Mutagenesis, Vol. 18, No. 3, 235-242,
May 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Noscapine hydrochloride-induced numerical aberrations in cultured human lymphocytes: a comparison of FISH detection methods and multiple end-points
1 Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521, USA and 2 Pfizer Inc., Groton, CT 06340, USA
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The cytokinesis block in vitro micronucleus (MN) assay in combination with CREST staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes allows mechanistic information on the induction of numerical chromosomal aberrations to be obtained through a rapid and simple microscopic analysis. These techniques can now be used to investigate relationships between the induction of chromosomal loss, non-disjunction and polyploidy by aneuploidy-inducing agents. In the present study, we treated 72 h cultured lymphocytes for the last 24 h of culture with various concentrations of the cough medicine noscapine hydrochloride (NOS) (3.9–120 µg/ml) in the presence of either cytochalasin B (CYB) (3 µg/ml) or 5-bromo-2'-deoxyuridine (BrdU) (1 µM). Using the CREST staining modified MN assay in the CYB-treated cultures, we detected significant increases in CREST-positive but not CREST-negative MN in both binucleated and, to a lesser extent, mononucleated cells, demonstrating the ability of this compound to induce chromosomal loss. In addition, using FISH with chromosome 1- and 9-specific classical satellite probes, a significant induction of chromosomal non-disjunction in the binucleated lymphocytes and polyploidy in the mononucleated lymphocytes was seen, indicating that polyploidy induced by NOS may occur without progression through a normal anaphase and/or telophase. In the BrdU-treated cultures, a dose-dependent induction of hypodiploidy, hyperdiploidy and polyploidy was observed using FISH with a chromosome 9-specific
-satellite probe in the labeled cells. By comparison, in the unlabeled non-cycling cells, only a slight increase in hyperdiploidy/polyploidy but not hypodiploidy was seen. A comparison of the effects seen at different concentrations shows that at the lower effective concentrations, all three types of numerical aberrations, chromosomal loss, non-disjunction and polyploidy, contributed to the numerical aberrations seen, whereas at the highest concentration tested, polyploidy was the predominant alteration. These studies indicate that FISH in combination with CYB or BrdU immunfluorescent staining can be sensitive tools for the identification of aneuploidy-inducing agents. |
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Aneuploidy is a condition in which the chromosome number of a cell deviates from a multiple of the normal haploid complement and occurs when replicated chromosomes fail to accurately segregate between the two daughter cells during mitosis or meiosis. It may arise spontaneously or as a consequence of exposure to an aneuploidy-inducing agent. Despite the importance of aneuploidy for human health, genetic assays to detect aneuploidy-inducing agents are currently limited and not sufficiently validated (Aardema et al., 1998
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The most direct way to assess chromosomal aneuploidy is to evaluate chromosome numbers in metaphase cells. However, this method has the disadvantage that it is time consuming, prone to technical artifacts and restricted to actively dividing cells. One alternative is to measure the induction of micronuclei (MN), which can arise from either chromosomal breakage or chromosomal loss during cell division. Since the induction of MN can only occur in dividing cells, it is important to have a proliferation marker, particularly in cell culture systems like human lymphocytes where only a fraction of the total cell population proliferates following mitogenic stimulation. One of the methods to overcome this problem is to block cytokinesis using cytochalasin B (CYB) to distinguish dividing and non-dividing cells (Fenech and Morley, 1985). In conjunction with immunfluorescence staining using CREST (calcinosis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly and telangiectasia; a subform of scleroderma) serum or fluorescence in situ hybridization (FISH) with a pancentromeric DNA probe to characterize the origin of MN, the MN assay has become an easy and rapid method to identify agents that are clastogenic and/or have aneuploidy-inducing potential (Schuler et al., 1997). Although this assay is very sensitive for detecting chromosomal loss, it is unable to detect non-disjunction and chromosome gain. However, the cytokinesis blocked MN assay in combination with FISH using chromosome-specific probes allows the measurement of both chromosomal loss and non-disjunction events at the same time (Marshall et al., 1996; Zijno et al., 1996a,b; Elhajouji et al., 1997; Sgura et al., 1997; Minissi et al., 1999). As numerical aberrations can be induced by different mechanisms, it is important to determine the inter-relationships between the various end-points. It has been shown that induction of non-disjunction often occurs at lower concentrations than induction of chromosomal loss (Marshall et al., 1996; Zijno et al., 1996a,b; Elhajouji et al., 1997; Sgura et al., 1997; Minissi et al., 1999) and, therefore, a NOAEL or threshold based upon the MN assay may not be adequate. On the other hand, recent publications have indicated that both the absolute and relative frequencies of non-disjunction, chromosomal loss and polyploidy may be influenced by a modification of cell division by CYB itself (Zijno et al., 1996a; Minissi et al., 1999).
A valuable alternative, especially to exclude synergistic effects of aneuploidy-inducing agents and CYB, could be the use of 5-bromo-2'-deoxyuridine (BrdU) as a proliferation marker. Surprisingly, despite the fact that BrdU is routinely used in the analysis of sister chromatid exchange, only a few publications using BrdU as proliferation marker in the MN assay have been reported (Norppa et al., 1990; Montero-Montoya et al., 1997). The availability of a monoclonal anti-BrdU antibody now allows the detection of BrdU at low concentrations and below those associated with genotoxic effects or interference with cell division (Pinkel et al., 1985). Therefore, BrdU could become a valuable option to improve the detection of numerical aberrations in the MN assay when the use of CYB is not desirable and when using FISH with chromosome-specific DNA probes.
The aims of this paper are to compare FISH detection methods and multiple end-points for the detection of numerical aberrations in cultured human lymphocytes. In addition, the inter-relationships between the induction of non-disjunction, chromosomal loss and polyploidy have been investigated. Noscapine hydrochloride (NOS), an isoquinoline alkaloid used in a variety of cough suppressants, was chosen as test compound because of its ability to induce high levels of polyploidy and aneuploidy over a wide range of concentrations in vitro (Ishidate, 1988; Gatehouse et al., 1991; Mitchell et al., 1991; Schuler et al., 1999). In this study, we treated cultured human lymphocytes with various concentrations of NOS in the presence of either CYB or BrdU. In the CYB-treated cells, we performed the in vitro MN assay scoring both the binucleated and mononucleated cells. In addition, binucleated cells were evaluated for non-disjunction events and mononucleated cells for the induction of polyploidy using chromosome-specific classical satellite probes for chromosomes 1 and 9. In comparison, the BrdU cultures were evaluated for induction of hyperdiploidy in replicated and non-replicated interphases as well as metaphases using FISH with a chromosome 9-specific -satellite probe in combination with BrdU immunfluorescent staining. The different FISH methods in this study were compared and the advantages of CYB and BrdU as proliferation markers in cultured human lymphocytes are discussed.
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Cell culture
Peripheral blood was collected by venipuncture from healthy male volunteers between 21 and 35 years of age in heparinized vacutainers. All blood samples were taken in accordance with a protocol approved by the Institutional Review Board at the University of California–Riverside. Cultures of isolated lymphocytes were established in RPMI 1640 medium (Gibco BRL) supplemented with 5% fetal calf serum (Hyclone, Logan, UT), 5% iron-supplemented calf serum (Hyclone), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Mediatech, Washington, DC) and 2.36% phytohemagglutinin type M solution (Gibco BRL). All cells were maintained at 37°C in 5% CO2 and 100% humidity.
Treatment conditions and detection of numerical aberrations in BrdU-treated cultures
Isolated lymphocytes were grown as 2 ml cultures at an initial cell density of 0.5x106 cells/ml for 47.5 h in each of two separate experiments. Cultures were centrifuged for 7 min at 300 g and the cell pellet resuspended in complete medium containing 1 µM BrdU (Sigma Chemical Co., St Louis, MO). NOS (CAS no. 128-62-1; Sigma) was added immediately in a final DMSO concentration of 0.1%. The number of cells for all treatments and respective controls were counted 21 h after treatment using an automated cell counter (Coulter). Growth index was calculated by dividing the total number of cells for each treatment at this time point with the total number of cells at the beginning of the treatment period. Relative growth was determined by dividing the growth index of the NOS-treated cultures by the growth index of the respective control. After aliquots were taken, colcemid (50 ng/ml) was added to the remaining cell suspension and all cultures were harvested by centrifugation at the end of the 24 h treatment period. Cells were swollen with a hypotonic KCl solution (75 mM) for 30 min at room temperature, fixed with two or three changes of 3:1 (v/v) methanol:acetic acid, dropped onto pre-cleaned glass slides, air dried and stored under N2 at -20°C.
For the detection of hyperdiploidy and polyploidy in replicating and non-replicating interphases as well as metaphases, we developed a FISH technique using the -satellite region of chromosome 9 (for probe generation see Hasegawa et al., 1995) in combination with BrdU immunfluorescence to distinguish between cycling and non-cycling cell populations. The target DNA was denatured for 2–5 min in 70% formamide, 2x SSC at 70°C and slides were dehydrated in an ethanol series (70, 85 and 100%) for 2 min each at room temperature. The slides were placed on a slide warmer set at 40°C, 10 µl of the denatured hybridization cocktail was applied and the slides were hybridized overnight at 37°C. The hybridization cocktail contained 55% formamide, 10% dextran sulfate, 1x SSC, 1 µg sheared herring sperm DNA, 20–100 ng biotinylated -satellite probe for chromosome 9 and deionized water as required, all in a total volume of 10 µl. Post-hybridization washes consisted of three washes in 0.1x SSC at 65°C for 5 min each and one wash in PX buffer (0.1 M phosphate buffer, pH 8.0, 0.2% w/v Triton-X-100) at room temperature. The biotinylated probe was detected using fluoresceinated avidin [5 µg/ml in PX buffer with 5% non-fat dry milk supernatant (PXM); Vector Laboratories, Burlingame, CA] followed by biotinylated anti-avidin D IgG (5 µg/ml in PXM; Vector Laboratories) and another layer of fluoresceinated avidin. BrdU incorporation was detected using a mouse anti-BrdU antibody (1:3.5 dilution in PBS containing 0.5% Tween 20; Roche Diagnostics, Indianapolis, IN) for 30 min at 37°C and Texas Red goat anti-mouse IgG (10 µg/ml in PXM; Roche Diagnostics) for 20 min at room temperature. Between incubations, slides were washed for 6 min each in PX buffer at room temperature with three changes of washing solution. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) at 1µg/ml in a phenylenediamine antifade.
After staining BrdU-treated cells using the differential staining procedure described above, replication indexes were determined by counting the number of first, second and third division metaphases in 200 consecutive metaphase cells for each experiment. Replication indices (RI) were determined using the method of Ivett and Tice (1982) with RI = [(1x1M) + (2x2M) + (3x3M)]/n, where 1M, 2M and 3M are the number of first, second and third division metaphases and n is the total number of cells scored. Relative RI were determined by dividing the RI of the treatments by the RI of the control for each experiment. Mitotic indices were determined by scoring 1000 cells for the presence of mitotic figures for each experiment. Relative mitotic indices were determined by dividing the mitotic indices of the treatments by the mitotic indices of the control. For metaphase, BrdU-labeled and unlabeled interphase cells, the number of hybridization signals was determined. Cells showing less than two hybridization signals for chromosome 1 were classified as hypodiploid, cells with more than two hybridization signals for chromosome 1 were classified as hyperdiploid or polyploid. All scoring was performed on coded slides.
Treatment conditions and detection of numerical aberrations in CYB-treated cultures
Isolated lymphocytes were grown as 2 ml cultures at an initial cell density of 0.5x106 cells/ml for 47.5 h in each of two separate experiments. Cultures were centrifuged for 7 min at 300 g and resuspended in complete medium containing 3 µg/ml CYB (Sigma). NOS was added immediately in a final DMSO concentration of 0.1%. CYB cultures were harvested 24 h later by centrifugation at 600 r.p.m. for 5 min using a Shandon cytocentrifuge and fixed in 100% methanol for 10 min at room temperature. CYB slides were stored desiccated in a N2 atmosphere at -20°C. CREST immunfluorescent staining was performed as described previously (Eastmond and Tucker, 1989). A total of 500 mononucleated and 500 binucleated cells for each experiment were analyzed for the presence of MN using the scoring criteria described earlier (Countryman and Heddle, 1976; Eastmond and Tucker, 1989) and the nuclear division index (NDI) was determined by scoring 200 cells from each experiment for the presence of mononucleated, binucleated, trinucleated and tetranucleated cells. The NDI was calculated using the method of Eastmond and Tucker (1989) with NDI = [M1 + (2xM2) + (3xM3) + (4xM4)]/n, where M1–M4 represent the number of cells with one to four nuclei, respectively, and n is the total number of cells scored. The relative NDI was determined by dividing the NDI of each treatment by the respective control.
The method for the generation of classical satellite probes for chromosomes 1 and 9 has been described earlier (Eastmond et al., 1994; Hasegawa et al., 1995; Rupa et al., 1995) and standard conditions were used for dual labeling fluorescence in situ hybridizations with both chromosome-specific probes (Trask and Pinkel, 1990). Briefly, hybridizations were performed in 55% formamide, 10% dextran sulfate, 1x SSC using 1 µg sheared herring sperm DNA, 20–100 ng FluoroGreenTM-labeled classical satellite probe for chromosome 1 (Oncor, Gaithersburg, MD), 20–100 ng digoxigenin-labeled classical satellite for chromosome 9 and deionized water as required, all in a volume of 10 µl. Following post-hybridization washes in 50% formamide, 2x SSC at 42–44°C, nuclei were counterstained with DAPI (1 µg/ml) in a phenylenediamine antifade solution.
All slides were scored using a Nikon fluorescence microscope at 1250x magnification. The scoring was performed on coded slides and a minimum of 500 mononucleated and, if not otherwise specified, 500 binucleated cells per dose for each of two separate experiments. A triple bandpass filter (no. P/N 61002; Chroma Technology Corp., Brattleboro, VT) was used to simultaneously visualize the yellow-green (FluoroGreenTM; classical satellite chromosome 1), red (Cy3; classical satellite chromosome 9) and blue (DAPI counterstain) fluorescence. In binucleated cells, only cells that showed a combined total of four hybridization signals for each of the two chromosomes were included in the evaluation. Cells with an unequal number of hybridization signals in both daughter nuclei were classified as having been formed by non-disjunction. In addition, the frequency of micronucleated cells was determined. In mononucleated cells, the number of hybridization signals for each chromosome was counted. Cells with more than two hybridization signals for both chromosomes were considered to be polyploid and cells with more than two hybridization regions for only one chromosome were classified as hyperdiploid.
Statistical analysis
Dose-related increases in the frequency of MN, hypodiploidy, hyperdiploidy and polyploidy induced by NOS were determined using the Cochran–Armitage test for trend in binomial proportions (Margolin et al., 1986). Following a positive response in the trend test, a one-tailed Fisher exact test was used to compare each treatment with the respective control. Critical values were determined using a 0.05 probability of type I error.
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Influence on markers of proliferation
The influence of NOS on various cell growth and cytotoxic end-points is shown in Figure 1
. To facilitate comparison, all parameters have been expressed as a percentage of control values. Consistent with an interference with cell growth, four of the proliferation markers showed dose-related decreases whereas one, mitotic index, exhibited an increase above the control level. Although the responses for each of the end-points were quite similar with the exception of the mitotic index, growth inhibition seems to be one of the most sensitive proliferation markers.
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Induction of MN, non-disjunction and polyploidy in CYB-blocked lymphocytes
For the detection of NOS-induced chromosomal loss, we used the MN assay in CYB-blocked lymphocytes in combination with CREST immunfluorescent staining. NOS increased the formation of MN in both binucleated and mononucleated cells in a dose-related fashion, as illustrated in Figure 2a and b
(P < 0.05, Cochran–Armitage trend test). The highest frequencies of MN in both cell types were seen at the 60 µg/ml concentration with a frequency of 14% in binucleated and 4% in mononucleated cells. In the binucleated cells, a statistically significant increase in total micronucleated cells, total MN and signal-positive MN could be detected at 15 µg/ml and higher concentrations (Figure 2a P, < 0.05, Fisher’s exact test). On the other hand, an induction of signal-negative MN, indicative of chromosomal breakage, was not seen (P > 0.05, Cochran–Armitage trend test). It should also be mentioned that because of the low nuclear division indices at the two highest concentrations (Figure 1
), only 100 binucleated cells for each experiment were scored at 60 µg/ml, and at 120 µg/ml almost no binucleated cells could be evaluated. In comparison, in the mononucleated cells significant increases in MN were only seen at 30 and 60 µg/ml, with much lower induction frequencies than in binucleated cells (Figure 2a and b). No significant induction was seen at the highest tested concentration (P > 0.05, Fisher’s exact test).
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To obtain a more complete picture of the induction of numerical aberrations in the CYB modified MN assay, we evaluated the induction of MN and non-disjunction in binucleated cells as well as the induction of polyploidy in mononucleated cells using chromosome-specific FISH with classical satellite probes for chromosomes 1 and 9. Examples of segregation patterns seen using this technique are illustrated in Figure 3a
–d. Figure 3a shows a normal untreated interphase and a metaphase lymphocyte, each with two copies of chromosomes 1 and 9. Figure 3b displays a CYB-blocked binucleated lymphocyte with a large MN containing two copies of chromosomes 1 and 9. A binucleated cell with one copy of chromosome 9 in one daughter nucleus and three copies in the other, indicating the occurrence of a non-disjunction event, is seen in Figure 3c. A large polyploid mononucleated lymphocyte showing three hybridization regions for chromosome 1 and four signals for chromosome 9 is illustrated in Figure 3d. The results obtained using this technique are summarized in Figure 2c and Table I. A significant increase in both non-disjunction and polyploidy could be seen at concentrations between 15 and 60 µg/ml (Figure 2c; P < 0.05, Fisher’s exact test). At 120 µg/ml, only a few binucleated cells could be found, essentially restricting the analysis to mononucleated cells. Although a significant induction of polyploid cells could be observed at this concentration (P < 0.05, Fisher’s exact test), the frequencies are much lower than at 60 µg/ml, most likely due to the inability of many cells to escape the NOS-induced mitotic arrest. The frequencies of non-disjunction events and MN induction involving chromosomes 1 and 9 are compared in Table I. Significant increases in both non-disjunction and MN were noticed between 15 and 60 µg/ml. However, for both chromosomes, higher frequencies of non-disjunction than MN were observed. In addition, at the highest concentration evaluated, a significant increase in binucleated cells showing non-disjunction events for both chromosomes 1 and 9 simultaneously was detected. In comparison, only a small number of micronucleated cells showed chromosomal loss for both chromosomes simultaneously. The frequencies of non-disjunction and MN for each chromosome were quite similar despite the small sample size, indicating that both chromosomes are equally involved in NOS-induced chromosomal loss and non-disjunction.
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Induction of hypodiploidy, hyperdiploidy and polyploidy in BrdU-labeled lymphocytes
For the detection of numerical aberrations induced by NOS, chromosome-specific FISH for chromosome 9 was performed in combination with BrdU immunfluorescent labeling to distinguish actively replicating from non-replicating cells. Illustrations of metaphase and interphase cells following hybridization are shown in Figure 3e and f
. A second division metaphase with one labeled and one unlabeled sister chromatid is shown in Figure 3e. A total of 1000 metaphases regardless of their division stage from each of two separate experiments were evaluated for the presence of hypodiploid, hyperdiploid and polyploid cells. The results are presented in Figure 4a. NOS induced a statistically significant increase in hyperdiploid and polyploid cells (P < 0.05, Cochran–Armitage trend test). A statistically significant increase in hypodiploidy could not be detected in the metaphase preparations, probably due to a high background level of hypodiploidy in the control (P > 0.05, Cochran–Armitage trend test). Significantly higher frequencies of hyperdiploidy were observed at concentrations between 15 and 60 µg/ml and of polyploidy between 7.8 and 120 µg/ml (P < 0.05, Fisher’s exact test). A statistically significant induction of polyploidy but not hyperdiploidy was seen at the highest concentration tested (120 µg/ml). When combining hyperdiploid and polyploid cells, significant increases in cells with three and four copies of chromosome 9 could be seen even for 3.9 µg/ml (8 per 1000 cells) when compared with the control (1 per 1000 cells).
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The BrdU labeling technique made it possible to distinguish between replicating and non-replicating interphase cells. An example of a labeled interphase cell is shown in Figure 3f
. In BrdU-labeled interphase cells, a significant dose-related increase in both hyperdiploidy/polyploidy and hypodiploidy was observed (Figure 4b; P < 0.05, Cochran–Armitage trend test) with the first significant increase at 15 µg/ml for both parameters (P < 0.05, Fisher’s exact test). It should be mentioned in this context that interphase FISH using a single chromosome probe is not able to distinguish hyperdiploid from polyploid cells. The maximum hyperdiploidy/polyploidy induction could be detected at the highest tested concentration but the maximum for hypodiploidy was seen at 60 µg/ml. These results indicate that NOS induces primarily polyploidy at 120 µg/ml and is consistent with the metaphase analysis which showed that 120 µg/ml NOS induces almost exclusively polyploidy. In unlabeled cells that had not passed through an S phase during treatment, a much weaker effect was seen than in the labeled cells. A significant increase in hyperdiploid/polyploid cells could only be seen at the 60–120 µg/ml concentrations. This increase is most likely due to G2 cells that had passed through one abnormal mitosis since start of treatment with NOS but did not undergo an S phase. Induction of hypodiploid cells could not be detected in this cell population and the overall frequencies of cells with no and one hybridization signal were quite high even in the control. The relatively high frequencies of cells with one hybridization region in lymphocyte cultures were likely due to an elevated percentage of unstimulated lymphocytes and indicates that hypodiploidy as well as hyperdiploidy/polyploidy frequencies as detected by FISH are highly influenced by replication and cell division.
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Discussion |
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In this study we compared FISH detection methods and multiple end-points for the detection of the aneuploidy-inducing agent NOS in cultured human lymphocytes. The results show that the CYB modified micronucleus assay as well as the BrdU assay can be used with high sensitivity to detect numerical chromosomal aberrations in cultured human lymphocytes. However, metaphase analysis combined with FISH was somewhat more sensitive, an important consideration for identifying thresholds and NOAELs. The results show that NOS can induce chromosomal loss, hyperdiploidy and hypodiploidy at relatively low concentrations. A comparison of the chromosomal alterations seen at different NOS concentrations showed that at the intermediate concentrations, all three types of numerical alterations, chromosomal loss, non-disjunction and polyploidy, contributed to the aberrations seen, whereas at the highest concentration polyploidy became the predominant alteration, indicating that the severe spindle damage at this concentration leads exclusively to the induction of polyploidy.
As indicated above, we used various cytotoxic indicators to investigate the influence of NOS on the cell cycle and showed that all of the proliferation end-points were significantly influenced by increasing NOS concentrations. To better illustrate the relationship between the different end-points, the results observed at the 30 µg/ml concentration will be used as an example. Following a 24 h treatment with 30 µg/ml NOS, cell growth was reduced by 47% as compared with the respective control. Of the remaining cells, 64% had gone through at least one S phase and 67% of the metaphases were in their second division, indicating that they had gone through two separate S phases. Despite the remarkable reduction in growth, the frequency of cells in metaphase was 48% higher than in the respective control. Interestingly, the nuclear division index is only slightly lower at this concentration than in the control. These results indicate that NOS delays the cell cycle specifically at the mitotic stage, but rather than inducing a complete mitotic block, it slows down the progression through mitosis, leading to an increase in mitotic cells. It should also be mentioned that NOS itself can lead to multinucleation at higher concentrations (P.Muehlbauer, data not shown), indicating that some caution should be used in interpreting the nuclear division index data.
The induction of aneuploidy and polyploidy by chemical or physical agents is restricted to proliferating cells and can only take place in cells that pass through mitosis into the next cell cycle. In cultured human lymphocytes, for example, only the 40–60% proliferating cells that undergo mitosis during chemical treatment are susceptible to aneuploidy-inducing agents. Restricting analysis to proliferating cells should therefore substantially increase the sensitivity in measuring the induction of aneuploidy. In this study we have compared the usefulness of several proliferation markers to improve the sensitivity of the micronucleus assay and chromosome-specific FISH to detect aneuploidy-inducing agents. Restricting analysis to cells in metaphase is one of the possibilities to overcome the proliferation problem. The main advantages are that only proliferating cells are counted and that counting of hybridization regions on individual chromosomes is very easy. This may be reflected in the results in this study, where significant increases in numerical aberrations could be seen at lower concentrations in metaphase cells than with the standard interphase approaches. In addition, numerical aberrations are easily distinguishable from breaks and translocations affecting the region targeted by the DNA probes. However, restricting analysis to metaphase cells also has several drawbacks. Lack of optimal harvest times and techniques in metaphase preparation and analyses can result in an under- or overestimation of the loss or gain of chromosomes. In this study the aneuploidy frequencies detected in the metaphase analysis were somewhat more variable, with lower maximum frequencies of numerical aberrations than those seen with the interphase approaches. A possible explanation for the lower aneuploidy frequencies observed at higher concentrations could be a NOS-induced mitotic delay, where cells remain in mitosis for a longer time period and are unable to make it to the second mitosis during the 24 h treatment interval. This is supported by the lower replication indices observed at these concentrations. In addition, compounds that delay cell cycle progression or that block cells in certain stages of the cell cycle lead to low mitotic yields, making the evaluation of metaphases rather time consuming, if not impossible. The evaluation of metaphase cells, particularly using FISH, can also become obscured by artifacts like premature chromatid separation in metaphase cells or the counting of anaphase cells, which can be falsely interpreted as hyperdiploid or polyploid cells.
A good alternative to scoring metaphases is the use of cytokinesis blocked interphase cells. This technique can be used to detect chromosomal loss, non-disjunction and polyploidy with the advantage that hybridization artifacts, mostly due to probe overlap, doublets and breakage within the hybridization region, can easily be avoided by restricting analysis to binucleated cells with the correct number of hybridization regions (Zijno et al., 1994, 1996b). It has been shown by several authors that non-disjunction in binucleated cells is the most sensitive end-point in this assay (Marshall et al., 1996; Zijno et al., 1996a,b; Elhajouji et al., 1997; Sgura et al., 1997; Minissi et al., 1999). However, one of the most interesting findings in our study is the unexpected high frequencies of polyploidy and MN in the mononucleated cells of the CYB cultures even at NOS concentrations that showed little or no influence on the frequency of binucleated cells. In a similar study with nocodazole in whole blood cultures, increases in MN and polyploidy in mononucleated cells were found, but only at concentrations that significantly reduced the frequency of binucleated cells (Elhajouji et al., 1998). These results indicate that in the presence of NOS or nocodazole, lymphocytes can undergo chromatid separation without normal anaphase/telophase and thus produce polyploid cells as well as MN. As a consequence, the common model that MN formation and polyploidy development require mitotic cells to undergo telophase seems to be incorrect. A comparison of the different alterations seen at different concentrations shows that following NOS treatment, the first significant increase in polyploidy was seen at the same concentration at which the frequencies of MN and non-disjunction were first significantly elevated. This indicates that at the same NOS concentration, both cells with severe spindle damage, resulting in the formation of polyploid cells, as well as cells with dislocated or misaligned chromosomes, leading to the formation of aneuploid cells, exist. In fact, in an earlier study in whole blood cultures we could show that NOS induced both apolar metaphase cells and anaphase/telophase lagging at similar concentrations (Schuler et al., 1999). At the highest NOS concentration tested in this study, polyploidy became the predominant effect and neither induction of MN nor non-disjunction were seen. At this highest concentration, the formation of binucleated cells did not occur, indicating that NOS does not induce a perfect mitotic block. Affected cells enter the following interphase through mitotic slippage, producing tetraploid cells. These tetraploid cells can proliferate, entering the next mitotic cycle and become octaploid with longer treatments (M.Schuler and D.A.Eastmond, unpublished results). The presented results show that cells do not have to undergo a cell division in order to become polyploid or form MN, but have to pass through mitosis into the next cell cycle.
Recent studies have indicated that CYB can have an influence on absolute frequencies of MN and polyploidy as well as the relative frequencies of chromosomal loss, non-disjunction and polyploidy (Zijno et al., 1996b; Minissi et al., 1999). For example, Antoccia et al. (1993) did not observe micronucleated cells when human fibroblasts were treated with both colchicine and CYB. When comparing the induction of MN by vinblastine in cultures with and without CYB, increased mitotic indices and higher polyploidy frequencies were seen in the CYB cultures, suggesting a synergistic effect of CYB and vinblastine. A similar result was seen by Minissi et al. (1999) in whole blood cultures treated with colchicine, where CYB cultures showed lower frequencies of chromosomal laggards and MN but higher frequencies of polyploidy when compared with cultures without CYB. The higher frequencies at lower concentrations seen for the induction of non-disjunction when compared with chromosomal loss are probably caused by the absence of the actin ring during late anaphase leading to a shorter distance between the poles and, as a consequence, to a change in segregation patterns. As a result, the different alterations seen in the CYB assay, although being useful, have to be interpreted with caution.
A valid alternative, especially when interactions of test compound and CYB are of concern, could be the use of BrdU as the proliferation marker, as no influence of this compound on segregation of chromosomes during mitosis would be expected. Indeed, in these short-term cultures BrdU incorporation does not increase the frequencies of aneuploidy and polyploidy above control levels that have been seen historically (Eastmond et al., 1995). In addition, Norppa et al. (1990) demonstrated a good correlation between the BrdU and CYB techniques, since a high proportion of the cells that are BrdU labeled would be binucleated when CYB is used as the proliferation marker. However, cultivation of cells in the presence of high concentrations of BrdU is known to affect cellular functions and can lead to a variety of chromosomal alterations (for a review see Zwanenburg et al., 1984). BrdU incorporation can also lead to increased melting temperatures of DNA (Augenlicht et al., 1974; David et al., 1974), which could lead to problems when using in situ hybridization techniques in highly BrdU-substituted DNA. It should be mentioned in this context that most of the adverse effects have been seen at high BrdU concentrations or in very sensitive cell lines. By using immunhistochemistry with fluorescent labeled antibodies, BrdU concentrations can be lowered to reduce or eliminate many of the adverse effects related to the incorporation of BrdU into cellular DNA. In addition, when working with aneuploidy-inducing agents that induce polyploidy at high concentrations, it seems that BrdU labeling may give the most reliable estimate of cycling and non-cycling cells. In fact, this study shows that the strongest dose–response effect with the highest absolute frequencies of abnormal cells was seen in replicating interphase cells. Even at NOS concentrations that caused toxicity or a delay in the cell cycle a sufficient number of BrdU-labeled cells was available for evaluation. The main disadvantage of the BrdU technique is that even though it is a very good marker of proliferation, it is an indirect indicator of cells that have undergone mitosis. Cells could, for example, pass through an S phase and never complete the subsequent mitosis as a result of cell cycle delay.
In conclusion, our results show that FISH methods in combination with proliferation markers like CYB and BrdU allow the sensitive detection of various end-points of numerical chromosomal aberrations in cultured human lymphocytes. The use of antibodies against internal proliferation markers, like proliferating cell nuclear antigen (PCNA) or Ki67, in combination with FISH may be valid alternatives and will be evaluated in future studies.
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3 To whom correspondence should be addressed. Tel: +1 909 787 4497; Fax: +1 909 787 3087; Email: david.eastmond{at}ucr.edu
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Received on March 19, 2002; accepted on September 13, 2002.
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