Aging and sister chromatid exchange : I. The effect of aging on mitomycin-C induced sister chromatid exchange frequencies in mouse and rat bone marrow cells in vivo

Utilizing the differential staining of chromatids containing BUdR, it was demonstrated that frequencies of mitomycin-C induced sister chromatid exchanges decline with age. The concomitant increase in chromosomal aberrations suggest an altered response to DNA damage with aging.     

Sister chromatid exchanges in Tradescantia paludosa

A modified fluorescence-plus-Giemsa technique is described that allows differential staining of sister chromatids in root tip cells from cuttings of Tradescantia paludosa. With this staining technique, chromatids with both DNA strands unsubstituted are differentiated from chromatids containing 5-bromouracil in place of thymine in one of the strands of the DNA duplex. The baseline level of sister chromatid exchanges was shown to be dependent on the concentration of 5-bromodeoxyuridine in the treatment solution, the mean frequency being 43.5 sister chromatid exchanges per cell for the experimental protocol suggested.     

Sister Chromatid Exchanges in Tradescantia Paludosa

A modified fluorescence-plus-Giemsa technique is described that allows differential staining of sister chromatids in root tip cells from cuttings of Tradescantia patudesa. With this staining technique, chromatids with both DNA strands unsubstituted are differentiated from chromatids containing 5-bromouracil in place of thymine in one of the strands of the DNA duplex. The baseline level of sister chromatid exchanges was shown to be dependent on the concentration of 5-bromodeoxyuridine in the treatment solution, the mean frequency being 43.5 sister chromatid exchanges per cell for the experimental protocol suggested.     

Differential staining of chromatids reveals substitution of BUdR for thymine in “old” DNA strands

Metaphases collected from cultures grown for three cell cycles in 5-bromodeoxyuridine (BUdR) and then for one or two further cell cycles without BUdR show persistence of differentially FPG-stained chromatids. The cell cycle length is not altered by the presence of BUdR. After removal of BUdR, the cells synthesize DNA and incorporate mainly thymine, as demonstrated by density gradient analysis of DNA. Our observations suggest that chromatids with T-B DNA stain lightly after removal of BUdR, in contrast with their dark staining when cultures are maintained in BUdR. Thus, in any experimental condition, there is a correspondence between the nature (T-DNA or B-DNA) of the “old” DNA strands and the FPG-staining (dark or light) of the chromatids.     

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml.Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR.To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml.On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

The mechanism of differential sister chromatid fluorescence as studied with the GC-specific DNA-ligand mithramycin

Chromosomes of human blood cells exposed to BUdR for two cell cycles showed an R-band pattern of fluorescence without lateral differentiation after staining with the GC-specific DNA-fluorochrome mithramycin. Differential sister chromatid fluorescence could be induced by a mild near-ultraviolet irradiation pretreatment which was without effect in Feulgen staining. Thus, besides the primary alteration of DNA structure caused by incorporation of BUdR, secondary structural alterations, probably mediated via chromosomal proteins, are required in order to obtain differential mithramycin-fluorescence of sister chromatids. The differential staining pattern was similar to that achieved with the AT-specific DNA-fluorochrome DAPI. Therefore, it may be concluded that the base specificity of fluorochromes does not play any part in the production of differential fluorescence of sister chromatids by this method.     

Reverse sister chromatid differential staining by Coomassie Brilliant Blue R-250

A sister chromatid differential staining pattern is observed if chromosomes replicate for two cycles in the presence of 5-bromodeoxyuridine (BUdR) and are subsequently stained in Hoechst 33258, irradiated with black light, and then stained in Coomassie Brilliant Blue R-250. In this pattern the chromatids containing DNA that is bifilarly substituted with BrdUrd are darkly stained and the chromatids with DNA that is unifilarly substituted are lightly stained. This staining pattern is the reverse of that found when slides are stained in Hoechst plus Giemsa. Slides stained with either Giemsa or Coomassie Blue can be destained and restained repeatedly with the other stain to alternate the pattern observed.     

Random Segregation of Chromatids at Mitosis in Saccharomyces Cerevisiae

Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.     

Sister chromatid exchanges in isolabeling

Isolabeling observed by autoradiography in sister chromatids at the second or later metaphases after incorporation of ³H-thymidine has sometimes been ascribed to an exchange between the multiple DNA duplexes in polynemic sister chromatids. An analysis reported here on the frequency and size of isolabeled regions in chromosomes of the rat kangaroo shows that all isolabeling can be accounted for by sister chromatid exchanges coupled with the image spread that can occur in tritium autoradiographs. Hence, in this case it becomes unnecessary to postulate binemy or polynemy to explain isolabeling.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation.     

Stimulation of Herpesvirus saimiri Expression in the Absence of Evidence for Type C Virus Activation in a Marmoset Lymphoid Cell Line

Nucleic acid base analogues were used to examine a Herpesvirus saimiri (HVS)-infected marmoset lymphoid cell line (MLC-1) for possible association with type C viruses. Synthetic templates poly(rA).d(pT)(10) and poly(dA).d(pT)(10) were used to detect RNA-directed DNA polymerase activity in 100-fold concentrated tissue culture fluids. HVS was monitored by immunofluorescence for early, late, and membrane antigens. MLC-1 cells were exposed to 30 mug of 5-bromo-2'-deoxyuridine (BUdR) per ml for 24 h and examined daily. Similar experiments used 5-iodo-2'-deoxyuridine (IUdR) (20 mug/ml) for 30 h or IUdR (20 mug/ml) for 3 days followed by 2% dimethyl sulfoxide for 4 days. Results of these experiments failed to show any type C virus-like polymerase; however, HVS expression was greatly stimulated. BUdR and IUdR enhanced expression of HVS-associated antigens five- to sevenfold, with maximal stimulation being observed 3 to 4 days after removal of the analogue. IUdR-dimethyl sulfoxide treatment was generally less effective. Although more cells showed HVS antigens, the treatments did not increase cell-free infectious virus. The data suggest that HVS-infected lymphoid cells can be stimulated to express virus in a manner similar to that of the Epstein-Barr virus in Burkitt's lymphoma cells. No evidence of type C virus was found in stimulated cultures.     

Analyse des échanges de chromatides dans les cellules somatiques humaines

A new technique (BUdR treatment followed by acridine orange staining) allowing a differentiation of sister chromatids is described. A statistical analysis of 91 human karyotypes gives an estimate of the frequency of exchanges. The mean of sister chromatid exchanges is 27,3 and the minimum number is 11 per cell. — The frequency of these exchanges is proportional to the relative length of each chromosome, and the accumulation of several exchanges in some segments evokes the possibility of a negative interference. — The analysis of endomitoses treated with BUdR during at least two generations is not in disagreement with the model of semi-conservative replication of chromosomal DNA, but the modifications of the chromatids may result from a completely different process. — The frequency of endomitoses is increased by the treatment. These endomitoses allow a very precise analysis of the evolution of the sister chromatid exchanges, during two successive cellular generations.

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Travail de l'E.R.A. N 47 du C.N.R.S. et C.E.A. Contrat N 293.     

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 x 10(-2) M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 microgram per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.     

Mitotic inter-homologue junctions accumulate at damaged DNA replication forks in recQ mutants

Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation. Received: 3 July 1996 / Accepted: 4 October 1996     

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.     

Epstein-Barr Virus-associated Antigens in Non-producing Clones of Human Lymphoblastoid Cell Lines

NUCLEIC acid hybridization suggests that the Epstein-Barr virus (EBV) genome may be present in human lymphoblastoid cell lines that are free of detectable EBV^1,2. We describe here a plentiful appearance of EBV-associated early antigens (EA) and the viral capsid antigen (VCA) in non-producing Raji and NC-37 cell lines when exposed to 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine (IUdR). These antigens were synthesized in all the Raji and NC-37 clones exposed to BUdR or IUdR, strongly suggesting that a complete, but unexpressed, EBV genome exists in the cells of these non-producing lines.     

Differential gene activity visualized on sister chromatids after replication in the presence of 5-azacytidine

Mitotic chromosomes with sister chromatids bearing differentially active ribosomal gene clusters were recovered from human lymphocytes exposed to 5-azacytidine. The hypothesis was that the differential activity was determined by the hypomethylation of one of the two sister chromatids. The verification was carried out by labeling the 5-azacytidine-substituted chromatid with BUdR, and then checking the location of active clusters by specific staining techniques. Data obtained confirmed that the chromatid bearing the active cluster was indeed the 5-azacytidine-substituted one.     

Immunohistochemical Double Staining with Immunogold-Silver and Alkaline Phosphatase to Identify Nuclear Markers of Cellular Proliferation

To facilitate cell kinetics studies of brain tumors labeled with thymidine analogs, we developed a new method to identify nuclei labeled sequentially with bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) by double staining with immunogold-silver and alkaline phosphatase. Patients received an intraoperative infusion of BUdR: excised tumor specimens were immediately labeled with IUdR in vitro. fixed with 70% alcohol, embedded in paraffin, and cut into 6 pm sections. The sections were incubated first with BR-3. a monoclonal antibody that recognizes only BUdR, and then with IU-4. a monoclonal antibody that recognizes both BUdR and IUdR: sections were counterstained with hematoxylin to identify unlabeled nuclei. Nuclei labeled only with IUdR stained red, whereas those labeled with BUdR or with both BUdR and IUdR stained black against a red background: unlabeled nuclei stained blue. This method was the most efficient differential staining technique to identify nuclei labeled only with IUdR and those labeled with BUdR among unlabeled nuclei.