Download Detection of chromosome 2 and chromosome 7 within X-ray

Mutagenesis vol.12 no.2 pp.55-59, 1997
Detection of chromosome 2 and chromosome 7 within X-rayor colchicine-induced micronuclei by fluorescence in situ
hybridization
K.Wuttke, CStreffer and W.-U.MUIler1
Institut fllr Medizinische Strahlenbiologie, Universitatsklinikum Essen,
D45122 Essen, Germany
'To whom correspondence should be addressed
The occurrence of chromosome 2 and chromosome 7 within
micronuclei of binucleated lymphocytes induced by X-rays
or colchicine was scored using the whole chromosome
painting technique. The observed frequency of involvement
in micronucleus formation was compared with the yield
that would be expected theoretically, when the random
participation of each chromosome is assumed. No difference
was observed between the expected and observed inclusion
of chromosome 2 or chromosome 7 into micronuclei after
X-ray exposure (2.5 Gy). This was also the case for
chromosome 2, but not for chromosome 7 after colchicine
treatment (0.04/0.06 ug/ml); chromosome 7 was detected
approximately 1.5 times more frequently in micronucleus
formation than would be expected from the assumption of
a random distribution.
Introduction
Genetic changes are central to the initiation and progression
of neoplasias. A marker of chromosomal damage on the
cytogenetic level is the formation of micronuclei: one possible
fate of acentric fragments induced by clastogens as well as of
lagging chromosomes induced by spindle poisons is micronucleation, which occurs after mitosis during nuclear membrane formation. The cytokinesis block method using
cytochalasin B allows detection of cells which have undergone
division, as binucleated cells, and micronuclei occurring in
such cells can be used effectively to detect clastogenic or
aneugenic effects (Mtlller and Streffer, 1994).
Independent of the micronucleus-inducing agent, it is
assumed that all chromosomes participate in aberrations in a
random way depending on their DNA content. In general,
this appears to be true. However, there are some indications
that cytogenetic damage is not always distributed randomly,
but that some chromosomes are involved more frequently than
others (e.g. Brogger, 1977; Hayata and Dutrillaux, 1985; Tawn,
1988; Natarajan et ai, 1992; Hando et ai, 1994; Richard
et ai, 1994; and additional literature cited in these papers).
To test this possibility, we combined the cytokinesis block
micTonucleus assay with the whole chromosome painting
technique for chromosomes 2 and 7 of human lymphocytes
after exposure to X-rays or to the spindle poison, colchicine.
This allowed the simultaneous determination of the chromosome 2 and chromosome 7 status of micronuclei induced by
clastogenic (X-rays) or aneuploidy-inducing (colchicine)
agents.
Materials and methods
Cell culture and treatment
Lymphocytes were isolated from the blood of a female donor by density
centrifugation over Ficoll-Hypaque Qymphocyte separation medium;
© UK Environmental Mutagen Society/Oxford University Press 1997
Fresenius, Bad Homburg, Germany). They were cultured at a density of
0.5X106 cells/ml in Roswell Park Memorial Institute (RPMI) 1640 medium
(Gibco, Eggenstein, Germany) supplemented with 259b fetal calf serum and
antibiotic/antimycotic solution (Gibco). The lymphocytes were stimulated by
phytohaemagglutinin (PHA; Gibco). The cells were cultivated in multi-well
tissue culture plates (Falcon) in an incubator (Heraeus, Osterode, Germany)
at 37°C and 5% CO?.
Depending on the experimental procedure, the lymphocytes were either
irradiated with 2.5 Gy X-rays (240 kV, 0.5 mm copper filter, dose rate 1 Gy/
min) shortly before PHA stimulation or treated with colchicine (Sigma,
Deisenhofen, Germany) 44 h after PHA stimulation. Due to inter-individual
variation in the sensitivity of the cells to the spindle poison, the lymphocytes
were exposed to different concentrations of colchicine (0.02, 0.04, 0.06 or
0.08 ug/ml). Fluorescence in situ hybridization was performed with those
slides revealing the highest frequency of micronuclei (0.04/0.06 |ig/ml).
Induction of binucleated cells
Cytochalasin B (Sigma) was added to the culture at a final concentration of
5 |ig/ml 44 h after the initiation of the culture, and the cells were harvested
24 h later.
Slide preparation
Cells were transferred to precleaned slides using a cytocentrifuge (48 g, 5 min)
and air-dried for 2 h. They were then fixed in cold methanol for 15 min and
air-dried overnight. The slides were stored at -20°C till processed for
hybridization.
Whole chromosome painting by fluorescence in situ hybridization (FISH)
FISH was performed using probes specific for whole chromosomes. The slides
were hybridized simultaneously with Spectrum Green-conjugated DNA probes
specific for chromosome 2 (Vysis-Company, Stuttgart-Fasanenhof, Germany)
and Spectrum Orange-conjugated DNA probes specific for chromosome 7
(Vysis-Company). The fluorescent dye 4,6-diamidino-2-pbenylindole (DAPI;
150 ng/ml) was used to counterstain all chromosomes. In general, hybridization,
washes and detection of bound probes were carried out according to the probe
manufacturer's instructions, except for a few modifications. As the probes
were developed for the detection of chromosomes in metaphase, pretreatment
of the slides prior to denaturation was found to be necessary for the staining
of chromosomes in interphase. Briefly, slides were pretreated by RNase
(100 Ug/ml; Boehringer) for 1 h at 37°C, washed three times in 2X sodium
chloride/sodium citrate (SSC) at room temperature, pretreated with pepsin
(50 ilg/ml; Sigma) for 7 min at 37°C, washed twice in phosphate-buffered
saline (PBS) and once in PBS + 50 mM MgCl2 at room temperature, fixed
in 1% formaldehyde for 10 min at room temperature and washed in PBS at
room temperature. Afterwards, the slides were dehydrated in ethanol (70, 90,
100, 100%; 3 min each) and air-dried. The DNA in the denaturation mix was
denatured at 73°C for 5 min, and applied to slides that were denatured at
71°C for 5 min in 70% formamide/2x SSC (pH 7-8) and dehydrated in a
70, 85, 100% ethanol series. The slides were covered by a 22X22 mm
coverslip and sealed with rubber cement Hybridization was performed at
37°C overnight in a moist chamber. The slides were air-dried after extensive
washing of the slides in 50% fonnamide/2X SSC (pH 7-8) at 45°C for 30
min, followed by one wash in 2X SSC for 10 min and one wash for 5 min
in 2X SSC + 0.1% MM0.
Scoring procedure
Microscopic analysis was performed on a Leica Diaplan fluorescence microscope that was connected to a laser scanning unit at a magnification of X1000.
Both main nuclei and micronuclei had to be visible with DAPI to exclude
artefacts. By changing the filter sets, the Spectrum Orange and Spectrum
Green signals were clearly visible within the main nuclei. From time to time,
chromosomes 2 and 7 were also stained in metaphase spreads to act as
controls for the specificity of the method. At the start of die study, some
signals in main nuclei and micronuclei were scanned to become familiar with
the differentiation of artefacts. A total of 1000 X-ray-induced micronuclei
and 1000 colchicine-induced micronuclei were analysed. These two groups
of 1000 micronuclei were pooled from two blood samples obtained at different
times from die same person; both samples showed the same result
55
K.Wuttke et al
A
D*t»«tl*n of
mm* «T i n h u w n
ttotlon of • ( r i M t n t
of ohrMOtMt 2
within a «I or- anuo I «u*
• ff • blnuolaatsd ly»phooyU
Fig. 1. (A) Detection of chromosome 2 (green fluorescence) and chromosome 7 (red fluorescence) in a metaphase spread of a human lymphocyte. (B)
Detection of a fragment of chromosome 2 within a micronucleus of a binuclcated lymphocyte (green fluorescence); theredfluorescenceindicates the location
of chromosome 7.
Statistical analysis
As a measure of the statistical uncertainty of the observed frequency of
involvement of the individual chromosomes in micronucleus formation, 95%
confidence intervals were calculated (Sachs, 1984). Derivation of the expected
values is explained in the Results section.
Results
Figure 1A shows a metaphase spread of a human lymphocyte
painted simultaneously with chromosome-specific DNA probes
for chromosome 2 and chromosome 7. As expected, four
chromosomes are brightly labelled. In Figure IB, the painting
of these chromosomes in the interphase nuclei of a binucleated
lymphocyte is shown. Within the micronucleus of this cell, a
fragment of chromosome 2 was made visible.
Different fixation techniques for the binucleated cells were
tested to find out the one that gave the best basis for a good
signal after whole chromosome painting. Ah" fixation protocols
which included the use of acetic acid led to a chromosome
paint of minor quality, whereas the use of absolute methanol
(-20°C) after cytocentrifugation of the cells onto slides gave
good results. However, in this case, the attachment of the cells
to the surface of the slides was not good enough, so that cells
could be lost during denaturation. A post-fixation procedure
after RNase and pepsin treatment (see Material and methods)
was found to be necessary. According to this scheme, the
hybridization led to good chromosome painting for interphase
chromatin as well.
The micronucleus assay and the subsequent fluorescence
in situ hybridization protocol using whole chromosome painting allowed the determination of the composition of
micronuclei with regard to the involvement of (fragments of)
chromosome 2 and chromosome 7. The participation of these
specific chromosomes was investigated after exposure of
human lymphocytes to clastogenic X-ray irradiation (2.5 Gy)
or after treatment with the aneuploidy-inducing spindle poison
colchicine (0.04/0.06 (ig/ml). The total number of analysed
micronuclei induced by X-rays or colchicine was 1000 in
each case.
The theoretical expected frequency of involvement in
56
micronucleus formation of the chromosomes under study can
be calculated as follows: the chance of a target being damaged
by X-rays depends on its size. Consequently, for radiationinduced micronuclei, the length of the different chromosomes
must be considered. As the relative length of the individual
chromosomes in metaphase may not be representative for the
corresponding chromosomes in interphase, a more reliable
indicator is the relative DNA content of the chromosomes
which is correlated with their length. According to Mendelsohn
et al. (1973), the relative content of the DNA of chromosomes
2 and chromosomes 7 is 8.0 and 5.3 respectively as a percentage
of the total of the diploid female genome. Consequently,
the theoretical frequency expected of involvement of these
chromosomes in micronucleus composition is 80 for chromosome 2 and 53 for chromosome 7 per 1000 analysed
micronuclei (see Figure 2).
For cholchicine-induced micronuclei, the probability of
the single chromosomes becoming involved in micronucleus
formation is independent of their length because of the spindledamaging action of the agent. We also have to consider that
we cannot distinguish between the two copies of each autosome.
Consequently, the expected frequency of chromosome integration in this case is based on the assumption of a random
involvement, 1000/23 = 44 (see Figure 2; for obvious reasons,
no confidence interval can be given here).
After exposure to X-rays, good agreement was found
between the expected and observed frequencies for both
chromosome 2 and chromosome 7 (P > 0.05) (see Figure 2).
Table I shows the classification of the analysed micronuclei
with regard to their chromosome 2 and chromosome 7 status.
The quotient resulting from the division of the quantity of
chromosome 2-positive micronuclei by the one of chromosome
7-positive micronuclei (70/49) is 1.43 and is in good accordance
with the quotient calculated from the relative DNA contents
of both chromosomes (8.00/5.30 = 1.51).
After colchicine treatment of the cells, similar values for
expected and observed frequencies of involvement were found
for chromosome 2 (Figure 2). In contrast, this was not the
Detection of chromosomes 2 and 7 in micronuclei using FISH
Table I. Classification of the analysed micronuclei induced by X-rays or colchicine with regard to their chromosome 2 and chromosome 7 status
Signal-negative micronuclei after
Total no. analysed micronuclei after
colcichine
X-rays
Chromosome 2
Chromosome 7
Ratio of chromosome 2/7
1000
1000
X-rays
1000
1000
930
951
colchicine
965
933
Signal-positive micronuclei after
X-rays
colchicine
obsiexp.
obs ./exp.
70/80
49/53
1.43/1.51
35/44
67/44
0.52/1.00
obs. = observed; exp. = expected (in the case of X-rays based on the relative DNA content of the chromosomes according to Mendelsohn er aL, 1973, in the
case of colchicine based on the number of chromosomes).
Table IL Classification of the signal-positive micronuclei with regard to their distribution per binucleated lymphocyte
Chromosome 2
Chromosome 7
No. cases with 1 signal positive micronucleus
X-rays
colchicine
X-rays
colchicine
X-rays
colchicine
70
49
35
67
66
45
33
57
2
2
1
5
no. of signal-positive micronuclei
per 1000 micronuclei
X-rays
80
colchicine
-
HE- •
80 - -
—
40 -
--
20 -
—
—
n
obs.
axp.
chromosome 2
2 signal positive micronuclei
Total no. siginal-positive micronuclei after
obs.
exp.
chromosome 7
obs.
exp.
obs.
exp.
chromosome 2 chromosome 7
Fig. 2. Comparison between the theoretically expected (exp.) and the
experimentally observed (obs.) frequency of involvement of chromosome 2
and chromosome 7 in the formation of micronuclei induced by X-rays (2.5
Gy) or colchicine (0.04/0.06 Hg/ml). (Bars indicate the 95% confidence
intervals).
case for chromosome 7; comparing the frequency of observed
(67) and expected (44) chromosome 7-positive micronuclei,
a significant over-involvement of this chromosome in micronucleus formation described by a factor of 1.5 was detected
(Figure 2).
The ratio obtained from the signal-positive micronucleus
values for chromosome 2 and chromosome 7 after colchicine
treatment (35/67 = 0.52) (Table I) deviates strongly from the
expected value of 1 which should be found if both chromosomes
had been involved in micronucleus formation to the same
degree.
Table II shows the classification of only the signal-positive
micronuclei with regard to their distribution per binucleated
lymphocyte. The most frequent finding was that only one
chromosome 2- or one chromosome 7-positive micronucleus
per binucleated lymphocyte was observed. After X-ray exposure, two cases were found with two signal-positive micronuclei
per binucleated lymphocyte for chromosome 2 and chromo-
some 7 respectively. Also after colchicine treatment, one
binucleated cell was observed with two signal-positive
micronuclei for chromosome 2 and, interestingly, even five
binucleated cells with two chromosome 7-positive micronuclei.
Discussion
In this study, we compared the frequency found experimentally
of involvement of chromosome 2 and chromosome 7 in
micronucleus formation with the one that would be expected
theoretically, when a participation of each individual chromosome on the basis of randomness is assumed. As micronucleiinducing agents, we used the clastogen X-ray irradiation
(2.5 Gy) and the aneuploidy-inducing spindle poison colchicine
(0.04/0.06 ng/ml).
We have specifically focused on chromosome 2 and chromosome 7 because: (i) the non-random occurrence of exchanges
involving chromosome 7 in healthy control individuals (Tawn,
1988) hints at a special genetic fragility of this chromosome;
(ii) the non-random chromosomal abnormalities of chromosome 7 have been shown to be of interest in the development
of a number of different malignancies such as malignant
gliomas, neuroblastoma, prostate cancer, transformed lymphoblasts and transformed liver epithelial cells (e.g. Steel et aL,
1980; Bigner et aL, 1986, 1987; Rey et aL, 1987; Cremer
et aL, 1988; Llombart-Bosch et aL, 1989; Takahashi et aL,
1994; Steadman et aL, 1994). Steadman et aL (1994) showed
that in transformed rat liver epithelial cells the non-random
occurrence of the chromosomal abnormality involving chromosome 7 is located near the proto-oncogene c-myc; (iii) chromosome 2 is representative of the large sized chromosomes and
thus is expected to be involved more frequently in damaging
events than the shorter chromosomes.
The results obtained after X-ray exposure clearly show
that neither of the two analysed chromosomes is included
preferentially into micronuclei, but that their participation in
the expression of cytogenetic damage is merely random (Figure
2). The correlation between the relative DNA content (and
thus the relative length) of the chromosomes and the probability
of the chromosomes being damaged by X-rays is obvious
(Table I). As X-rays are known to damage chromosomes
57
K-Wuttke et al
mainly by inducing chromosome breakage resulting in two
acentric fragments (e.g. Evans, 1988; WeiBenborn and Streffer,
1991), the occurrence of two micronuclei with a signal from
the same chromosomes (Table II) within a binucleated lymphocyte is not surprising; their packing into different micronuclei
can be expected because of the spatial location of the fragments
belonging to the same damaged chromosome.
The targets of the damaging action of colchicine are the
microtubules which form the mitotic spindle. By this
mechanism, lagging chromosomes appear during anaphase;
these cannot be distributed into the daughter-cells and so the
incorporation of these chromosomes into micronuclei during
telophase is the result. Hence, micronuclei induced by
colchicine are expected to be composed of whole chromosomes. The colchicine concentration chosen in this study
(0.02-0.08 |i.g/ml) does not lead to a total disruption of the
spindle; consequently, the cleavage of the cells was still
possible and induced micronuclei could be expressed.
Based on knowledge of the effects of colchicine, one would
expect that a damaged spindle fibre would probably affect all
chromosomes with equal frequency. However, the results of
this study are not in accordance with this assumption; while
the observed involvement of chromosome 2 in micronucleus
formation corresponds with the theoretical frequency, we found
a significant over-involvement of chromosome 7 (Figure 2,
Table I). Why should chromosome 7 fail to align on the
remaining spindle more frequently than other chromosomes?
At present, we are not able to give a satisfactory explanation.
Instead, we have developed three hypothetical explanations:
(i) it is known that different parts of the cytoskeleton are
linked together and that their functions are co-ordinated (for
review see Alberts et al, 1983). There is evidence that
cytoplasmic microtubules co-ordinate the various parts of the
cytoskeleton responsible for complex intracelluar movements.
They influence the distribution of actin filaments as well as of
intermediate filaments, which play a structural and tensionbearing role in the cell. The principal filaments of the cytoskeleton are extensively interconnected by a three-dimensional
network as well as by soluble, diffusing factors. One component
of the nuclear matrix, in which the chromosomes are postulated
to be anchored, is the non-chromatin network, a three-dimensional system of filaments with associated granular structures
(Nickerson et al, 1987). There is no evidence, but it is
imaginable, that these two filament systems interact (especially
during mitosis when the nuclear envelope is disappearing and
the spindle enters the nuclear area), so that the colchicineinduced disruption of the microtubules might affect the network
of the nuclear matrix. Depending upon the anchorage location
of chromosome 7 within the matrix, this chromosome may be
predisposed for a failure in aligning on the spindle; (ii) the
relative number of inserting microtubules per kinetochore of
the different chromosomes has not been investigated up to
now. If each chromosome is not attached by the same amount
of spindle fibres, one explanation for our finding with chromosome 7 could be that this chromosome might contain a
relatively low number of microtubules, so that the probability of
this chromosome becoming a laggard as a result of colchicineinduced disruption of the spindle is relatively high; (iii) it may
be possible that chromosome 7 has a number of nucleotide
sequences similar to sequences within other chromosomes, so
that this chromosome is especially prone to hybridize with
areas of other chromosomes. With this mechanism, its chance
58
of aligning on a spindle with reduced numbers of microtubules
might be modified.
We are aware that our attempts to explain the principal
finding of the over-involvement of chromosome 7 in micronucleus formation are speculative. Further investigations are
in progress to clarify whether such a non-random participation
in the expression of cytogenetic damage can also be found for
other chromosomes, maybe for those with similar size as
chromosome 7.
The results of Ntisse et al (1992a,b) confirm our observations
for chromosome 2 and chromosome 7 generally; the DNA
distribution of radiation-induced micronuclei measured by flow
cytometry did not show any peaks, leading to the conclusion
that the composition of radiation-induced micronuclei can be
explained by random breakage of chromosomes. In contrast,
by using the spindle poison 2-chlorobenzylidene malonitrile,
the authors described a DNA distribution of the induced
micronuclei with several peaks, suggesting a correspondence
with the DNA distribution of specific chromosomes which
unfortunately have not been defined precisely.
Looking at the classification of the signal-positive
micronuclei with regard to their distribution per binucleated
cell after colchicine-treatment (Table II), the occurrence of
two signal-positive micronuclei per binucleated lymphocyte
for chromosome 2 and chromosome 7 respectively, is surprising; after colchicine treatment, each micronucleus should be
composed of entire chromosomes. For chromosome 7 the
highest number of binucleates with two micronuclei per cell
containing this chromosome was found. It can be excluded that
each of the two micronuclei enclosed one of the homologues of
chromosome 7, for the corresponding signal was also detected
in the main nucleus. If the chromatids of the lagging chromosomes began to separate, one would then see two distinct
signals that could have been included into different micronuclei.
Another explanation can be derived from the similar results
found by other investigators; according to Sternes and Vig
(1989), there appears to be a loose correlation between the
formation of acentric fragments and kinetochore-negative
micronuclei after colchicine treatment. It is also possible
that some of the kinetochore-negative micronuclei actually
represent the whole chromosome-micronuclei, in which the
antigen for the antikinetochore-antibody is altered, so that the
kinetochore fluorescence is not detectable. However, studies
using in situ hybridization for centromere identification showed
only ~50-70% centromere-positive micronuclei (Adler, 1993;
Hayashi et al, 1994). Thus, micronuclei induced by colchicine
arose predominantly, but not exclusively, by whole lagging
chromosomes. Whether or not the remaining fraction of
micronuclei is due to chromosome breakage remains to be
determined.
In conclusion, our results (referring to chromosome 2 and
chromosome 7) and those cited from the literature (referring
to the entire genome of a cell) indicate a quite unexpected
trend in the answer to our initial question about the comparative
influence of clastogenic and aneuploidy-inducing agents on
the participation of individual chromosomes in the expression
of micronuclei. Whereas after exposure to ionizing radiation,
chromosome-specific damage would be imaginable, and after
treatment with a spindle poison an effect with equal frequency
on the individual chromosomes would be expected, quite the
opposite must be postulated now. However, further thorough
studies on specific chromosomes and an investigation of
Detection of chromosomes 2 and 7 In micronuclei using FISH
possible spindle poison mediated damaging mechanisms on
individual chromosomes are needed.
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Received on June 24, 1996; accepted on October 23, 1996
59