Chromosome segregation from cell hybrids
IV. Movement and position of segregant set chromosomes in early-phase
interspecific cell hybrids
PAULA A. ZELESCO* and JENNIFER A. MARSHALL GRAVES
Department of Genetics and Human Variation, La Trobe University, Bnndoora, Victoria 30S3, Australia
* Author for correspondence
Summary
We searched for evidence of aberrant movement
or position of segregant set chromosomes in Cbanded and G-11 -banded early-phase hamstermouse and hamster-human cell hybrids that had
been prepared with minimal disruption. No evidence was obtained for an increased frequency of
multipolar mitosis, delayed or precocious metaphase congression or anaphase segregation, or for
exclusion of chromosomes from the daughter
nuclei. However, in hamster-human hybrids,
segregant set (human) chromosomes were observed to assume a central position within a ring
of hamster chromosomes on the metaphase plate.
Such non-random positioning may imply that the
centromeres of segregant chromosomes make aberrant, or simply less efficient, attachments to the
spindle in hybrid cells. This aberrant position
may perhaps result indirectly in chromosome loss
by interfering with the normal processes of replication, repair or transcription.
Introduction
Two distinct movements of chromosomes characterize normal mitotic behaviour; congression at the metaphase plate and anaphase migration to opposite poles of
the spindle. Both movements are effected by correct
interaction of centromeres with spindle fibres. If segregant set chromosomes were to fail to attach to spindle
fibres, attached less efficiently, or became prematurely
disengaged, aberrant positioning or movement of
segregant chromosomes should be observed at metaphase and anaphase in hybrid cells. The exclusion of
segregant chromosomes from the reconstituted
daughter nuclei might also be apparent as micronuclei
at telophase.
We used C-banding and G-11-banding to determine
the parental origin of chromosomes in hamster-mouse
and hamster—human cell hybrids. We screened populations of early-stage hybrids for aberrant metaphase
congression and anaphase and telophase segregation
and for non-random positioning of segregant chromosomes.
Interspecific cell hybrids retain a virtually intact set of
chromosomes of one parent (the 'retained set') while
preferentially losing chromosomes of the other parent
(the 'segregant set'). The mitotic spindle has long been
thought to be involved in this directional chromosome
segregation (Migeon, 1968; Handmaker, 1973), and
there are several observations consistent with the idea
that loss occurs at mitosis (Schall & Rechsteiner, 1978;
Dawson & Graves, 1984). Loss of groups of chromosomes could result from aberrant interactions between
segregant set chromosomes and the spindle fibres of the
hybrid cells (Handmaker, 1971, 1973). We have recently shown that segregation is independent of hybrid
spindle constitution (Zelesco & Graves, 1987). There
remains the possibility that aberrant interactions between spindle and centromere result from intrinsic or
induced differences in efficiencies with which retained
and segregant set chromosomes engage the spindle.
Journal of Cell Science 89, 49-56 (1988)
Printed in Great Britain @ The Company of Biologists Limited 1988
Key words: mitosis, cell hybrids, chromosome segregation,
chromosome movement, chromosome position.
49
Materials and methods
Parent cell lines
Rodent lines were: Bio, a TK-deficient Chinese hamster
line; Cmd4, an HPRT-deficient Chinese hamster line; and
LTA, a TK-deficient mouse line (described in full by Hope &
Graves, 1978; Zelesco & Graves, 1987). Human lines were:
EUE, an HPRT-deficient HeLa derivative; and Daudi
(Klein el al. 1967), a human suspension line obtained from
Dr N. Hoogcnraad, La Trobe University, Victoria.
Cell culture
Cells were routinely cultured in Dulbecco's modified Eagle's
medium (DME, GIBCO) supplemented with 10% foetal calf
serum (Flow, Australia), 60jUgml~' penicillin (Sigma) and
SO^fgml"' streptomycin (Glaxo, Australia).
Cell fusion
Disaggregated parental cells were mixed in a 1:1 ratio and
fused in suspension with polyethylene glycol, MT1000 (BDH
or Ajax, Australia) diluted to a 50% (w/v) solution in serumfree DME. Fused cells were cultured in 100mm diameter
Petri dishes (Corning) in HAT medium (Szybalski el al.
1962) containing 10~4 M-hypoxanthine, 4xlO~ ? M-aminopterin and 1-6X 10~5 M-thymidine (Sigma), in a humidified
CO2 incubator. Populations of hamster-mouse fused cells
were harvested for cytological study, but for hamster-human
fusions, because of the low frequencies of hybrids, macroscopic colonies were isolated by suction with a micropipette,
transferred to individual microscope-slide growth chambers
(Lab-Tek, Miles Laboratories, Australia) and propagated
until sufficient cells were present for cytological study.
Cytological preparations
Petri dishes containing 20-40 hamster-mouse hybrid colonies were sampled at random between 10 and 20 days after
fusion. Cells were trypsinized and gently fixed without
colchicine or hypotonic treatment, by slowly adding a few
drops of fixative (3:1 (v/v), methanol:acetic acid) to the
suspension in a centrifuge tube. Cells were centrifuged gently
and resuspended in fresh fixative twice, then slides were
prepared by spreading the suspension gently onto dry microscope slides and air drying. Slide chambers containing
individual hamster-human hybrids were sampled 22-27 days
after fusion. Cells were gently fixed in situ by removal of half
the medium, and addition of fixative, which was subsequently replaced twice and aspirated; slides were then air
dried.
Staining methods
Mouse and hamster chromosomes were differentiated by Cbanding (Levershae/ al. 1980). Hamster and human chromosomes were differentially stained (magenta and pale blue,
respectively) by a modification of the G-ll banding technique (Bobrow & Cross, 1974; A. Dobrovic, Flinders
University, Australia). Slides were placed in 2XSSC (SSC is
OlSM-NaCI, 0-015 M-sodium citrate) at 58-62°C for Smin,
rinsed in double-distilled water, incubated in 4 % Giemsa
(Harlcco) in 10"2M-NaOH at 37°C for 9min, rinsed and air
dried. Cells were examined using a Leitz Dialux 2 microscope
50
P. A. Zelesco and jf. A. M. Graves
and photographed using Kodak technical pan, Agfa Copex or
Word FP4.
Results
Characteristics of hybrids
Hamster-mouse hybrids were recovered
from
Cmd4 X LTA fusions at frequencies of 2x 10~5 to
4X 10~ s . Several hamster-mouse hybrids were isolated
and propagated individually in order to determine the
direction and extent of chromosome segregation in this
hybrid combination. Table 1 shows that some hybrids
segregated about half the hamster complements and
retained one set (or sometimes almost two sets) of
mouse chromosomes. However, the reverse pattern
was also observed in several hybrids. Thus the mass
populations of early hybrids would have contained
mixtures of hybrids segregating hamster, and hybrids
segregating mouse, chromosomes.
Hybrids were recovered from Bio X EUE and Bio X
Daudi crosses at much lower frequencies (approx.
10~ 6 ). All retained hamster chromosomes (1, or sometimes 2 sets) and segregated human chromosomes.
Established BIo-EUE hybrids retained two to eight
human chromosomes, but early-phase hybrids appeared to have more. An exact count of human
chromosomes in G-ll-banded early hybrids was difficult because, in the absence of colchicine and hypotonic treatment, chromosomes often overlapped. However, a careful analysis of 30 and 21 (respectively)
favourable metaphase cells showed that BIo-EUE
early hybrids retained an average of 17, and BIoDaudi early hybrids retained 12 (Table 1).
Table 1. Mean chromosome numbers in hybrid and
parental cells
Hamster—mouse hybrids
Mean number of chromosomes
Mouse
Hamster
Hamster parent (Cmd4)
Mouse parent (LTA)
21
Hybrid subclonc: a
b
c
d
e
f
g
h
15
17
12
13
12
9
10
13
26
20
38
45
65
79
84
91
Hamster
Human
Hamster—human hybrids
Hamster parent (Bio)
Human parent (Daudi)
50
22 (mode)
(EUE)
BIo-EUE hybrid
Bio—Daudi hybrid
36
22
46
54
17
12
Table 2. Frequency of multipolar mitosis
Cell line
Cmd4
LTA
Cmd-LTA hybrids
Bio
EUE
Daudi
BIo-EUE hybrids
Bio—Daudi hybrids
No. cells
%
Multipolar
mitosis
100
100
50
100
100
100
100
100
2
1
2
4
15
5
17
0
Chromosome movement in early-phase hybrids
If chromosomes are lost from hybrid cells as the result
of delayed congression or anaphase separation, we
would expect to detect, at high frequencies, segregant
chromosomes preferentially displaced from the metaphase plate, lagging at anaphase, and forming micronuclei at telophase. If loss results from formation of trior tetrapolar spindles, multipolar mitoses should be
frequent. We studied mitotic cells from hamstermouse and hamster-human hybrids that had been
fixed in situ or in suspension without colchicine or
hypotonic pretreatment, in order to preserve the spatial
relationships of chromosomes.
Multipolar mitosis. Since a newly formed hybrid cell
might be expected to possess four rather than two
mitotic centres, it is possible that tripolar and tetrapolar mitoses are frequent, and result in aberrant
segregation and chromosome loss. We observed the
frequencies of multipolar mitoses in the early-phase
hamster-mouse and hamster-human hybrids. Table 2
shows that the frequency of multipolar mitoses in
hamster-mouse hybrids was extremely low, and of the
order of the frequency in parental cells. The frequency
among early hamster-human hybrids was higher, but
hardly more than the frequency among the EUE
parents.
Metaphase congression. Metaphase spreads were
selected for analysis only if they were unbroken, were
oriented to give either a polar or lateral view, and were
differentially stained. These rigorous scoring criteria
were felt to be essential for a valid analysis, although
they left rather small sample sizes for hamster-human
early hybrids; only two of 12 samples (BIo-EUE fixed
at 26 days, and BIo-Daudi at 24 days) contained cells
deemed to be scorable. Chromosomes were scored as
delayed in congression if they either lay within or were
outside the ring in radial spreads, and had no attachment with the equatorial plate in a lateral view. It was
reasoned that, when a three-dimensional cell is viewed
in two dimensions, lagging chromosomes within the
space occupied by the spindle will usually appear
within the ring in a polar view, and detached from the
band of chromosomes in an equatorial view. Lagging
chromosomes outside this space will also be detached
from the band in an equatorial view, but will appear
outside the ring in a polar view. In hamster-mouse
hybrids, chromosomes could be identified unequivocally as to parental origin by their strongly C-banding
centromeric regions (Fig. 1A). In G-11-banded hamster-human hybrids, hamster chromosomes stained
magenta and human chromosomes pale blue (Fig. IB).
Table 3 shows the proportion of metaphase parental
and hybrid cells with one or more lagging chromosomes. A high proportion of hamster-mouse hybrid
cells possessed laggards; however, this proportion was
no greater than the proportion of parental cells with
laggards (Table 3); also, both mouse and hamster
chromosomes were represented among laggards, frequently within the same cell (Fig. 1A). Among the
scorable hamster—human hybrid cells, too, it was clear
that hamster chromosomes lagged as frequently as
human (Table 3, Fig. IB).
Anaphase movement. There were few anaphase figures in either hybrid type; all were scored. Table 4
shows that both mouse and hamster chromosomes mav
Table 3. Frequency of metaphase spreads with lagging chromosomes
Jo Cells with laggards*
Cell line
Cmd4
LTA
Cmd-LTA hybrids
Bio
EUE
Daudi
BIo-EUE hybrids
Bio—Daudi hybrids
No. cells
Hamster
Mouse
Total
20
16
60
55
50
56
49
55
56
67*
No. cells
Hamster
Human
50
50
50
23
19
26
30
30
30
30
16
43
16
Unidentified
9
Total
26
30
30
52*
16*
* Cells having both types of laggards are included in both subtotals.
Mitosis in cell hybrids
51
Fig. 1. Mctaphase figures from untreated
early hybrids showing chromosomes
lagging in congression. (A) Polar view of
a C-banded early mouse-hamster hybrid
cell, showing one mouse chromosome
(filled arrow) and one hamster
chromosome (open arrow) distant from
the ring of chromosomes. (B) Lateral
view of a g-11-stained early
hamster-human hybrid cell, showing
displacement of both hamster
chromosomes (open arrows) and human
chromosomes (filled arrows) from the
equatorial plate.
Fig. 2. C-banded anaphase figure from
early mouse-hamster hybrid, showing
lagging hamster chromosome (open
arrow) and mouse chromosomes (filled
arrows).
Fig. 3. G-11-stained polar view of an
early hamster-human hybrid. The
chromosomes in the peripheral ring are
all magenta stained (hamster) and those
within the ring (with one exception) are
all pale blue (human).
1A
lag at anaphase in hamster-mouse hybrids, often in thi
same cell (Fig. 2); hovever, the frequency of laggard
is no greater in hybrids than in parental cells. Then
were too few scorable hamster—human anaphase fig
tires to permit any meaningful analysis.
Inclusion in daughter nuclei. If segregant chromo
somes were delayed in their anaphase movement ant
are lost because they are excluded from daughte
nuclei, we would expect to observe a higher frequenc
of micronuclei among early telophase figures in hybri<
cells than in parental cells. We scored all available earl;
telophase figures for hamster—mouse and hamsterhuman hybrids. Hamster-mouse hybrids did not shov
an increase in micronucleus frequency (Table 5). No
did the BIo-Daudi hybrids; however, BIo-EUE di<
contain 6% cells with micronuclei (compared to 0 am
2 % in parent cells); this difference is non-significant a
shown by a yf text.
Thus, our studies of metaphase, anaphase am
telophase have provided no evidence that chromosom
movement is impaired in any way in early-phas
hamster—mouse or hamster—human cell hybrids.
Chromosome position in early hybrids
C-banded populations of hamster-mouse early hybrid
were examined for any evidence of non-randon
chromosome positioning at metaphase. There were n
cells, out of 23 radial and 37 side-on views, in which th
mouse and hamster chromosome sets were spatiall
separated (side-by-side or concentric); mouse am
52
P. A. Zelesco and J. A. M. Graves
1B
Table 4. Frequency of anaphase cells ivith lagging chromosomes
% Cells with laggards
Cell line
No. cells
Hamster
15
11
15
47
Cmd4
LTA
Cmd—LTA hybrids
19
10
13
3
0
Bio
EUE
Daudi
BIo-EUE hybrids
Bio—Daudi hybrids
Table 5. Frequency of telophase cells with
micron uclei
Cell line
Cmd4
LTA
Cmd—LTA hybrids
Bio
EUE
Daudi
BIo-EUE hybrids
Bio—Daudi hybrids
No. cells
% Cells with
micronuclci
100
100
100
100
100
100
100
100
3
0
0
0
2
0
6
0
hamster chromosomes were always intermixed within
metaphase spreads.
A completely different result was obtained when the
positions of hamster or human sets in 40 G-11 -banded
early hamster-human hybrids were examined. In all 40
scorable hybrid cells (representing both crosses), the
pale-blue human chromosomes all lay nearer the centre
of radial mitoses, usually completely within a ring of
magenta-stained hamster chromosomes (Fig. 3). The
positions of hamster and human chromosomes in
metaphase figures viewed side-on were consistent with
this interpretation, in that a blue core was seen to be
encased by superimposed magenta chromosomes. The
outermost regions of the metaphase plate contained
only magenta chromosomes. This clearly non-random
arrangement of chromosomes was seen not to be related
to chromosome size, since some group A human
chromosomes were central while some group IV hamster chromosomes (which are less than half the size)
were peripheral. Nor did the staining difference reflect
merely the positions of chromosomes, since occasional
hamster chromosomes within the ring were conspicuously magenta stained, while occasionally a bluestained human chromosome was observed among the
peripheral chromosome ring.
Mouse
46
73
46
Hamster
Human
100
Total
47
73
60*
Total
4
0
4
100
Discussion
We made light-microscope studies of the movement
and position of chromosomes in the early phase of two
types of cell hybrids. Both sets of hamster—human
hybrids that we studied showed rapid preferential
segregation, invariably of human chromosomes, while
the hamster-mouse hybrids showed a more gradual
loss of either hamster or mouse chromosomes in
different clones. Hamster-mouse hybrids derived from
different combinations of cell lines have been described
that segregate mouse (Scaletta el al. 1967) or hamster
chromosomes (Graves, 1975); however, it is unusual to
find both directions of segregation among hybrids from
a single cross. Evidently the (unknown) factors that
determine direction of segregation must be rather
evenly balanced in these hybrids (and in similar
hybrids described by Marin & Pugliatti-Crippa, 1972),
so that direction may be established by variations
between fused cells; perhaps the dosage of some factor,
shown to be important by fusions between polyploid
lines (Graves & McMillan, 1984), depends on the cycle
phase of the cell at the time of fusion in these cell
combinations.
We studied dividing cells fixed with the minimum of
disruption early in the generation of hamster-mouse
and hamster-human hybrids, in order to test the
hypothesis that chromosome segregation results from
multipolar mitosis, delayed metaphase congression,
anaphase lagging or exclusion from the telophase
nucleus. From the numbers of chromosomes lost, and
the numbers of cell generations that elapsed between
fusion and fixation, we could roughly estimate the level
of abnormal mitotic events required to account for
segregation. Populations of hamster-mouse hybrids
fixed 10-20 days post-fusion could have undergone up
to 20 cell generations. Individual hamster-human
hybrids isolated as colonies of a few hundred cells, then
propagated for a further 7 days could have undergone
up to 15. Both these figures are likely to be large
overestimates, since we have assumed cell generation
Mitosis in cell hybrids
53
times equivalent to those of the parent lines; however,
the cycle times of interspecific hybrids are initially very
much longer than those of either parent (Graves &
Koschel, 1980). Even so, the loss of up to 12 hamster or
24 mouse chromosomes over 20 cell generations would
be expected to give rise to an observable abnormality in
more than half the mitotic mouse-hamster hybrid
cells, while the loss of about 35 human chromosomes
over 15 cell generations should be detectable as an
average of 2-3 abnormalities per cell. We observed
nothing like this level of mitotic abnormality. We found
no evidence that multipolar mitosis, delayed metaphase
congression, anaphase segregation or micronucleus
formation were any more frequent than in parental
cells. Nor did segregant chromosomes appear to be
involved more frequently in such irregularities than
retained chromosomes. It is possible that irregularities,
such as multipolar mitoses (Oftebro, 1968) and asynchrony (Johnson & Rao, 1970), may have already had
their effect in the very earliest divisions, and so were
not detected in the hybrids studied here. From our
findings, however, we must conclude that aberrant
mitosis or delayed movement of segregant chromosomes cannot account for chromosome segregation.
hamster-human hybrid metaphase cells were clearly
non-random. Segregant (human) chromosomes were
observed always to occupy a central position on the
metaphase plate, within a ring of hamster chromosomes, and this arrangement was independent of
chromosome size.
Other observations of non-random arrangements of
genomes in mouse-human cell hybrids have been made
(Rechsteiner & Parsons, 1976; Rogers et al. 1983), but
these were reported to be transient and considered,
therefore, to be unrelated to progressive chromosome
segregation. It is likely that in this study the use of
disruptive preparative procedures (Colcemid and
hypotonic treatment) and the identification of mouse
and human chromosomes by morphology alone may
have obscured a non-random arrangement. Genome
separation has also been reported for interspecific
sexual hybrids; for instance, chick-quail hybrids
(Bammi et al. 1966) and barley-rye hybrids (Finch et
al. 1981; Schwarzacher-Robinson et al. 1987). In
interspecific barley hybrids, segregant set chromosomes were observed to occupy positions outside a ring
of retained set chromosomes (Finch, 1983); quite the
opposite pattern to that reported here.
This finding was unexpected, for two reasons. First,
interspecific barley hybrids (sexual, not somatic),
which segregate chromosomes from one parental set
during early embryogenesis, have a high frequency of
chromosome lagging (Bennett et al. 1976), suggesting
that segregation in this system does result from delayed
mitotic movement. Thus, it appears that there is only
limited analogy between chromosome segregation in
mammalian somatic hybrids and interspecific plant
hybrids. Second, our failure to observe abnormal
chromosome movement leaves in question the fate of
segregated chromosomes. If they are not lost during
early mitoses, they may be fragmented and degraded
during interphase. We did observe some signs of
fragmentation and degradation in early hybrids and
have frequently noted cells of established hybrids with
multiple rearrangements, or fuzzy puddles of staining
material, which may represent the discarded chromosomes; however, these are hard to quantify. Another
possibility is that segregant chromosomes may fail to
undergo replication during the S phase.
There are two different ways in which the aberrant
positioning we observed might be related to segregation. Human chromosomes may occupy an aberrant
central position on the metaphase plate as a result of
aberrant, or at least less efficient interactions between
centromeres and spindle fibres. We have recently
demonstrated (Zelesco & Graves, unpublished data),
that one parental form of /3-tubulin is repressed or
under-expressed in hamster-mouse hybrids; thus it is
possible that there is a limited supply of spindle fibres
in interspecific hybrids. If there were an intrinsic
difference (due to centromeric DNA sequences) or a
difference induced (e.g. by DNA modification, see
Drahovsky et al. 1980, 1981; Finch, 1983) in the
efficiency with which the hamster and human centromeres bind to spindle fibres, human chromosomes
might engage fewer (or more) than hamster, and the
resultant of the unequal torsion may force the human
set into a central position on the spindle. Aberrant
interactions between segregant set chromosomes and
the spindle may be directly responsible both for the
aberrant positioning of human chromosomes, which
we observed, and for aberrant movement resulting in
segregation. However, we did not observe this latter
effect.
If the position of a chromosome on the metaphase
plate is directly or indirectly related to its chance of
elimination, a non-random arrangement of segregant
set and retained set chromosomes might be expected in
interspecific hybrids. To test this hypothesis, we studied the relative positions of segregant and retained
chromosomes in C-banded and G-11-banded hamster-mouse and hamster-human hybrids. Although
we detected no non-random positioning of mouse and
hamster chromosomes in hamster-mouse hybrids, the
positions of hamster and human chromosomes in all
54
P. A. Zelesco and jf. A. M. Graves
Perhaps, then, aberrant positioning of segregant set
chromosomes, such as we and others have observed,
has a more direct relationship to segregation. Bennett
(1984) has described a strong tendency for parental
genomes in interspecific and intergeneric hybrid plant
cells to separate concentrically, and has provided
evidence from reconstructed nuclei that it is the
parental genome that occupies the peripheral domain
that is predisposed for mitotic chromosome elimination. The positions of chromosomes in interphase cells
is now thought to be highly ordered, and to have some
functional significance (Agard & Sedat, 1983). Metaphase positioning may reflect this interphase organization. Bennett (1984) has demonstrated a correlation in
hybrid plant cells between gene activity, including
suppression of nucleolar-organizing regions, and intranuclear chromosome or genome position. In a somatic
cell hybrid, chromosomes of the retained set may
assume their normal relative positions, while those of
the segregant set cannot be accommodated in a normal
spatial arrangement. A breakdown in the normal arrangements of segregant set chromosomes may result in
a disruption of the normal synthetic activities of these
chromosomes. This could be responsible for the
repression of ribosomal RNA (Onishi et al. 1984),
histones (Ajiro et al. 1978) and tubulin (Zelesco and
Graves, unpublished data) as well as, perhaps, affecting the stability of A" chromosome inactivation and the
conformation of the inactive A' (Gartlere^ al. 1985). It
could also, conceivably, impede normal DNA replication or repair of segregant chromosomes, and lead to
their subsequent loss from daughter cells. This hypothesis does not contradict those observations that suggest
that the rate of segregation is related to number of cell
divisions (Schall & Rechsteiner, 1978), since failure of
replication or repair could be limited to the S phase of
the cycle. This hypothesis could be tested directly by
studying DNA synthesis in rodent and human chromosomes of early-phase hybrids.
This work was funded by grants from the Australian
Research Grants Scheme and the National Health and
Medical Research Council. Some of this work was completed
while Dr Zelesco held a Commonwealth Postgraduate Research Scholarship.
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{Received 21 Mav 1987 - Accepted, in revised fonn,
12 October 19S7)