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1.
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. 相似文献
2.
It has been shown repeatedly that exposure of G(1) cells unifilarily labelled with 5-bromodeoxyuridine (BrdU) to X-rays leads to sister chromatid exchanges (SCE) when the cells are allowed to grow for one further cycle in the absence of BrdU. It has been suggested that damage induced by ionizing radiation does not lead to 'true' SCE and that the observed SCE are 'false', resulting from structural chromosomal aberrations, especially interstitial inversions. We used a painting probe for the p14 region of human chromosome 3 and anti-BrdU antibodies to analyse the frequency of radiation-induced SCE in that chromosome. This method allowed us to discriminate between para- and pericentric 'true' and 'false' SCE. Our results indicate that most radiation-induced SCE do not result from inversions. 相似文献
3.
Chinese hamster ovary (CHO) cells were exposed to the restriction endonuclease AluI in the presence of 1.1 M glycerol. Chromosomal aberrations and sister chromatid exchange (SCE) were scored in first post-treatment metaphases following recovery times of 6-18 h. At all recovery times chromosomal aberrations were induced by the enzyme. In AluI-treated damaged cells significant elevations of SCE frequencies were found after recovery times of 12-18 h. These results indicate that SCE, unlike chromosomal aberrations, are induced only in late G1 and early S phase of the cell cycle. The lesions producing SCE are postulated as DNA single-strand breaks and gaps induced by AluI in canonical structures of DNA and in DNA-RNA hybrids. 相似文献
4.
The method of choice to differentiate sister chromatids is to incorporate BrdU in replicating DNA. The disadvantage of BrdU
is that its spontaneous or induced radicalization may itself lead to sister chromatid exchanges. Biotin-labelled dUTP is a
widely used thymidine analogon for labelling isolated DNA. Its chemical structure suggests that, in contrast to BrdU, it does
not give rise to radical formation. We electroporated proliferating Chinese hamster ovary (CHO) cells in the presence of biotin-dUTP
which was subsequently detected in metaphase cells with TRITC-conjugated avidin. Microscopic analysis of second mitoses after
labelling revealed a clear differential staining of sister chromatids. Thus substitution of thymidine with biotin-dUTP is
another method to analyse SCE.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
5.
Chinese hamster primary embryonic cells (at G1 phase) were treated with 1.0 or 3.0 microg/ml bleomycin and chromosomal aberrations in first division metaphases were analysed by fluorescence in situ hybridization (FISH) using arm-specific painting probes for chromosomes 3, 4, 8 and 9. We observed that bleomycin induced all classes of chromosome-type aberrations very efficiently. The interesting findings were: (i) the frequency of induced interstitial translocations (i.e. insertions) was approximately equal to that of reciprocal translocations; (ii) the frequency of induced pericentric inversions was higher than that of centric rings. In our earlier studies, we found that X-rays induced a low frequency of interstitial translocations in comparison with reciprocal translocations and equal frequencies of centric rings and pericentric inversions. These data suggest that bleomycin differs from X-rays with respect to the induction of some specific types of aberrations. The results of a chi2 test examining the hypothesis that formed aberrations among the chromosomes or chromosome arms are randomly distributed on the basis of their relative lengths revealed a differential involvement of these chromosomes in the aberrations following exposure to bleomycin. In general, chromosome 8 was found to be more involved in induced aberrations than expected, chromosome 4 was randomly involved, whereas chromosomes 3 and 9 were less involved. This study demonstrates the utility of arm-specific painting probes for efficient detection of a large variety of chromosomal aberrations induced by bleomycin. 相似文献
6.
The effect of post-treatment with inhibitors of DNA synthesis (hydroxyurea, aphidicolin) and repair (caffeine, 3-aminobenzamide) on the frequencies of chromosomal aberrations and sister chromatid exchange (SCE) induced by mitomycin C and decarbamoyl mitomycin C both in Chinese hamster cells and in human lymphocytes in vitro has been studied. The data show that in the case of Chinese hamster and human lymphocytes mitomycin C-treated cells there is an increased frequency of both chromosomal aberrations and SCE after a G2 post-treatment with the inhibitors, while no increase is observed for decarbamoyl mitomycin C-treated cells. Since SCE are DNA synthesis-dependent phenomenon, an increase in the frequency of SCE also in the G2 phase might suggest that after mitomycin C treatment there is a residual DNA synthesis still going on very late in the cell cycle. 相似文献
7.
W W Au J P O'Neill W Wang H E Luippold R J Preston 《Teratogenesis, carcinogenesis, and mutagenesis》1984,4(6):515-522
The induction of chromosome aberrations, sister chromatid exchanges (SCE), cytotoxicity, and 6-thioguanine-resistant mutation by neocarzinostatin (NCS) in Chinese hamster ovary cells was analyzed. It was observed that within the same concentration range of 0.01-0.1 mu/ml NCS, the drug induced a significant increase in all analyzed end-points except in SCE frequencies. There was no increase in SCE frequencies even when the cells were treated at the G1/S border in the first cell cycle and when aberrations were observed in the same cell showing a second cycle differential staining pattern. Our study indicates that the cellular damage induced by NCS leads to expression in chromosome aberrations, cytotoxicity, and mutagenicity but not in sister chromatid exchanges. 相似文献
8.
P. Morales-Ramírez V. L. Cruz-Vallejo T. Vallarino-Kelly R. Rodríguez-Reyes 《Somatic Cell and Molecular Genetics》1995,21(1):33-41
The efficiency of mitomycin C or gamma rays to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation before S. The SCE frequencies induced by MMC in the first division (before BrdU incorporation) were significantly lower in the early G1 compared to the late G1, indicating some repair of SCE-inducing lesions. In the second division (after BrdU incorporation), there was no difference between SCE induced in early and late G1, indicating that MMC-induced lesions in such conditions are very persistent and not repairable during G1. The radio induced SCE frequency at early G1 was significantly lower than that observed in late G1, in cells irradiated after BrdU incorporation, suggesting that half of gamma ray-induced DNA lesions that elicit SCEs were repaired. 相似文献
9.
Chromosomal aberrations induced by in vitro irradiation: comparisons between human sperm and lymphocytes 总被引:3,自引:0,他引:3
B F Brandriff L A Gordon L K Ashworth A V Carrano 《Environmental and molecular mutagenesis》1988,12(2):167-177
Types and frequencies of structural aberrations in human sperm and lymphocyte chromosomes from one donor were compared after in vitro irradiation with 100, 200, and 400 rad in order to determine if cells with dramatically different chromatin configurations are similarly affected and to investigate the feasibility of using lymphocytes as surrogates for germ cells in risk estimation. Sperm chromosomes were analyzed after fusion with eggs from the golden hamster. Total frequencies of induced aberrations were similar in the two cell types. However, the relative frequencies of rejoined lesions (dicentrics), compared with unrejoined lesions (chromosome breaks and acentric fragments), were different. At the three doses tested, a constant ratio of 5 dicentrics in lymphocytes for every dicentric in sperm was induced. Conversely, for every chromosome break or acentric fragment induced in lymphocytes, 1.7 such events were induced in sperm at the three doses tested. 相似文献
10.
Neocarzinostatin induces chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells 总被引:1,自引:1,他引:0
Chinese hamster ovary (CHO) cells were treated with neocarzinostatin(NCS) and analyzed for chromosomal aberrations and sister chromatidexchanges (SCE). After treatment the cells were recovered for9, 20, 26 or 30 h. NCS induces chromosomal aberrations and SCE.SCE were much more frequent in cells with chromosome type aberrationsat 20 h recovery time than in those with chromatid type aberrationsat 9 h recovery time. In second post-treatment cells at 26 or30 h recovery time NCS induced chromosomal aberrations but onlyfew SCE. 相似文献
11.
Tunca B Egeli U Zorluoglu A Yilmazlar T Yerci O Kizil A 《Cancer Genetics and Cytogenetics》2000,119(2):139-145
The expression frequency of common fragile sites induced by aphidicolin (Apc), bromodeoxyuridine (BrdU), and caffeine was evaluated on prometaphase chromosomes obtained from the peripheral blood lymphocytes of 32 patients with colon cancer, 30 of their clinically healthy family members and 30 age-matched normal controls. The proportion of damaged cells (P < 0.001), the mean number of chromosomal aberrations and the expression frequencies of fragile sites were significantly higher in the patient and relative groups compared to the control group. Our findings show an increased genetic instability in patients with colon cancer and their first-degree relatives. In addition, common fragile sites can be used as a suitable marker for determining genetic predisposition to cancer. 相似文献
12.
The frequencies of chromosomal aberrations and sister chromatid exchange (SCE) were measured in lymphoblastoid cell lines (LCLs) and in phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated lymphocytes from males with X-linked lymphoproliferative (XLP) syndrome, their obligate carrier mothers, and control subjects. We observed an increased frequency of chromosomal aberrations including increased polyploidy in LCLs derived from families with XLP with time in culture. The SCE rate in LCLs (mean of 3.89 SCEs per cell) was much lower than that in PHA- or PWM-stimulated lymphocytes: PWM-stimulated lymphocytes showed 9.58 SCEs per cell and PHA-stimulated cells had 11.38 SCEs per cell. A greater number of chromosomal gaps and breaks in the D-group chromosomes of LCLs of affected males and carrier females were identified compared to the number expected, based on chromosomal length and the number of aberrations seen in PHA-stimulated cell cultures. No differences in the frequency of SCEs or chromosomal aberrations were found in control subjects and affected males or carrier females in the peripheral lymphocytes stimulated by PHA. Phenotypes of XLP appear to arise from failure of immune responses to Epstein-Barr virus (EBV) and not from intrinsic chromosomal breakage or instability. 相似文献
13.
It has been suggested not only that sister chromatid exchange (SCE) induction might serve as a qualitative indicator of mutagenesis, but also that induced SCE frequencies are linearly related to induced mutation frequencies. The consistency of the relationship between SCEs and mutations was tested in the present work. Confluent Chinese hamster ovary (CHO) cells were exposed to ethylnitrosourea (ENU) and then held at confluency for various times prior to initiation of SCE and mutation assays. Cells held at confluency are typically thought to be arrested in their progression through the cell cycle, so that "S-dependent" processes such as fixation of mutations and formation of SCEs will not occur, while DNA repair processes might continue to operate. If repair processes reduce the number of SCE-inducing lesions during the holding period and, hence, reduce the subsequently determined SCE frequencies, then mutation frequencies should similarly be reduced if SCEs and mutations are related. It was observed, however, that induced SCE frequencies decreased exponentially with holding time, while mutation frequencies remained constant. Qualitatively similar results were obtained in log-phase cells. Cell cycle analysis demonstrates that confluent CHO cells can cycle, and ways are considered in which this might affect SCE and mutation frequencies. It is concluded that the decline in SCE frequency (with time) cannot be attributed solely to the presence of cycling cells in confluent cultures. It appears, therefore, that at least some forms of ENU-induced DNA damage that can lead to SCE were repaired and as such are distinct from those forms that are mutagenic. Thus SCEs are not necessarily related to mutations, because the two events may represent manifestations of different forms of DNA damage. Whether or not this represents a universal phenomenon that would hold true for agents other than ENU remains to be determined. 相似文献
14.
Specificity of arsenite in potentiating cytogenetic damage induced by the DNA crosslinking agent diepoxybutane. 总被引:4,自引:0,他引:4
In the present study, the induction of sister chromatid exchanges (SCEs) and chromosomal aberrations were measured in normal human lymphocytes treated with low concentrations of arsenite alone (0.5-2.0 microM) and arsenite in combination with the potent DNA crosslinking agent diepoxybutane (DEB). Experiments were carried out with lymphocytes from blood donors with different sensitivities to SCE induction by DEB. Arsenite, beginning at concentrations as low as 1 microM, increased SCE frequencies; chromosomal aberration frequencies were increased at 2 microM of arsenite. DEB treatments alone increased SCE frequencies and chromosomal aberrations. The yields of chromatid deletions and exchanges in lymphocytes exposed to both arsenite and DEB were markedly increased above the levels expected if the effects of the two agents had been simply additive. The frequencies of chromatid deletions were 4- to 8-fold greater than expected and chromatid exchanges were increased 7- to 40-fold. Chromatid exchanges detected in cells treated with arsenite and DEB were predominately incomplete exchanges. The most dramatic increases in chromatid aberrations were observed in lymphocytes from an individual sensitive to SCE induction by DEB, indicating that individuals may vary in their sensitivity to the co-clastogenic effects of arsenite. At concentrations that dramatically affect aberrations, arsenite had no effect on the induction of SCEs by DEB. These studies suggest a specific interaction of arsenite with the induction or repair of DNA damage produced by DEB that leads to chromosomal aberrations but not to SCEs. Based on the selective chemical reactivity of low concentrations of arsenite with proteins containing vicinal dithiols and the occurrence of these groups within DNA repair proteins, it is proposed that the specific co-clastogenic effects of arsenite may be mediated by its interference with DNA repair activities. 相似文献
15.
Russell J. DuFrain 《Environmental and molecular mutagenesis》1983,5(6):813-824
Streptonigrin (NSC-45383), a direct-acting clastogen which induces SCEs in vivo and chromosome aberrations both in vivo and in vitro, was evaluated for SCE induction in both G0 and stimulated rabbit lymphocytes. Determinations were made for 16 cultures from seven female rabbits. These included controls as well as cells exposed to 90 μg/kg in vivo, cells pulse-treated with 50 ng/ml in vitro, and a culture continuously exposed to 5 ng/ml in vitro. For all cultures the SCE/ cell frequency was determined from 20 complete (44 chromosome) metaphases and, in selected cultures, SCEs on individual chromosomes (880 per culture from 20 cells) were enumerated to determine SCE/chromosome frequency and the chromosomal distribution of SCEs. Analysis of variance and least significant difference tests of the √x transformed SCE/cell data show that cells exposed to Streptonigrin while dividing have significantly higher (P<0.01) frequencies (over double the control 5.3 SCE/cell value) whereas treated G0 cells were not significantly different from the controls. Dispersion analysis of both SCE/cell and SCE/ chromosome data confirms the adequacy of the Poisson distribution for spontaneous or baseline but not streptonigrin-induced SCEs. 相似文献
16.
N. V. Chervonskaya 《Bulletin of experimental biology and medicine》1970,70(6):1435-1437
The preservation of radiation injuries to chromosomes was studied during cultivation of irradiated human diploid cells. In the first subculture after irradiation the number of cells with chromosomal aberrations was 2.5 times greater than in the control culture. After two subcultures of the irradiated strain, on the eighth day after irradiation, cells bearing unstable chromosomal aberrations and tetraploid cells induced by irradiation disappeared from the population.Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Kraevskii.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 70, No. 12, pp. 89–92, December, 1970. 相似文献
17.
Transmission of gamma-ray-induced unstable chromosomal aberrations through successive mitotic divisions in human lymphocytes in vitro 总被引:1,自引:0,他引:1
Transmission of unstable chromosomal aberrations through successive mitotic divisions M1-M4 was analysed in 3 Gy gamma-ray-irradiated G0 human lymphocytes in vitro, harvested at 50, 72 and 96 h. Bromodeoxyuridine (BrdU) incorporation and subsequently the fluorescence plus Giemsa (FPG) method allowed the cell cycle status of each cell scored to be ascertained. Higher dicentric frequencies were observed in cells within the same post-irradiation division derived from extended culture times, indicating either a delay in cell cycle progression of aberrant cells or different lymphocyte sub-populations. The yield of complete dicentrics decreased by a factor of two in passing from M1 to M2 and showed further reductions of 26 and 44%, respectively, in moving from M2 to M3 and from M3 to M4. In the case of conventionally stained metaphases, wherein the cell cycle status does not enter the picture, the dicentric frequencies showed a reduction in yield of 39.6% at 72 h and 52.1% at 96 h compared with 50 h, because the cells analysed comprise a mixture of metaphases in different cell cycles. In the FPG stained first division metaphases, 92-100% of dicentrics analysed at 50, 72 and 96 h and in the conventionally stained metaphases, 90-94, 72-84 and 54-80% of dicentrics analysed at 50, 72 and 96 h respectively were complete dicentrics (with fragments). In the M1, M2, M3 and M4 metaphases analysed, 92-100, 50-89, 20-70 and 10-50% of dicentrics, respectively, were complete dicentrics and 55-75, 53-68, 43-57 and 36-50% excess acentrics, respectively, were seen in cells with complete dicentrics. Interindividual variation was observed in cell cycle kinetics, radiation-induced chromosomal aberrations and micronucleus frequencies. A salient feature of the delayed effects was the induction of giant cells and the mirror dicentrics observed in them. The reduction in dicentric frequencies due to either cell cycle delay or cell death in successive generations is aided by the multiplicity of aberrations. Bridge-breakage-fusion (BBF) events mediated by dicentric chromosomes may also be instrumental in the perpetuation of chromosomal instability. 相似文献
18.
In vivo persistence of sister chromatid exchanges (SCE) induced by gamma rays in mouse bone marrow cells 总被引:1,自引:0,他引:1
The sister chromatid exchange (SCE) frequencies induced in bone marrow cells by in vivo irradiation with gamma rays before or after bromodeoxyuridine (BrdUrd) incorporation were compared. The frequency of SCE at different postirradiation times was also measured in bone marrow cells in vivo, irradiated before BrdUrd incorporation. Increased sensitivity to SCE induction by radiation was found in cells after BrdUrd incorporation for one cycle when compared with cells irradiated before BrdUrd incorporation. The increased SCE frequency persisted for at least 72 hr after the initial irradiation, implying that the gamma ray-induced lesion(s) capable of eliciting an SCE are persistent and cannot be easily repaired. 相似文献
19.
《Mutation Research/DNA Repair》1990,235(2):129-135
The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied.The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-β-d-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions.Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations.Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage. 相似文献
20.
The aims of the present study were to establish the following: (i) the course of sister chromatid exchange (SCE) induction by ethylnitrosourea (ENU) in the first, second and third divisions as a function of the exposure time; (ii) the persistence of SCE-inducing lesions and the determination of whether or not they are always involved in SCE formation; (iii) the effect of bromodeoxyuridine (BrdU) incorporation on the induction and persistence of SCE. Three-way differential staining of sister chromatids in murine bone marrow cells in vivo was used in the present study. The results indicate the following: (i) SCE induction in each cell division depends on the course of DNA duplication, suggesting that SCE occurs at the replication fork; (ii) the cell population under study could be considered synchronous and had a cell cycle duration of nearly 9 h; (iii) in the second and third cell divisions ENU preferentially induced SCE in the cycle in which the exposure occurred; (iv) lesions induced by exposure to ENU did not cause SCE at the same site in subsequent divisions; (v) ENU was also capable of producing a long-lasting induction of SCE in BrdU-unsubstituted DNA; (vi) the sensitivity to SCE induction by the mutagen increases nearly proportionally to BrdU incorporation into DNA. 相似文献