105
Journal of Cell Science 103, 105-115 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
Inhibitors of topoisomerases do not block the passage of human
lymphocyte chromosomes through mitosis
A. T. SUMNER
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
Summary
Cultured human lymphocytes have been treated with a
number of topoisomerase inhibitors, to see whether
topoisomerase II is involved in the process of chromosome segregation at anaphase. Results were assessed by
examination of cytogenetical preparations of spread
chromosomes. Four effects were observed, although no
inhibitor produced all four effects. These effects were:
inhibition of entry into mitosis; chromosome breakage
and rearrangement; inhibition of chromosome condensation; and inhibition of chromosome segregation.
Evidence for the last was ambiguous. Although there
was evidence that separation of chromatids was affected
when cells were treated with colchicine as well as
topoisomerase II inhibitors (most notably with nalidixic
acid, which resulted in complete fusion of the chromatids), no evidence was obtained to show that, in the
absence of colchicine, cells treated with inhibitors could
not proceed through anaphase normally. The topoisomerase I inhibitor, camptothecin, differed from the
topoisomerase II inhibitors in not showing any effect on
chromosome condensation or any significant effect on
segregation.
Introduction
n , an accumulation of chromosomes at the metaphase
stage of mitosis might be expected; as described by
Uemura and Yanagida (1986) for fission yeast, the
mitotic spindle would pull but fail to separate chromosomes. Inhibitors of topoisomerase II are used extensively in cancer chemotherapy (D'Arpa and Liu, 1989),
and their effects on cells have therefore been studied
extensively; their main effects on the cell cycle appear
to be blockage in interphase, induction of chromosome
damage, and inhibition of chromosome condensation
(Huang et al. 1973; Pommier et al. 1988; Rowley and
Kort, 1989), but an unequivocal effect on chromosome
segregation has not yet been reported.
Experiments in which mammalian cells have been
treated with topoisomerase II inhibitors (Charron and
Hancock, 1991; Downes et al. 1991) may be inconclusive, because many of these inhibitors cause extensive
chromosome damage which may itself, independently
of an action on topoisomerase II, lead to an appearance
of malsegregation. In the present experiments, I have
concentrated on studying the behaviour of the chromosomes during mitosis, rather than relying on signs of
non-disjunction, such as micronuclei, and strands of
chromatin connecting daughter nuclei, which could
arise from a variety of causes.
It is to be expected that the enzyme topoisomerase II
would be involved in mammalian chromosome segregation, both on theoretical grounds, and by analogy
with the situation in lower eukaryotes. It is believed
that the daughter DNA molecules resulting from
replication are catenated, and therefore require the
action of topoisomerase II to disentangle them (Hsieh
and Brutlag, 1980; Sundin and Varshavsky, 1981;
Murray and Szostak, 1985). Extensive experiments with
yeast topoisomerase II mutants indicate that the
enzyme is needed for disjunction of chromosomes, both
at mitosis (di Nardo et al. 1984; Uemura and Yanagida,
1986; Uemura et al. 1987; Holm et al. 1989) and at
meiosis (Rose et al. 1990). However, results from
experiments on yeasts may not necessarily be applicable
to higher organisms, and there is no clear evidence yet
that topoisomerase II is involved in chromosome
segregation in mammalian cells. In any case, the
chromosomes of yeasts are too small for studying some
of the processes of segregation easily.
In the present study, the possible involvement of
topoisomerase II in mammalian chromosome segregation has been investigated using a number of
inhibitors of topoisomerase II, and studying the effects
of these on chromosomes at the different stages of
mitosis. It was reasoned that if chromosome segregation were prevented by inhibition of topoisomerase
Key words: chromosome condensation, chromosome
damage, chromosome segregation, mitosis, topoisomerases.
Materials and methods
Human peripheral blood lymphocyte cultures were grown
106
A. T. Sumner
Table 1. Topoisomerase inhibitors used
Inhibitor
Source
Stock solution
Topoisomerase I inhibitor
Camptothecin
Sigma
1 mg/ml in DMSO (2.874 mM)
Topoisomerase II inhibitors
Nalidixic acid, Na salt
Ethidium bromide
Ellipticine
Daunomycin
Mitoxantrone
Amsacrine (m-AMSA)
Etoposide
Sigma
BDH
Sigma
Sigma
Cyanamid of Gt. Britain Ltd
Warner-Lambert/Parke-Davis
Bristol-Myers Squibb
5% in distilled water
0.4 mg/ml in distilled water (1 mM)
1 mg/ml in 10 mM HCI (4 mM)
10 /ig/ml in distilled water (20 /iM)
0.44 mg/ml in distilled water (1 /tM)
2 mg/ml in DMSO (5 mM)
3 mg/ml in DMSO (5 mM)
according to standard procedures for approximately 72 h. Five
hours before the cultures were harvested, a sample of a stock
solution of a topoisomerase inhibitor was added to each
culture, to produce the desired concentration. One set of
cultures had colchicine (0.5 /ig/5 ml culture) added at the same
time as the inhibitor; the other set of cultures did not have
colchicine added. The topoisomerase inhibitors used are
listed in Table 1. Control cultures had no inhibitor added, but
if the inhibitor was dissolved in dimethyl sulphoxide
(DMSO), all the cultures in that experiment, including the
control, had the same amount of DMSO added (including the
vehicle for the inhibitor), as some results appeared to indicate
that DMSO alone might influence the proportions of cells at
the different stages of mitosis (see Fig. 2, below).
At the end of the culture period, the cultures were treated
with hypotonic solution (0.075 M potassium chloride), fixed in
methanol/acetic acid (3:1, v/v), and spread on slides according
to standard procedures. They were stained with Giemsa, and
the numbers of cells at the different stages of mitosis counted,
using light microscopy. Examples of the different categories
of mitotic cells used for this classification are shown in Fig. 1.
Certain specimens were prepared for, and analysed by,
scanning electron microscopy, as described by Sumner (1991).
Results
Inhibition of chromosome segregation
The primary purpose of the experiments described in
this paper was to determine whether topoisomerase II
inhibitors would prevent normal segregation of human
chromosomes. This question was tackled in two ways.
In cultures lacking colchicine, it was expected that, if
segregation were blocked, there should be an accumulation of metaphases. On the other hand, prolonged (5
h) culture in the presence of colchicine normally causes
a substantial proportion of the metaphases to display
chromosomes with divergent chromatids (Fig. le),
taken to be an indication of segregation of the
chromosome arms. Although mitotic cells were classified into one of seven stages (Fig. 1), the important
distinction was taken to be that between chromosomes
with unseparated or parallel chromatids (Fig. la-d),
and chromosomes with divergent, or completely separated (anaphase and telophase), chromatids (Fig. le-g).
It should be noted that chromosomes with divergent
chromatids were largely confined to cultures treated
with colchicine, and anaphases were normally found
only in cultures not treated with colchicine. Telophases
were very rarely found, even in cultures not treated
with colchicine. This may well have been because
fixation in methanol/acetic acid followed by spreading
on slides disrupts the cell membrane and dissolves the
cytoplasm, and the two daughter telophase nuclei could
therefore become widely separated from each other.
The proportions of mitotic cells in the different stages
of mitosis are shown in Figs 2,3 and 5-10. It must be
emphasised that these histograms give no indication of
the numbers of mitotic cells, and although no attempt
was made to measure the mitotic index, it was clear that
all treatments reduced the number of mitotic cells to a
substantial degree. The results obtained with each
inhibitor will now be described in turn.
Camptothecin (Fig. 2)
Wben applied to lymphocyte cultures at concentrations
between 0.5 fjM and 15 /JM in the absence of colchicine,
camptothecin reduces the number of mitotic cells to a
very low level. Variations in the proportions of cells at
different mitotic stages shown in the histogram are
therefore unlikely to be of any significance. In the
presence of colchicine, camptothecin appears to reduce
the number of mitotic cells somewhat, but has no effect
on the proportions of cells at the different stages of
mitosis.
Nalidixic acid (Fig. 3)
Nalidixic acid, in the presence or absence of colchicine,
causes a moderate reduction in the number of mitotic
cells. In the absence of colchicine, the proportions at
the different mitotic stages do not appear to be changed
significantly. In the presence of colchicine, however,
some remarkable changes take place. Metaphases with
divergent chromatids, which comprise 37% of the
control, are virtually eliminated by either 2.15 mM or
4.3 mM nalidixic acid. There is a substantial increase in
the proportion of prophase cells, but most interestingly,
most of the metaphase cells, which in the controls have
parallel chromatids (Fig. 4b), have what are here
termed fused chromatids after nalidixic acid treatment
(Fig. 4a). Although the chromosomes have the typical
shape of metaphase chromosomes, there is no sign of a
split between the chromatids. This appearance was only
found as a very rare variant on control slides, or indeed
after treatment with any other compound.
Chromosome segregation
107
J
*
g
••*• •T«
Fig. 1. Light micrographs defining the classification of the
different stages of mitosis used in this study, (a) Early
prophase: long tangled chromosomal threads occupying a
circular, nucleus-shaped area; (b) late prophase: chromosomes
still elongated, but individually recognisable, and occupying a
more irregular area; (c) metaphase with fused chromatids:
contracted chromosomes with no visible space between
chromatids; (d) metaphase with parallel chromatids;
(e) metaphase with divergent chromatids in some or all
chromosomes; (f) anaphase: separation of centromeres of many
or all chromosomes, with or without separation of the daughter
groups of chromosomes; (g) telophase: complete separation and
rounding up of daughter groups of chromosomes. Bars, 10 j<m.
108
A. T. Sumner
Control
OMSO
0.575(1 M
2.87pM
U.4(iM
Camptotnecin concentration
Control
DMSO
O.S75MM
2 3 7 (JM
U.<MM
Camptothedn concentration
S3 Prophases
H Early metaphases
•
Late meta-telophases
25 k
2.15 mU
4.3 mU
Nahfodc add concentration
Contra
2.15 mU
Unll
NaBdfcdc a d d concentration
Fig. 2. Histograms showing
the proportions of mitoses
in prophase, early
metaphase (with fused or
parallel chromatids), and
late meta-telophase
(metaphases with divergent
chromatids, anaphases, and
telophases), after treatment
of cultures with
camptothecin at the
concentrations indicated, in
the presence (a) or absence
(b) of colchicine.
Fig. 3. Histograms showing
the proportions of mitoses
at different stages after
treatment of cultures with
nalidixic acid, in the
presence (a) or absence (b)
of colchicine. For further
explanation, see the legend
to Fig. 2.
m Prophases
M Earty metaphases
i—i Late meta-tetophases
Fig. 4. Scanning electron micrographs of metaphase chromosomes: (a) from a culture treated with nalidixic acid, showing
the lack of any splitting between the chromatids ("fused chromatids"); (b) from a control culture, grown without nalidixic
acid, showing normal separation of the chromatids. Bars, 10 ixm.
Chromosome segregation
Control
1nM
5jiM
10|iM
20 nM
Control
1 ^M
S |iM
W pM
Ethidium concentration
Ethidium concentration
S3 Prophases
H Early metaphases
• Late meta-telophases
Ethidium (Fig. 5)
Treatment of cultures with ethidium does not produce
any significant reduction in the total number of mitotic
cells, but does increase the proportion of mitotic cells
classified as prophase, in both the presence and absence
of colchicine. In the absence of colchicine, there is a
reduction in the proportion of metaphases with parallel
chromatids. In the presence of colchicine, there is a
steady decrease in the proportion of the later stages of
mitosis, predominantly metaphases with divergent
chromatids, as the concentration of ethidium is
increased from zero to 20
Ellipticine (Fig. 6)
The results with ellipticine are generally similar to those
obtained with ethidium, except that there is a substantial reduction in the number of mitotic cells as the
ellipticine concentration is increased (40 and 100 /xM
ellipticine resulted in virtually complete suppression of
mitosis).
Daunomycin (Fig. 7)
At concentrations between 4 and 100 nM, daunomycin
has little effect on the number of mitotic cells, or on the
proportions at different stages of mitosis. However, at
Control
4pM
20|*l
EHIpBclne concentration
BS Prophases
M Early metaphases
i—i Late meta-teloptiases
Control
20 i
109
Fig. 5. Histograms showing
the proportions of mitoses
at different stages after
treatment of cultures with
ethidium bromide, in the
presence (a) or absence (b)
of colchicine. For further
explanation, see the legend
to Fig. 2.
concentrations of 0.4 fjM and above, mitosis is
suppressed almost completely.
Mitoxantrone (Fig. 8)
Treatment of cultures with mitoxantrone reduces the
number of mitotic cells, and indeed largely suppresses
mitosis in the absence of colchicine at the highest
concentrations tested (1 and 2 /JM). In the absence of
colchicine, there is no significant alteration in the
proportions of the different mitotic stages. In the
presence of colchicine there is a suppression of
prophase with increasing mitoxantrone concentration,
and a slight increase in the proportion of metaphases
with divergent chromatids.
Amsacrine (Fig. 9)
Amsacrine, at concentrations from 1-5 JJM, almost
completely suppresses mitosis in the absence of colchicine. The same concentrations of amsacrine, in the
presence of colchicine, cause a small reduction in the
number of mitoses, and completely suppress prophases.
No substantial changes in the proportions of later stages
were found.
Etoposide (Fig. 10)
Etoposide, at concentrations from 1-20 /iM, almost
4|AI
Ellipticine concentration
20j*l
Fig. 6. Histograms showing the
proportions of mitoses at
different stages after treatment
of cultures with ellipticine, in
the presence (a) or absence (b)
of colchicine. For further
explanation, see the legend to
Fig. 2.
110
A. T. Sumner
%
(a)
100
Control
4nM
20nM
Control
100nU
4nM
20nM
100nM
Daunomydn concentration
Daunomydn concentration
m Prophases
H Earty metaphases
CD Late meta-tetophaaes
25 -
Control
02\M
0.6 (iM
Control
Mltoxantrone concentration
Effl Prophases
I B Early metaphases
CD Late meta-telophases
Control
1(lM
0.2 jiU
o.S^iM
1^iM
Mitoxantrone concentration
2MM
Fig. 7. Histograms showing
the proportions of mitoses
at different stages after
treatment of cultures with
daunomycin, in the
presence (a) or absence (b)
of colchicine. For further
explanation, see the legend
to Fig. 2.
Fig. 8. Histograms showing
the proportions of mitoses
at different stages after
treatment of cultures with
mitoxantrone, in the
presence (a) or absence (b)
of colchicine. For further
explanation, see the legend
to Fig. 2.
20J1U
2 |iM
Amsacrlne concentration
Etoposide concentration
ra Prophases
M Early metaphases
CD Late meta-telophases
ra Prophases
M Early metaphases
CD Late meta-telophases
Fig. 9. Histogram showing the proportions of mitoses at
different stages after treatment of cultures with amsacrine
in the presence of colchicine. For further explanation, see
the legend to Fig. 2.
Fig. 10. Histogram showing the proportions of mitoses at
different stages after treatment of cultures with etoposide
in the presence of colchicine. For further explanation, see
the legend to Fig. 2.
completely suppresses mitosis in the absence of colchicine. In the presence of colchicine, there was some
reduction in the total number of mitoses, and at the
highest concentration of etoposide (20 fjM) complete
elimination of prophases. The proportion of metaphases with divergent chromatids increased slightly.
Inhibition of chromosome condensation
Although the primary purpose of this study was to
investigate the effect of topoisomerase II inhibitors on
chromosome segregation, incidental observations were
made on various other points. Most of the inhibitors
Chromosome segregation
111
Fig. 11. Highly extended metaphase chromosomes seen after treatment with (a) ethidium, (b) amsacrine. Bars, 10 j/m.
tested appeared to inhibit chromosome condensation,
reflected in an increase in the number of mitoses with
long chromosomes showing a chromomeric structure.
This was particularly clear with amsacrine and daunomycin (Fig. 11), but was not observed with camptothecin or etoposide, and the effect was small with nalidixic
acid. In the case of ethidium, and perhaps also
ellipticine, this effect is shown as an increase in the
proportion of prophase cells.
Chromosome damage
Several inhibitors caused chromosome damage (Fig.
12). This was particularly noticeable with amsacrine
and etopside, but was also induced by mitoxantrone,
daunomycin, and the topoisomerase I inhibitor camptothecin. No chromosome damage was noticed in the
experiments with ethidium, ellipticine or nalidixic acid.
Discussion
The experiments described in this paper were designed
to test the effects of topoisomerase inhibitors on
mammalian chromosome segregation in two ways.
Firstly, treatment of cultures with inhibitors in the
absence of colchicine might be expected to lead to an
accumulation of metaphase cells: the spindle might
pull, but fail to separate, the chromosomes (cf. Uemura
and Yanagida, 1986). Secondly, in the presence of
colchicine, metaphases will accumulate in the absence
of the spindle, which has been destroyed by the
colchicine; however, under these conditions a characteristic divergence of the chromatids is seen, which is
taken to be a manifestation of the segregation of the
chromatids (but not of the centromere). The 5 h
treatment with colchicine, and with inhibitors of
topoisomerases, was chosen to maximise the effects just
described. However, since the G2 phase of human
lymphocyte cultures is in the region of 3 h (Kikuchi and
Sandberg, 1964; Younkin, 1975), cells blocked in
metaphase after 5 h treatment are likely to be derived
mainly from cells in G2 at the start of treatment, with a
proportion of S phase cells. Although it has been
assumed that these cells form a uniform population in
their response to inhibitors, there is no direct evidence
for this, and indeed the procedure could conceivably
select a population of mitotic cells that shows reduced
susceptibility to topoisomerase inhibitors. Even if this
were so, however, the principle behind the present
experiments would not be invalidated, although higher
concentrations of reagents might be needed to produce
an observable effect. In practice, there is a limit to the
extent to which concentrations of inhibitors can be
increased, as the highest concentrations tested often
caused almost complete elimination of mitotic cells
(results not shown), and in many cases produced
extensive chromosome damage before this limit was
reached.
Four main effects of topoisomerase inhibitors on
mitosis can be recognised in the present experiments
(Table 2). The first is inhibition of entry into mitosis,
seen as a loss of prophases, and a reduction in the total
112
A. T. Sumner
Fig. 12. Metaphases showing damaged chromosomes after treatment with (a) mitoxantrone, (b) amsacrine. Bars, 10
number of mitoses. This was a common, but not
universal effect, which has already been described for
many inhibitors (Grieder et al. 1977; Rowley and Kort,
1989; del Bino et al. 1991; Zucker and Elstein, 1991).
The second effect is inhibition of chromosome condensation. This effect has also been reported previously
(Wright and Schatten, 1990; Charron and Hancock,
1990), and is expected on theoretical grounds, although
measurements will be required to assess the degree of
inhibition of condensation caused by the different
compounds. It should be noted that the topoisomerase I
inhibitor, camptothecin, does not appear to inhibit
chromosome condensation. A third effect induced by
several, but not all, the compounds tested is the
induction of chromosome damage; again there have
been previous reports of this phenomenon (Huang et
al. 1973; Kao-Shan et al. 1984; Pommier et al. 1988;
Charron and Hancock, 1991).
The main effect sought in this study, the inhibition of
chromosome segregation, has been the most difficult to
establish. Evidence that it is occurring comes from a
reduction in the proportion of metaphases with divergent chromatids, and a reduction in the proportion of
anaphases. These effects are, however, only seen with
certain compounds. Most interesting, however, is the
observation of metaphase chromosomes with fused
chromatids after treatment with nalidixic acid. All these
observations suggest that chromosome segregation
might be inhibited. On the other hand, there is no
evidence that any of the topoisomerase II inhibitors
tested caused an accumulation of cells in metaphase,
and in fact most caused some reduction, often very
Table 2. Summary of effects of topoisomerase inhibitors on mitotic chromosomes
Camptothecin
Nalidixic
acid
Inhibition of entry into mitosis
Loss of prophases
Reduction in number of mitoses
+
+
_
—
Inhibition of chromosome
condensation
Increase in prophases
Elongated chromosomes
—
—
+
Inhibitor
Chromosome breakage and
rearrangement
Inhibition of segregation
Fused chromatids
Accumulation of metaphases
Reduction in metaphases with
divergent chromatids
Reduction of anaphases
—
_
- +
(+)
Ethidium
bromide
Ellipticine
Daunomycin
Mitoxantrone
(+)
+
_
—
4,
+
+
(+)
(+)
+
(+)
+
—
++
—
—
—
_
++
_
_
(+)
_
—
—
_
+
+
+
—
Amsacrine
Etoposide
+
+
•+
—
+
—
++
—
—
+
+
+
—
w
_
—
^-
_
_
+
—
—
(+)
_
—
—
- , No discernable effect; ( + ) , possible slight effect; + , moderate effect; + + , strong effect.
+
+
+
_
_
+
Chromosome segregation
substantial, in the number of mitoses. This is most
easily interpreted as a block in G2, as already described
by several authors (Grieder et al. 1977; Rowley and
Kort, 1989; del Bino et al. 1991; Zucker and Elstein,
1991), but with little or no effect on cells already in
mitosis, which apparently pass through mitosis more or
less normally. The results described in this paper do
not, therefore, support the hypothesis that topoisomerase II is involved directly in the segregation of
mammalian chromosomes at mitosis. This is in agreement with the results of Rowley and Kort (1989), who,
however, used somewhat non-specific reagents. Wright
and Schatten (1990) also failed to find an effect of
teniposide on mitotic segregation, although this compound inhibited meiotic segregation, in the clam
Spisula.
The assay used in this study for assessing inhibition of
segregation is similar to that described by Miller and
Adler (1989), although the method used here was
devized independently. Miller and Adler used their
method to study spindle poisons, and it might be argued
that, in the present study, when microtubules were not
destroyed by the use of colchicine, the spindle could still
function to pull the daughter chromosomes apart at
anaphase, breaking any DNA connections between
them mechanically. Such a process would be expected
to lead to damaged chromosome fragments, which
might be visible at anaphase, and appear as micronuclei
at the next interphase. The latter point has not been
studied yet, but no evidence was found in any of the
experiments described here for faulty segregation at
anaphase. However, Wilson et al. (1984) reported the
induction of micronuclei by several topoisomerase II
inhibitors.
Downes et al. (1991) have recently reported that
inhibitors of topoisomerase II do, in fact, interfere with
chromosome segregation in mammalian cells, and that
high concentrations of etoposide or amsacrine prevent
anaphase separation of chromatids. There are two
possible explanations of the difference between the
results of Downes et al. and those reported in this
paper. Firstly, they used much higher concentrations of
topoisomerase II inhibitors (20-60 /xM etoposide,
instead of 1-20 ^M used here, and 40 fiM amsacrine,
instead of 1-5 ^M used here), although treatment was
for a much shorter time. It might simply be that the
concentrations used in my experiments were inadequate to produce an effect on segregation. However,
the concentrations used in the present experiments
were comparable with those reported in the literature,
and it was not practicable to use higher concentrations
as these completely eliminated mitotic cells. On the
other hand, even 1 fjM amsacrine or etoposide induce
substantial chromosome damage if used before mitosis.
In fact, Silvestrini et al. (1970) showed that treatment of
cultures with high concentrations of Adriamycin caused
the accumulation of metaphases showing extensive
chromosomal damage, while Charron and Hancock
(1991) have reported defective segregation after treatment with teniposide, due to the inability of rearranged
chromosomes to segregate normally. Indeed, Downes
113
et al. (1991) found a similar phenomenon themselves
using a low concentration of etoposide. However,
Downes et al. (1991) in their critical experiments
applied the topoisomerase inhibitors only during mitosis, thus eliminating the problem of chromosome
damage. They point out, nevertheless, that effects on
segregation are only perceptible at concentrations of
etoposide that are lethal after a period comparable with
the duration of mitosis, and the possibility must be
borne in mind that their results could be affected by the
unhealthy condition of the cells rather than being
entirely due to inhibition of topoisomerase II.
In the present experiments, the effects of several
topoisomerase II inhibitors have been compared. It was
felt that, since most of them have side-effects other than
inhibition of topoisomerase II, it would be necessary to
look for effects common to several compounds to be
certain that an effect on topoisomerase II was being
observed. In addition, different compounds act on
topoisomerase II in different ways. For example,
Adriamycin, a compound closely related to daunomycin, also inhibits cytochrome c oxidase in mitochondria
(Goormaghtigh et al. 1982). Mitoxantrone appears to
inhibit mainly the 180 kDa form of human topoisomerase II, which does not vary significantly in amount
during the cell cycle, but is largely without effect on the
170 kDa form (Harker et al. 1991). The latter does vary
in amount during the cell cycle, and is therefore more
likely to be the form responsible for mitotic events. In
addition mitoxantrone inhibits polymerisation of microtubules (Ho et al. 1991), and therefore might be
expected to cause metaphase arrest independently of
any action on topoisomerase II. Nalidixic acid, as well
as having some inhibitory effect on topoisomerase II, is
in fact primarily an ATPase inhibitor (Gallagher et al.
1986). Since it is the only compound tested that
promotes chromatid fusion (strictly speaking, the
inhibition of chromatid separation) this phenomenon,
although potentially interesting in its own right, is
unlikely to be the result of inhibition of topoisomerase
II. Even compounds such as etoposide and VM-26
(teniposide), which are regarded as being among the
most specific topoisomerase II inhibitors, also inhibit
phosphorylation of histones, a process associated with
chromosome condensation, although it is not clear
whether this is a direct effect (Lock and Ross, 1990;
Roberge et al. 1990). Amsacrine, another topoisomerase II inhibitor regarded as being highly specific, also
inhibits aldehyde oxidase (Gormley et al. 1983). It
seems, therefore, that it is not possible to rely on one
single topoisomerase II inhibitor to investigate the
importance of topoisomerase II in cellular processes.
In conclusion, a variety of compounds reported to
inhibit topoisomerase II induce four effects on human
mitotic cells: inhibition of entry into mitosis; chromosome breakage and rearrangement; inhibition of
chromosome condensation; and inhibition of chromosome segregation. None of the compounds tested
produced all these effects; this may have been due to
differences in their actions on topoisomerase II, or the
use of inappropriate concentrations of the compounds.
114
A. T. Sumner
There is also the possibility that some inhibitors were
also acting on substrates other than topoisomerase II.
Evidence that the inhibitors tested were inhibiting
segregation was least satisfactory, since although there
was some evidence that segregation was inhibited in
cells treated with colchicine, it appeared that in the
absence of colchicine cells could pass through mitosis
without difficulty. In this context it is worth noting that
Koshland and Hartwell (1987) found that the daughter
DNA molecules in a yeast minichromosome were
decatenated before anaphase, possibly shortly after S
phase. If this finding could be extrapolated to mammalian chromosomes, there would be no reason to
expect that topoisomerase II inhibitors would have any
effect on the anaphase separation of chromosomes. The
proposal by Sumner (1991) that topoisomerase II acts
on centromeric satellite DNA at the start of anaphase to
produce chromosome segregation would not, therefore, be supported; instead it would seem more likely
that initiation of segregation is due to the action of a
"chromatid separase" (Ostergren and Andersson,
1973) on the proteins of centromeric heterochromatin.
The results reported here do not, therefore, agree
completely with those described for yeasts (di Nardo et
al. 1984; Uemura and Yanagida, 1986; Uemura et al.
1987; Holm et al. 1989) and more recently for
mammalian cells (Downes et al. 1991), in which
segregation appears to be defective if topoisomerase II
is inhibited. It should be noted, however, that the
studies just cited did not investigate individual chromosomes, and the possibility that abnormal segregation
might be a consequence of the induction of chromosomal damage must be borne in mind.
I thank Sheila McBeath, George Spowart and Eric
Thomson for setting up blood lymphocyte cultures, and
Cyanamid of Great Britain Ltd, Warner-Lambert/ParkeDavis (Mr J R Britten) and Bristol-Myers Squibb (Dr P
Eggleton) for gifts of mitoxantrone, amsacrine and etoposide,
respectively. Lesley Campbell typed the manuscript, and
Sandy Bruce prepared the photographic illustrations with
their customary skill, and I must also thank Paul Perry for his
advice and help in preparing the histograms by computer.
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{Received 21 February 1992 - Accepted, in revised form,
23 June 1992)