Colcemid and the Mitotic Cycle

Journal of Cell Science 102, 387-392 (1992) 387 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 been impacted by, recent and important findings on the control mechanisms by which the cell monitors progress The precise segregation of replicated chromosomes to through, and ultimately exits, mitosis (e.g. see Hartwell daughter cells during mitosis depends on the formation and Weinert, 1989; Murray and Kirschner, 1989). of a bipolar spindle composed primarily of microtubules The aim of this commentary is to oultine the process (MTs). Since MTs are highly dynamic structures whose of C-mitosis in plant and animal cells with an emphasis spatial organization is critical for proper spindle on new data that provide possible explanations for why function, physical and chemical agents that interfere various cell types behave differently during mitosis in with MT behavior invariably disrupt mitosis. Perhaps the presence of drugs that disrupt MT function. the most notable of these agents is colchicine, derived Although our focus is on colchicine/Colcemid, many of from plants of the genus Colchicum, which has long the conclusions may be applicable to similar drugs that been known to be a potent inhibitor of cell division disrupt mitosis through their action on MTs. 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 The 'mitotic block' related but less-toxic compound demecolcine (Colce- mid), has been mostly elucidated and other drugs (e.g. Over a wide range of concentrations colchicine and podophyllotoxin, steganacin, vinblastine, Nocodazole) Colcemid do not affect the rate at which cells enter have been discovered that interfere similarly with mitosis (reviewed by Eigsti and Dustin, 1955; Sluder, mitosis through their action on MTs (e.g. see Eigsti and 1979). When applied well before nuclear envelope Dustin, 1955; Deysson, 1968; Mareel and DeMets, breakdown (NEB) and in a sufficient concentration 1984). these drugs completely inhibit the formation of spindle The functional basis of how colchicine and Colcemid MTs. As a result, during NEB the chromosomes are disrupt the spindle is now well understood (e.g. see released into the cytoplasm where they remain ran- Taylor, 1965; Wilson et al., 1976; Dustin, 1978; Mareel domly dispersed throughout the prolonged period of C- and DeMets, 1984). However, much of our knowledge mitosis (Fig. 1). It is noteworthy that the chromosome of how mitosis proceeds in the presence of these drugs condensation cycle (see Mazia, 1987) continues during (C-mitosis; Levan, 1938) is based on cytological C-mitosis (Fig. 2), so that over time the chromosomes examinations of fixed cells conducted prior to 1955 may become quite condensed, reducing their regular (summarized by Eigsti and Dustin, 1955; Dustin, 1978). length by 1-1.5x (Ludford, 1936; Bajer, 1959; reviewed Although these pioneering studies provided fundamen- by Eigsti and Dustin, 1955; Mazia, 1961). During the tal data regarding the effects of colchicine/Colcemid on later stages of condensation the sister chromatids spindle formation in plants and animals, and estab- usually separate along their length, except in the lished much of the terminology still used to characterize centromeric region, to form X-shaped chromosomes or the process of C-mitosis, few addressed the ultimate "C-pairs" (for plants, see Levan, 1938; Ostergren, 1943; fate of C-mitotics in animal tissues. Moreover, those Mole-Bajer, 1958; for animals, see Ludford, 1936; that did failed to reach a consensus concerning the ex- Stubblefield, 1964; Cooke et al., 1987; Figs 1,2). tent that colchicine/Colcemid permanently blocks cells In his classic 1938 paper on the effects of colchicine 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 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 389

Levan states "the prophases arrive at metaphase and rest", "blocked or arrested in mitosis", "C-mitotic are kept at that state for a long period...". This arrest", "halted at metaphase", etc., colchicine, Colce- statement was based on Strasburger's (1884; see page mid and drugs with similar actions do not permanently 120 of Wilson, 1925) terminology of the time, which block plant and many animal cells in mitosis. Rather, separated the mitotic cycle into prophase, metaphase, when compared with controls, most drug-treated cells anaphase and telophase without an intervening stage of invariably spend a significantly greater period of time prometaphase. The impetus for establishing "prometa- (up to 10-fold; Eigsti and Dustin, 1955) in (prometaphase" as a distinct stage of mitosis occurred between phase of) mitosis prior to entering interphase of the the publication of Schrader's first (1944) and second next cell cycle. (1953) books on mitosis, well after Levan's initial The prolongation of the mitotic period during C- studies. As first emphasized by Nebel and Ruttle in 1938 mitosis is not a unique response to the destruction of the (see also Ostergren, 1943), and more recently by Sluder spindle by colchicine and similar drugs. On the (1979, 1988), C-mitotics are blocked in prometaphase contrary, concentrations of Colcemid or vinblastine not metaphase. Indeed, after prolonged periods in C- that have little or no discernable effect on spindle mitosis, recovering sea urchin cells still require the formation in sea urchins (Sluder, 1988) or HeLa-S3 cells same 10 minute prometaphase interval to construct a (Jordon et al., 1991) significantly prolong mitosis (sea spindle and congress chromosomes that is normally urchins) or may even permanently arrest the cells at required in untreated controls (Sluder, 1979; see also true metaphase (HeLa). Similarly, prolongation of the Brinkley et al., 1967). Regardless, the erroneous notion mitotic period is not a unique response to Colcemid or that colchicine/Colcemid blocks the mitotic cycle at other drugs that disrupt MTs; the duration of prometa- metaphase is still perpetuated as evidenced by the phase in untreated cells is greatly extended by the continued widespread use of the terms "metaphase presence of mal-oriented chromosomes (Mazia, 1961; arrest", "C-mitotic metaphase", "maintained in meta- Zirkle, 1970; Rieder and Alexander, 1989), and/or by phase", "held in metaphase", "colchicine (or C)- the absence of normal spindle bipolarity (Sluder and metaphase", "metaphase-blocked", etc. Begg, 1983; Hunt et al., 1992). A clear distinction between a mitotic block at It has been proposed by Hartwell and Weinert (1989) prometaphase and metaphase should not be viewed as a that cells possess control mechanisms, termed "check- trivial matter. It becomes increasingly important as points", which function to ensure that the events of the molecular-genetic and cell-free systems are used to cell cycle are properly coordinated. The fact that the dissect more closely, and to define, the sequence of onset of anaphase is considerably delayed by partial or biochemical events comprising mitosis. Indeed, the total disruption of the spindle (as in C-mitosis), term "metaphase arrest" is commonly used to charac- treatments that minimally compromise MT function, by terize various somatic cell mutants blocked in mitosis, the lack of spindle bipolarity, and/or by mal-oriented and to describe the outcome of experimental treatments chromosomes on a bipolar spindle, reveals that the on mitotic cells, even under conditions in which spindle process of spindle formation is "monitored" by such a formation is largely or completely inhibited. These surveillance checkpoint. As emphasized by Mazia "metaphase arrested" cells contrast sharply with those (1961,1987), and more recently by others (Hartwell and oocytes that are naturally arrested at true metaphase I Weinert, 1989; Murray and Kirschner, 1989), this or II of meiosis with fully fomed spindles (reviewed by checkpoint appears to control cell entry into anaphase, Longo, 1973), and those (few) somatic cells that can be and passage through this point triggers a cascading induced by various treatments to arrest permanently in series of events that allow a rapid escape from mitosis, mitosis with fully formed (e.g. see Shoji-Kasai et al., advancing the cell to interphase of the next cell cycle. 1987; Jordan et al., 1991) or nearly fully formed (Hirano It has recently become clear that the nuclear and et al., 1988) spindles. 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 Escaping the mitotic block required for the catalytic activity of the protein kinase, p34cdc2 (Solomon et al., 1990). Cyclin synthesis drives Most, if not all plant cells undergo repeated cell cycles cells into mitosis (Murray and Kirschner, 1989), while in the presence of colchicine (e.g. see Levan, 1938; the initiation of anaphase and the cells' subsequent exit Nebel and Ruttle, 1938; Eigsti and Dustin, 1955), a fact from mitosis is coincident with the rapid proteolytic that has been widely utilized for generating polyploid destruction of these proteins (Evans et al., 1983; strains of commercially valuable crops. Similarly, many reviewed by Murray and Kirschner, 1989; Whitfield et types of animal cells, including some from Chinese al., 1990). More specifically, in somatic cells cyclin A hamsters (Stubblefield, 1964), newts (Fig. 1), rat appears to reach peak levels just before NEB, and is kangaroos (Jensen et al., 1987), mice (Kung et al., then degraded during prometaphase as the spindle 1990), humans (Chamla et al., 1980) and sea urchins forms. By contrast, the cyclin B level remains high until (Sluder, 1979), are capable of completing one or more the metaphase-anaphase transition, at which time it rounds of C-mitosis in the presence of the drug. Thus, drops precipitously. Importantly, cyclin A is degraded contrary to the implications of such common terms as but cyclin B levels remain high throughout the "mitotic arrest", "stathmokinesis", "metaphase ar- 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 similar to the time of anaphase in untreated cells (Mole- monopolar spindles (Hunt et al., 1992). Together these Bajer, 1958). data strongly support the argument that passage There is currently no consensus concerning the extent through the spindle-formation surveillance checkpoint to which C-anaphase occurs in animal cells (e.g. see is triggered by declining levels of cyclin B. If true, it will Levan, 1954; Mazia, 1961; Rao and Engelberg, 1966; become important to elucidate how the life expectancy Mclntosh, 1979), and there are several obvious reasons of cyclin B is determined by the "state of microtubules for this confusion. Unlike plants, the ultimate fate of and form of the spindle" (Hunt et al., 1992). The recent individual chromosomes during C-mitosis in animals is isolation of mitotic arrest-deficient (mad) mutants in difficult to follow clearly because most cells progress- yeast (Hoyt et al., 1991; Li and Murray, 1991), in which ively round throughout this process. Moreover, few the cells fail to arrest at mitosis in response to loss of investigators have studied the course of C-mitosis in MT function, offers a promising approach for under- animal cells with the explicit goal of determining standing how the cell monitors spindle formation. whether the chromatids disjoin. Not all animal cells ultimately pass through C-mitosis C-anaphase figures are seen in many types of animal and enter the next cell cycle in the presence of drugs cells when assayed by using squashed or dropped that disrupt MT function. For example, cells of certain chromosome preparations. These include, but are not mammalian lines (including HeLa S3, Vero, Tera2) die limited to, grasshopper spermatogonium (Sokolow, after 72 h in C-mitosis (see references quoted by Eigsti 1939), human lymphocytes (Gabarron et al., 1986), and Dustin, 1955; Kung et al., 1990), possibly from their mouse carcinoma (Ludford, 1936), ascites tumor inability to synthesize mRNA (Dustin, 1959). In some (Levan, 1954), Chinese hamster ovary (Stubblefield, cases, a significant proportion of the cells within a 1964), rat kangaroo kidney epithelia (Vig, 1981) and mitotically arrested population escape the block while Drosophila neuroblasts (Gonzalez et al., 1991). (See others die in mitosis (i.e. the block is leaky; e.g. see Eigsti and Dustin (1955), for additional references on Shoji-Kasai et al., 1987; Jordan et al., 1991). Kung et al. C-anaphase in chromosome spreads of animal cells.) (1990) have recently shown that the ability of a cell type Studies on premature centromere separation (e.g. see to survive C-mitosis is somewhat species-specific, and is Fitzgerald et al., 1975), and the sequence of centromere positively correlated with its ability to degrade cyclin B separation (e.g. see Vig, 1981), reveal that the harsh during the prolonged mitotic period. Although these preparative treatments used for these analyses (hypo- experiments do not distinguish whether cyclin B tonic swelling, fixation in acetic acid/ethanol, squashing degradation causes, or simply results from, the bio- or dropping onto slides) are not likely to induce chemical changes leading to escape from mitosis, the chromatid separation artificially. former does provide a possible molecular basis for why C-anaphase has also been clearly demonstrated in sea some cells are truly "arrested" in mitosis by colchicine urchin embryos fixed and lightly flattened between two or Colcemid while others can ultimately advance to coverslips (Sluder, 1979). Moreover, C-anaphase fig- interphase. Clearly, "the stringency of the [spindle ures represent approx. 1-2% of all mitotics in PtK formation surveillance checkpoint]...varies among dif- cultures fixed after 18 h in a concentration (20 fxM) of ferent cell lines" (Kung et al., 1990). 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 time- Chromatid disjunction in the absence of a lapse video light microscopy of similarly treated newt spindle lung cells (Fig. 1). With respect to these findings it is noteworthy that individual chromosomes within the In actively cycling cells the initiation of anaphase, and cytoplasm of PtK (Brenner et al., 1980) and newt thus exit from mitosis, is signaled by the disjunction of (Rieder and Alexander, 1989) cells, which fail to attach replicated chromatids. In some types of cells the to the normally forming spindle, still separate their chromatids of each replicated chromosome separate at chromatids at the onset of anaphase. Chromatid the centromeric region near the end of the C-mitotic disjunction also occurs during monopolar mitosis in period. This "C-anaphase" (Levan, 1938) phenomenon newts (Rieder et al., 1986) and sea urchins (Mazia et appears to occur in all plant cells (reviewed by Levan, al., 1981). 1954; Eigsti and Dustin, 1955), where it has been The mechanism responsible for chromatid separation especially well characterized owing to the absence of remains mysterious. It is clear from studies on C- rounding during the division process (Mole-Bajer, 1958; mitotics that it is not dependent on antagonistic pulling Lambert, 1980). In Haemanthus each pair of replicated forces, generated by the spindle, that act on sister chromosomes requires a 1-2 min period to separate (see kinetochores within the centromeric region. This Fig. 6 of Lambert, 1980), and all chromatids of the conclusion contrasts sharply with those models for genome separate in near but not perfect synchrony (see chromatid separation in yeast, generated to explain the Eigsti and Dustin, 1955; Mole-Bajer, 1958; Lambert, apparent need for spindle MT-dependent forces during 1980) in the complete absence of MTs (Lambert, 1980). DNA decatenation by topoisomerase II (Holm et al., Shortly after separation the chromatids begin to swell 1985, 1989; Uemura and Yanagida, 1986). In some and undergo telophase events to form a 4N or polyploid animal cells chromatid disjunction exhibits a close tem- restitution nucleus. The total duration of C-anaphase is poral coupling to the Ca2+-mediated inactivation of the C-mitosis 391 p34cdc2/cyclin B complex and the destruction of cyclin B particular cell type can exit C-mitosis depends on its (Hunt et al., 1992; Shamu and Murray, 1992). It also ability to overcome the spindle-formation surveillance probably requires DNA topoisomerase II activity checkpoint in the absence of a spindle, an ability that (Downes et al., 1991; Shamu and Murray, 1992) and may depend on whether the cell can ultimately degrade perhaps the modification of INCENP (Cooke et al., cyclin B while in C-mitosis. C-mitotics capable of 1987) and CLiP (Rattner et al., 1988), proteins unique passing through this checkpoint normally advance to to that region of the centromere spanning the sister interphase by way of a C-anaphase. C-anaphase is kinetochores. In this context it is noteworthy that indicated by the separation of sister chromatids and this chromatids maintain firm centromeric connections event does not depend on forces generated by the prior to C-anaphase, after becoming separated along spindle. the remainder of their length (see above). Thus the processing of chromatin that leads to chromatid We thank Drs. G. Sluder, S.P. Alexander, S.S. Bowser and J.G. Ault for their scientific comments, and Ms. S. Nowo- separation is multi-phasic (i.e. the decatenation and grodzki for editorial assistance. This work was supported, in subseqeunt separation of chromosome arms and telo- part, by grants from the National Institutes of Health, meres occurs well before that of the centromeres). General Medical Sciences R01-40198 (to C.L.R.) and R01- It remains to be determined whether C-anaphase is a 43264 (to R.E.P.), by grant no. 2725 from the Council for characteristic feature of C-mitosis in all animal cells. Tobacco Research (to R.E.P.), and by American Cancer Statements that it does not occur must be re-evaluated Society grant JFRA 62121 (to R.E.P.). in the context of those considerations that tend to mask its appearance. However, as discussed above some cell References types ultimately die in C-mitosis, apparently because they cannot degrade cyclin B to initiate those anaphase Bajer, A. S. (1959). 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