosomal level. These results show that RAD51-gene conversion can participate in induced SCE but that alternative pathways should exist.

The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

BackgroundBloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10) or PML (promyelocytic leukemia) nuclear bodies, where it associates with TOPIIIα, and to the nucleolus.ResultsThis report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities.ConclusionThe helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

Reduction of diepoxybutane-induced sister chromatid exchanges by glutathione peroxidase and erythrocytes in transgenic Big Blue® mouse and rat fibroblasts

We have investigated the effect of glutathione peroxidase (GSH-Px) and mammalian erythrocytes (RBCs) on spontaneous and diepoxybutane (DEB)-induced sister chromatid exchange (SCE) in primary Big Blue® mouse (BBM1) and Big Blue® rat (BBR1) fibroblasts. DEB is the putative carcinogenic metabolite of 1,3-butadiene (BD) for which inhalation exposure yields a high rate of malignancies in mice but not in rats. BD is metabolized differently in mice and rats, producing much higher levels of DEB in mice than in rats, which may partly explain the different carcinogenic responses. However, other factors may contribute to the observed differences in the rodent carcinogenic response to BD. DEB is a highly reactive compound. Upon epoxide hydrolysis, DEB can covalently bind to DNA bases. Likewise, DEB generates reactive oxygen species that, in turn, can either damage DNA or produce H 2O 2. Reduced glutathione (GSH) is known to play a role in the metabolism and detoxification of DEB; and GSH is reduced by GSH-Px in the presence of H 2O 2. GSH-Px is a constitutive enzyme that is found at high concentrations in mammalian RBCs. Therefore, we were interested in examining the role of RBCs and GSH-Px on DEB-induced SCE in rat and mouse cells for detection of possible differences in the species response. Transgenic BBM1 and BBR1 fibroblasts were treated with either 0, 2 or 4 μM DEB plus 0, 2 or 20 units of GSH-Px with and without 2×10 8 species-specific RBCs. DEB effectively induced SCEs in both rat and mouse cells. The relative induction of SCEs in both cell types was comparable. Both GSH-Px and RBCs alone and in combination were effective in significantly reducing DEB-induced SCEs in both mouse and rat fibroblasts, although there was more variability in the SCE response in rat cells. The present study suggests that GSH-Px may be important in the detoxification of DEB-induced DNA damage that results in the formation of SCEs.

Negative regulation of alkylation-induced sister-chromatid exchange by poly(ADP-ribose) polymerase-1 activity

One of the earliest responses to DNA damage in eukaryotic cells is activation of poly(ADP-ribose) polymerase-1 (PARP-1), a DNA strand break-dependent nuclear enzyme which covalently modifies proteins with poly(ADP-ribose). Here, we show that conditional over-expression of PARP-1 in stably transfected hamster cells, which causes cellular over-accumulation of poly(ADP-ribose) by several-fold, strongly suppresses alkylation-induced sister-chromatid exchange (SCE), while cytotoxicity of alkylation treatment is slightly enhanced. Viewed together with the known potentiation of SCE by abrogation of PARP-1 activity, our results provide evidence that PARP-1 activity is an important regulator of alkylation-induced SCE formation, imposing a control that is strictly negative and commensurate with the level of enzyme activity.

Poly(ADP-ribosyl)ation: a posttranslational protein modification linked with genome protection and mammalian longevity.

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by the 113-kDa enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and, to a lesser extent, by several other recently described polypeptides. The catalytic function of PARP-1 is directly stimulated by DNA strand breaks, thus making poly(ADP-ribosyl)ation one of the immediate cellular responses to oxidative and other types of DNA damage. Poly(ADP-ribosyl)ation plays an important role in the recovery of proliferating cells from certain types of DNA damage, and this has been linked mechanistically with an involvement in DNA base-excision repair. Furthermore PARP-1 activity is necessary to maintain genomic stability under conditions of genotoxic stress and is actually a key regulator of alkylation-induced sister-chromatid exchange formation, imposing a control that is strictly negative and commensurate with the enzyme activity level. Finally, there is a positive correlation between the poly(ADP-ribosyl)ation capacity of mononuclear leukocytes of various mammalian species and species-specific life span. Likewise, lymphoblastoid cell lines derived from human centenarians display a higher poly(ADP-ribosyl)ation capacity than controls. In conclusion, PARP-1 may be viewed as a factor that is responsible for downregulating the rate of genomic instability events, which are provoked by the constant attack by endogenous and exogenous DNA-damaging agents, in such a way as to tune them to a level which is just appropriate for the life span potential of a given species.

Sister chromatid exchange formation in mammalian cells is modulated by deoxyribonucleotide pool imbalance.

The formation of sister chromatid exchanges (SEC) is closely related to DNA replication which is dependent on the proper balance of deoxyribonucleic triphosphates. Since DNA precursor imbalances can influence DNA metabolism, the relationship between 5-bromodeoxyuridine (BrdUrd)-induction of SCE and nucleotide pool imbalance was examined in Syrian hamster fetal cells (HFC) and mouse 3T6 cell line. In exponentially growing HFC, deoxyadenosine and deoxyguanosine stimulated the formation of SCE, while deoxycytidine (dCyd) prevented the dose dependent increase of SCE caused by BrdUrd. Cell growth inhibition produced by amino acid deprivation resulted in a significant increase in SCE as compared to exponentially growing cultures. This SCE enhancement was reduced by dCyd in HFC and did not occur in mouse 3T6 clone deficient in deoxycytidine deaminase. The modulation of SCE by DNA precursors is consistent with the induction of BrdUrd mutagenesis in mammalian cells and raises the possibility that common DNA changes are responsible for the induction of SCE and mutation in mammalian cells.

Characterization of new transgenic Big Blue® mouse and rat primary fibroblast cell strains for use in molecular toxicology studies

We have established and characterized primary mouse and rat cell strains for studies designed to complement in vivo gene mutation assays using the Big Blue® mouse or rat. Primary fibroblast cell strains, designated BBM1 and BBR1, were derived from a transgenic male Big Blue® B6C3F1 mouse and from a male Big Blue® Fischer-344 rat, respectively. Both BBM1 and BBR1 are genetically stable and mostly diploid. Both cell strains have low spontaneous frequencies of mutation at the lacI and cII loci as well as low frequencies of sister chromatid exchange and micronuclei formation. In addition, N-ethyl-N-nitrosourea (ENU) induces mutations at the cII locus in both BBM1 and BBR1 cells. These new primary Big Blue® mouse (BBM1) and rat (BBR1) fibroblast cell strains represent useful new models for molecular toxicology studies. Environ. Mol. Mutagen. 34:90–96, 1999 Published 1999 Wiley-Liss, Inc.