Mutagenesis, Vol. 14, No. 4, 417-426,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
The application of comparative genomic hybridization and fluorescence in situ hybridization to the characterization of genotoxicity screening tester strains AHH-1 and MCL-5
Center for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales–Swansea, Swansea SA2 8PP, UK
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Abstract |
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AHH-1 TK+/– is a human B cell-derived lymphoblastoid cell line that constitutively expresses a high level of the cytochrome CYP1A1. The MCL-5 cell line was developed by transfection of AHH-1 with cDNAs encoding the human cytochrome P450s, CYP1A2, CYP2A6, CYP2E1, CYP3A4 and microsomal epoxide hydrolase carried in plasmids. The metabolic components of these cell lines make them a useful screening tool for use in mutagenicity studies. Although AHH-1 and MCL-5 are closely related, the two cell lines show differences which cannot be attributed to transfection. In the present study both cell lines were investigated for chromosome stability by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) using whole chromosome probes and telomeric probes. Amplification in chromosomes 4q, 3q and 9p was observed in both cell lines. To compare the cell lines directly, AHH-1 and MCL-5 DNAs were co-hybridized on the same metaphases using a modified CGH technique. The only difference observed between AHH-1 and MCL-5 was the degree of amplification involving the subtelomeric region of chromosome 4; the additional telomeric region (4q) was translocated onto chromosome 11 and/or chromosome X. FISH was use to show the presence of isochromosomes 3q and 9p in both cell lines with a chromosome number of 48 or higher. These data demonstrate that CGH and FISH with chromosome-specific probes are able to resolve complex karyotypes and to highlight subchromosomal regions involved in rearrangements and potential chromosome fragile sites. Analyses such as those described here may be of considerable value in the determination of the stability of a variety of the cell lines used in the mutagenicity testing of chemicals.
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Introduction |
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Comparative genomic hybridization (CGH) is a powerful molecular and cytogenetic technique that provides an overview of genetic imbalance within the entire genome (Kallionemi et al., 1992
, 1994). Based on a modified in situ hybridization technique, CGH can provide a comprehensive analysis of the entire genome, by screening for DNA sequence copy number changes and localizing those chromosomal regions gained or lost on a metaphase template. In this assay, normal human metaphases are competitively hybridized with differentially labelled total genomic test DNA and normal reference DNA. The hybridization kinetics of the two DNAs are independent, so the ratio of binding of the two probes is proportional to the ratio of the copy number of the sequences in the DNA samples to a specific locus. Any deviation from a 1:1 ratio will be recognized by analysis of hybridization patterns and can be quantified by computation. This procedure offers many advantages: it does not require samples with high mitotic index or cell culturing and information on the whole genome is provided in a single hybridization using a small amount of DNA (1 µg) (Kallionemi, 1994).
Despite its extensive applicability, this technique has some sensitivity limits in the analysis of cell lines or tumour tissues, including the following: (i) the detection of gained or lost DNA regions in relation to chromosome sites. Due to the repetitive nature of the centromeres and telomeres, copy number changes in these regions are hardly detected. (ii) The influence of the size of the region amplified or deleted and the deviation in the copy number. Zitzelsberger et al. (1997) established that the lowest detection limit of an over-represented DNA sequence is 0.25 Mb and that the smaller the amplicon the higher the copy number must be in order to be detected (
20 copies). Maximum resolution of 2 Mb for loss of one homologue and 1 Mb for loss of both homologues has been verified by the same authors. (iii) The percentage of the cell population with the same copy number deviation, because of the characteristic mosaicsm of tumours and unstable cell lines.
In this study, CGH, chromosome painting and fluorescence in situ hybridization (FISH) analysis using telomeric probes were used to characterize the karyotype of two lymphoblastoid cell lines: AHH-1 and MCL-5. Although FISH with chromosome-specific composite DNA probes (i.e. chromosome painting) provides considerable information about interchromosomal rearrangements, it is unable to recognize intrachromosomal aberrations (Lichter et al., 1988; Pinkel et al., 1988; Natarajan et al., 1991, 1992; Tucker et al., 1993). Moreover, classic G-banding is not able to detect subtelomere translocations because most of the chromosome ends stain negatively. In many cases, FISH with subtelomeric or telomeric probes is the best method to detect cytogenetically cryptic subtelomere abnormalities (Kingsley et al., 1997). For simplicity, when referring to the above-mentioned in situ hybridization method we will use the term telomeric fluorescence in situ hybridization (Telo-FISH). Human telomeric regions are characterized by a single tandem repeat (TTAGGG)n and an additional complex of subtelomeric repeats (Moyzis et al., 1988; Brown et al., 1990; Cross et al., 1990). Recently a complete set of human telomeric probes has been isolated and characterized (Nyng et al., 1996).
The AHH-1 and MCL-5 cell lines used in the present study were developed by Crespi et al. (1990, 1991) for genotoxicity studies. AHH-1 TK+/–, a human B lymphoblastoid cell line, was derived from the RPMI 1788 cell line (Freedman et al., 1979), isolated from a healthy male donor and spontaneously transformed by Epstein-Barr virus (EBV) (Genetest Corporation Manual; Crespi et al., 1991). The AHH-1 cell line constitutively expresses a high level of native CYP1A1. MCL-5 was developed by transfection of cDNAs of the human cytochrome P450s, CYP1A2, CYP2A6, CYP2E1, CYP3A4 and microsomal epoxide hydrolase into the AHH-1 cell line (Crespi et al., 1991). Thus, these cell lines are a useful screening tool in genotoxicity studies to investigate mutagenic potency in complex pathways (Crespi and Thilly, 1984; Crespi et al., 1991). Both cell lines have been investigated by classic cytogenetics and by chromosome painting. Lippoli Doepker et al. (1998), using classical karyotyping techniques, have reported that MCL-5 presents a modal chromosome number of 48. The two extra chromosomes found were two isochromosomes, one derived from the long arm of chromosome 3 [i(3q)] and the other from the short arm of chromosome 9 [i(9p)]. The same group also found balanced translocations involving some autosomic chromosomes and chromosome X. Doherty (1996), using chromosome painting, counted a modal chromosome number of 48 (the two extra chromosomes were identified as chromosomes 3 and 9) and also observed that many chromosomes were involved in balanced and unbalanced translocations. In order to resolve the discrepancies between the results obtained by Lippoli Doepker et al. (1998) and by Doherty (1996) and to asses the stability of the two cell lines, we performed CGH together with FISH to analyse the karyotypes.
Combining these approaches, it has been possible to identify amplifications, translocations and chromosome rearrangements that are frequently undetectable by conventional chromosome banding methods and to investigate the chromosome stability of the cell lines under study.
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Materials and methods |
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Cell lines
AHH-1 TK+/– is a human B cell-derived lymphoblastoid cell line that constitutively expresses a high level of the cytochrome CYP1A1. The MCL-5 cell line was developed by transfection of AHH-1 with cDNAs encoding the human cytochrome P450s, CYP1A2, CYP2A6, CYP2E1, CYP3A4 and microsomal epoxide hydrolase carried in plasmids (Crespi et al., 1991
). HF12 (human fibroblast 12) is a male primary diploid fibroblast cell line provided by the MRC Genome Stability Research Group (Harwell, UK). The human lymphocytes (HL) were donated by a healthy 31-year-old female donor.
Cell culture
AHH-1 TK+/– cells were cultured in suspension in RPMI-1640 medium (Gibco, Paisley, UK) supplemented with 9% horse serum (Sera Labs, Sussex, UK) in 80 cm2 flasks. MCL-5 cells were grown in RPMI-1640 containing 2 mM L-histidinol and 9% horse serum (both from Genetest, Cambridge, UK). The cell density was maintained at 2x105–106 cells/ml and the cells were not subcultured for longer than a 5 week period. HF12 cells were grown in DMEM (Gibco) supplemented with 15% fetal calf serum (Labtech, Uckfield, UK) in 25 cm2 flasks. Subculturing was carried out when cells reached confluency. The medium used for lymphocyte culture was RPMI-1640 supplemented with 20% fetal bovine serum (Labtech).
DNA extraction
Total genomic DNA was extracted from a minimum of 1x107 cells by a procedure based on separating proteins from DNA by salt precipitation (DNA extraction kit; Stratagene, La Jolla, CA). Extracted DNA was resuspended in enzyme-free water and quantified by spectroscopy.
Cytogenetics
HF12 cells were routinely cultured at a density of 2x105 cells/ml in a total volume of 10 ml. Two cell cycles later (48 h) the cells were treated with 0.03 µg/ml colcemid (Gibco) final concentration for 90 min. Metaphase cells were harvested by centrifugation, the supernatant discarded and the pellet of cells resuspended in pre-warmed hypotonic solution (0.56% KCl) for 10 min. After centrifugation, pre-chilled, freshly prepared Carnoy's fixative (3:1 methanol:acetic acid) was used to fix the cells in two stages of 30 and 10 min, respectively. The cell pellet was finally resuspended in 1 ml of fixative and the cells dropped onto clean chilled slides.
Telo-FISH
Freshly prepared slides were washed in 2xSSC for 2 min at room temperature, dehydrated through an ethanol series and maintained at 37°C. Chromoprobe coverslips, carrying the telomere biotin- and digoxigenin-labelled specific DNA probes of the selected chromosomes, were placed onto the slide and the hybridization performed according to the kit protocol (Chromoprobe-T; Cytocell, Oxford, UK). Stringent post-hybridization washes were performed using the formamide-free system. Immunological steps of detection of biotinylated probe were the same as described in the protocol kit. The slides were counterstained with the DAPI-Antifade solution provided with the kit.
Chromosome painting
Fresh slides were prepared by dropping the fixed pellet of metaphase cells onto ice-cold, polished slides. The protocol used to perform the FISH with whole chromosome probes for chromosomes 3, 4 and X was adapted from the protocol provided by Cambio. The counterstain, following the washes, was DAPI (5 µg/ml; Cambio, Cambridge, UK) added to mountant Vectashield (Vector Laboratories, Burlingame, CA).
Comparative genomic hybridization
CGH was performed according to the protocol of Kallioniemi et al. (1994) with a few modifications.
DNA Probe for CGH. Normal and test DNAs were prepared using 1 µg directly labelled with either fluorescein-12-dUTP (Amersham, Little Chalfont, UK) or rhodamine-12-dUTP (Vysis, Richmond, UK) by nick translation (Vysis kit system). The reaction times and the amounts of enzyme were modified in order to produce fragment lengths in genomic probes of between 600 and 2000 bp, as confirmed by migration on a 1% agarose gel. Aliquots of 200 ng of normal DNA and 400 ng of test DNA were simultaneously hybridized to normal metaphase chromosomes in the presence of Cot-1 (10 ng) for 48–72 h at 37°C. Cot-1 DNA is added to avoid the large ratio change at the heterochromatic regions characterized by repeated sequences. The slides were washed according to the protocol of the Vysis kit. Counterstaining with 0.01 ml of DAPI solution (5 µg/ml) permits interactive karyotyping. Images were captured with a Cohu Integrating Camera and a computerized six position ludl filter wheel with discrete filter sets for an Olympus microscope to allow overlapped registration of the three dyes: Texas red, FITC and DAPI. CGH analysis was carried out using PowerGene 760 karyotyping (Probe & CGH Software System). Five representative metaphases were analysed fully and the results from these were studied separately and combined to produce an average fluorescence ratio for each chromosome. Confidence intervals have been calculated and displayed for the mean profiles.
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Results |
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CGH analysis
Based on previous data obtained in our laboratory (Doherty, 1996
), the karyotype of the cell lines AHH-1 and MCL-5 used was initially interpreted as 48, XY, +3, +9. The primary data from CGH consist of intensity ratios of the fluorochromes as a function of their position on the chromosomes: the over- and under-represented DNA segments were quantified by computation of FITC/TRITC image ratios and average ratio profiles. Profiles from several copies of the same chromosome in different metaphases were averaged to reduce the fluctuation in image signal. Confidence intervals were calculated and displayed for the mean profile. A copy number change can be considered significant when the profile is outside the limits 0.75–1.25.
CGH of AHH-1 DNA (in green) versus normal control DNA (in red) (Figure 1).
CGH analysis of the AHH-1 DNA revealed extensive amplification involving the long arm of chromosome 3 (3q), the short arm of chromosome 9 (9p) and a region of chromosome 4 (4q). The fluorescence ratios (Figure 1a) and the amplification–deletion map (Figure 1b) clearly show these genomic imbalances. It can also be seen that large regions of heterochromatin (e.g. chromosome 1 and 9 centromeres) and some telomeric regions can, as indicated earlier, be problematical in this assay and are therefore excluded from this interpretation. The average green:red ratio of chromosomes 3 and 9 was 2:1 and suggested duplication of one of these chromosomal homologues (Figure 1c and d).
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CGH of MCL-5 DNA (in green) versus normal control DNA (in red) (Figure 2
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The results obtained by analysing the derived cell line MCL-5 by CGH also indicated that chromosomes 3, 4 and 9 were amplified in the same chromosomal region as in the AHH-1 cell line (Figure 2). Although chromosome 3 presented a lower level of amplification in the MCL-5 cell line corresponding to the region 3q12–q21 (Figure 2). This finding might suggest a deletion in this region and would confirm the cytogenetic data obtained by Lippoli Doepker (1998) regarding chromosome 3. Although considerably more variation was seen in this cell population, no other regions of the genome indicated a fluorescence ratio outside the range of significance. The CGH amplification–deletion map of MCL-5 versus normal control DNA reveals inconsistencies in the abnormalities found compared with AHH-1 (Figures 1b and 2
). In the amplification–deletion map the right and left lines next to the respective chromosomes are the results concerning any single analysed metaphase cell. These data have been confirmed by repeated experiments and could be due to variation in the copy number change along the length of the chromosomes in MCL-5 cells.
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CGH of AHH-1 DNA (in green) versus MCL-5 DNA (in red) (Figure 3
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In order to compare the lymphoblastoid cell lines directly, the CGH technique was modified, omitting the control DNA. The DNAs of AHH-1 and MCL-5, labelled in green and red, respectively, were co-hybridized onto the same metaphases. The results obtained using this method showed no apparent differences in chromosome 9 between the two cell lines. The only difference observed was the degree of amplification involving the paracentromeric region of chromosome 3 and the subtelomeric region of chromosome 4 (Figure 3a). In Figure 3b–d chromosome 4 CGH profiles obtained after the three different CGH analyses are compared showing that the chromosomal region 4p31–35 in AHH-1 has a higher level of amplification than in MCL-5. The differences in the degree of amplification might be due to different copy number deviation and to the different size of the amplicons involved in the two cell lines.
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FISH analysis
FISH with whole chromosome 3, 4, 11 and X probes and with specific telomeric DNA probes for the short arm (labelled in green) and the long arm (labelled in red) of chromosomes 3, 4 and 9 was performed on AHH-1 and MCL-5 metaphase chromosomes.
Chromosome 3 and 9 analysis.
Chromosome painting showed the presence of three chromosomes hybridizing with the chromosome 3 DNA probe (Figure 4a). A similar result was found by FISH with the chromosome 9 DNA probe. Dual colour FISH with telomeric probes for both chromosomes under study was used to assess the composition of those chromosomes with positive FISH signals. Telo-FISH demonstrated the presence of two chromosomes 3 with intact ends and of one isochromosome 3q (Figure 4b) and gave evidence of a normal pair of chromosome 9 plus an isochromosome 9p (Figure 4c). The isochromosomes 3q and 9p were detected in 100% of cells with a modal chromosomal number of 48.
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Chromosome 4 analysis. To interpret the data obtained by CGH regarding the composition of chromosome 4, chromosome painting and Telo-FISH using chromosome 4-specific probes was carried out. These analyses permitted us to locate intrachromosomal rearrangements involving chromosome 4 in both cell lines. The chromosome painting approach showed that genetic material from chromosome 4 was translocated onto an unidentified chromosome, although the normal autosomic pair of chromosome 4 appeared intact (Figure 5a
). This observation may explain the amplification found in the long arm of chromosome 4 by CGH analysis. In situ hybridization with differentially labelled 4p and 4q probes supported our hypothesis and demonstrate that the region involved in the translocation is the distal part of the long arm (Figure 5b). The percentages of AHH-1 and MCL-5 cells with constitutional variation of chromosome 4 and chromosome X are summarized in Table I and three karyotypic groups could be also identified. The data refer to 120 cells scored for each cell line. Eighty-two per cent of AHH-1 cells had two normal chromosomes 4 plus a derived chromosome involving the translocation of the subtelomeric region of chromosome 4 and an unknown chromosome (Table I, group 2) and 13% had two derived chromosomes of the latter type (group C). The percentages of the same groups of cells in MCL-5 were, respectively, 86 and 0.8%. A small percentage of cells also contain apparently random rearrangements (Figure 5c).
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Chromosome 4, 11 and X analysis. The unidentified chromosome implicated in the translocation with chromosome 4 above appeared consistently the same and similar to chromosome 11. Chromosome painting with a chromosome 11 probe permitted us to demonstrate that in all the cells with one derived chromosome, in both cell lines, the distal part of the long arm of chromosome 4 is translocated onto the long arm of chromosome 11 and results in der(11)t(11;4) (Figure 5d
). The same region 4q31–35 is, in some cases, also translocated onto the short arm of chromosome X, generating the derived chromosome der(X)t(X;4) (Figure 5e). In all the cells with a double translocation of chromosome 4, both the observed derived chromosomes are present. Chromosome painting with an X DNA probe showed that, in both cell lines, chromosome X is either whole or rearranged with chromosome 4 or with an unidentified chromosome (Figure 5f) at a different frequency (see Table II).
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Karyotypic groups found in MCL-5 and AHH-1 cell lines
Three karyotypic groups of cells were detected in the AHH-1 and MCL-5 cell populations by FISH and CGH. Both cell lines presented an aneuploid karyotype with a modal chromosome number of 48. Ninety per cent of the AHH-1 cells had 48 chromosomes, whereas only 65% of MCL-5 cells presented the same modal chromosomal number. In all cells with 48 chromosomes, the isochromosomes 3q and 9p were present.
The percentage of cells belonging to each karyotypic group is represented in Tables I and II. The nomenclature suggested by the ISCN (International System for Human Cytogenetic Nomenclature, 1995) is applied to characterize them. The telomeric probes used were documented by Nyng et al. (1996). Table I shows the distribution of chromosome 4 constitution in AHH-1 and MCL-5. Note that, in the following descriptions, ish indicates in situ hybridization and wcp indicates whole chromosome probe.
Group A. 48, XY, ish, i(3)(q10) (wcp3+B47a2+B35c1), +i(9)(p10) (wcp9+34H2+2241a4). Cells with an isochromosome 3q and an isochromosome 9p. Normal pair of chromosome 4.
Group B. 48, XY, ish, i(3)(q10) (wcp3+B47a2+B35c1), +der(11)t(11;4), +4 (wcp 4+wcp11+B31+cT55), +i(9)(p10) (wcp9+34H2+2241a4). The distal segment of chromosome 4 (4q31) has been translocated onto the long arm of chromosome 11 generating der(11)t(11;4). Also present was one isochromosome 3q and one 9p and two normal chromosome 4. The resulting net imbalance is a trisomy for 4q31 tel.
Group C. 48, XY, ish, i(3)(q10) (wcp3+B47a2+B35c1), +der(11)t(11;4)x2, +4(wcp4+wcp11+B31+cT55), +i(9)(p10)(wcp9+wcpX+34H2+2241a4). The distal segment of chromosome 4 (4q31) has been translocated onto the long arm of chromosome 11 and onto the short arm of chromosome X, generating two derived chromosomes: der(11)t(11;4) and der(X)t(X;4). Two normal chromosomes 4 are present and additional chromosome 4 material in t(11;4) and t(X;4). The resulting net imbalance translates into a 4-fold amplification of the subtelomeric region of chromosome 4.
Table II shows the distribution of chromosome X constitution in AHH-1 and MCL-5 cells. In this analysis we refer to the most consistent karyotypic groups of both cell lines (Table I, groups A and B).
Group A'. 48, XY, ish, i(3)(q10) (wcp3+B47a2+B35c1), +der(11)t(11;4), +4 (wcp4+wcp11+B31+cT55), +i(9),(p10) (wcp9+34H2+2241a4), +X(wcpX). The distal segment of chromosome 4 (4q31) has been translocated onto the long arm of chromosome 11 generating der(11)t(11;4). Chromosome X present a normal constitution.
Group B'. 48, XY, ish, i(3)(q10) (wcp3+B47a2+B35c1), +der(11)t(11;4), +4 (wcp4+wcp11+B31+cT55), +i(9),(p10) (wcp9+34H2+2241a4), +der(X)t(X;?)(wcpX). The distal segment of chromosome 4 (4q31) has been translocated onto the long arm of chromosome 11 generating der(11)t(11;4). Genetic material of an unidentified chromosome is translocated onto the short arm of chromosome X generating der(X)t(X;?).
Group C'. 48, XY, ish, i(3)(q10) (wcp3+B47a2+B35c1), +der(?)t(?;4)x2, +4(wcp4+B31+cT55), +i(9)(p10) (wcp9+34H2+2241a4). The distal segment of chromosome 4 (4q31) has been translocated onto the long arm of chromosome 11 and onto the short arm of chromosome X, generating two derived chromosomes: der(11)t(11;4) and der(X)t(X;4).
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Discussion |
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Cells expressing human metabolic enzymes, such as AHH-1 and MCL-5, may be considered as convenient models for determing the key enzymes involved in metabolic activation (Styles et al., 1994
) and to assess the cytogenetic effects of procarcinogen treatment. In these lymphoblastoid cell lines reactive metabolites are generated intracellularly and the genetic end-point is detected within the same cell without the problems of toxicity and stability imposed by the use of extracellular metabolizing systems. Our main interest in this study was to determine the karyotypic stability of these cell lines and to develop procedures to analyse other cell lines.
CGH in conjunction with FISH using probes for chromosomes 3, 4, 9 and X allowed us to describe abnormalities in both cell lines. CGH was able to detect amplification of the entire long arm of chromosome 3, of the subtelomeric region of chromosome 4 and of the entire short arm of chromosome 9. CGH was able to demonstrate that the amplification of chromosomes 3 and 9 shown by Doherty (1996) with chromosome painting was limited to the long arm of chromosome 3 and the short arm of chromosome 9. This was confirmed by Telo-FISH with chromosome-specific probes, which are differentially labelled on the p (green) and on the q (red) arms. Thus, it was possible to detect the presence of two normal pairs of chromosomes 3 and 9 plus two isochromosomes derived, respectively, from the long arm of chromosome 3 (i[3q]) and the short arm of chromosome 9 (i[9p]). Since the two cell lines are related, as one is derived from the other, we used a modified CGH procedure in order to compare them directly to highlight any differences in the level of amplification at various subchromosomal regions. This allowed us to demonstrate a different degree of amplification in the distal part of chromosome 4. The CGH mean ratio obtained by labelling AHH-1 in green and MCL-5 in red shows a deviation from the 1:1 ratio in the profiles of chromosomes 3 and 4 obtained by the analysis of 10 metaphases and displays a slight amplification in the 3q12–q21 and in the 4q35 regions (Figure 3a and b). The ratio profile lies on the conventional significance borderline, expressing an amplification of 0.25-fold. However, as we are comparing two cell lines with a similar percentage of altered DNA cells, then the conventional threshold could be considered artificially high. The interpretational difficulties concerning the deletion of 3q12–q21 are due to the presence of the isochromosome 3q, which results in an excess copy number of this sequence in a homologous chromosomal region. Further studies would be useful by carrying out a reverse hybridization in order to confirm the under-representation involving the 3q region. Although in the karyotypic analysis performed by Lippoli Doepker et al. (1998) no rearrangements were detected in chromosome 4, they identified a deletion in the region 3q12–q21. These data are in agreement with our CGH results. Chromosome counting and FISH analysis were performed on 120 metaphases (Table I) to assess the involvement of chromosomes 4, 11 and X in amplification and possible translocations. The modal chromosome number was 48. In both cell lines the largest group of cells carried a derived chromosome der(11)t(11;4) where the region 4q31–34 is over-represented once. Moreover, 13% of AHH-1 cells had the same region repeated twice due to the presence of a second derived chromosome, der(X)t(X;4). Only one out of 120 MCL-5 cells showed this rearrangement. The latter results explain the different level of amplification involving the distal part of the long arm of chromosome 4 obtained by the modified CGH between the two cell lines. Chromosome X was often involved in rearrangements either with chromosome 4 or with some other unidentified chromosomes. Wherever chromosome 4 was doubly amplified, chromosome X was also always involved in a translocation.
In order to confirm our hypothesis regarding the involvment of the amplified telomeric region 4q in the translocation and the presence of the two entire chromosome 4, FISH with telomeric probes for the p and q arms of chromosome 4 was performed. This last approach confirmed our hypothesis.
The mechanism involved in the formation of the derived chromosome is unknown and different hypotheses could be considered. It is unclear if the amplification occurred before or after translocation. Since we found the reciprocal translocated chromosome in a very low percentage of cells, it could be hypothesized that one copy of chromosome 4 was duplicated during cell division due to non-disjunction, to give rise to two normal chromosomes. One homologue of chromosome 4 then went through a balanced translocation resulting in a chromosome 4 with the distal part belonging to another chromosome and a derived chromosome with the terminal part of the long arm of chromosome 4. Whereas the derived chromosome was stably inherited in the daughter cells, the reciprocal product was lost. This event could be due to the presence of the normal chromosome 4 pair being more stable in mitotic segregation and successful in any competition for binding the spindle fibres. The duplication and non-disjunction theory has also been proposed by Lippoli Doepker et al. (1998) to justify the hyperploidy of the two cell lines due to the presence of the two isochromosomes.
Summarizing our findings, the AHH-1 and MCL-5 cultures show a modal chromosome number of 48, including the isochromosomes i(3q) and i(9p). Moreover, we confirm a consistent translocation event involving chromosome 4 with chromosome 11 and with chromosome X. We have also demonstated that the two cell lines present a different but constant degree of amplification involving the distal part of the long arm of chromosome 4. This new finding has been proved by reverse hybridization of chromosome 4 telomeric probes onto AHH-1 and MCL-5 metaphases. In the karyotypic data reported by Lippoli Doepker et al. (1998) regarding the MCL-5 cell line, genetic material of an unidentified chromosome was translocated onto chromosome 11 in the region 11q23 and onto chromosome X in the region Xp22.3. Digital FISH image analysis obtained in our experiments evaluated with the CGH Software System permitted us to locate the translocation of chromosome 4 material onto chromosome 11 at 11q23.1 and to chromosome X at Xp22.3. This observation could suggest the presence of fragile sites in these regions.
Based on these results we can deduce that although the two cell lines are closely related they have different chromosome stabilities. Whereas the AHH-1 strain presents an apparent stability in its karyotype, MCL-5 cells appear relatively more unstable. This is further supported by our finding that 65% of the MCL-5 cells had 48 chromosomes, whereas 90% of AHH-1 cells have the same modal chromosome number, which could suggest a lack of stability in the former. Lippoli Doepker et al. (1998), using classic cytogenetics (G-banding) to examine the karyotypic stability of these cells, also investigated the micronucleus (MN) and sister chromatid exchange (SCE) frequencies and reported a MN frequency significantly higher than previous data obtained in our and in other laboratoties (White et al., 1992; Styles et al., 1994; Doherty et al., 1996). For these reasons we agree with Lippoli Doepker et al. (1998) in suggesting caution when using these cell lines in genotoxicity studies. Furthermore, if we take into consideration the theory of jumping translocations or the presence of fragile sites in chromosomes 11 and X, karyotypic instability is more strongly supported. The characterization of the karyotypic groups that we performed was based specifically on the analysis of a limited range of chromosomes. Further in-depth analyses, using different combinations of whole chromosome and telomeric probes, could provide more details about the involvement of all chromosomes in possible rearrangements.
The major advantage of CGH is its ability to scan the entire genome starting from just a few nanograms of genomic DNA without requiring any previous knowledge of the genetic aberrations and any specific probes. This technique is not able to detect changes that are not present in a substantial proportion of the cells under study and cannot reveal any other aberrations that do not involve a copy number change. Moreover, it is very difficult to identify deletions in some regions that are amplified in the other homologous chromosome. Chromosome painting is a powerful technique for detecting chromosomal aberrations such as translocations, insertions, multiple rearrangements and numerical deviations. FISH with telomeric probes (Telo-FISH) focuses on structural rearrangement that might be intrachromosomal, such as inversions, and that otherwise wouldn't be detected. Moreover, it provides information about the integrity of the telomeres and, in some cases, of the whole chromosome structure. By combining these various methods it is possible to dissect and describe complex karyotypes. We recommend that cells should be screened before use in mutagenicity studies to confirm the chromosomal stability of the subculture. Indeed, this approach could be used to pre-screen any cell culture for chromosomal stability before its use in genotoxicity studies.
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Acknowledgments |
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The work described here has been supported in part by funds from Astra Pharmaceuticals and the Environmental Programme of the European Union.
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Notes |
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1 To whom correspondence should be addressed. Tel: +44 1792 295361; Fax: +44 1792 195447; Email: bacorso{at}swansea.ac.uk
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References |
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Received on December 16, 1998; accepted on March 16, 1999.
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