Journal of Cell Science 105, 563-569 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
563
Topoisomerase II inhibition prevents anaphase chromatid segregation in
mammalian cells independently of the generation of DNA strand breaks
Duncan J. Clarke, Robert T. Johnson and C. Stephen Downes*
Cancer Research Campaign Mammalian Cell DNA Repair Research Group, Department of Zoology, University of
Cambridge, Downing Street, Cambridge CB2 3EJ, UK
*Author for correspondence
SUMMARY
Yeast temperature-sensitive mutants of DNA topoisomerase II are incapable of chromosome condensation
and anaphase chromatid segregation. In mammalian
cells, topoisomerase II inhibitors such as etoposide (VP16-123) have similar effects. Unfortunately, conclusions
drawn from work with mammalian cells have been limited by the fact that the standard inhibitors of topoisomerase II also generate DNA strand breaks, which when
produced by other agents (e.g. ionizing radiation) are
known to affect progression into and through mitosis.
Here we show that the anti-tumour agent ICRF-193,
recently identified as a topoisomerase II inhibitor oper-
INTRODUCTION
During replication the double-stranded DNA molecules of
sister chromatids inevitably become concatenated. These
concatenations must be separated in preparation for
anaphase chromatid segregation; Cook (1991) has recently
reviewed the structural complexities of this decatenation.
The only known eukaryotic enzyme capable of decatenating double-stranded DNA is DNA topoisomerase II (EC
5.99.1.3); see Osheroff et al. (1991) for a review of its
mechanism.
In budding and fission yeast, temperature-sensitive Top2
mutants have demonstrated the essential role of this enzyme
in decatenating sister chromatids before anaphase commences (di Nardo et al., 1984; Holm et al., 1989; Uemura
and Yanagida, 1986; Uemura et al., 1987). When grown at
the non-permissive temperature, Top2 mutants of
Schizosaccharomyces pombe are unable to complete the
final stages of chromosome condensation (Uemura et al.,
1987). If Top2 cells are shifted to the non-permissive temperature after condensation is complete, the cells attempt
anaphase but fail to separate their chromatids. The cells do,
however, continue with septation, though full cytokinesis
is prevented by chromatin bridges joining the two daughters (Uemura and Yanagida, 1986; Uemura et al., 1987).
When cytokinesis is blocked, as in the Top2-cdc11 double
ating by a non-standard mechanism, generates neither
covalent complexes between topoisomerase II and DNA,
nor adjacent DNA strand breaks, in mitotic HeLa. However, the drug does prevent anaphase segregation in
HeLa and PtK2 cells, with effects similar to those of
etoposide. We therefore conclude that topoisomerase II
function is required for anaphase chromosome segregation in mammalian cells, as it is in yeast.
Key words: topoisomerase II, ICRF-193, anaphase, chromatid
segregation
mutant, the cells re-enter interphase with a single nucleus
(Uemura and Yanagida, 1986). Yeast topoisomerase II is
therefore required for chromosome condensation and chromatid segregation, but is not needed for progression through
the later stages of mitosis.
In higher eukaryotes, definitive proof of mitotic topoisomerase II functions is not yet possible, since topoisomerase
II mutants have not been isolated. As a substitute, specific
topoisomerase II inhibitors such as the non-intercalating
epipodophyllotoxins, etoposide (VP-16-123) and teniposide
(VM-26), or intercalating inhibitors such as m-AMSA, have
been employed. Treating mammalian cells with such
inhibitors produces a G2 delay (Kalwinsky et al., 1983;
Tobey et al., 1990). Events in cell division are also affected
in a manner parallel to yeast Top2 mutations. Wright and
Shatten (1990) found that meiotic chromosome condensation and segregation could be inhibited with teniposide in
Spisula solidissima (surf clam) oocytes. Similarly, Downes
et al. (1991) showed that etoposide inhibits mitotic segregation in mammalian cells (HeLa and PtK2), but like the
aforementioned yeast Top2 mutations, does not prevent
cytokinesis. More recently, Shamu and Murray (1992) elegantly showed that etoposide inhibits chromatid separation
in an in vitro system derived from sperm nuclei in a Xeno pus egg extract.
Data obtained with topoisomerase II inhibitors have not
564
D. J. Clarke, R. T. Johnson and C. S. Downes
been totally unequivocal, however. Sumner (1992) has
shown that low doses of a variety of topoisomerase II
inhibitors do not prevent the separation of chromatids that
occurs in human lymphocytes arrested for a prolonged
period in mitosis with Colcemid. In this system, it is not
possible to employ high doses of topoisomerase inhibitors,
such as have been found to be necessary to prevent
anaphase disjunction in other cells and cell-free systems
(Downes et al., 1991; Shamu and Murray, 1992), since such
concentrations are toxic over prolonged periods; the low
doses employed may not be adequate to inhibit topoisomerase II fully in the lymphocyte system. Alternatively,
Sumner suggests that effects seen in other systems may be
a consequence of DNA damage induced by high doses of
the inhibitors used.
Such damage is an inevitable consequence of the mode
of action of all the topoisomerase II inhibitors used by previous investigators (etoposide, teniposide, m-AMSA). All
these act by arresting topoisomerase II at the transition state,
when it is covalently linked to DNA at sites of DNA
double-strand breaks, thus generating what are known as
cleavable complexes. This prevents the topoisomerase IImediated re-ligation reaction, which is the last step of
decatenation; and ultimately double-stranded DNA breaks
result. Such DNA damage is known to induce G2 delay,
and so the G2 delay seen in response to topoisomerase II
inhibitors could be explained by a cell cycle arrest in
response to DNA damage (Lock and Ross, 1990). More
speculatively, the effects of topoisomerase II inhibitors at
anaphase could be analogous to the disturbed segregation
that has long been known to occur in mitotic mammalian
cells after X-irradiation (Perk, 1941).
However, a different class of topoisomerase II inhibitors
has now become available: the bisdioxopiperazines, a class
of antitumor agents which were originally developed in the
1970s in the ICRF laboratories, Lincoln’s Inn Fields,
London. Since many antineoplastic drugs have chelating
properties, bisdioxopiperazines were designed as derivatives of the calcium chelator EDTA, with increased ability
to penetrate cell membranes. ICRF-193 is the mesodimethyl derivative of ICRF-159, which was itself produced
from ICRF-154, the bis-cyclic imide of EDTA (Creighton
et al., 1969). Early attempts to characterise the cytotoxicity of these agents showed that ICRF-159 prevented complete mitosis, resulting in the formation of tetraploid cells.
Creighton and Birnie (1970) found that ICRF-159 reduced
the gross rate of DNA synthesis in cultured cells; and
Creighton (1979) showed that progression through S-phase
and mitosis is delayed. ICRF-159 also delays BHK-21S
cells in G2 (White and Creighton, 1976; White and
Creighton, 1977).
The mechanism of action of the ICRF bisdioxopiperazines has recently become clearer. Tanabe et al. (1991)
showed that ICRF-193 inhibits calf thymus topoisomerase
II, as measured by decatenation of Crithidia fasciculata
kinetochore DNA, without effect on topoisomerase I or
DNA polymerases α and β. The drug inhibited topoisomerase II without generating topoisomerase II-DNA cleavable complexes, and suppressed the formation of etoposideinduced cleavable complexes. Ishimi et al. (1992) also
demonstrated total inhibition of topoisomerase II-mediated
decatenation of replicated P4 phage DNA (in HeLa extract)
by 12.5 µM ICRF-193. Ishida et al. (1991) found that in
RPMI 8402 human leukaemic cells, ICRF-154 does not
form cleavable complexes but does cause G2 delay. They
also observed prophase blocked cells with very tangled
chromatin after treating asynchronous cells with 10 µM
ICRF-193 for 15 hours.
We have investigated the action of ICRF-193 on mitotic
events in HeLa and PtK2 cells, arguing that if topoisomerase
II is required for mitotic decatenation, ICRF-193 should
have effects similar to those of etoposide (Downes et al.,
1991).
MATERIALS AND METHODS
Chemicals and cells
ICRF-193 stock, a generous gift from Dr Andrew Creighton, was
dissolved at 2 mg/ml in DMSO. Etoposide was dissolved as a 34
mM stock in DMSO. The membrane-permeant Ca2+ chelator
BAPTA-AM (bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid,
acetoxymethyl ester) was dissolved at 20 mM in DMSO. All
stocks were stored at −20°C. In each experiment, drug-free controls containing DMSO only showed no effects. [Methyl3H]thymidine (45.5 Ci/mmol) was obtained from Amersham International.
HeLa (human adenocarcinoma) and PtK2 cells (Potorus tri dactylis, rat kangaroo) were grown as monolayers in Eagle’s minimal essential culture medium supplemented with 10% foetal calf
serum, penicillin and streptomycin at 37°C, in an atmosphere of
5% CO 2.
Mitotic synchrony
Logarithmically growing HeLa were incubated with 2 mM thymidine for 24 h to arrest cells in S-phase. Thymidine-containing
medium was removed and cells were allowed to continue through
S-phase for 6 hours, after which a 9 h automatic nitrous oxide
treatment (80 psi) was employed to achieve a mitotic block (Rao,
1968; Downes et al., 1987). Mitotic populations were isolated by
shake-off and mitotic purity analysed after cytocentrifugation. For
the clonal survival and cleavable complex assay, populations with
over 97% mitotic cells were used.
Clonal survival assay
Mitotic HeLa (isolated as described above) were treated for 1 h
with ICRF-193, or DMSO only for control, washed thoroughly
with warm PBS, then incubated with fresh medium for 7 days.
Each dose was tested at least in triplicate.
Cleavable complex assay
Logarithmically growing HeLa were prelabelled for 48 h with 0.05
µCi/ml [3H]thymidine, then arrested in mitosis as above. Samples,
consisting of 2×105 mitotic cells in medium, were incubated for
15 min with ICRF-193 or etoposide; or for 15 min with ICRF193 then with added etoposide for a further 15 min (DMSO concentration never >0.1%). Cells were pelleted by centrifugation,
and medium removed, then cleavable complexes of inhibited
topoisomerase II and DNA were prepared by a modification
(Squires et al., 1991) of the method of Liu et al. (1983). Cells
were lysed in alkaline sucrose solution (5% w/v sucrose, 10 mM
Na2EDTA, 0.1 M NaOH, 0.15 M NaCl) for 25 min on ice. Following neutralisation with 0.15 M K2HPO4, lysates were sonicated to reduce the size of the DNA. DNA cross-linked to protein was precipitated with 100 mM KCl, 1% SDS and 0.5 mg
Topoisomerase II function in mitosis
565
herring sperm DNA. The samples were vortexed, incubated on ice
for 10 min, then the cross-linked precipitates were isolated as
described by Liu et al. (1983). Radioactive counts in the precipitated samples were expressed as percentages of the total radioactivity in aliquots taken from each lysate before potassium/SDS
precipitation.
Passage through mitosis
Suspensions of mitotic HeLa were placed in poly-L-lysine-coated
wells of Flow multiwell immunoassay slides (approximately 30
µl/well) in normal medium or medium containing ICRF-193 or
BAPTA-AM (DMSO never >0.5%). Slides were incubated under
normal conditions for 10-90 min, then fixed in −20°C methanol
for 4 min. After being air dried, cells were stained with toluidine
blue and mounted with DPX (Analar); or for laser confocal
microscopy, cells were stained with propidium iodide (50 mg/ml
with 50 mg/ml DNase-free ribonuclease, 10 min) and mounted
with P-phenylamine diamine (1 mg/ml in 90% glycerol/10%
PBS). Photographs were taken on Kodak Technical Pan film with
a Nikon FE2 camera. Passage through anaphase/telophase was
examined in samples of 200-400 cells for each drug concentration, accumulating data from three experiments.
PtK2 time-lapse study
A heated (37°C) and CO2-equilibrated (5%) chamber surrounding
a Nikon Diaphot inverted microscope was used to study living
PtK2 cells during cell division. The DMSO concentration never
exceeded 0.1%. Cells were filmed with a Hitachi Denshi KPC500E/K video camera and a Sony VO-9850P videocassette
recorder. Still photographs were taken at intervals on Ilford pan
F film.
RESULTS
Lethality of ICRF-193 in mitosis
Fig. 1 shows clonal survival for mitotic HeLa, treated with
ICRF-193 for 1 hour (beginning immediately after the cells
were released from a nitrous oxide metaphase block), relative to control cells treated with medium containing
DMSO only (DMSO never >0.1%). At higher concentrations the potency of ICRF-193 levels off. HeLa are a particularly aneuploid cell line consisting of near-triploid and
near-tetraploid populations. The lethality of ICRF-193 may
only reflect killing of the cells with fewer chromosomes,
since these cells should be more vulnerable to the effects
of nondisjunction. Alternatively, mitotic cells that respond
to ICRF-193 by generating only one post-mitotic daughter
may survive at high doses of ICFR-193. It is also possible
that the nitrous oxide synchrony affects a sub-population of
cells, rendering them especially sensitive to topoisomerase
II inhibition. We do not believe that the ICRF-193 resistant population corresponds to cells that had begun
anaphase early, since HeLa cells released from a nitrous
oxide block (which disorganises microtubules) require
about 30 minutes to reform their metaphase plates and begin
anaphase, while the ICRF-193 was added immediately after
release.
Effects of ICRF-193 on cleavable complex
formation
The generation of topoisomerase II-DNA cleavable complexes by etoposide and ICRF-193 has been investigated in
Fig. 1. Clonal survival of mitotic synchronised HeLa cells treated
with ICRF-193 for 1 hour. Error bars show standard error.
mitotic HeLa (Fig. 2a). In our assay we find that a 15 minutes incubation with 1-3 µM etoposide traps around 40%
of the [3H]thymidine-labelled DNA. Under the same conditions ICRF-193 (up to 7 µM) fails to increase the radioactivity in the precipitates above background levels. We conclude, therefore, that ICRF-193 does not covalently link
topoisomerase II molecules to DNA. We have also been
unable to detect DNA strand breaks in mitotic HeLa treated
with ICRF-193 (data not shown).
When a prior 15 minute incubation with ICRF-193 is followed by a 15 minute incubation with etoposide, there is a
substantial suppression of etoposide-induced cleavable
complexes (Fig. 2b). This is consistent with ICRF-193
inhibiting topoisomerase II and thus preventing etoposide
from blocking active transition states. These results are in
complete agreement with previous work in cell free systems (Tanabe et al., 1991; Ishimi et al., 1992) and with
RPMI 8402 human leukaemic cells (Ishida et al., 1991), in
which the mechanism of action of ICRF-193 has already
been more thoroughly determined.
ICRF-193, but not BAPTA-AM, affects passage of
HeLa through anaphase
To quantify the effects of ICRF-193 on chromosome segregation, mitotic HeLa were incubated for various times on
poly-L-lysine-coated multiwell slides. After fixation and
staining, the frequency of chromosomes lagging in
anaphase and of abortive anaphases (or of cytokinesis
attempted in the absence of anaphase segregation) were
scored. Examples of such aberrant anaphases are shown in
Fig. 3. To confirm that the material defined as chromatin
contained DNA, parallel slides were stained with propidium iodide for fluorescence microscopy (Fig. 3e and i). Fig.
4 collates data on anaphase disruption from three separate
experiments. Clearly, very low concentrations of the drug
(175-350 nM) are sufficient to increase the frequency of
lagging figures, and very considerable inhibition of segregation is apparent at 1.75-7 µM ICRF-193 (50-75%
abortive anaphases).
These effects of ICRF-193 parallel those already reported
for etoposide, but cannot easily be attributed to DNA
damage induced by the drug. However, ICRF-193 is a
derivative of a calcium-chelating agent, and may act on
intracellular calcium levels as well as inhibiting topoisomerase II. The role of cellular calcium ions in the control
566
D. J. Clarke, R. T. Johnson and C. S. Downes
a
b
Fig. 2. (a) Cleavable complex formation in mitotic HeLa cells
after 15 min incubation with ICRF-193 (filled squares) or
etoposide (filled circles). (b) Inhibition of etoposide-induced
cleavable complex formation in mitotic HeLa by ICRF-193; 15
min incubation with no ICRF-193 (filled circles), 1.75 µM ICRF193 (open squares), 3.5 µM ICRF-193 (open circles) or 7 µM
ICRF-193 (open triangles) followed by 15 min incubation with
ICRF-193 and added etoposide. The data presented here were
confirmed in a subsequent experiment.
of mitotic events is disputed, but there is no doubt that
changes in levels of free calcium can correlate with, though
not necessarily cause, mitotic events (Hepler, 1989; Preston
et al., 1991; Zhang et al., 1992). One might argue that
ICRF-193 opposes anaphase progression by sequestering
intracellular calcium. To test this hypothesis, we have
treated mitotic HeLa, on release from arrest at metaphase
by nitrous oxide, with the potent calcium chelator BAPTA,
in the esterified form that can pass through cell membranes.
HeLa possess adequate intracellular esterases to convert the
molecules taken up into free BAPTA, which is trapped in
the cells (Preston et al., 1991). No inhibition of anaphase
segregation by BAPTA-AM, at concentrations up to 40 µM,
was seen (data not shown). We therefore doubt that the
anaphase inhibition caused by ICRF-193 is attributable to
any calcium chelating activity it may possess.
Effects of ICRF-193 on unsynchronised, mitotic
PtK2 cells
The effects of inhibiting topoisomerase II by ICRF-193
during mitosis (without prior synchronisation) were studied
in more detail by time-lapse video analysis. For this, PtK2
cells were used, since they do not round up during mitosis
Fig. 3. Effects of ICRF-193 during mitosis in HeLa cells. Control
anaphase and telophase cells (a) anaphase/telophase laggards
typical of low dose (0.175-1.75 µM) ICRF-193 (b, c and e)
abortive anaphases at higher concentration, 1.75-7 µM, (d and i)
and formation of hyperploid cells (f, g and h). Preparations were
toluidine blue stained for light microscopy (a-d, f-h) or propidium
iodide stained for confocal microscopy (e and i). Bar, 10 µm.
and hence can be observed easily. ICRF-193 (1.4 µM) was
given to selected prophase or metaphase cells and the
effects on cycle progression monitored. Several video
sequences were recorded and still photographs were taken
of four mitotic cells treated with ICRF-193. Eight other
mitotic cells were observed during the time-lapse analysis,
which were not recorded photographically. The sequence
of still photographs in Fig. 5 shows typical responses by a
metaphase cell (cell 1, on the right) and a prophase cell
(cell 2, on the left) to ICRF-193. Cell 2 took 16 minutes to
reach metaphase; metaphase itself lasting for 18 minutes.
In the abortive anaphases which ensued, chromatid separation did not occur completely. Cell 1 began anaphase after
12 minutes and after 30 minutes a highly abnormal cytokinesis was attempted. In control cells, cytokinesis usually
follows complete chromatid separation; this clearly has not
happened in this case. Anaphase was accompanied by apoptotic blebbing typical of abnormal cell division (Johnson et
al., 1975), and during the next 10 minutes half of the cell
Topoisomerase II function in mitosis
567
sis taking approximately 30 minutes (cell 1, which was
already in metaphase when the cell was identified) and 44
minutes (cell 2). We note that no effect of ICRF-193 on
the adjacent interphase cells can be observed.
DISCUSSION
Fig. 4. Bar chart showing the proportion of normal HeLa
anaphase/telophase cells (unshaded), anaphase/telophase laggards
(stippled) and abortive anaphases (black) with various doses of
ICRF-193; mean of three experiments.
was destroyed. The chromatin of the intact daughter cell
began to decondense after 45 minutes. Cell 2 also undergoes an abnormal anaphase, although in this case the chromatin appears to have separated into several portions. In
the PtK 2 time-lapse studies of Downes et al. (1991) control
cells took an average of 27 minutes to progress from early
metaphase to the onset of cytokinesis; cells treated with 60
µM etoposide during prophase took an average of 41 minutes. These data are comparable with the results we have
obtained using 1.4 µM ICRF-193: metaphase to cytokine-
This work establishes that inhibiting mitotic topoisomerase
II activity in mammalian cells with ICRF-193 prevents
anaphase chromatid segregation, without inflicting measurable damage on DNA. This effect is the same as that previously reported for agents such as etoposide, which binds
to topoisomerase II and arrests its action after it has created DNA strand breaks; or m-AMSA, which intercalates
into DNA and prevents the resealing of DNA strand breaks
formed by topoisomerase II (Downes et al., 1991). The
common element in the action of all these agents is topoisomerase II inhibition. The effects of ICRF-193 show that
topoisomerase II activity is required for anaphase segregation in mammalian cells, as it is in yeast, in conditions
where interpretation is not complicated by side effects of
DNA damage. We cannot formally exclude the possibility
that ICRF-193 inflicts a small number of DNA lesions in
mitotic mammalian cells, too few to be distinguished from
background in our assays. However, other workers have
consistently failed to detect ICRF-193-induced DNA
damage in other systems. And with other inhibitors of topoisomerase II, which act by creating cleavable complexes and
DNA strand breaks, considerable amounts of damage can
be inflicted without much retarding mitotic progression
Fig. 5. Effects of ICRF-193 on mitotic PtK2 cells. Photographs taken at the following time points after addition of 1.4 µM ICRF-193: (a
to i) 0, 10, 12, 16, 32, 34, 38, 42 and 65 min. Arrows: cell 1, a metaphase cell; cell 2, a prophase cell. Cell 1 begins anaphase after 12 min,
and after 30 min a highly abnormal cytokinesis is attempted. Cell 2 takes 16 minutes to reach metaphase; metaphase itself lasts for 18
min. In the following abortive anaphase, chromatid separation does not occur completely. Bar, 40 µm.
568
D. J. Clarke, R. T. Johnson and C. S. Downes
(Downes et al., 1991; Shamu and Murray, 1992). This work
leaves open the question of whether the previously reported
action of ICRF bisdioxopiperazines in delaying progression
through G2 into mitosis is also a direct effect of topoisomerase II inhibition.
The action of ICRF-193 in inhibiting segregation (Figs
4-6) is consistent with previous work using etoposide. To
quantify the effects of ICRF-193 on chromosome segregation during anaphase, we have scored lagging chromosomes
and aborted anaphases. ICRF-193 is far more potent than
etoposide, significantly increasing the frequency of
anaphase laggards at 350 nM and causing over 40% of
anaphases to abort at 1.75 µM. Concentrations of 60-80 µM
or 10-30 µM etoposide would be required to inhibit topoisomerase II to this extent in HeLa cells (Downes et al.,
1991) or in the Xenopus extract system of Shamu and
Murray (1992), respectively.
The exact topoisomerase II target of ICRF-193 is not
known. In mammalian cells there are two kinds of topoisomerase II; the 170 and 180 kDa proteins, encoded by
genes on chromosomes 17 and 3, respectively. Expression
of the 170 kDa protein increases markedly in G2 and mitosis (Woessner et al., 1991) and it has been identified as an
integral component of mitotic chromosomes prepared from
primary chicken fibroblasts or from Chinese hamster cells
(Earnshaw et al., 1985; Earnshaw and Heck, 1985; Charron and Hancock, 1990). In Escherichia coli, segregation
of replicated, catenated DNA, and subsequent separation of
the circular supercoiled chromosomes at cell division, are
known to be effected by a special enzyme, topoisomerase
IV, which like the bacterial topoisomerase II is a class 2
topoisomerase capable of decatenation (Adams et al., 1992).
It would be satisfyingly symmetrical if the mammalian 170
kDa protein turned out to be analogous to the bacterial
topoisomerase IV.
The 170 kDa species is known to be inhibited preferentially by merbarone, a topoisomerase II inhibitor, which
appears to act in a similar way to ICRF-193, without generating DNA breaks (Drake et al., 1989). The relative
effects of ICRF-193 on the 170 and 180 kDa species of
topoisomerase II have not been investigated, nor is it known
whether merbarone inhibits mitosis. We would predict that
in mitotic cells the target of ICRF-193 would be the 170
kDa species, and that merbarone should likewise inhibit
anaphase segregation.
In time-lapse studies of PtK2 cells, we observe that cells
given ICRF-193 in prophase appear to complete prophase
without delay: we have not observed problems in the late
stages of chromatin condensation such as are seen in the
Top2 yeast mutants (di Nardo et al., 1984; Uemura et al.,
1987; Holm et al., 1989); and in in vitro systems (Wood
and Earnshaw, 1990; Adachi et al., 1991; Hirano and
Mitchison, 1991). Time-lapse studies using etoposide to
inhibit topoisomerase II similarly failed to provide evidence
for topoisomerase II function in prophase (Downes et al.,
1991). However, Ishimi et al. (1992) demonstrated that
ICRF-193 blocks the late, decatenatory stages of SV40
chromosome replication; and Ishida et al. (1991) observed
extremely tangled chromatin (which inhibited cycle progression through prophase) in prophase after lymphocytes
were incubated with 10 µM ICRF-193 for 15 hours. We
interpret these data as showing that decatenation is largely
completed in S-phase and G2. Short pulses of ICRF-193 or
etoposide during prophase presumably fail to perturb condensation since sufficient decatenation has already taken
place.
The time-lapse studies with PtK2 confirm the effects of
ICRF-193 in unsynchronised cells. This eliminates the possibility that the effects seen in synchronised mitotic HeLa
are due to the nitrous oxide treatment which destablises the
microtubules of the spindle apparatus during synchrony;
such destabilisation could arguably result in aberrant
anaphase segregation in some cells after the block is
released. When given in prophase or metaphase to unsynchronised PtK2 cells, ICRF-193 prevents anaphase chromosome segregation. We suppose that residual catenations
between sister chromatids, which are not enough to prevent
chromosome condensation, are present at least until
metaphase. Although the period from full metaphase until
the onset of cytokinesis is prolonged, cycle progression is
not blocked and cytokinesis continues, albeit aberrantly.
Hence, as in yeast, mammalian cells need topoisomerase II
for chromatid segregation, but neither topoisomerase II
activity nor chromatid segregation are necessary for the terminal stages of cell division. Previous work with etoposide
left this conclusion uncertain (although probable) since
etoposide-induced DNA strand breaks could conceivably
have caused aberrant cycle progression, with uncoupling of
segregation and cytokinesis.
In a very recent paper, Sumner (1992) reported that
inhibitors of topoisomerases do not block the separation of
human lymphocyte chromatids during prolonged colcemid
arrest. He correctly points out that the inhibitors of topoisomerase II previously used have a variety of non-specific
effects, including the production of DNA damage. This criticism does not apply to the topoisomerase II inhibitor ICRF193. Sumner used very low concentrations of etoposide and
other drugs in an attempt to limit the affects of DNA
damage; unfortunately these low concentrations, which had
little or no effect on chromatid segregation, may not have
adequately inhibited topoisomerase II either.
We have here confirmed that topoisomerase II action is
required for anaphase chromatid segregation in mammalian
cells. In the absence of segregation, cytokinesis is not prevented. Hence, there is apparently no mammalian ‘decatenation complete?’ checkpoint for exiting from mitosis.
We thank Charles ffrench-Constant for use of the time-lapse
equipment, Tony Mills for instruction in confocal microscopy,
Paul Smith for etoposide, Tim Cheek for BAPTA-AM, and especially Dr Andrew Creighton, Department of Rheumatology, St.
Bartholomew’s Hospital, London, for a generous gift of ICRF193 and information about its history. We are grateful to the
Cancer Research Campaign, of which R.T.J. is a Fellow, for continued support, and to the Medical Research Council for a studentship to D.J.C.
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D. J. Clarke, R. T. Johnson and C. S. Downes
(Received 9 February 1993 - Accepted 4 March 1993)