Colcemid and the Mitotic Cycle
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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 disjunc- tion (i.e. anaphasic separation) of replicated chromo- somes. 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 (prometa- phase" 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.