Mutagenesis, Vol. 15, No. 3, 251-255,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Segregation of sex chromosomes in human lymphocytes
1 Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, FIN-00250 Helsinki, Finland, 2 Department of Anatomy, Embryology and Genetics, University of Zaragoza, Zaragoza, Spain and 3 Department of Genetics and Microbiology, Autonomous University of Barcelona, Barcelona, Spain
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Abstract |
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Centromeric FISH was used to investigate the segregation of sex chromosomes in human lymphocytes. The aim of the study was to evaluate the effects of cell culture, cytokinesis block, age and sex on segregation and to compare the behaviour of the X and Y chromosomes. In uncultured T lymphocytes of five elderly women, the mean frequencies of nuclei hyperdiploid and hypodiploid for the X chromosome were not significantly affected by culturing the cells or by cytokinesis block. In cultured binucleate lymphocytes of two age groups of men, the X chromosome showed significantly higher mean frequencies of hyperdiploidy, hypodiploidy and reciprocal gain and loss than the Y chromosome. Reciprocal gain and loss of the Y chromosome was statistically significantly higher in the older than the younger men. In four women, studied in the same series, the rates of X chromosome aneuploidy did not significantly differ from those obtained in men. In conclusion, malsegregation of the X chromosome is common in lymphocytes of both men and women and more frequent than Y chromosome malsegregation. However, there is no clear sex difference for X chromosome reciprocal gain and loss. This would suggest that the high loss of the X chromosome in women, documented in metaphase studies, is due to micronucleation.
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Introduction |
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Aneuploidy is the most common known cause of mental retardation, spontaneous abortions and congenital malformations (Abruzzo and Hassold, 1995
; Hassold et al., 1996) and also plays an important role in carcinogenesis (Tucker and Preston, 1996). Despite these associations, little is known about the mechanistic origin of aneuploidy (Hassold et al., 1996).
Traditionally, much of our understanding of somatic aneuploidy has been based on metaphase analysis, which is time consuming, prone to technical artefacts and indicative of only the proliferating cell population (Guttenbach et al., 1995). Lately, these problems have been overcome by fluorescence in situ hybridization (FISH) with chromosome-specific centromeric DNA probes, which allows rapid and direct identification of specific aneuploidies in a large number of interphase nuclei (Eastmond et al., 1995; Guttenbach et al., 1995). Moreover, application of in situ hybridization to cells blocked in cytokinesis by cytochalasin B (Cyt-B) allows one to accurately estimate the occurrence of reciprocal gain and loss of a chromosome (Zijno et al., 1994). Also, pre-existing aneuploid nuclei can be distinguished from those arising in vitro (Zijno et al., 1996a).
Since the early 1960s, the rate of chromosome loss in cultured human lymphocytes has been known to increase with advancing age (Jacobs et al., 1961). Studies of banded metaphases showed that the loss most frequently involved the X chromosome in females and the Y chromosome in males (Fitzgerald and McEwan, 1977; Galloway and Buckton, 1978; Nowinski et al., 1990). More recently, studies using FISH with chromosome-specific centromeric probes in interphase lymphocytes of women highlighted the excessive over- representation of the X chromosome in micronuclei (MN) (Guttenbach et al., 1994; Hando et al., 1994; Richard et al., 1994; Catalán et al., 1995; Surrallés et al., 1996a,b) and the high rate of X chromosome loss and malsegregation (Guttenbach et al., 1995; Zijno et al., 1996a), with a strong tendency towards increased frequencies with ageing. The few studies available on men pointed to a clear age-dependent micronucleation (Guttenbach et al., 1994; Nath et al., 1995; Catalán et al., 1998) and loss (Guttenbach et al., 1995) of the Y chromosome and increased malsegregation (Zijno et al., 1996b; Carere et al., 1999) and micronucleation (Hando et al., 1997; Catalán et al., 1998; Carere et al., 1999) of the X chromosome. However, until now, no studies have simultaneously analysed malsegregation of the X and Y chromosomes in men, which would allow a comparison of the sex chromosomes and yield information on the involvement of the active X in aneuploidy.
In the present study, two sets of experiments were performed. In the first, the frequencies of X chromosome aneuploidy in female T lymphocytes cultured in the presence or absence of Cyt-B were compared with in vivo frequencies obtained from uncultured cells, to evaluate the effects of cell culture and cytokinesis block on aneuploidy. In the second set of experiments, X and Y chromosome aneuploidy and segregation behaviour were simultaneously analysed in binucleate cultured lymphocytes of two age groups of men. To allow comparison between sexes, X chromosome abnormalities were concurrently studied in women.
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Materials and methods |
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Cell preparation and in situ hybridization
For the first set of experiments, five non-smoking women, 47–60 years of age (53.6 ± 4.9 years, mean ± SD), were chosen. To ascertain that the same cell population was studied in vivo and in vitro, T lymphocytes were immunomagnetically sorted from suspensions of uncultured and cultured (72 h, with and without 6 µg/ml Cyt-B) cells and directly spun onto slides with a cytocentrifuge. The slides were hybridized with an
-satellite DNA probe, which specifically detects a centromeric region of the X chromosome (DXZ1, biotin label; Oncor, Gaithersburg, MD). T lymphocyte isolation, cell culture, slide preparation and FISH were carried out as previously described in a report on MN (Surrallés et al., 1996a).
The second set of experiments was performed with four women and 10 men, distributed equally between two age groups: <30 years and >50 years. Isolated lymphocytes (mononuclear cells) were cultured for 65 h with and without 6 µg/ml Cyt-B and were spun onto slides with a cytocentrifuge. The slides were hybridized with the biotin-labelled DXZ1 probe and slides from the male donors were simultaneously hybridized with a digoxigenin-labelled Y chromosome-specific centromeric -satellite probe (DYZ3; Oncor). The details of the FISH procedure have been published previously (Catalán et al., 1998) when describing MN analysis.
The labelling efficiency of the probes used was checked for 200 nuclei in each case and the analysis was performed only when >90% of the nuclei on the slide showed appropriate labelling.
Slide scoring and statistical analysis
In both sets of experiments, the number of hybridization signals for the X and (in men) Y chromosomes was analysed from the nuclei of 2000 mononucleate (uncultured T cells and cell cultures without Cyt-B) or binucleate (cultures with Cyt-B) cells per donor and cell type. The analyses were done by two trained microscopists working independently, each scoring half of the material in each experiment.
From the different types of FISH signal distributions, the frequency of hyperdiploidy [HRD (%)] and the frequency of reciprocal gain and loss [RGL (%)] were calculated. The HRD (%) reflects, in both mononucleate and binucleate cells, the frequency of nuclei with a number of FISH spots (X or Y) higher than normal. The RGL (%), which only applied to binucleate cells where both daughter nuclei remained together, is the frequency of binucleate cells with X chromosome distributions of 3/1 and 4/0 cells in women and X or Y chromosome distributions of 2/0 in men. Thus, only those cells where the total number of spots was `correct' (i.e. 4 X spots in women and 2 in men and 2 Y spots in men) were included in RGL (%). Although some other distributions, such as 2/1 and 2/0 in women and 1/0 in men, might also result from reciprocal gain and loss, if spots overlap (Eastmond et al., 1995) they were not included in RGL (%).
We also evaluated the frequency of apparent hypodiploidy [HOD (%)], i.e. nuclei that showed less than two X chromosomes in women and no X or Y chromosomes in men. We are well aware that overlap and fusion of hybridization signals may largely explain the high number of `hypodiploids', diploid cells being classified as hypodiploid (Eastmond and Pinkel, 1990; Eastmond et al., 1995; Zijno et al., 1996b); this phenomenon could be a problem, especially when only single colour FISH (X chromosomes in women) is used. Hypodiploidy analysis was included to allow comparison with some earlier studies on X and Y loss in mononucleate interphase cells (Guttenbach et al., 1995).
To yield comparable hyper- and hypodiploidy data for cytokinesis-blocked and untreated cells, the unit of observation was in both cases the single nucleus. Without Cyt-B all intact nuclei were considered, whereas all nuclei belonging to intact binucleate cells were scored with Cyt-B.
Comparison among the different types of cells in the first set of experiments and between the sex chromosomes and age groups of men in the second set were performed by multifactorial analysis of variance. The sex effect in the second set was evaluated using the two-tailed t-test (Statview SE + Graphics v.1.03).
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Results |
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Results on the distribution of X chromosome signals in cultured and uncultured T cells of the five older women included in the first set of experiments are shown in Tables I and II
. Apparent X hypodiploid nuclei were clearly more common than X hyperdiploid nuclei. Multifactorial analysis of variance on the frequencies of hyperdiploid or `hypodiploid' nuclei showed no significant differences attributable to cell culture, individuals or use of Cyt-B.
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In the two age groups of men in the second set of experiments, both the X and Y chromosomes were detected simultaneously in the same cells (Table III
). This allowed us to evaluate the number of probable polyploid binucleate cells, defined as containing more than two copies of all chromosomes and, therefore, of both sex chromosomes. Only 24 apparently polyploid cells were found in a total of 20 000 cells analysed, which gives a low polyploidy rate.
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The older group of men showed somewhat higher mean frequencies of X hyperdiploid nuclei, `hypodiploid' nuclei and cells with reciprocal gain and loss of the X chromosome than the younger group (Table III
). However, no statistically significant age effects were observed for any class of X chromosome missegregation in the men in the analysis of variance. The mean frequency of binucleate cells with reciprocal gain and loss of the Y chromosome was 2.2 times higher (P = 0.001) in the older than the younger men, but the age difference was not significant for the mean frequencies of Y hyperdiploid or `hypodiploid' nuclei.
In multifactorial analysis of variance on men, there was a statistically significant (P < 0.01) difference between the X and Y chromosomes for the frequency of hyperdiploid and `hypodiploid' nuclei as well as for reciprocal gain and loss, the X chromosome showing higher malsegregation rates.
In the four women of the second set of experiments (Table II), the mean frequency of hyperdiploid nuclei was higher and that of `hypodiploid' nuclei lower than was observed in the first set of experiments (Table II), but the data should not be directly compared due to different scorers and cell types (lymphocytes versus T lymphocytes). The mean frequency of X chromosome reciprocal gain and loss was similar as in the first series and the 3/1 distribution predominated, although a few 4/0 cells were also seen.
When the X aneuploidy frequencies of the women in the second set were divided by two, to take into account their two X chromosomes, men actually appeared to show slightly more X hyperdiploid nuclei, X hypodiploid nuclei and cells with reciprocal gain and loss of the X chromosome than women. However, the effects were not statistically significant (two-tailed t-test).
In the analysis of binucleate cells (Tables II and III), most of the abnormal X and Y distributions were `illegitimate', as they involved either too few signals (mostly 2/1 and 1/1 for X in women and 1/0 and 0/0 for X and Y in men) or too many signals (mostly 3/2 for X in women and mostly 2/1 for X and Y in men), instead of the expected total number of 4 X signals in women and 2 X and 2 Y signals in men.
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Discussion |
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We found no significant differences in the frequency of nuclei hyperdiploid or hypodiploid for the X chromosome in women between uncultured T cells and T cells cultured with or without Cyt-B, which suggests that cultured lymphocytes reflect in vivo aneuploidy rates. This finding agrees with that of Guttenbach et al. (1995), who could not find differences between resting and cultured lymphocytes in X and Y hypodiploidy. Our previous analysis of MN in the same subjects (Surrallés et al., 1996a
) did, however, show a higher frequency of MN harbouring the X chromosome in the cultured than the quiescent T lymphocytes. This may indicate that cell culture affects X micronucleation in women even if no significant effects are observed in the frequencies of hyper- or hypodiploid nuclei.
The X+ MN or other types of X missegregation formed in vitro do not markedly affect X aneuploidy rates probably because aneuploidy of in vivo origin is so frequent. Binucleate cells with an illegitimate number of X chromosome signals (more or less than 4), which apparently are mostly derived in vivo (Zijno et al., 1996a), were the most prevalent types of aneuploidy. Our data indicate that less than half of all X hyperdiploid nuclei and a minority of apparent hypodiploid nuclei seen in female lymphocyte cultures were formed by reciprocal gain and loss in the first in vitro division, much of the rest being of in vivo origin. On the other hand, Carere et al. (1999) observed that in binucleate lymphocytes of men reciprocal gain and loss of the X chromosome (an in vitro event) is more frequent than balanced hyperdiploids (a probable in vivo event).
In the present study, binucleate cells with reciprocal gain and loss of the X chromosome constituted ~1% of female binucleate cells. These figures closely follow the frequencies of X+ MN reported earlier from the same experiments (Surrallés et al., 1996a; Catalán et al., 1998). Our results agree with those of Zijno et al. (1996a), who concluded that X chromosome loss (MN) and reciprocal gain and loss (`non-disjunction') occur at similar rates in binucleate female lymphocytes.
The correct interpretation of hypodiploidy data remains unclear. The high frequencies of hypodiploid interphase cells may partly be due to overlap of FISH signals (Eastmond and Pinkel, 1990; Eastmond et al., 1995), but the clear age-dependent increase in X and Y loss (Guttenbach et al., 1995; Mukherjee et al., 1996) also suggests that hypodiploidy analysis yields meaningful data. Accordingly, the present results showed a higher hypodiploidy rate for the X than the Y chromosome in men, which agrees with our data on hyperdiploidy and reciprocal gain and loss of these chromosomes.
We could not demonstrate any effects on sex chromosome segregation for Cyt-B-induced cytokinesis block. Similarly, Zijno et al. (1996b) saw no increase in hyperdiploid nuclei in binucleate cells. These results contrast with our previous findings on MN in the same cultures, where a higher frequency of MN containing the X chromosome was seen when binucleate cells were compared with cells cultured without Cyt-B (Surrallés et al., 1996a; Catalán et al., 1998).
As the X chromosome was the only chromosome we detected in women, we could not distinguish hyperdiploidy from polyploidy (Eastmond et al., 1995). However, polyploids in women do not explain the difference in hyperdiploidy between men and women, since after correction for the number of X copies, the rate was higher (although not significantly) in men. Undetected polyploidy was not an important source of error probably because of the low frequency of polyploid cells (0.12% in men) in comparison with sex chromosome hyperdiploidy.
The lack of a significant difference between the sexes in the frequency of X chromosome reciprocal gain and loss was an unexpected finding. In our earlier paper on MN of the same donors (Catalán et al., 1998), we found a clearly higher frequency of X+ MN in women than in men and, therefore, also expected an elevated rate of gains and losses. Metaphase studies have likewise indicated a higher total rate of X malsegregation in women than men (Fitzgerald et al., 1975; Fitzgerald and McEwan, 1977) and Zijno et al. (1996a) observed a five times higher frequency of X malsegregation (`non-disjunction' and MN combined) in women than men. Our findings seem to suggest that X chromosome micronucleation and reciprocal gain and loss originated by different mechanisms. It would appear that the high X chromosome loss in women is primarily due to excessive micronucleation of the X chromosome rather than non-disjunction.
This is the first study where the aneuploidy of both sex chromosomes was simultaneously analysed in binucleate lymphocytes of men. The X chromosome showed higher frequencies of hyperdiploidy, reciprocal gain and loss and apparent hypodiploidy than the Y chromosome. This is in agreement with our previous observation of a higher frequency of X+ MN than Y+ MN in the same men (Catalán et al., 1998). The different rates of reciprocal gain and loss of the X and Y chromosomes may be explained by chromosome-specific mechanisms of non-disjunction, as has been suggested for germ cells (Abruzzo and Hassold, 1995; Hassold et al., 1996). In fact, our frequencies of reciprocal gain and loss of the Y chromosome were similar to the previously reported frequencies of Y+ MN (Catalán et al., 1998), while reciprocal gain and loss was clearly more prevalent than MN for the X chromosome of the men. As these classes of X missegregation showed similar rates in women, it could be that non-disjunction affects the homologous X chromosomes similarly, but micronucleation would preferentially concern the inactive X (which only women have). This hypothesis would explain why females have a clearly higher frequency of X chromosome loss in metaphase and X+ MN but similar rates of X reciprocal gain and loss in comparison with men. Although the preferential involvement of the inactive X in aneuploidy has been investigated in several studies (Fitzgerald et al., 1975; Abruzzo et al., 1985; Tucker et al., 1996; Surrallés et al., 1996b), conclusions have been contradictory and further experiments are thus required.
An effect of age was observed for Y chromosome reciprocal gain and loss, in accordance with our finding on a clear age-related increase in Y+ MN in the same subjects (Catalán et al., 1998). Similar (non-significant) trends were seen in the frequencies of Y hyper- and hypodiploid nuclei.
Carere et al. (1999) observed a positive correlation with age for both hyperploidy and reciprocal gain and loss of the X chromosome. Also in our study, the older men had more X hyperdiploid nuclei and reciprocal gain and loss than the young, although the effect was not statistically significant. Previously, Zijno et al. (1996b) described an age-dependent increase in X missegregation, combining X reciprocal gain and loss and micronucleation. They could not find an association between age and the rate of putative pre-existing monosomic and trisomic cells, in agreement with our finding of a lack of an age effect in X chromosome hypodiploidy. An age-dependent increase was described for the frequency of X+ MN in men (Hando et al., 1997; Catalán et al., 1998; Carere et al., 1999).
In conclusion, the present study suggests that the baseline frequencies of X hyperdiploidy and apparent hypodiploidy in female lymphocytes are not markedly affected by cell culturing or by the use of Cyt-B. Malsegregation of the X chromosome is common in lymphocytes of both men and women and more frequent than Y chromosome malsegregation. The reciprocal gain and loss of the Y chromosome shows an age effect, which together with the previously shown age-dependent formation of Y+ MN indicates a chromosome-specific mechanism of malsegregation for the Y chromosome. There does not seem to be any clear sex difference for X chromosome hyperploidy or reciprocal gain and loss, which would suggest that the high loss of the X chromosome in women is due to micronucleation.
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Acknowledgments |
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We thank Dr C.Moreno for statistical advice. J.S. and J.C. were supported by the Human Capital and Mobility Programme of the Commission of the European Communities (contract ERBCHBGCT940537) and the European Science Foundation, respectively. J.S. currently holds a `Contrato de Incorporación de Doctores' from the Spanish Ministry of Education and Culture.
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Notes |
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4 Present address: Laboratory of Medical Genetics, Helsinki University Central Hospital, Helsinki, Finland
* To whom correspondence should be addressed. Tel: +358 9 4747336; Fax: +358 9 4747208; Email: hannu.norppa{at}occuphealth.fi
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References |
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Received on October 18, 1999; accepted on January 21, 2000.
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