Sister chromatid exchange and cell cycle in fibroblasts of Bloom's syndrome

Summary Sister chromatid exchange (SCE) has been studied in the fibroblasts of five Bloom's syndrome patients, one heterozygote, and two normal individuals. The high frequency of SCE already known in the lymphocytes of Bloom's syndrome was also found in the fibroblasts of all five patients. However, populations with low and high frequency of SCE were not found. In addition, chromosome aberrations appeared with a lower frequency.The cell cycle duration in the Bloom's fibroblasts appeared to be similar to that in the normal cell line, and the difference in the growth pattern appeared to be due to the variation in the mitotic index. The cell cycle lasted about 24 h in at least four of the Bloom's lines studied during the present experiments.     

Disparate effects of 5-bromodeoxyuridine on sister-chromatid exchanges and chromosomal aberrations in Bloom syndrome fibroblasts

The existence of a high frequency of spontaneous sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) has thus far been supported by data on a small number of BS cell lines. To examine the cause of baseline SCEs more broadly, the frequencies of SCEs, as well as chromosomal aberrations (CAs) in 4 additional BS fibroblast strains were compared, under different assay and cell culture conditions, with those of normal cells in the range of approximately 0.9-90% 5-bromodeoxyuridine (BrdUrd) substitution into template DNA. SCEs at low levels of BrdUrd substitution were detected by an extremely sensitive immunofluorescent technique. From approximately 0.9% to 4.5% BrdUrd substitution, the SCE frequency in BS cells remained constant, at a level (40/cell) 8 times higher than that of normal cells. As BrdUrd substitution increased further, the SCE frequency in BS cells increased almost linearly, reaching 70-100 per cell at approximately 90% substitution, while the SCE increment in control fibroblasts was less than 5 per cell. Analysis of SCEs in 3 successive replication cycles similarly revealed that the SCE increment in BS cells depended on BrdUrd only at a high BrdUrd substitution level. In contrast to data on SCEs, CA induction by incorporated BrdUrd in BS cells was only slightly higher than that in normal cells. Thus, BS cells are extremely sensitive to BrdUrd for SCE induction, but much less so for CA induction.     

Suppression of the frequencies of sister chromatid exchanges in Bloom's syndrome fibroblasts by co-cultivation with Chinese hamster cells

Summary The effect of co-cultivation of Bloom's syndrome fibroblasts with Chinese hamster ovary cells (CHO) on the incidence of sister chromatid exchanges (SCEs) was studied. The results show that suppression of the frequency of SCEs in Bloom's syndrome cells occurs only if cell to cell contact is present with CHO cells, without any effect on the SCE frequency in the latter.It is suggested that possible genetic heterogeneity between different Bloom's syndrome patients can be studied using the method of co-cultivation.     

Cytogenetic investigations in a new case of Bloom's syndrome

Summary Cytogenetic studies of an 8-year-old caucasian girl with typical but mild manifestation of Bloom's syndrome showed a characteristic increase of homologous chromatid translocations and prematurely condensed chromosomes. The average frequency of sister chromatid exchanges (SCE) in lymphocytes with 133 was much higher than in skin fibroblasts with 49. The inter- and intrachromosomal distributions of SCE in lymphocytes were analyzed.Prof. Dr. H.-R. Wiedemann to his 60th birthday.     

Functional interactions between BLM and XRCC3 in the cell

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIalpha suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)-induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.     

Effects of mitomycin C on sister chromatid exchange in normal and Bloom's syndrome cells

Bloom's syndrome lymphocytes, which are characterized by a high incidence of sister chromatid exchanges (SCE: 80.6 per cell), were treated with mitomycin C (MMC) and the effect of the chemical on SCE frequency compared with that in normal cells. Raising the concentration of MMC from 1 X 10(-9) to 1 X 10(-7) g/ml led to about 10-fold increase (61.7 SCE per cell) in the SCE frequency over the base line in normal lymphocytes (6.4 SCE per cell), though chromosome aberrations remained at a relatively low frequency. MMC caused about a two-fold rise in SCE in cells of Bloom's syndrome (128.8 SCE at 10(-9) g/ml; 139.3 SCE at 10(-8) g/ml). The frequency of chromosome aberrations in Bloom's syndrome cells at concentrations of MMC of 1 X 10(-9) and 1 X 10(-8) g/ml was 0.350 and 0.825 per cell, respectively, and low when compared to the increased number of SCE. The increased frequency of SCE in normal and Bloom's syndrome cells is in contrast to the reported findings with cells from Fanconi's anemia and xeroderma pigmentosum. The distribution of SCE in MMC-treated normal cell correlates with that of spontaneous SCE in cells of Bloom's syndrome.     

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

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

SCEs are induced by replication of BrdU-substituted DNA templates, but not by incorporation of BrdU into nascent DNA

After 3 rounds of DNA replication in the presence of BrdU, third-division metaphase cells can be scored for the frequencies of SCEs that occurred during cycles 1 and 2, and also for the frequency of SCE during cycle 3. This procedure was used to resolve the issue of SCE induction by replication of BrdU-substituted DNA templates versus induction by BrdU incorporation into nascent DNA. It was observed that third-cycle SCE frequencies in CHO are dependent upon the amount of BrdU that was present during cycles 1 and 2 and are independent of the BrdU concentration during the third cycle. It is therefore BrdU serving as a template, rather than BrdU being incorporated, that initiates the SCE event. A model is proposed that produces reasonable fits to the observed data. It also predicts a true background or spontaneous SCE frequency of 3 per cell per cycle as previously reported by Heartlein et al. (Mutation Res., 107 (1983) (103-109). The predicted single twin ratio is higher than that reported by Wolff and Perry (Exp. Cell Res., 93 (1975) 23-30), and possible explanations for this discrepancy are discussed.     

The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes

AA8 Chinese hamster ovary cells were treated with halogenated nucleosides analogues of thymidine, namely CldU, 5-iodo-2'-deoxyuridine (IdU), and 5-bromo-2'-deoxyuridine (BrdU), following different experimental protocols. The purpose was to see whether incorporation of exogenous pyrimidine analogues into DNA could interfere with normal chromosome segregation. The endpoint chosen was endoreduplication, that arises after aberrant mitosis when daughter chromatids segregation fails. Treatment with any of the halogenated nucleosides for two consecutive cell cycles resulted in endoreduplication, with a highest yield for CldU, intermediate for IdU, and lowest for BrdU. The frequency of endoreduplicated cells paralleled in all cases the level of analogue substitution into DNA. Our results seem to support that thymidine analogue substitution into DNA is responsible for the triggering of endoreduplication. Besides, the lack of any effect on endoreduplication when CldU was present for only one S-period strongly suggest that it is the nature of template, and not nascent DNA, that plays a major role in chromosome segregation. Taking into account that topoisomerase II cleaves DNA at preferred sequences within its recognition/binding sites, the likely involvement of the enzyme is discussed.     

Characterization of a potent catenation activity of HeLa cell nuclei

Using an assay which measures catenation of a supercoiled DNA template, we have characterized and quantitated a potent activity identified in crude fractions of HeLa cell nuclei. Catenation requires Mg-ATP and a DNA-condensing agent, polyvinyl alcohol. A filter-binding or agarose gel assay can be used to quantitate activity. In this reaction, DNA topoisomerase I relaxes the input supercoiled DNA to provide DNA topoisomerase II, a strongly favored template for catenation. DNA topoisomerase II preferentially catenates relaxed DNA over supercoiled DNA by a factor of 100. One molecule of DNA topoisomerase II is able to catenate about 20 circles of relaxed DNA/min at 30 degrees C but only 0.16 circle of supercoiled DNA/min at 30 degrees C. The purified HeLa topoisomerase I can also catenate DNA under these assay conditions, yet in an ATP-independent fashion. It is much less efficient than topoisomerase II; one molecule of topoisomerase I catenates only about 3.8 X 10(-3) molecules of supercoiled DNA/min at 30 degrees C with a DNA template containing 5% nicked circles. This remarkable difference between the two enzymes allows quantitation of DNA topoisomerase II activity seen in the presence of excess topoisomerase I. Unlike Escherichia coli topoisomerase I (omega), catenation by the HeLa topoisomerase I is not stimulated by gapped circles.     

Incorporated bromodeoxyuridine enhances the sister-chromatid exchange and chromosomal aberration frequencies in an EMS-sensitive Chinese hamster cell line

The mutant Chinese hamster cell line, EM9, is characterized by a high baseline sister-chromatid exchange (SCE) frequency, increased sensitivity to cell killing, and a defect in DNA strand-break repair. The molecular basis for this pleotrophic phenotype is not known. We examined, at the chromosomal level, the increased sensitivity of this mutant to incorporated BrdUrd. By varying the amount of BrdUrd in template DNA and measuring the frequency of SCEs and chromosomal aberrations, we demonstrated the enhanced sensitivity of EM9 to BrdUrd present in the template strand of DNA. Our results show that a 6-fold increase in SCEs occurs due to DNA replication over a BrdUrd-substituted template relative to a dThd-substituted template. With regard to aberration production in EM9, there is a significant enhancement of aberrations and a specific bias toward damage for the chromatid with Brdurd in the template strand. While these cells share some phenotypic properties with cells from patients with Bloom's syndrome, the genotypic similarities have not yet been established.     

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase . Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.     

Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome.

The clinical radiosensitizer bromodeoxyuridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.     

DNA synthesis in Bloom's syndrome fibroblasts

The relationship between relative rates of DNA synthesis and DNA content in Bloom's syndrome fibroblasts (BS cells) was investigated by flow cytometry. The cells were pulse labelled with 5-bromo-2'-deoxyuridine (BrdU). The BrdU content and cellular DNA content of individual BS cells were simultaneously measured by flow cytometry in which the cells were double-stained by a FITC-conjugated anti BrdU monoclonal antibody (mAb) for the BrdU content (green) and by PI (propidium iodide) (red) for total DNA content. Their red fluorescence histograms were analysed by a microcomputer to evaluate the cell fractions of each S compartment. The BrdU uptake in the early S phase of BS cells was lower than that of normal cells (fibroblasts from skin of a normal human), whereas the uptake in the middle and late S phase was essentially the same as that of normal cells. The early S phase in BS cells accounted for over 50% of the S phase cells. These findings suggest that, in comparison with normal cells, the rate of DNA synthesis in the early S phase of BS cells is lower, but is identical to controls in the middle and late S phases.     

Normal uracil-DNA glycosylase activity in Bloom's syndrome cells

Cells from patients with Bloom's syndrome, a rare human disease with autosomal recessive mode of inheritance, exhibit cytological abnormalities involving DNA metabolism. Bloom's syndrome is characterized by a greatly increased cancer frequency which may reflect a specific defect in DNA repair and replication. Evidence has recently been presented of the existence in Bloom's syndrome of an abnormality of the DNA ligase involved in semiconservative DNA replication. Another abnormality, in the excision-repair pathway of Bloom's syndrome cells, is reportedly due to an aberrant immunological reactivity of the DNA-repair enzyme uracil-DNA glycosylase. In this investigation we show, however, that the catalytic activity of uracil-DNA glycosylase appears to be normal in Bloom's syndrome lymphoblastoid cells.     

DNA synthesis in Bloom's syndrome fibroblasts

Abstract. The relationship between relative rates of DNA synthesis and DNA content in Bloom's syndrome fibroblasts (BS cells) was investigated by flow cytometry. The cells were pulse labelled with 5-bromo-2'-deoxyuridine (BrdU). The BrdU content and cellular DNA content of individual BS cells were simultaneously measured by flow cytometry in which the cells were double-stained by a FITC-conjugated anti BrdU monoclonal antibody (mAb) for the BrdU content (green) and by PI (propidium iodide) (red) for total DNA content. Their red fluorescence histograms were analysed by a microcomputer to evaluate the cell fractions of each S compartment. The BrdU uptake in the early S phase of BS cells was lower than that of normal cells (fibroblasts from skin of a normal human), whereas the uptake in the middle and late S phase was essentially the same as that of normal cells. The early S phase in BS cells accounted for over 50% of the S phase cells. These findings suggest that, in comparison with normal cells, the rate of DNA synthesis in the early S phase of BS cells is lower, but is identical to controls in the middle and late S phases.     

Epidermal growth factor-induced topoisomerase(s). Intracellular translocation and relation to DNA synthesis

We have used epidermal growth factor (EGF) to investigate the relationship between eukaryotic topoisomerases and DNA synthesis. We found that EGF stimulates topoisomerase activity in human fibroblasts and Swiss/3T3 mouse fibroblasts. The first increase is seen in the cytoplasm, followed by increased activity in the nucleus. The nuclear increases correspond to increases in DNA synthesis. A type II topoisomerase is stimulated as indicated by the ATP dependence of the relaxing reaction and by the formation of catenanes. We have also found that the topoisomerase activity in the cytoplasm is sedimentable indicating that it is either membrane-associated or in a supramolecular complex. The stimulation of topoisomerase activity by EGF may represent a key step in the process by which EGF induces DNA synthesis and cell division.     

Sister-chromatid exchanges and gene mutations are induced by the replication of 5-bromo- and 5-chloro-deoxyuridine substituted DNA

The thymidine (dT) analogue 5-chlorodeoxyuridine (CldU) induces 7–8-fold more sister-chromatid exchanges (SCE) than does 5-bromodeoxyuridine (BrdU) at equal substitution for dT in Chinese hamster ovary cells in culture. This difference facilitates study of the mechanism of induction of SCE by these analoques. Cultures were incubated with either BrdU or CldU for one cell cycle, followed by incubation in the presence of dT alone or BrdU or CldU for the second cell cycle and the SCE frequency determined in M2 cells. The results suggest that the induction of SCE is dependent only on the replication of the analogue-substituted DNA during the second cell cycle. Additional studies employed cultures grown in the presence of BrdU or CldU for 7 days to obtain mainly bifilarly substituted DNA, followed by 2 rounds of replication in the presence of dT alone. The SCE frequencies were approximately twice those found in cultures which had undergone the usual 2 rounds in the presence of the analogue; this is consistent with the replication of twice the amount of analogue-substituted DNA. Furthermore, such long-term growth in the presence of BrdU or CldU also results in concentration-dependent increases in the frequency of 6-thioguanine-resistant mutants, suggesting that gene mutations also result from the replication of analogue-substituted DNA.     

Hypersensitivity of Bloom's syndrome fibroblasts to N-ethyl-N-nitrosourea

Fibroblast cells from two Japanese patients with Bloom's syndrome (BS) and normal donors were studied for the inactivation of colony-forming ability and the induction of sister-chromatid exchanges (SCEs) after N-ethyl-N-nitrosourea (ENU) treatment. The reduction of ENU-induced SCEs as a function of post-treatment incubation time was also compared between BS and normal fibroblasts. BS cells were approximately 4 times more sensitive than normal cells to the lethal effect of ENU and remarkably hypersensitive to the SCE induction by ENU. The post-treatment incubation of ENU-treated normal cells in the fresh medium resulted in a time-dependent decrease of the SCE level until 6 h after which time the SCE level remained the plateau of about 50% of the initial level. In contrast, the ENU-induced SCEs in BS cells decreased much more slowly with post-treatment incubation time and its half life was 24 h. These results collectively support the view that BS cells may be defective in the rapid repair of certain type(s) of DNA damages induced by ENU.     

RecA-like activity in mammalian cell extracts of different origin

The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.