387
Journal of Cell Science 102, 387-392 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
COMMENTARY
Colcemid and the mitotic cycle
CONLY L. RIEDER*
Wadsworth Center for Labs and Research, P.O. Box 509, Albany, NY 12201-0509, USA and Department of Biomedical Sciences, State
University of New York, Albany, NY 12222, USA
and ROBERT E. PALAZZO
Marine Biological Laboratory, Woods Hole, MA 02543, USA
*Author for correspondence at Wadsworth Center for Labs and Research
Introduction
The precise segregation of replicated chromosomes to
daughter cells during mitosis depends on the formation
of a bipolar spindle composed primarily of microtubules
(MTs). Since MTs are highly dynamic structures whose
spatial organization is critical for proper spindle
function, physical and chemical agents that interfere
with MT behavior invariably disrupt mitosis. Perhaps
the most notable of these agents is colchicine, derived
from plants of the genus Colchicum, which has long
been known to be a potent inhibitor of cell division
through its effects on spindle MT assembly (reviewed
by Eigsti and Dustin, 1955; Dustin, 1978; Sluder, 1991).
Over the years the action of colchicine, and the closely
related but less-toxic compound demecolcine (Colcemid), has been mostly elucidated and other drugs (e.g.
podophyllotoxin, steganacin, vinblastine, Nocodazole)
have been discovered that interfere similarly with
mitosis through their action on MTs (e.g. see Eigsti and
Dustin, 1955; Deysson, 1968; Mareel and DeMets,
1984).
The functional basis of how colchicine and Colcemid
disrupt the spindle is now well understood (e.g. see
Taylor, 1965; Wilson et al., 1976; Dustin, 1978; Mareel
and DeMets, 1984). However, much of our knowledge
of how mitosis proceeds in the presence of these drugs
(C-mitosis; Levan, 1938) is based on cytological
examinations of fixed cells conducted prior to 1955
(summarized by Eigsti and Dustin, 1955; Dustin, 1978).
Although these pioneering studies provided fundamental data regarding the effects of colchicine/Colcemid on
spindle formation in plants and animals, and established much of the terminology still used to characterize
the process of C-mitosis, few addressed the ultimate
fate of C-mitotics in animal tissues. Moreover, those
that did failed to reach a consensus concerning the extent that colchicine/Colcemid permanently blocks cells
in mitosis, or whether these drugs inhibit the disjunction (i.e. anaphasic separation) of replicated chromosomes. Both of these issues are germane to, and have
been impacted by, recent and important findings on the
control mechanisms by which the cell monitors progress
through, and ultimately exits, mitosis (e.g. see Hartwell
and Weinert, 1989; Murray and Kirschner, 1989).
The aim of this commentary is to oultine the process
of C-mitosis in plant and animal cells with an emphasis
on new data that provide possible explanations for why
various cell types behave differently during mitosis in
the presence of drugs that disrupt MT function.
Although our focus is on colchicine/Colcemid, many of
the conclusions may be applicable to similar drugs that
disrupt mitosis through their action on MTs.
The 'mitotic block'
Over a wide range of concentrations colchicine and
Colcemid do not affect the rate at which cells enter
mitosis (reviewed by Eigsti and Dustin, 1955; Sluder,
1979). When applied well before nuclear envelope
breakdown (NEB) and in a sufficient concentration
these drugs completely inhibit the formation of spindle
MTs. As a result, during NEB the chromosomes are
released into the cytoplasm where they remain randomly dispersed throughout the prolonged period of Cmitosis (Fig. 1). It is noteworthy that the chromosome
condensation cycle (see Mazia, 1987) continues during
C-mitosis (Fig. 2), so that over time the chromosomes
may become quite condensed, reducing their regular
length by 1-1.5x (Ludford, 1936; Bajer, 1959; reviewed
by Eigsti and Dustin, 1955; Mazia, 1961). During the
later stages of condensation the sister chromatids
usually separate along their length, except in the
centromeric region, to form X-shaped chromosomes or
"C-pairs" (for plants, see Levan, 1938; Ostergren, 1943;
Mole-Bajer, 1958; for animals, see Ludford, 1936;
Stubblefield, 1964; Cooke et al., 1987; Figs 1,2).
In his classic 1938 paper on the effects of colchicine
Key words: colcemid, mitotic cycle, microtubules, cell cycle.
388
C. L. Rieder and R. E. Palazzo
Fig. 1. Sequential phase-contrast photomicrographs, taken from a time-lapse video light-microscopic recording, of a newt
lung cell proceeding through C-mitosis in the presence of 20 fjM Nocodazole. The chromatids comprising each chromosome
are well separated along their length, except in the centromere region, in C. C-anaphase is initiated between D and E,
during which time the chromatids of each chromosome disjoin in the centromeric region (e.g., cf. centromere regions noted
by arrows in C-E). Approximately 30 min later (G) the chromatids undergo telophase changes that lead to the formation
of a restitution nucleus (H). Bar in H, 50 /jm.
Fig. 2. Schematic drawing of
the chromosome cycle during
C-mitosis. After nuclear
envelope breakdown (A-B) the
chromosomes continue to
thicken and shorten. Over
time the two chromatids
comprising each chromosome
become separated along their
length (C-D), but remain connected in the centomere region (E). During C-anaphase the chromatids completely disjoin (F)
to form "pairs of skis". After a short time, relative to the duration of C-mitosis, the chromatids undergo telophase
decondensation (G) to form ultimately a micronucleated restitution nucleus (H).
C-mitosis
Levan states "the prophases arrive at metaphase and
are kept at that state for a long period...". This
statement was based on Strasburger's (1884; see page
120 of Wilson, 1925) terminology of the time, which
separated the mitotic cycle into prophase, metaphase,
anaphase and telophase without an intervening stage of
prometaphase. The impetus for establishing "prometaphase" as a distinct stage of mitosis occurred between
the publication of Schrader's first (1944) and second
(1953) books on mitosis, well after Levan's initial
studies. As first emphasized by Nebel and Ruttle in 1938
(see also Ostergren, 1943), and more recently by Sluder
(1979, 1988), C-mitotics are blocked in prometaphase
not metaphase. Indeed, after prolonged periods in Cmitosis, recovering sea urchin cells still require the
same 10 minute prometaphase interval to construct a
spindle and congress chromosomes that is normally
required in untreated controls (Sluder, 1979; see also
Brinkley et al., 1967). Regardless, the erroneous notion
that colchicine/Colcemid blocks the mitotic cycle at
metaphase is still perpetuated as evidenced by the
continued widespread use of the terms "metaphase
arrest", "C-mitotic metaphase", "maintained in metaphase", "held in metaphase", "colchicine (or C)metaphase", "metaphase-blocked", etc.
A clear distinction between a mitotic block at
prometaphase and metaphase should not be viewed as a
trivial matter. It becomes increasingly important as
molecular-genetic and cell-free systems are used to
dissect more closely, and to define, the sequence of
biochemical events comprising mitosis. Indeed, the
term "metaphase arrest" is commonly used to characterize various somatic cell mutants blocked in mitosis,
and to describe the outcome of experimental treatments
on mitotic cells, even under conditions in which spindle
formation is largely or completely inhibited. These
"metaphase arrested" cells contrast sharply with those
oocytes that are naturally arrested at true metaphase I
or II of meiosis with fully fomed spindles (reviewed by
Longo, 1973), and those (few) somatic cells that can be
induced by various treatments to arrest permanently in
mitosis with fully formed (e.g. see Shoji-Kasai et al.,
1987; Jordan et al., 1991) or nearly fully formed (Hirano
et al., 1988) spindles.
Escaping the mitotic block
Most, if not all plant cells undergo repeated cell cycles
in the presence of colchicine (e.g. see Levan, 1938;
Nebel and Ruttle, 1938; Eigsti and Dustin, 1955), a fact
that has been widely utilized for generating polyploid
strains of commercially valuable crops. Similarly, many
types of animal cells, including some from Chinese
hamsters (Stubblefield, 1964), newts (Fig. 1), rat
kangaroos (Jensen et al., 1987), mice (Kung et al.,
1990), humans (Chamla et al., 1980) and sea urchins
(Sluder, 1979), are capable of completing one or more
rounds of C-mitosis in the presence of the drug. Thus,
contrary to the implications of such common terms as
"mitotic arrest", "stathmokinesis", "metaphase ar-
389
rest", "blocked or arrested in mitosis", "C-mitotic
arrest", "halted at metaphase", etc., colchicine, Colcemid and drugs with similar actions do not permanently
block plant and many animal cells in mitosis. Rather,
when compared with controls, most drug-treated cells
invariably spend a significantly greater period of time
(up to 10-fold; Eigsti and Dustin, 1955) in (prometaphase of) mitosis prior to entering interphase of the
next cell cycle.
The prolongation of the mitotic period during Cmitosis is not a unique response to the destruction of the
spindle by colchicine and similar drugs. On the
contrary, concentrations of Colcemid or vinblastine
that have little or no discernable effect on spindle
formation in sea urchins (Sluder, 1988) or HeLa-S3 cells
(Jordon et al., 1991) significantly prolong mitosis (sea
urchins) or may even permanently arrest the cells at
true metaphase (HeLa). Similarly, prolongation of the
mitotic period is not a unique response to Colcemid or
other drugs that disrupt MTs; the duration of prometaphase in untreated cells is greatly extended by the
presence of mal-oriented chromosomes (Mazia, 1961;
Zirkle, 1970; Rieder and Alexander, 1989), and/or by
the absence of normal spindle bipolarity (Sluder and
Begg, 1983; Hunt et al., 1992).
It has been proposed by Hartwell and Weinert (1989)
that cells possess control mechanisms, termed "checkpoints", which function to ensure that the events of the
cell cycle are properly coordinated. The fact that the
onset of anaphase is considerably delayed by partial or
total disruption of the spindle (as in C-mitosis),
treatments that minimally compromise MT function, by
the lack of spindle bipolarity, and/or by mal-oriented
chromosomes on a bipolar spindle, reveals that the
process of spindle formation is "monitored" by such a
surveillance checkpoint. As emphasized by Mazia
(1961,1987), and more recently by others (Hartwell and
Weinert, 1989; Murray and Kirschner, 1989), this
checkpoint appears to control cell entry into anaphase,
and passage through this point triggers a cascading
series of events that allow a rapid escape from mitosis,
advancing the cell to interphase of the next cell cycle.
It has recently become clear that the nuclear and
cytoplasmic events that lead to mitosis are regulated, in
part, by the sequential synthesis and accumulation of
"cyclin" proteins A and B. These proteins are cofactors
required for the catalytic activity of the protein kinase,
p34cdc2 (Solomon et al., 1990). Cyclin synthesis drives
cells into mitosis (Murray and Kirschner, 1989), while
the initiation of anaphase and the cells' subsequent exit
from mitosis is coincident with the rapid proteolytic
destruction of these proteins (Evans et al., 1983;
reviewed by Murray and Kirschner, 1989; Whitfield et
al., 1990). More specifically, in somatic cells cyclin A
appears to reach peak levels just before NEB, and is
then degraded during prometaphase as the spindle
forms. By contrast, the cyclin B level remains high until
the metaphase-anaphase transition, at which time it
drops precipitously. Importantly, cyclin A is degraded
but cyclin B levels remain high throughout the
prolonged prometaphase exhibited by C-mitotics (Kung
390
C. L. Rieder and R. E. Palazzo
et al., 1990; Whitfield et al., 1990) and cells containing
monopolar spindles (Hunt et al., 1992). Together these
data strongly support the argument that passage
through the spindle-formation surveillance checkpoint
is triggered by declining levels of cyclin B. If true, it will
become important to elucidate how the life expectancy
of cyclin B is determined by the "state of microtubules
and form of the spindle" (Hunt et al., 1992). The recent
isolation of mitotic arrest-deficient (mad) mutants in
yeast (Hoyt et al., 1991; Li and Murray, 1991), in which
the cells fail to arrest at mitosis in response to loss of
MT function, offers a promising approach for understanding how the cell monitors spindle formation.
Not all animal cells ultimately pass through C-mitosis
and enter the next cell cycle in the presence of drugs
that disrupt MT function. For example, cells of certain
mammalian lines (including HeLa S3, Vero, Tera2) die
after 72 h in C-mitosis (see references quoted by Eigsti
and Dustin, 1955; Kung et al., 1990), possibly from their
inability to synthesize mRNA (Dustin, 1959). In some
cases, a significant proportion of the cells within a
mitotically arrested population escape the block while
others die in mitosis (i.e. the block is leaky; e.g. see
Shoji-Kasai et al., 1987; Jordan et al., 1991). Kung et al.
(1990) have recently shown that the ability of a cell type
to survive C-mitosis is somewhat species-specific, and is
positively correlated with its ability to degrade cyclin B
during the prolonged mitotic period. Although these
experiments do not distinguish whether cyclin B
degradation causes, or simply results from, the biochemical changes leading to escape from mitosis, the
former does provide a possible molecular basis for why
some cells are truly "arrested" in mitosis by colchicine
or Colcemid while others can ultimately advance to
interphase. Clearly, "the stringency of the [spindle
formation surveillance checkpoint]...varies among different cell lines" (Kung et al., 1990).
Chromatid disjunction in the absence of a
spindle
In actively cycling cells the initiation of anaphase, and
thus exit from mitosis, is signaled by the disjunction of
replicated chromatids. In some types of cells the
chromatids of each replicated chromosome separate at
the centromeric region near the end of the C-mitotic
period. This "C-anaphase" (Levan, 1938) phenomenon
appears to occur in all plant cells (reviewed by Levan,
1954; Eigsti and Dustin, 1955), where it has been
especially well characterized owing to the absence of
rounding during the division process (Mole-Bajer, 1958;
Lambert, 1980). In Haemanthus each pair of replicated
chromosomes requires a 1-2 min period to separate (see
Fig. 6 of Lambert, 1980), and all chromatids of the
genome separate in near but not perfect synchrony (see
Eigsti and Dustin, 1955; Mole-Bajer, 1958; Lambert,
1980) in the complete absence of MTs (Lambert, 1980).
Shortly after separation the chromatids begin to swell
and undergo telophase events to form a 4N or polyploid
restitution nucleus. The total duration of C-anaphase is
similar to the time of anaphase in untreated cells (MoleBajer, 1958).
There is currently no consensus concerning the extent
to which C-anaphase occurs in animal cells (e.g. see
Levan, 1954; Mazia, 1961; Rao and Engelberg, 1966;
Mclntosh, 1979), and there are several obvious reasons
for this confusion. Unlike plants, the ultimate fate of
individual chromosomes during C-mitosis in animals is
difficult to follow clearly because most cells progressively round throughout this process. Moreover, few
investigators have studied the course of C-mitosis in
animal cells with the explicit goal of determining
whether the chromatids disjoin.
C-anaphase figures are seen in many types of animal
cells when assayed by using squashed or dropped
chromosome preparations. These include, but are not
limited to, grasshopper spermatogonium (Sokolow,
1939), human lymphocytes (Gabarron et al., 1986),
mouse carcinoma (Ludford, 1936), ascites tumor
(Levan, 1954), Chinese hamster ovary (Stubblefield,
1964), rat kangaroo kidney epithelia (Vig, 1981) and
Drosophila neuroblasts (Gonzalez et al., 1991). (See
Eigsti and Dustin (1955), for additional references on
C-anaphase in chromosome spreads of animal cells.)
Studies on premature centromere separation (e.g. see
Fitzgerald et al., 1975), and the sequence of centromere
separation (e.g. see Vig, 1981), reveal that the harsh
preparative treatments used for these analyses (hypotonic swelling, fixation in acetic acid/ethanol, squashing
or dropping onto slides) are not likely to induce
chromatid separation artificially.
C-anaphase has also been clearly demonstrated in sea
urchin embryos fixed and lightly flattened between two
coverslips (Sluder, 1979). Moreover, C-anaphase figures represent approx. 1-2% of all mitotics in PtK
cultures fixed after 18 h in a concentration (20 fxM) of
nocodazole sufficient to deplete the cells of MTs (C.L.
Rieder and R.W. Cole, unpublished). We have also
observed the process of C-anaphase directly by timelapse video light microscopy of similarly treated newt
lung cells (Fig. 1). With respect to these findings it is
noteworthy that individual chromosomes within the
cytoplasm of PtK (Brenner et al., 1980) and newt
(Rieder and Alexander, 1989) cells, which fail to attach
to the normally forming spindle, still separate their
chromatids at the onset of anaphase. Chromatid
disjunction also occurs during monopolar mitosis in
newts (Rieder et al., 1986) and sea urchins (Mazia et
al., 1981).
The mechanism responsible for chromatid separation
remains mysterious. It is clear from studies on Cmitotics that it is not dependent on antagonistic pulling
forces, generated by the spindle, that act on sister
kinetochores within the centromeric region. This
conclusion contrasts sharply with those models for
chromatid separation in yeast, generated to explain the
apparent need for spindle MT-dependent forces during
DNA decatenation by topoisomerase II (Holm et al.,
1985, 1989; Uemura and Yanagida, 1986). In some
animal cells chromatid disjunction exhibits a close temporal coupling to the Ca2+-mediated inactivation of the
C-mitosis
p34cdc2/cyclin B complex and the destruction of cyclin B
(Hunt et al., 1992; Shamu and Murray, 1992). It also
probably requires DNA topoisomerase II activity
(Downes et al., 1991; Shamu and Murray, 1992) and
perhaps the modification of INCENP (Cooke et al.,
1987) and CLiP (Rattner et al., 1988), proteins unique
to that region of the centromere spanning the sister
kinetochores. In this context it is noteworthy that
chromatids maintain firm centromeric connections
prior to C-anaphase, after becoming separated along
the remainder of their length (see above). Thus the
processing of chromatin that leads to chromatid
separation is multi-phasic (i.e. the decatenation and
subseqeunt separation of chromosome arms and telomeres occurs well before that of the centromeres).
It remains to be determined whether C-anaphase is a
characteristic feature of C-mitosis in all animal cells.
Statements that it does not occur must be re-evaluated
in the context of those considerations that tend to mask
its appearance. However, as discussed above some cell
types ultimately die in C-mitosis, apparently because
they cannot degrade cyclin B to initiate those anaphase
events that allow them to exit the mitotic cycle (Kung et
al., 1990; Whitfield et al., 1990; Hunt etal., 1992). Since
the initiation of anaphase is normally heralded by
chromatid separation, cells that are unable to exit Cmitosis may never disjoin their chromatids. In such cells
spindle formation would be necessary for chromatid
separation (i.e. anaphase) only because it is a prerequisite for initiating cyclin B degradation to allow passage
through the spindle-formation surveillance checkpoint,
not because chromatid separation is based on forces
generated by the spindle (e.g. see Gonzalez et al.,
1991).
Although chromatid disjunction is normally temporaly coincident with cyclin B destruction, it may not be
mediated, even indirectly, by declining cyclin B levels
but by some other independent signal. Under these
circumstances cells would be able to separate their
chromatids without necessarily initiating those other
events of anaphase that allow them to exit mitosis.
Reports that certain mutant human cells appear to
remain arrested for considerable periods of time in
mitosis, with some or all of their chromatids disjoined
(Fitzgerald et al., 1975; Rudd et al., 1983; Gabarron et
al., 1986), argue in favor of this hypothesis. By contrast,
it is also possible that the events of anaphase that allow
the cell to exit mitosis can occur in the absence of
chromatid separation. Such a "relief of dependence"
(Hartwell and Weinert, 1989) is suggested by the
observation that treatments that inhibit chromatid
separation in mammalian cells do not necessarily
prevent exit from mitosis (Downes et al., 1991).
Conclusions
We have reviewed the evidence that, for many cells,
disruption of the mitotic spindle with Colcemid,
colchicine and similar drugs delays but does not inhibit
progression through the mitotic cycle. Whether a
391
particular cell type can exit C-mitosis depends on its
ability to overcome the spindle-formation surveillance
checkpoint in the absence of a spindle, an ability that
may depend on whether the cell can ultimately degrade
cyclin B while in C-mitosis. C-mitotics capable of
passing through this checkpoint normally advance to
interphase by way of a C-anaphase. C-anaphase is
indicated by the separation of sister chromatids and this
event does not depend on forces generated by the
spindle.
We thank Drs. G. Sluder, S.P. Alexander, S.S. Bowser and
J.G. Ault for their scientific comments, and Ms. S. Nowogrodzki for editorial assistance. This work was supported, in
part, by grants from the National Institutes of Health,
General Medical Sciences R01-40198 (to C.L.R.) and R0143264 (to R.E.P.), by grant no. 2725 from the Council for
Tobacco Research (to R.E.P.), and by American Cancer
Society grant JFRA 62121 (to R.E.P.).
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Note added in proof
While this manuscript was at the printers Gosh and
Paweletz (Exp. Cell Res. 200, 215-217, 1992) reported
that okadaic acid inhibits sister chromatid separation in
HeLa cells without inhibiting exit from mitosis. As a
result, at the next mitosis diplochromosomes are
formed that contain 4 unseparated chromatids. These
data support the hypotheses that phosphatase 1 activity
is required for sister chromatid separation, and that
chromatid separation is not an obligatory event for
escape from mitosis.