Mutagenesis, Vol. 15, No. 4, 303-310,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Spontaneous and X-ray-induced chromosomal aberrations in Werner syndrome cells detected by FISH using chromosome-specific painting probes
Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands and 1 Laboratory of Molecular Genetics, National Institute on Aging, National Institute of Health, Nathan Shock Drive, Baltimore, MD 212224, USA
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Abstract |
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Werner syndrome (WS) is a rare autosomal disorder characterized by premature aging exhibiting chromosome instability and predisposition to cancer. Cells derived from WS patients show a variety of constitutionally stable chromosomal aberrations as detected by conventional chromosome banding techniques. We have employed the fluorescence in situ hybridization (FISH) technique using painting probes for 12 different chromosomes to detect stable chromosome exchanges in three WS cell lines and three control cell lines. WS cell lines showed increased frequencies of both stable and unstable chromosome aberrations detected by FISH and Giemsa staining, respectively. One WS lymphoblastoid cell line (KO375) had a 5/12 translocation in all the cells and ~60% of the cells had an additional translocated chromosome 12. A high frequency of aneuploid cells was found in all the WS cell lines studied. Though WS cells are known to be chromosomally unstable, unlike other chromosome instability syndromes they are not sensitive to mutagenic agents. We studied the frequencies of X-ray-induced chromosomal aberrations in two WS cell lines and found an ~60% increase in the frequencies of fragments and no consistent increase in the frequencies of exchanges.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Werner syndrome (WS) is a rare, autosomal recessive disorder characterized by the appearance of premature aging, early onset of cataract, osteoporosis, diabetes mellitus, atherosclerosis, leg ulcers, hyperkeratosis and high frequencies of neoplasia. The neoplasms are predominantly of mesenchymal origin and are the principal cause of death in these patients (Beauregard and Gilchrest, 1987
).
Fibroblast-like (FL) cells from WS patients have been found to grow more slowly, reach a senescent morphology more rapidly and demonstrate a markedly reduced in vitro lifespan compared with that of cells from age-matched normal individuals (Salk et al., 1981; Salk, 1982; Scappaticci et al., 1982). WS lymphoblastoid cell lines have also been found to grow poorly (Salk et al., 1981; Salk, 1982).
Cytogenetic studies employing chromosome banding techniques revealed a variety of non-constitutional, stable chromosome rearrangements, ranging from partial deletion of a chromosome to multiple translocations involving several chromosomes in the same cell (Hoehn et al., 1975; Salk et al., 1981, 1982; Scappaticci et al., 1982; Stefanini et al., 1989). Although structurally abnormal, the number of chromosomes has been found usually to be 46 (pseudodiploid) and rearrangements could be passed to daughter cells, thus generating clones of cells with identical cytogenetic markers. This characteristic cytogenetic pattern of pseudodiploidy with multiple, variable, stable chromosome rearrangements that are clonal has been called `variegated translocation mosaicism' (VTM) and is considered to be a characteristic feature of WS (Hoehn et al., 1975; Salk et al., 1981).
Since chromosome instability and cancer predisposition are both characteristic of WS, this disorder has been classified as a `chromosome instability syndrome' similar to Bloom syndrome (BS), ataxia telangiectasia (AT) and Fanconi anaemia (FA) (German, 1983). Increased sensitivity to mutagens has been demonstrated in cells derived from AT, FA and BS patients, whereas very limited data are available for WS cells.
Recently, application of the technique of fluorescence in situ hybridization (FISH) with chromosome-specific painting probes has increased the efficiency of detection of numerical and structural chromosome rearrangements. In the present study we employed FISH using chromosome-specific painting probes for 12 chromosomes to analyse the chromosome constitution of two lymphoblastoid and one fibroblast cell line from three WS patients. The chromosomal radiosensitivity of two lymphoblastoid cell lines was also studied using FISH with chromosome-specific painting probes for chromosomes 1, 4, 5 and 12.
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Materials and methods |
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Cell cultures and culture conditions
Three cell lines from three patients with WS were used: AG3141, a fibroblast cell line, passage 12; DJG, a lymphoblastoid cell line transfected with Epstein–Barr virus (EBV); KO375, a lymphoblastoid cell line transfected with EBV. As controls the following cell lines were used: GM38A, a normal fibroblast cell line, passage 23; AG9387, a normal lymphoblstoid cell line transformed with SV40; SNW646, a normal lymphoblastoid cell line transformed with EBV. Cell lines KO375, DJG and SNW646 were originally obtained from Dr George Martin (Seattle). Cell lines AG9387 and AG3141 were obtained from the Corriell Cell Repository (New Jersey).
The cultures were grown in 10 ml plastic flasks in RPMI 1640 medium supplemented with 15% fetal calf serum (Gibco) (heat inactivated for 30 min at 56°C), 2 mM glutamine and antibiotics.
Irradiation
Three lymphoblastoid cell cultures (two WS cell lines, DJG and KO375, and one control cell line, SNW646) were irradiated in plastic flasks with 2 Gy X-rays using an Andrex SMART 225 machine operating at 200 kV, 4 mA, at a dose rate of 2 Gy/min. These three cell lines were chosen for comparison as they were all transformed by EBV. Non-irradiated cultures served as controls. 5-Bromodeoxyuridine (10 µM; Sigma) was added after irradiation to facilitate scoring of exclusively first division metaphases. The cultures were incubated in plastic flasks at 37°C in a 5% CO2 atmosphere for 24 h, the last three in the presence of demecolcine (0.1 µg/ml), and cells were harvested. The cells were subjected to hypotonic shock (0.075 M KCl, 37°C), fixed in methanol:acetic acid (3:1) and cytological preparations were made in the routine way. For conventional cytogenetic analyses, some of the slides were stained with Giemsa. The remaining preparations were stored at –20°C until processed for in situ hybridization.
Staining of chromosomal preparations
For analysis using light microscopy, the slides were stained in 5% aqueous Giemsa solution for 8 min. For identification of second division cells, the slides were stained according to the fluorescence plus Giemsa technique (Perry and Wolff, 1974). Before in situ hybridization, the slides were stained with Hoechst 33258 [1 mg/ml in phosphate-buffered saline (PBS); Sigma] for 15 min in the dark, followed by incubation with 100 µl of the same solution under a coverslip exposed to UV light (Philips TLD 18W/08, distance ~6 cm) for 20 min at 55°C. Slides were then washed with PBS- Mg, Ca and dehydrated through an ethanol series (70, 90 and 100%).
In situ hybridization
Commercially produced (Cambio, UK) composite probes for chromosomes 1–5, 8, X, 12, 18, 19, 21 and 22 were employed. Replicate slides from all cell lines were painted using two- or three-colour FISH with a cocktail of one FITC-labelled chromosome-specific probe, one biotin-labelled chromosome-specific probe and, in the case of three-colour FISH, equal amounts of FITC- and biotin-labelled chromosome-specific probes. The FITC-labelled chromosome probes painted green, the biotin-labelled probes painted red (Texas red) and the FITC+biotin-labelled probes painted yellow/orange. The following combinations were used: 1-biotin/3-FITC+biotin/X-FITC; 2-biotin+FITC/4-biotin/8-FITC; 2-biotin/8-FITC; 1-biotin/X-FITC; 3-FITC/4-biotin; 5-biotin/12-FITC; 18-FITC/19-biotin; 21-FITC/22-biotin. Between 71 and 788 cells were analysed for these different combinations from the different cell lines. For analysis of the chromosome constitution a centromeric probe was not required. In this case the centromeres were visualized by differential DAPI staining. The slides from the irradiated cells were painted with probes specific for chromosomes 1, 4, 5 and 12. One set of these slides was painted with a cocktail of a FITC-labelled chromosome-specific probe for chromosome 1 and a biotin-labelled chromosome-specific probe for chromosome 4 together with a pancentromeric probe comprising equal amounts of biotin- and FITC-labelled probe (Cambio, UK). In this case chromosome 1 was painted green, chromosome 4 was painted red and the centromeres were painted yellow/orange. Another set of the slides were painted with a cocktail of a biotin-labelled chromosome-specific probe for chromosome 5 and a FITC-labelled chromosome-specific probe for chromosome 12 together with a pancentromeric probe comprising equal amounts of biotin- and FITC-labelled probe (Cambio, UK). In this case chromosome 5 was painted red, chromosome 12 was painted green and the centromeres were painted yellow/orange.
The in situ hybridization procedure employed was essentially similar to that proposed by Pinkel et al. (1986). Before in situ hybridization the slides were incubated with RNase (100 µg/ml in 2x SSC) for 1 h at 37°C, washed three times for 5 min each in 2x SSC and for 5 min in PBS and then incubated in pepsin (0.005%) in 10 mM KCl for 10 min at 37°C. After washing with PBS containing 50 mM MgCl2 they were post-fixed with 1% formaldehyde in PBS–/MgCl2 for 10 min at room temperature and dehydrated.
The whole chromosome probes were denatured, reassociated for 60–90 min at 37°C, applied to the slides and hybridization was performed overnight at 42°C. When the pancentromeric probe was applied in addition to the whole chromosome probes, a sequential hybridization reaction was performed. After hybridization of the whole chromosome probes overnight at 42°C, the slides were washed twice for 5 min each in 50% formamide, 2x SSC at 42°C, followed by two 5 min washes in 2x SSC at 42°C, and dehydrated. The centromeric probe was denatured, applied to the washed slides and hybridized overnight at 37°C.
Amplification of fluorescein was achieved with two layers of rabbit anti-FITC and FITC-conjugated goat anti-rabbit IgG (Cambio, UK). Amplification of biotin was achieved with two layers of Texas red–avidin and biotinylated goat anti-avidin (Cambio, UK). Antibodies were diluted in 15% blocking protein (Cambio, UK) in 4x SSC, 0.05% Tween 20. Each round of amplification was carried out in a humidified chamber at 37°C for 20 min, followed by three 5 min washes in 4x SSC, 0.05% Tween 20. Slides were dehydrated and embedded with Vectashield mounting medium (Vector Laboratories) containing 0.15 µg/ml DAPI (Serva) counterstain.
Scoring of aberrations
Fluorescence microscopy was performed on a Zeiss Axioskop equipped with a triple filter for simultaneous visualization of chromosomes painted red (biotinylated probes detected with Texas red), green (fluorescein-labelled probes detected with FITC), yellow [fluorescein:biotin (1:1)-labelled probes simultaneously detected with FITC and Texas red] and blue (DAPI counterstaining). All types of aberrations involving the painted chromosomes were scored in the first post-irradiation division. These included dicentrics, rings, excess chromosome fragments (not associated with dicentrics and rings), translocations and complex aberrations, which are defined as exchanges involving three or more breaks in two or more chromosomes (Savage and Simpson, 1994). Depending on the FISH pattern, translocations were classified as: reciprocal translocations, represented by two bi-colour monocentric chromosomes; incomplete/terminal `one-way' translocations, whose reciprocal event could not be detected; interstitial translocations (insertions). The incomplete/terminal translocations were classified into three types: a painted chromosome (usually truncated) with terminal acentric DAPI signal and accompanied by a completely painted acentric fragment; a DAPI stained chromosome with a terminal acentric painted signal; a painted chromosome with a terminal acentric DAPI signal. These three types of incomplete/terminal translocations correspond to FISH patterns of apparently simple incomplete/terminal exchanges I, II and III, respectively, in the nomenclature of Simpson and Savage (1996). Dicentrics were classified as: complete reciprocal dicentrics, seen as a bi-colour dicentric chromosome accompanied by a bi-colour acentric fragment; two types of incomplete dicentrics, one represented by a bi-colour dicentric chromosome with a single colour painted acentric fragment and the other seen as a bi-colour dicentric not accompanied by a painted or bi-colour acentric fragment. These two types of incomplete dicentrics correspond to patterns IV and VI, respectively, of Simpson and Savage (1996). Bi-colour acentric fragments (type V; Simpson and Savage, 1996) were also recorded, but were not included in the total counts of either the dicentrics or translocations. The term incomplete is used, although it is realized that some of these exchanges are in fact complete, the reciprocal event being beyond the resolution of the FISH assay (Boei et al., 2000; Fomina et al., 2000). The complex aberrations were analysed, on the one hand, using the system of Savage and Simpson (1994) wherever possible and, on the other hand, they were split into the above described aberrations and included in the total counts. Examples of chromosome aberrations involving the painted chromosomes are shown in Figure 1.
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Results |
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The results obtained from conventional Giemsa analyses of the chromosome constitution of one fibroblast and two lymphoblastoid WS cell lines are presented in Tables I and II
. All three WS cell lines showed a high incidence of hypo- and hyperdiploidy (Table I), ranging from 28 to 54%. The highest frequency of hyperdiploidy was found in KO375 cells. The three WS cell lines also showed the presence of tetraploid cells. The frequency of tetraploid cells was found to be highest (~24%) in one of the WS lymphoblastoid cell lines, DJG. The other lymphoblastoid cell line had only one tetraploid cell in 100. In addition to tetraploid cells the WS fibroblast cell line AG3141 had a low frequency (3–4%) of octoploid cells. Analysis of the unstable chromosome aberrations detected in Giemsa stained slides revealed a low frequency of aberrant cells, as well as dicentrics, chromosome fragments and chromatid fragments, in the two WS lymphoblastoid cell lines. A higher frequency of aberrant cells and dicentrics was observed in the WS fibroblast cell lines (Table II).
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The results on the chromosome constitution of the WS and control cell lines obtained after painting chromosomes 1–5, 8, X, 18, 19, 21 and 22 are presented in Table III
. A low frequency of dicentrics and translocations was found in all cell lines analysed. The frequencies of dicentrics, translocations and chromosome fragments were found to be highest in the WS fibroblast cell line. In fact, the frequency of 7.15 dicentrics observed in this cell line corresponds well with the frequency of 7.32 dicentrics observed with conventional Giemsa staining (Table II
), indicating that dicentrics are randomly distributed within the genome. In this cell line one dicentric involving chromosome 3 was observed in 436 cells, four dicentrics involving chromosome 19 in 114 cells and four dicentrics involving chromosome 22 in 117 cells. Since these 12 painted chromosomes represent ~67% of the human genome (Morton, 1991), any dicentric involving the non-painted chromosomes would be included in this observed frequency. Out of four octoploid cells, one cell was found with two dicentrics involving chromosome 19. Translocations were observed involving six of the painted chromosomes (1, 3, 5, X, 12 and 19). The frequency of translocations was found to be lower than the frequency of dicentrics in the WS fibroblast cell line. However, it was 2-fold higher compared with the control fibroblast cell line GM38A. The highest frequency of chromosome fragments was also found for the WS fibroblast cell line. The two lymphoblastoid cell lines had no dicentrics involving the painted chromosomes.
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An interesting finding was the chromosome constitution of the WS cell line KO375. All cells of this cell line had a reciprocal translocation between chromosomes 5 and 12 (Figure 1A
). Analysis of 397 KO375 cells from slides painted with biotin-labelled chromosome 5 and FITC-labelled chromosome 12 probes revealed that 144 cells (36.27%) were diploid and carried a reciprocal translocation t(5,12). The remaining cells were hyperdiploid carrying the same reciprocal translocation plus one or more additional chromosomes: in 239 cells (60.20%) the additional chromosome was the translocated chromosome 12 (Figure 1B); in four cells (1.0%) it was the translocated chromosome 5; in three cells (0.76%) three extra chromosomes were found, one translocated chromosome 5 and two translocated chromosomes 12; in five cells (1.30%) the additional chromosome was the normal chromosome 12; in one cell (0.25%) four extra chromosomes were observed, one translocated chromosome 5 and three translocated chromosomes 12; in one cell (0.25%) three extra translocated chromosomes 12 were observed. Only the normal chromosome 5 was never seen as an additional chromosome in hyperdiploid cells. These observations are in good agreement with the incidence of hyperdiploid cells found by conventional Giemsa analysis. This being the chromosome constitution of the WS cell line KO375, a very low frequency of translocations and chromosome fragments comparable with the control level was found. A similar low frequency of translocations and chromosome fragments was obtained for the other WS lymphoblastoid cell line, DJG. For the three WS cell lines, the fibroblasts showed the highest frequencies of chromosome aberrations and chromosome 19 was most frequently involved in dicentrcis, translocations and chromosome fragments. The three WS cell lines showed an increased frequency of aneuploid cells compared with the controls. Trisomy was observed for most of the painted chromosomes. The highest frequency of trisomic cells was found in the WS lymphoblastoid cell line DJG, which also showed the highest incidence of tetraploid cells.
The results on frequencies of chromosome aberrations induced in WS lymphoblastoid cells by 2 Gy X-rays detected by conventional Giemsa staining are presented in Table IV. Both WS lymphoblastoid cell lines showed a slight increase in the frequencies of aberrant cells (~25%) and chromosome fragments (~60%) as compared with the control. The increase in dicentric frequency was more pronounced in the WS cell line DJG (~30%) than in KO375 cells (~12%). The results on X-ray-induced chromosome aberrations involving chromosomes 1, 4, 5 and 12 in WS lymphoblastoid cells are presented in Table V. In the KO375 cell line dicentrics and translocations were recorded separately for the normal chromosome 5 and translocated chromosome 5 and the normal chromosome 12 and the translocated chromosome 12. At the time this experiment was performed ~90% of the cells had two copies of the translocated chromosome 12. Variable differences in the dicentric and translocation frequencies were observed between WS and normal cells involving chromosomes 4, 5 and 12. Both WS lymphoblastoid cell lines showed an increase in the frequency of translocations involving chromosome 1. This increase was by a factor of 1.4 in DJG and 1.7 in KO375 in comparison with irradiated control cells. KO375 also showed an increase in the frequency of dicentrics by a factor of 1.3 compared with irradiated control cells. Table VI presents the observed and expected frequencies of translocations and dicentrics involving chromosomes 1, 4, 5 and 12. The genomic frequencies of dicentrics and translocations were calculated using the formula GF = P/2.05xf(1 – f) (Lucas et al., 1992), where P is the frequency of aberrations detected for the painted chromosome and f is the fraction of the genome painted. Genomic frequency estimates were based on DNA content of these four chromosomes according to Morton (1991). Since two-colour painting was used, independent values of the frequencies of dicentrics and translocations for the whole genome could be calculated based on each painted chromosome. For each cell line the average genomic frequency from the four chromosomes was used to estimate the expected frequency of dicentrics and translocations. In the case of cell line KO375 a correction was made for the additional translocated chromosome 12. In this cell line the frequencies of dicentrics/translocations for chromosomes 5 and 12 were assumed to be equivalent to the frequency of dicentrics/translocations for both the normal and translocated chromosomes 5 and 12, respectively. Of the four chromosomes studied, only chromosome 1 responded with higher frequencies of aberrations than expected based on the DNA content in both lymphoblastoid cell lines, whereas in the control cells no difference between the observed and expected frequencies of aberrations involving chromosome 1 was found.
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Table VII
shows a comparison of the frequencies of chromosome aberrations involving chromosomes 1, 4, 5 and 12 in diploid and tetraploid cells from the WS cell line DJG induced by 2 Gy X-rays. The frequencies of both dicentrics and translocations involving chromosomes 1 and 4 were increased by a factor of 2, whereas the increase in dicentrics and translocations involving chromosomes 5 and 12 were increased by a factor of almost 3 in tetraploid cells. In 100 tetraploid cells from this cell line 106 dicentrics were observed in Giemsa stained slides, which is twice as high as the frequency of 53 dicentrics in diploid cells (Table IV).
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Table VIII
presents a more detailed analysis of the chromosome aberrations involving chromosomes 1, 4, 5 and 12. The chromosome aberrations were classified into: translocations (reciprocal, terminal/incomplete and insertions); dicentrics (reciprocal and incomplete); apparently simple incomplete translocations; apparently simple incomplete dicentrics (those not participating in the complexes); complex. No difference was observed in the relative proportions of apparently simple translocations and dicentrics and complex aberrations between WS and normal cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Werner syndrome cells have been shown to exhibit chromosomal instability and variegated translocation mosaicism (Salk et al., 1981
).These earlier studies were carried out by conventional chromosome banding techniques. Since the FISH technique using chromosome painting probes has high resolution in detecting chromosome exchanges, especially translocations, in the current study we employed this technique in three WS cell lines, one fibroblast and two lymphoblastoid. There was a high prevalence of aneuploidy in the cell line KO375, in which ~40% of cells were trisomic, and in cell line DJG, in which 40% of cells were tetraploid. These observations confirm earlier reports (Salk et al., 1981). Regarding the structural aberrations, frequencies of unstable aberrations (dicentrics and fragments, detected by Giemsa staining and FISH) and translocations (detected by FISH) were low in WS cells except for KO375 cells. All KO375 cells had a 5/12 translocation and ~60% had an additional translocated chromosome 12, as well as other translocations involving other painted chromosomes at low frequency. This cell line was very unstable and confirms the variegated translocation mosaicism character of WS cells.
Though WS cells exhibit chromosome instability, unlike other chromosome instability syndromes, such as FA and BS, they do not express hypersensitivity to mutagens, such as UV light, mono- and bifunctional alkylating agents, bleomycin, etc. (Arlett and Harcourt, 1980; Gebhart et al., 1988; Stefanini et al., 1989; Saito and Moses, 1991). The molecular defect in WS patients has been identified as mutations in consensus helicase domains (Chang-en Yu et al., 1997). Other human recessive disorders, such as xeroderma pigmentosum complementation groups D and B, trichothiodystrophy and Cockayne syndrome B, have mutations in helicases and are known to be sensitive to mutagenic carcinogens such as UV light and ionizing radiation. Since no studies have been carried out on the sensitivity of WS cells to ionizing radiation, it was considered of interest to evaluate induction of chromosomal aberrations by X-rays. In general, WS cells were slightly more sensitive than the control cells, especially for radiation-induced chromosome fragments (~60%), which may indicate a defect in the repair of X-ray-induced damage. The exact nature of these fragments (terminal or interstitial) is not known. FISH in combination with telomere-specific probes may throw some light on the nature of these breaks and reveal any differences between control and WS cells. There was no obvious difference between the WS cells and control cells with regard to the relative frequencies of radiation-induced incomplete and complete exchanges. Thus, it appears that WS cells are not highly sensitive to ionizing radiation or other DNA damaging agents, in spite of mutations in the helicase domains.
At the individual chromosome level, there appear to be differences in response between WS and control cells. Chromosome 1 responded with higher frequencies of exchanges than expected, on the basis of its DNA content in WS cells and not in control cells. The presence of both diploid and tetraploid cells in the same culture of DJG cells provided an opportunity to compare the induction of radiation-induced exchanges in these two types of cells. For chromosomes 1 and 4, an ~2-fold increase in the frequencies of exchanges was found. However, chromosomes 5 and 12 responded with an ~3-fold increase in the frequencies of exchanges in tetraploid cells. This points to some inherent inter-individual variations among the chromosomes which may lead to an increased or decreased response to radiation. The basis for these differences is not well understood.
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Acknowledgments |
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These investigations were supported in part by a contract from the European Union Nuclear Safety programme to A.T.N.
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Notes |
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2 To whom correspondence should be addressed. Tel: +31 71 5276154; Fax: + 31 71 5221615; Email: natarajan{at}rullf2.medfac.leidenuniv.nl
3 Present address: National Centre for Radiation Biology and Radiation Protection, Sofia 1756, Bulgaria
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References |
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Received on August 26, 1999; accepted on January 21, 2000.
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