Mutagenesis, Vol. 14, No. 3, 283-286,
May 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Comparison of AluI-induced frequencies of dicentrics and translocations in human lymphocytes by chromosome painting
1 MGC, Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, Leiden, The Netherlands, 2 Division of Human Cytogenetics and Quantitative Microscopy, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay, 3 Department of Genetics, University of Essen, Essen, Germany and 4 J.A.Cohen Institute of Radiopathology and Radiation Protection, Inter-University Institute, Leiden, The Netherlands
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Abstract |
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It has been shown repeatedly that following irradiation of human lymphocytes in the G0 stage, more translocations are induced than dicentrics. To check the role of DNA double-strand breaks (DSB) alone for the induction of symmetrical and asymmetrical chromosome aberrations, the frequencies of induced exchange aberrations by the restriction enzyme AluI were analyzed. The enzyme was introduced into cells using the pellet pipetting technique. Frequencies of induced translocations and dicentrics were determined using a chromosome painting assay with chromosome-specific DNA libraries for chromosomes 1, 4 and X (representing 16.8% of the human genome). The number of translocations detected was ~3-fold higher than the number of dicentrics, indicating that the increased frequency of translocations compared with dicentrics found in irradiated human lymphocytes does not result from DNA lesions other than DSB but from differential processing of DSB.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Exposure of cells to ionizing radiation and radiomimetic agents leads to the formation of a variety of symmetrical (translocations) and asymmetrical (dicentrics) chromosomal exchanges. Conventional cytogenetic techniques are efficient in identifying dicentrics. Translocations are detectable with banding techniques or, more recently, with the fluorescence in situ hybridization (FISH) technique using chromosome-specific DNA libraries (chromosome painting) and a pancentromeric DNA probe (Pinkel et al., 1986
; Cremer et al., 1990; Natarajan et al., 1992; Bauchinger et al., 1993). For ionizing radiation, most authors found that 1.5- to 3-fold more translocations than dicentrics were induced following irradiation of G0 lymphocytes (Cremer et al., 1990; Natarajan et al., 1992; Schmid et al., 1992; Bauchinger et al., 1993, Boei et al., 1996; Darroudi et al., 1998).
Ionizing radiation induces different DNA lesions, namely double-strand breaks (DSB), single-strand breaks, base damage and DNA–protein crosslinks. Among these, DSB were considered to be responsible for the induction of chromosomal aberrations (Natarajan and Obe, 1978; Natarajan et al., 1980; Darroudi et al., 1990; Obe et al., 1992). Furthermore, restriction enzymes (which induce only DSB) effectively induce chromosomal aberrations in Chinese hamster ovary cells, in a human lymphoblastoid cell line or in primary lymphocytes (Bryant, 1984, 1994; Natarajan and Obe, 1984; Darroudi and Natarajan, 1989; Columna et al., 1993; Martínez-López et al., 1995). It was found that restriction enzymes can be successfully internalized into human peripheral lymphocytes (HPL) by strongly pipetting cell pellets in the presence of the enzymes (Martínez-López et al., 1995).
In the present work, the involvement of DSB in the differential formation of symmetrical (translocations) and asymmetrical (dicentrics) chromosomal aberrations was investigated in HPL treated with the restriction enzyme AluI. The aberrations were analyzed by FISH using a cocktail of three chromosome-specific DNA libraries (chromosomes 1, 4 and X). Additionally, centromeres were visualized using a pancentromere-specific probe.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Blood cultures and preparations
Peripheral blood samples were collected from four healthy male donors. Heparinized whole blood (0.5 ml) was cultured in 10 ml round-bottom plastic tubes (Greiner) containing 4.0 ml of McCoy's 5A medium, 0.5 ml of heat-inactivated fetal calf serum (Gibco), 0.125 ml phytohaemagglutinin (Gibco) and antibiotics (100 U/ml penicillin and 125 µg/ml dihydrostreptomycin sulphate) (Hoechst and Seromed, respectively).
Cells were treated at 20 h culture time and were then recovered in complete medium supplemented with 5-bromodeoxyuridine (BrdUrd; Serva) at a final concentration of 10 µM for 40 h, including 3 h exposure to colcemid (final concentration 0.08 µg/ml; Ciba).
Lymphocytes were centrifuged and pellets were treated with KCl (0.075 M) for 15 min at room temperature. Lymphocytes were fixed three times with methanol:acetic acid (3:1), and cell suspensions were dropped onto ice-cold slides and air dried.
Treatment with AluI
After 20 h of culture, cell pellets were exposed to 20 U AluI (Gibco BRL). The enzyme was added to the pellet and cells were pipetted three times with a thin Pasteur pipette with a sharp (broken) rim. Recovery was in complete medium supplemented with 5-bromodeoxyuridine for 40 h. Control cells were treated either with 2 µl of AluI storage buffer (10 mM Tris–HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 0.5 mg/ml bovine serum albumin, 50% glycerol), which is the amount of buffer in which 20 U AluI was dissolved, or exposed to 20 U AluI without pipetting for ~10 min.
Fluorescence in situ hybridization (FISH)
Human chromosome-specific libraries were obtained from the American Type Culture Collection (ATCC). A cocktail of libraries for chromosomes 1, 4 and X was employed representing 16.8% of the male genome (Mendelsohn et al., 1973).
Probes were labeled by nick translation with biotin-16-dUTP, purified using Sephadex G50 gel, mixed with 50 µg of total human DNA and 25 µg of salmon sperm DNA and precipitated overnight in ethanol/sodium acetate at –20°C. The precipitated DNA was dried in a vacuum centrifuge and dissolved in a hybridization buffer containing 50% deionized formamide, 2x SSC, 40 mM phosphate buffer (pH 7) and 9% dextran sulphate.
FISH was carried out as described earlier (Pinkel et al., 1986; Natarajan et al., 1992). Briefly, slides were treated with RNase and pepsin and fixed with 1% formaldehyde in phosphate-buffered saline (PBS)/MgCl2. Slides were washed with PBS, dehydrated through an ethanol series and denatured with 70% deionized formamide, 2x SSC, 50 mM phosphate buffer (pH 7) at 80°C for 4 min. After denaturation, the slides were washed in a series of ice-cold ethanol (70, 90 and 100%). Labeled probes were denatured for 5 min at 70°C, chilled for 5 min on ice and then placed for 2 h in a water bath at 37°C for pre-annealing. Thereafter, the probes were mixed and applied to the slides in a total volume of 20 µl. The hybridization was carried out overnight in a moist chamber at 37°C, after which the slides were washed (three times) for 5 min with 50% formamide, 2x SSC (pH 7) at 42°C followed by 3x5 min washes in 0.1x SSC at 60°C. The slides were immunostained with avidin–FITC and goat anti-avidin antibody. The signal was amplified three times. The slides were dehydrated and counterstained with 1 µg/ml propidium iodide or DAPI in Vectashield mounting medium. In damaged metaphases stained with DAPI it was possible to discriminate between translocations and dicentric chromosomes, as the positions of the centromeres were clearly visible. Some slides were processed for FISH using chromosome painting probes and in addition a pancentromeric DNA probe.
Scoring of chromosomal aberrations
We analyzed differentially stained slides (Hill and Wolff, 1982) from control and AluI-treated cultures and scored 1000 metaphases from each donor for the occurrence of chromosomal aberrations in first and second post-treatment divisions using a Metaphase Finder System (MetaSystems).
The scoring of chromosomal aberrations in FISH metaphases was done with a Zeiss fluorescence microscope equipped with a triple filter combination (Omega). In accordance with the PAINT nomenclature system, only exchange aberrations involving both painted and unpainted chromosomes were considered (Tucker et al., 1995a). Aberrations scored were all types of translocations (reciprocal translocations, non-reciprocal translocations and insertions), dicentrics and half-painted fragments. If both counterparts of a translocation were visible, it was taken as a reciprocal translocation, if only one bi-coloured chromosome was present as a non-reciprocal translocation (Tucker et al., 1995a).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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AluI produces DSB at AG
CT sites and a large number of breaks are expected to occur in human DNA. No chromosomal aberrations were found in differentially stained control cells. The analysis of differentially stained treated cells revealed no aberrant metaphases in the second mitotic division (M2). Therefore, we can be sure that we are not scoring chromosomal aberrations in M2 in the painted chromosome preparations.
AluI (20 U) induced exclusively chromosome type aberrations (Table I). A low percentage of abnormal cells was found (3%) involving the three painted chromosomes (1, 4 and X) but a very high number of translocations and dicentrics were observed per aberrant cell, indicating an overdispersion of the intercellular distribution of aberrations, as described previously (Martínez-López et al., 1995).
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Most affected cells were heavily damaged showing many chromosomal rearrangements. Because of this, reciprocal translocations could not always be unambiguously identified and these were classified together with non-reciprocal translocations.
Reciprocal translocations are complementary events of complete dicentrics (dicentric + half-painted fragment), non-reciprocal translocations are complementary events of incomplete dicentrics (Savage, 1976). Therefore, we considered it adequate to pool reciprocal and non-reciprocal translocations to compare these data with the frequencies of dicentrics (complete and incomplete). Insertions were not taken into consideration.
At least one dicentric involving a painted chromosome per aberrant cell was found. The number of translocations (reciprocal and non-reciprocal) detected was ~3-fold higher than the number of dicentrics (complete and incomplete). Similar results were found using the streptolysin O method (Bryant, 1992) to introduce AluI into isolated human lymphocytes (unpublished data).
Figure 1 shows the number of translocations and dicentrics in 100 aberrant cells induced by 20 U AluI. The frequencies of translocations were 2.3- to 4.0-fold higher than the frequencies of dicentrics.
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Extra half-painted fragments (HPF) were counted separately. These HPF could represent rejoined fragments from terminal deletions and could therefore be taken into consideration as another possible counterpart of non-reciprocal translocations (Savage, 1976
). Even if we take into consideration the amount of HPF as complementary events of non-reciprocal translocations, the average frequencies of translocations are still higher than that of dicentrics, namely 1.5- to 2.8-fold (average 2.25-fold). In order to be sure that the observed excess of translocations was not due to misclassification of the exchange aberrations, some slides were painted with chromosome-specific DNA libraries and a centromere-specific probe. In this case, the frequencies of translocations involving chromosomes 3 and 8 were 2.0–3.3 times higher than that of dicentrics (data not shown), indicating that the above mentioned results cannot be explained by misclassification of aberrations.
Increased frequencies of X-ray-induced translocations as compared with dicentrics have been reported repeatedly (Cremer et al., 1990; Natarajan et al., 1992; Schmid et al., 1992; Bauchinger et al., 1993; Boei et al., 1996; Darroudi et al., 1998). The mechanism by which exchange type aberrations are produced has been an area of extensive speculation and research (Vyas et al., 1991; Savage 1993a,b; Durante et al., 1996; Darroudi et al., 1998). Of all lesions induced by DNA damaging agents, DSB have been implicated as the most important one (Natarajan et al., 1986). One of the approaches to understand the role of DSB in the induction of chromosome aberrations has been to damage chromosomal DNA with restriction enzymes, which exclusively induce DSB (Bryant, 1984; Natarajan and Obe, 1984; Darroudi and Natarajan, 1989). The sequence of events from DSB to dicentrics and translocations appear to be different (Darroudi et al., 1998). A question of interest is why one type of exchange aberration (translocations) should occur at higher frequencies than others (dicentrics). According to Straume and Lucas (1993), the higher frequency of translocations is a result of misscoring dicentrics for translocations. However, even when chromosome-specific DNA libraries were combined with a pancentromeric probe, consistently higher frequencies of translocations in relation to dicentrics following treatment of human lymphocytes with ionizing radiation was evident (Bauchinger et al., 1993; Boei et al., 1996; Darroudi et al., 1998).
In the present study, we have shown that the increased frequency of translocations compared with dicentrics found in irradiated HPL does not result from DNA lesions other than DSB but from differential processing of DSB.
It has been reported that the nuclear architecture could play an important role in the mode and kinetics of processing and repair of DNA damage (Oleinick et al., 1984; Mullenders et al., 1991; Savage, 1993a; Cremer et al., 1995; Tucker et al., 1995b). Studies using the method of premature chromosome condensation (PCC) in combination with FISH (Darroudi et al., 1998) or DNA repair inhibitors (ara-C, ara-A and 3-aminobenzamide) indicate that the formation of dicentrics and translocations are different (Natarajan et al., 1994).
In this respect, it would be of great interest to study the initial frequencies of breaks and chromosome exchanges and their repair kinetics with various restriction enzymes cleaving at specific sequences using PCC in combination with FISH in human lymphocytes.
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Acknowledgments |
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This work was supported financially, in part, by grants from CEC, Nuclear Safety Radiation Program (nos F14P-CT95-50001 and F14P-CT950011) to A.T.N. and by a fellowship from the Deutscher Akademischer Austauschdienst (DAAD) granted to W.M.-L.
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Notes |
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5 To whom correspondence should be addressed. Tel: +598 2 487 16 16; Fax: +598 2 487 55 48; Email. wlopez{at}iibce.edu.uy
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Received on June 19, 1998; accepted on November 20, 1998.
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