While much has been described about the control of mitotic cell cycles, the regulation of cell cycles that result in DNA levels greater than the diploid content (polyploid state) has attracted much interest only in recent years. This is probably due to the realization that polyploidy is prevalent in a variety of cells, including those of mammalian origin, and that the state of having duplicated chromosomes may affect cellular function. Polyploidy occurs in some cell types as a normal developmental process, while in others as a result of stress. Megakaryocytes, the platelet precursors, fall under the former category. Different cell cycles in which distinct phases are modified are responsible for the acquisition of the high ploidy-state in different lineages. In the current review, we will focus on the regulation of polyploidization in megakaryocytes, while also comparing it to other systems in which high ploidy is prevalent. Before proceeding, however, we will attempt to address a question often asked: what is the purpose of polyploidization? Does it present the cell with any benefit, or is it a state of disadvantage? We propose that the potential purpose of high ploidy may depend on the lineage in which it occurs. Polyploidization in all cell types allows an increase in metabolic output, cell mass, and cell size, without the need to devote energy to carry on all aspects of cellular division. In addition, the state of ploidy is likely to affect the expression profile of specific genes, as concluded from studies in the yeast system (Galitski et al., 1999). In the search for meaning in megakaryocyte polyploidy, we resort to the special function of this cell type. The megakaryocyte gives rise to platelets, which are important for thrombosis and hemostasis. Platelets fragment from this precursor cell due to elaborate changes in the cytoskeleton. High ploidy in megakaryocytes is associated with an increase in cell size, mRNA, and protein production (Brodsky and Uryvaeva, 1977; Hancock et al., 1993). The increase in these components may be important for the formation of extensive internal demarcation membranes and a surface-connected canalicular system, which are important for platelet fragmentation. While fragmenting, platelets carry all the precursor cell components except for nuclear material. Since the nucleus is not needed for platelet function, it would be quite wasteful to devote energy to allow cytoskeletal reorganization for the purpose of nuclear and cellular division. A single, large 32n megakaryocyte (2n representing a diploid state) yields approximately 3,000 platelets (Winkelmann et al., 1987), while several precursor diploid cells would have had to be generated in order to produce a similar number of platelets. The state of ploidy also seems to determine the reactivity of platelets produced (reviewed in Zimmet and Ravid, 2000), which may support the idea that, as in yeast, polyploidy impacts the profile of gene expression and hence, the repertoire of proteins in the cell. With this in mind, although high DNA content per cell may be of advantage in some specialized cells, one may envision it to be damaging to others. For example, transformed cells induced to become polyploid may produce vast amounts of a transforming factor, which may be released to further support the malignant phenotype. Understanding the regulation of the different cell cycles that lead to high ploidy, and hence being able to manipulate each, will provide a better basis for addressing the intriguing question: why polyploidy?
Polyploidy, recognized by multiple copies of the haploid chromosome number, has been described in plants, insects, and in mammalian cells such as, the platelet precursors, the megakaryocytes. Several of these cell types reach high ploidy via a different cell cycle. Megakaryocytes undergo an endomitotic cell cycle, which consists of an S phase interrupted by a gap, during which the cells enter mitosis but skip anaphase B and cytokinesis. Here, we review the mechanisms that lead to this cell cycle and to polyploidy in megakaryocytes, while also comparing them to those described for other systems in which high ploidy is achieved. Overall, polyploidy is associated with an orchestrated change in expression of several genes, of which, some may be a result of high ploidy and hence a determinant of a new cell physiology, while others are inducers of polyploidization. Future studies will aim to further explore these two groups of genes. J. Cell. Physiol. 190: 7–20, 2002. © 2002 Wiley-Liss, Inc.
NORMAL MECHANISMS RESPONSIBLE FOR THE MAINTENANCE OF A DIPLOID STATE
A regulated progression through a mitotic cell cycle involves replication of the entire genome exactly once in preparation for mitotic division (reviewed in Romanowski and Madine, 1996). Cell fusion experiments performed decades ago indicated that a G1 nucleus is induced to replicate early, whereas a G2 nucleus is unable to replicate without going through karyokinesis (Rao and Johnson, 1970). This suggested that the G2 nucleus must go through mitosis in order to regain its ability to replicate DNA and that the factors present in the S phase cell are able to induce replication only in the nucleus that had undergone cytokinesis. G1 egg extracts allow sperm or G1 HeLa nuclei to replicate once. Transfer of intact G2 HeLa nuclei or replicated sperm to fresh G1 extract is able to initiate re-replication only if the nuclear membranes are permeabilized, indicating that the nuclear membrane normally excludes factors that are able to promote DNA replication (Blow and Laskey, 1988; Leno et al., 1992). This activity is known in the literature as “licensing factor,” ensuring that DNA replication occurs only once per cycle. This is an important point in relation to the development of polyploidy, as it indicates that a mechanism which allows the cells to bypass this restriction and cell cycle arrest is necessary. Minichromosome maintenance (MCM) proteins have been described as replication licensing factors (Kearsey et al., 1996). The MCM proteins were originally found in Saccharomyces cerevisiae as mutants that were unable to maintain minichromosomes through repeated cell cycles. MCM2, through MCM7, have been isolated from a variety of eukaryotes including humans. A complex of MCM proteins has been found to bind to DNA, and is necessary for replication. This complex is displaced from chromatin during DNA replication and remains in the unbound state throughout G2. Re-formation of this complex on chromatin requires either permeabilization of the nuclear membrane or passage through mitosis. MCM proteins from several species have been found to be bound to DNA during G1 phase, displaced during S, and maintained unbound in the nucleus during G2 (Kimura et al., 1994; Madine et al., 1995; Kubota et al., 1997). Binding of MCM proteins to chromatin in Xenopus has been shown to require an additional activity known as “loading factor” (Madine et al., 1995). DNA replication in vertebrates depends on the binding of the origin recognition complex (ORC), Cdc6, the MCM, and Cdc45 (reviewed in Fujita, 1999; DePamphilis, 2000). The loading factor is linked to the cell cycle machinery through regulation by cyclin/Cdk (cyclin-dependent protein kinase) (Mahbubani et al., 1997). Cdc6 is required for recruitment of MCM proteins to the DNA prior to S phase and for initiation of DNA replication (Yew and Kirschner, 1997). Cdc6 is found in the nucleus during G1, and upon phosphorylation is translocated to the cytosol near the initiation of S-phase, perhaps to prevent its re-binding to the DNA. Mutation of Cdk sites in Cdc6, which prevent its nuclear export, neither inhibit DNA replication initiation nor lead to DNA re-replication and high ploidy (Pelizon et al., 2000). There are at least six classes of cyclins, designated A through G (Pines, 1993; Tamura et al., 1993). Cyclin B in association with p34Cdc2 (Cdc2 kinase) is fundamental to the regulation of the G2 to M transition in all eukaryotes thus far examined (reviewed in Nurse, 1994; Sherr, 1994). Cyclin A is also involved in the G2 to M transition and regulates progression through S phase (Girard et al., 1991; Pagano et al., 1992). G1 cyclins associate with specific Cdk subunits. Cyclin E associates primarily with Cdk2 (Dulic et al., 1992; Koff et al., 1992). The D-type cyclins appear to be induced earlier in G1 phase than in cyclin E and are capable of forming complexes with Cdk2, Cdk4, Cdk5, and Cdk6 (Xiong et al., 1992; Meyerson and Harlow, 1994). Transition between successive phases of the cell cycle are mediated by ordered pulses of Cdk activity. The activity of the different cyclin/Cdk complexes depends on alterations in kinase activity by phosphorylation or dephosphorylation of the kinase complex, the binding of Cdk inhibitors, and the rate of synthesis and degradation of the cyclins in a cell cycle-dependent fashion. Among the Cdk and Cdc2 inhibitors are the INK4 family composed of p16, p15, p18, and p19 which bind to Cdk4 and Cdk6 and prevent their association with D cyclins. The Cip/Kip family of inhibitors consists of p21, p27 (both widely expressed), and p57 (more tissue specific). They inhibit the kinase activity of most of the cyclin Cdk complexes (reviewed in Sherr, 1996). G1 and S phase as well as M phase regulators fluctuate in level during the cell cycle due to ubiquitin-mediated proteolysis (reviewed in Kornitzer and Ciechanover, 2000).
As we shall see below, the regulation of the endomitotic cell cycle in megakaryocytes may involve regulated expression of cyclins and kinases, protein degradation, and cell cycle inhibitors.
POLYPLIDY IS ACHIEVED BY DIFFERENT CELL CYCLES
Polyploidy can occur due to different types of cell cycles. A cell cycle that gives rise to multiple number of the normal haploid chromosome number in the cell (e.g., 2n in diploid cells or 1n in sperm nuclei) has been referred to as an endocycle, endoreplication, or endoreduplication. Throughout the literature, some of these definitions had been associated with cells that do not enter mitosis but rather proceed directly from a gap phase to a phase of DNA synthesis (cell cycles consisting of continuos S phase with no gap are rare). Polyteny is a special case of such an endoreduplication in which the duplicated chromosomes line up side by side, forming large polytene chromosomes. The broad definition of endocycling is further divided into sub-categories, based on the nature of the gap phase preceding re-entry into S-phase, i.e., which components of the M-phase exist. These include: Endomitosis, which according to its original definition involves M-phase entry with a block at prophase, without dissolution of the nuclear membrane (Geitler, 1953). This term is currently used, however, to describe the megakaryocytic cell cycle which involves nuclear envelope breakdown, entry to M-phase with no completion of anaphase B and cytokinesis (see below); C-mitosis, involving entry to M-phase, but blockage at metaphase following chromosome condensation and spindle formation; and Acytokinetic mitosis, during which the cells complete all aspects of mitosis except for cytokinesis, resulting in binucleate cells (reviewed in Zimmet and Ravid, 2000). Defining the structural feature of a cell cycle that yields high ploidy is essential for exploring its regulatory mechanisms. These cell cycle variations have been described in different cell types and species. Polyploidy was first recognized in plants in 1910 (Strassburger, 1910) and since then has been studied in a variety of plant species, including those undergoing an endomitotic cell cycle (reviewed in Edgar and Orr-Weaver, 2001). In insects, polyploidy has been described in arthropods and in Drosophila melagonster. Embryonic development of the fruit fly involves different mechanisms and degrees of polyploidy development in many cell types, including the development of polytene in the nurse cells (reviewed in Edgar and Orr-Weaver, 2001). Polyploidy is also found in various mammalian cells: in the liver, acytokinetic mitosis gives rise to cells with multiple nuclei, although some polyploid cells with single nuclei are also observed, particularly with aging (Brodsky and Uryvaeva, 1977; Kudryavtsev et al., 1993); a significant number of myocytes, particularly in the left ventricular wall, are plolyploid and under conditions of stress, hyperthrophy is associated with further polyploidization (Sandritter and Scomazzoni, 1964); polyploidy is also found in aortic vascular smooth muscle cells derived from hypertensive animals (Owens and Schwartz, 1982, 1983) and in smooth muscle of the uterus; there is also a significant increase in ploidy of thyreocytes and cells of the adrenal gland with age (Heiden and James, 1975; Auer et al., 1985). In the heart, vascular smooth muscle and in the endocrine organs, the exact nature of the M phase during the cell cycle has not been defined. Early events of mitosis seem to occur in polyploidizing aortic vascular smooth muscle cells (our unpublished data). Some of the trophoblast giant cells of the mammalian placenta become polyploid via a mechanism that is believed to resemble polytene formation in insects (Edgar and Orr-Weaver, 2001). Lastly, and the focus of this review, the mammalian megakaryocyte undergoes an endomitotic cell cycle during normal development.
STRUCTURAL FEATURES OF THE ENDOMITOTIC CELL CYCLE IN MEGAKARYOCYTES
Several studies have demonstrated that the cell cycle in polyploidizing megakaryocytes is composed of repeating rounds of DNA replication separated by short gaps (Odell and Jackson, 1968; Zhang et al., 1996). This excludes the possibility of polyploidy being achieved by means of a continuous DNA synthesis phase. Several early studies reported morphological observations of mitotic events in polyploid megakaryocytes (Goyannes-Villaescuca, 1969; Winkelmann et al., 1987). Nagata and co-workers have examined large numbers of primary murine megakaryocytes, cultured in the presence of the megakaryocyte-promoting cytokine, thrombopoietin (TPO), for the features of mitosis (Nagata et al., 1997). They demonstrated several polyploid cells with condensed chromosomes, spindles originating from multiple spindle poles, and multiple centrosomes. Breakdown of the nuclear membrane was also clear in these cells. They did not, however, observe signs which would indicate outward movement of the spindle poles or other features of late mitosis, concluding that the cells enter mitosis and proceed as far as anaphase A, but are then blocked from proceeding to anaphase B, telophase, and cytokinesis. A similar study published by Vitrat et al. took a comparable approach to study the morphological features of human megakaryocytes cultured with TPO (Vitrat et al., 1998). To overcome the rarity of cultured megakaryocytes (less than 1%) that are in M phase and in order to synchronize the cells, this group resorted to nocodazole treatment, after which they report approximately 20% of cells are blocked in “pseudo-metaphase.” Consistent with the other study, Vitrat et al. reported the observation of DNA condensation and the formation of a spherical mitotic spindle. In addition, chromatid separation and movement towards the spindle poles were demonstrated, suggesting that anaphase was in fact completed in these cells. In a later study in which nocodazole was not employed, it was concluded that in human, polyploizing megakaryocytes, the nuclear envelope dissolves, anaphase B and cytokinesis are skipped all together and the chromosomes do not segregate evenly (Roy et al., 2001) (Fig. 1).
MEGAKARYOCYTE DIFFERENTIATION AND PLOIDY
In vitro assays were developed over 3 decades ago to recognize early committed megakaryocytes in the marrow, the megakaryocyte colony-forming cells (MK-CFC). A population of megakaryocyte progenitors more primitive than MK-CFC was subsequently identified (the burst forming cells, MK-BFC), and characterized with a high proliferative potential. The MK-CFC cells express early markers of differentiation such as the glycoprotein GPIIb and the platelet factor four protein prior to switching to an endomitotic cell cycle (reviewed in Jackson et al., 1997). This endomitotic cell cycle results in cells that may possess a DNA content as high as 128n. The process of maturation takes place leading to fragmentation of polyploid cells (8n and higher) into platelets (Jackson et al., 1997) (Fig. 2). All stages of lineage proliferation, differentiation, and polyploidization have been shown to be stimulated by TPO. Several studies have indicated that while expression of differentiation markers can occur in proliferating megakaryocytic cell lines with no further increase in ploidy, proliferation-promoting transcription factors inhibit the shift to endomitosis. For instance, the expression of large T antigen, c-myc, or E2-F transcription factors in megakaryocytes of transgenic mice retains a larger fraction of the cells in a proliferative mode, resulting in a smaller fraction of cells with high ploidy (Ravid et al., 1993; Guy et al., 1996; Thompson et al., 1996). This suggested that the signal to begin endomitosis has to initially result in suppressing the expression of proliferation-promoting genes, and perhaps temporarily arresting cells at G0 prior to initiating an endomitotic cell cycle. In accordance with this suggestion, a survey of freshly isolated murine bone marrow cells indicated that only up to 15% of the megakaryocytes are actively cycling (Wang et al., 1995). TPO induces the recruitment of progenitor cells to the megakaryocytic lineage as well as induces the cells to become highly polyploid (reviewed in Kaushansky, 1995). Studies with cell lines indicated that differentiation in the megakaryocytic lineage could occur without subsequent polyploidization, but not vice versa, indicating that the signals generated by TPO to induce endomitosis are only sensed by a differentiated cell. It is not yet clear, however, at what exact point in the cell's life it is receptive to endomitosis-promoting signals. One may propose that the effect of TPO consists of two phases: TPO enhances the proliferation of the primitive MK-BFC and its subsequent differentiation to a megakaryocyte, thus leading to a temporary arrest at a G0 phase. The now fully differentiated cell will respond to TPO with a different set of signals than the undifferentiated one, and this would lead to endomitosis and high ploidy. In addition, it is possible that the progenitor cell has an intrinsic oscillator that dictates both the number of proliferation cycles prior to initiation of endocycling as well as the total number of endocycles. For instance, it has been concluded that a MK-CFC undergoes 1–8 cell divisions. Furthermore, in different species, the majority of megakaryocytes display as 16n, raising the possibility that the polyploidizing cells cycle up to 16n while only few progress to higher ploidy levels (Jackson et al., 1997). As reviewed below, the mechanisms by which TPO induces high ploidy are gradually being dissected.
REGULATION OF ENDOMITOSIS IN MEGAKARYOCYTES
Role of G1/S phase cyclins
Given the rarity of megakaryocytes in the marrow and the fact that the gap period in endomitotic megakaryocytes has been estimated to be less than 90 min in duration (Wang et al., 1995), finding sufficient polyploid mitotic megakaryocytes for study may be challenging indeed. Hence, several groups have used megakaryocytic cell lines to examine the regulation of the endomitotic cell cycle in this lineage. Potential alterations in G1 phase cyclins were evaluated initially in cell lines and in some cases, in vivo as well. Examination of the cyclin machinery has shown that cyclin D3 is expressed in both megakaryocytic cell lines and primary megakaryocytes, and is upregulated by ploidy-promoting factors such as TPO and phorbol ester (Wang et al., 1995; Zhang et al., 1996; Zimmet et al., 1997). Cyclin D3 overexpression in megakaryocytes of transgenic mice resulted in an increase in ploidy commensurate with that observed upon in vivo treatment with TPO (Zimmet et al., 1997). Cyclin D2 was not detectable in primary megakaryocytes, while cyclin D1 was expressed at low levels and moderately upregulated by TPO in vivo (Zimmet et al., 1997). Only moderate overexpression of cyclin D1 was achieved in megakaryocytes of transgenic mice by using the same tissue specific promoter employed to express elevated levels of cyclin D3. This led to a moderate increase in ploidy, suggesting that although cyclin D3 is unique in its high abundance in megakaryocytes, it is not unique in its ability to induce ploidy (Sun et al., 2001). These studies support a role for this G1 phase cyclin in promoting multiple cycles of endomitotic DNA synthesis and thus allowing the development of high-ploidy megakaryocytes in vivo.
D type cyclins are usually associated with Cdk4 and/or Cdk6 in mammalian cells, and they have been found to associate with Cdk2 as well (Xiong et al., 1992). To address the question, which Cdk is important for the function of cyclin D3 in promoting polyploidy in megakaryocytes, Datta et al. conducted studies with a phorbol ester-treated megakaryocytic cell line (HEL cells). They showed that in these cells, the major kinase subunit associated with cyclin D3 is Cdk2, and that this Cdk2/cyclin D3 complex displays highly elevated kinase activity in polyploid cells (Datta et al., 1998). As to other cyclins, cyclin E levels in polyploid HEL cells were found to be comparable to those during S phase of normal cycling cells (Datta et al., 1998). The kinase activity of cyclinE/Cdk2, however, is upregulated at an early stage of phorbol ester-promoted differentiation. In cultured human CD34+ cells, the mRNA level of cyclin E was reported to be slightly upregulated after differentiation into the megakaryocyte lineage (Furukawa et al., 2000). The functional aspect of cyclin E during megakaryocyte polyploidization was further addressed in a study using K562 cells which do not become polyploid upon phorbol ester treatment. After ectopic expression of cyclin E, these cells developed polyploidy upon phorbol ester treatment (Garcia et al., 2000). The authors concluded that part of the role of cyclin E involves sustaining cyclin A levels which are otherwise downregulated by phorbol ester treatment of K562 cells. Similarly, cyclin A is present in primary megakaryocytes (Garcia et al., 2000), and cyclin A/ Cdk2 kinase activity was increased in HEL cells that were induced to polyploidize (Datta et al., 1998). Taken together, augmented levels of cyclin E and cyclin A may also be important for facilitating the re-entry into S phase during endomitosis, although currently no animal model has been produced yet to verify this contention.
Role of cell cycle inhibitors
Human megakaryocytes in culture, as well as mouse splenic megakaryocytes have been reported to express p21 and p27 inhibitors (Zimmet et al., 1998; Taniguchi et al., 1999). During human hematopoiesis, the expression of p21 and p27 is absent when DNA synthesis takes place (Taniguchi et al., 1999). In the case of murine megakaryocytes, the detection of p21 was followed by in-situ hybridization in spleen sections, revealing that only a fraction (up to 15%) of the cells expressed p21 (Zimmet et al., 1998). It is possible that this method is not as sensitive as immunohistochemistry in detecting low levels of p21 in the cells. This is a relevant point since it has been proposed that the effect of this inhibitor on the cell cycle depends on its concentration. For instance, at high levels it causes cell cycle arrest at G1 and G2/M (Dulic et al., 1998), while at low concentration it facilitates G1 progression by mediating an increased association of cyclin D3 with Cdk4 (LaBaer et al., 1997; Cheng et al., 1999). TPO was shown to increase transcription of the p21 gene in a megakaryocytic cell line (Matsumura et al., 1997), and in accordance, others have reported that forced expression of p21 in two megakaryocytic cell lines yielded cells with polylobulated nuclei (Kikuchi et al., 1997; Matsumura et al., 1997). Although ploidy was not assessed by flow cytometry in this case, the authors suggested a role for p21 in promoting polyploidization in megakaryocytes. This notion was encouraged by an earlier publication of increased frequency of polyploid hepatocytes that occurred in vivo upon overexpression of p21 (Wu et al., 1996). Later studies also indicated that forced expression of p21 led to endoreduplication and to high ploidy in other cell types (Niculescu et al., 1998), but paradoxically, p21 elimination caused centriole overduplication and polyploidy in human hematopoietic cells (Mantel et al., 1999). It is thus reasonable to suggest that the effect of p21 not only depend on its concentration but also on the cell type in which it is examined. For instance, high levels of p21 may inhibit entry into S in all cell types including megakaryocytes, but at low levels, p21 may enhance Cdk4 activity and promote ploidy in cells in which there exists the programming to skip cytokinesis. In the case of megakaryocytes, future studies involving regulated expression of p21 in primary cells and in vivo are needed to examine these possibilities. It is relevant to note, however, that mice lacking p21 show no change in platelet counts (Deng et al., 1995), suggesting that ploidy level is not affected in this mouse model. This may be due to the functional redundancy of p21 and p27 in megakaryocytes, as forced expression of p27 in a human megakaryoblastic cell line exhibited a megakaryocytic morphology observed upon p21 overexpression (Matsumura et al., 1997). Unlike p21, however, p27 mRNA level was not augmented when primary human CD34+ cells were induced to differentiate into megakaryocytes (Furukawa et al., 2000). In a megakryocytic cell line, differentiation correlated with a downregulation of p27, which resulted in increased cyclin D3- and cyclin A-associated kinase activities (Datta et al., 1998). The consequences of simultaneous elimination of p21 and p27 in vivo on megakaryocyte ploidy have not yet been studied.
Regulation of early M phase
An important question related to cells undergoing endomitosis is whether the mitotic cyclins and kinases are altered to promote this cell cycle. Studies in megakaryocytic cell lines suggested that a reduction in activity of the cyclin B-dependent Cdc2 kinase is an important part of megakaryocyte polyploidization (Datta et al., 1996; Zhang et al., 1996). The work of Datta et al., carried out with a clone of HEL cells, had shown a reduction in Cdc2 kinase activity with polyploidy, even though levels of cyclin B1 in this system were apparently unaltered (Datta et al., 1996). Other investigations with synchronized megakaryocytic cell lines found cyclin B1, but not Cdc2, to be reduced in polyploidizing cells (Zhang et al., 1996). In all of these studies, cell cycle protein levels were determined by Western blot analyses of equally loaded proteins derived from diploid, actively proliferating cells, as compared to polyploidizing cells. In contrast to cyclin B1, the levels of cyclin A per micrograms protein in these latter cell lines were similar between diploid and polyploid cells, suggesting that the frequency of cells entering S phase in these populations was similar. Zhang et al. have also shown an increased destruction of cyclin B1, but not cyclin A, by the ubiquitin-proteosome pathway, both in a polyploidizing megakaryocytic cell line and in high ploidy primary murine megakaryocytes. These authors suggested that the anaphase promoting complex is functional in these cells and that the potentially accelerated or premature destruction of cyclin B may allow re-entry into the S phase of the cell cycle, thus promoting polyploidization (Zhang et al., 1998). In accordance with the above-described results in megakaryocytic cell lines, it is important to note that in yeast, activation of the degradation system, which lowers the levels of cyclin B, was found to be essential for re-entry into S phase in cells that do not enter anaphase due to an experimentally-induced mitotic blockage (Zachariae and Nasmyth, 1996). In mitotic cells, one of the licensing factors that permits cells to exit anaphase and to enter cytokinesis involves the degradation of cyclin B by the Anaphase Promoting Complex (APC) and inactivation of Cdc2 kinase (Adachi and Laemmli, 1994). This feature would be important in cells that undergo polyploidization by the endomitotic cell cycle. Cyclin B1 and Cdc2 were also examined in primary megakaryocytes. Cyclin B1 was detected in TPO-treated polyploidizing human megakaryocytes by immunohistochemistry, flow cytometry, immunoprecipitation, and Western blotting (Vitrat et al., 1998). The immunofluorescence microscopy studies showed association of cyclin B1 with the mitotic spindle in human as well as murine polyploid megakaryocytes during early mitosis (Nagata et al., 1997; Vitrat et al., 1998). Analysis by flow cytometry, involving DNA staining and reaction of the human cells with an antibody to cyclin B1, indicated that cyclin B1 is expressed in megakaryocytes of all ploidy classes. Careful examination of these flow cytometry data, however, revealed that cyclin B1 was not detectable during S phase, while the ratio of cyclin B1 to ploidy level (cyclin B/DNA content) was significantly greater in the low ploidy cells as compared to high ploidy ones (Vitrat et al., 1998). This may be due to the existence of a higher fraction of resting cells (in which cyclin B1 is never elevated) in the highly polyploid population. Alternatively, or in addition, this could reflect an accelerated degradation of cyclin B in the high ploidy cells in accordance with an earlier report on a megakaryocytic cell line (Zhang et al., 1998). In a very recent study with TPO-treated murine megakaryocytes, cyclin B1 levels were also followed by flow cytometry analysis of cells of different ploidy class (Crow et al., 2001), using a different antibody to cyclin B1 than the one used in the study of human cells (Vitrat et al., 1998). This investigation represents a unique effort to obtain large quantities of megakaryocytes selected from mouse bone marrow. The authors determined that on average, 36% of 8n-32n megakaryocytes expressed abundant cyclin B1 during G2/M and that the level of cyclin B1 per G2/M increased linearly with ploidy (after correction for cell size). This later conclusion contradicts somewhat the above-described reports on human samples. Crow et al. also reported that cyclin B expression oscillated normally in murine megakaryocytes undergoing endomitosis, although cyclin B1 never really disappeared. It is worth pointing out that the flow cytometry method of detection must rely on a careful choice of cyclin B antibody which should display specific staining by Western blotting, as all proteins (specific and others) will react with the antibody and display a positive signal in a ploidy class examined by flow cytometry. Western blot assays were also used to explore whether the levels of mitotic regulators are comparable in diploid and polyploid megakaryocytes of primary origin. Analyses of murine or human megakaryocytes indicated that high ploidy cells and diploid/tetraploid megakaryocytes or diploid, nonmegakaryocytic bone marrow cells, all contained similar levels of cyclin B1. Cdc2 levels in these populations were equivalent or somewhat reduced in the diploid cells (Vitrat et al., 1998; Bassini et al., 1999; Crow et al., 2001). In these investigations, however, the cells examined were not synchronized and the percentage of cycling cells in each population (diploid vs. polyploid) was not determined. This parameter might influence the profile of cyclins detected. Nevertheless, it has become clear that polyploidizing megakaryocytes contain significant levels of cyclin B1. Immunoprecipitation of cyclin B1-associated proteins showed a comparable level of H1 histone kinase activity in nocodazole-blocked human polyploid megakaryocytes as compared to the 2n/4n population, but Cdc2-associated ones were not examined in this study (Vitrat et al., 1998). A recent report on human primary, TPO-treated megakaryocytes that were subjected to immunohistochemistry, further supported the conclusion that cyclin B1 was associated with the mitotic spindle during polyploidization, but also indicated that it was undetectable at anaphase (Roy et al., 2001). This too sustained earlier findings (indicated as data not shown) that cyclin B1 is significantly reduced at anaphase of polyploidizing TPO-treated mouse megakaryocytes (Nagata et al., 1997). Roy et al., hence, deduced that the APC component responsible for cyclin B degradation is active in polyploidizing human megakaryocytes (Roy et al., 2001), as previously concluded with a megakaryocytic cell line and TPO-treated murine bone marrow cells (Zhang et al., 1998). Whether cyclin B1 degradation is accelerated in primary megakaryocytes undergoing endomitosis, as compared to proliferating ones, remains to be explored. Gu et al. examined non-TPO treated human bone marrow by immunohistochemistry and electron microscopy, and reported significant staining with an antibody to Cdc2 in all megakaryocytes and other bone marrow cells. Anti cyclin B1, on the other hand, showed negligible staining in megakaryocytes but strong staining in granulocytes, monocytes, and macrophages (Gu et al., 1993; Wang et al., 1995). Since only 15% of bone marrow megakaryocytes are actively involved in polyploidization (Wang et al., 1995), it is possible that the majority of cells examined were in a quiescent state.
Studies conducted with several cell systems and by the use of different methods of detection have led thus far to the following conclusion: it appears that although megakaryocytes undergoing an endomitotic cell cycle display cyclin B1 associated with the mitotic spindle, which would allow Cdc2 activation and chromosome condensation, cyclin B1 is undetectable at anaphase. Hence, it has been suggested that the proteosome components responsible for cyclin B degradation are functional during polyploidization. The extent to which the APC is active during late anaphase in megakaryocytes undergoing endomitosis, and whether this indeed facilitates re-entry into S phase in the absence of late anaphase and cytokinesis, awaits further examination. In addition, the current studies did not rule out the possibility that the rate of degradation of selected regulatory proteins, which are processed differently than cyclin B, is altered during endomitosis.
Regulation of late anaphase and cytokinesis
As detailed above, megakaryocytes are unique in that during the formation of a high ploidy state, the cells skip late anaphase and cytokinesis. In the search for potential mechanisms leading to high ploidy in this lineage, it is important to review some of the reports on regulators of cytokinesis or anaphase in different systems. Bhat et al. described the importance of a chromosome-associated protein for anaphase, the barren gene product in Drosophila, which interacts with Topoisomerase II in final chromatid segregation at anaphase (Bhat et al., 1996). In accordance with this observation, it has been demonstrated in a protozoan that a telomerase template mutation causes a block in anaphase (Kirk et al., 1997). In fission yeast, a mutation in the cut8+ gene, which encodes a protein kinase, was found to block anaphase (Samejima and Yanagida, 1994). In mammalian cells, the mitotic kinesin-like protein 1 is co-localized with Plk kinase, an analog of the Drosophilapolo gene product. This occurs during late M phase, thus implying the potential importance of kinesin and Plk kinase for late anaphase (Lee et al., 1995). As to address cytokinesis, several genes have been reported to be necessary for the proper function of the actin or microtubule cytoskeleton and cytokinesis, including the Pom1p protein kinase in yeast (Bahler and Pringle, 1998) and the human homolog of a tumor suppressor called adenomatous polyposis coli protein (Lee et al., 1995). Other genetic studies in yeast identified genes that are crucial for proper cytokinesis to occur, including the genes encoding Myo2p (a myosin heavy chain), a novel protein kinase, a GTP binding protein, a member of the ras superfamily of GTPases, and the multisubunit general transcription factor TFIID (Balasubramanian et al., 1998; Eng et al., 1998; Yamamoto and Horikoshi, 1998). These studies indicate that anaphase as well as cytokinesis depend on several regulators, and the mutation of each individual leads to an inability to enter anaphase or to follow cytokinesis. Any one of these above described gene products may be modified in polyploidizing megakaryocytes. In a search for such regulators, AIM-1 kinase, a member of the Aurora family of kinases, became a center of interest because of its downregulation in megakaryocytic cell lines treated with TPO and its lack of detection in primary murine, or human megakaryocytes undergoing polyploidization (Kawasaki et al., 2001; Zhang et al., 2001). In a study by Terada et al., the authors used a degenerate cloning method based on serine/threonine kinases (using the NRK-49F rat cell library) that revealed 12 new cDNAs, of which one was associated with M phase (Terada et al., 1998). Because of its sequence and potentially functional homology, it was termed: Aurora and Ipl1-like Midbody-associated protein (AIM-1). The Drosophila serine/threonine protein kinase Aurora is known to be required for progression through M phase. Females mutant aurora produce embryos in which centrosomes remain closely paired with no completion of cellular division (Glover et al., 1995). Thus, loss of function of this kinase leads to a failure of the centrosomes to separate and form a bipolar spindle. An analogous gene was identified in S. cerevisiae, encoding Ipl1 kinase. Loss of this kinase in yeast caused chromosome missegregation and cell death. In these cells, it was observed that unequal numbers of chromosomes segregated to the two poles (Francisco et al., 1994), reminiscent of what was described recently for poplyploidizing megakaryocytes (Roy et al., 2001). An additional protein that has sequence similarity to Aurora and Ipl1 is the human Aik/Ayk kinase. This kinase was found to be high at entry to M phase and localized to the spindle pole from prophase to anaphase (Kimura et al., 1997). A recent study identified myosin II regulatory light chain (MRLC) as a substrate for AIM-1 (Murata-Hori et al., 2000). This is very relevant, since MRLC is important in generating force in nonmuscle cells out, and is co-localized with AIM-1 at the cleavage furrow of dividing cells, which implies a role in cytokinesis. Studies in a subclone (Y10) of the L8057 murine megakaryoblastic cell line (Ishida et al., 1993), which is capable of expressing differentiation markers and of polyploidizing in response to TPO (Zhang et al., 1998), demonstrated that AIM-1 kinase mRNA was downregulated by this cytokine. The effect appeared post expression of differentiation markers and at the onset of polyploidization. This downregulation was found to be on the transcriptional level and not due to message destabilization (Zhang et al., 2001). Additional recent investigations demonstrated that overexpression of AIM-1 in megakaryocytic cell lines suppress ligand-induced polyploidization (Kawasaki et al., 2001). Studies in primary cells and in vivo should be pursued to further confirm a cause-effect relationship between AIM-1 suppression and induction of high ploidy.
Crucial choice of cell systems and methods to study endomitosis in megakaryocytes
Several lessons may be extracted from the above-described studies as to which systems and methods are essential tools for investigating endomitosis. The study with megakaryocytic cell lines involves a comparison of actively proliferating, diploid cells, and polyploidizing cells. When comparing the level of cell cycle proteins in these two populations, it is important to establish that the percentage of cycling cells is not significantly lower in the polyploid population, as this by itself will lead to a lower detection of cell cycle regulators in a nonsynchronized population. In some of the studies described with megakaryocytic cell lines (e.g., Zhang et al., 1998), cells were synchronized and DNA synthesis and G1 phase cyclins were determined to indicate a regulation of expression of selected cyclins in actively cycling cells. Pursuing such experiments with primary megakaryocytes has been technically challenging because of the rarity of this cell type in the marrow. Although the megakaryocytic cell lines used display typical differentiation markers and polyploidize in response to TPO, they are transformed in nature and the regulation of their endomitotic cell cycle may reflect a special case. Hence, the use of cell lines may only be a lead for obligatory studies with primary cells. Experience had thought that the study with primary cells has to be carried out, however, with TPO-stimulated samples, since the percentage of cycling megakaryocytes in freshly derived bone marrow is relatively low, up to 15% (Wang et al., 1995). Moreover, the low frequency of these cycling megakaryocytes (less than 0.01% of total bone marrow cells) renders the biochemical studies impossible to pursue. In the TPO-stimulated system too, it is important to establish the percentage of cycling cells in the diploid and polyploid populations in order to attribute changes in cell cycle modulators within each population to cell cycle regulation (and not as a mere reflection of cell cycle arrest). The challenge, however, would still be to obtain sufficient levels of extremely rare diploid megakaryocytes and the uncertainty about their cycling state. The latter could be assessed by determination of percentage of cells involved in DNA synthesis by in situ staining, and/or by quantitative measurements of a variety of cyclins in the sorted cells. Because of the paucity of diploid megakaryocytes in the marrow, investigators may also elect to compare these parameters in polyploidizing megakaryocytes and diploid bone marrow cells of other lineages, but the same caution has to be applied to interpretation. In choosing an assay to detect changes in cell cycle proteins in polyploidizing cells, several methods have been considered. Western blot analysis of proteins derived from sorted 2n–4n megakaryocytes as compared to > 4n cells has been pursued in several studies. The difficulty with this assay, which relies on cell sorting, is that the 2n–4n population of cells represents only 5–10% of total megakaryocytes and < 0.005% of bone marrow cells, hence calling for extreme caution with the determination of purity in the diploid population. Western blotting, however, presents the advantage of detecting a specific band of a correct molecular weight, regardless of cross reactivity of the antibody used with other proteins. This is certainly a concern with most of the antibodies used to detect cyclin B1, a center of attention in the study of megakaryocyte ploidy. As an alternative method, immunohistochemistry had proven most useful in detecting cyclins during endomitosis, as attention has been given to the cellular localization of a cyclin in relation to chromosome organization and cell cycle progression. In utilizing out immunostaining flow cytometry to determine cyclin levels in different ploidy states, special consideration has to be given to the quality of the antibody used. An antibody that reacts with several proteins (as indicated by Western blotting) may identify the specific and nonspecific proteins in different ploidy classes surveyed by flow cytometry, potentially leading to false conclusions. Competition with specific peptides may prove useful in these cases. Jacobberger et al. (1999) described the use of cyclin B antibody in immunofluorescence flow cytometry and the difficulties involved in it. The issues related to estimating the kinetics of cell cycle-related gene expression in G1 and G2 phases by the use of flow cytometry are also described in Jacobberger et al. (1999). In addition, while Western blotting evaluates the level of a specific protein per cellular microgram protein, flow cytometry evaluates a protein level within cells of different sizes. Since high ploidy cells are large in size, this has to be factored in the evaluation of a potential increase in the intensity of an antibody labeling. An increase in a cyclin level, after correction for size may indicate a lower rate of synthesis or higher degradation rate of the specific cyclin.
From what is described above, it appears that high ploidy is achieved by an orchestrated regulation of several processes. An interesting question would then be, how can ploidy be induced in mitotic cells of other lineages? One may suggest that a cellular programming that involves simultaneous abrogation of some stage of mitosis and cytokinesis, and upregulation of G1 phase components, such as cyclin D3, will induce ploidy more effectively than regulating either of these components singly.
REGULATION OF PLOIDY IN OTHER CELL TYPES: A COMPARISON TO THE MEGAKARYOCYTE
Genetic mutations that lead to DNA re-replication in yeast
Mutations affecting the cyclin B/cdc2 kinase
A large number of mutants involving the cdc2 kinase have been found to lead to DNA re-replication in yeast. Among these are temperature-sensitive mutants of cdc2 itself (Broek et al., 1991) and cdc13, the mitotic B-type cyclin in Saccharomyces. pombe (Hayles et al., 1994). In both of these cases, inactivation of the mutant gene during G2 is associated with a loss of cdc13-cdc2 kinase activity and the subsequent development of polyploidy. An identical phenotype is observed in S. cerevisiae mutants of cdc28, the budding yeast cdc2 homologue (Dahmann et al., 1995). The rum1 protein, which is a specific inhibitor of the cdc2 kinase in association with cdc13, has a similar effect when overexpressed in G2 cells (Correa-Bordes and Nurse, 1995). Overexpression of rum1 has no effect on a substantial portion of cdc2 kinase activity, most likely that associated with other B cyclins such as cig1 and cig2, suggesting that the Cdk activity which normally prevents re-replication is specific and requires particular partners. Rum1 is normally degraded through the ubiquitin-dependent proteosome pathway, to which it is targeted by phosphorylation at key residues by cyclin-cdc2 kinases (Benito et al., 1998). Mutation of these residues to alanine leads to stabilization of the protein and ensuing polyploidization. The S. pombe gene pop1, whose mutation leads to increased ploidy, also acts through this pathway. Pop1 is thought to have a role in the recognition and ubiquitination of both Rum1 and cdc18, an S-phase initiator. Mutants therefore accumulate high levels of both of these proteins. Another set of mutants from S. cerevisiae, the sim1 and sim2 mutations, also appears to allow a second round of DNA synthesis in G2 through a related mechanism (Dahmann et al., 1995). While the precise manner in which these proteins act is unknown (they may contain transcriptional repressive activity (Ema et al., 1996), the manner in which they promote re-replication is suggested by the observation that, while G2-arrested wildtype cells maintain high levels of the Clb5p-cdc28 kinase, these levels drop off quickly in sim1 and sim2 mutants. A similar phenotype is gained by overexpression of an inhibitor of the Clb5p-Cdc28 kinase, p40SIC1, in G2 cells. Either mutation of the SIM proteins or overexpression of p40SIC1 results in a loss of kinase activity that allows pre-replication complexes to re-form (Dahmann et al., 1995). Activation of the cdc2 kinase depends not only on binding to B cyclin regulatory subunits but also on dephosphorylation of key residues by cdc25 phosphatases, particularly cdc25C (Galaktionov et al., 1995). The 14-3-3δ gene was recently isolated as a p53-regulated element of the response to DNA damage (Hermeking et al., 1997). Overexpression of 14-3-3δ in human colon cancer cells resulted in growth arrest followed by polyploidization. Reports of 14-3-3δ binding and inactivation of cdc25C (Peng et al., 1997) have led to the speculation that overexpression of this protein leads to polyploidy through sequestration of cdc25C and subsequent failure to activate the cdc2 kinase. It is of interest to note that in some megakaryocytic cell lines, polyploidy was associated with downregulation of Cdc25, which leads to downregulation of Cdc2 kinase (Garcia and Cales, 1996).
Regulation of ubiquitin-mediated proteolysis in M phase: the APC
Entry into anaphase requires the ubiquitin-mediated proteolysis of several key proteins, including cyclin B (Zachariae and Nasmyth, 1996). As indicated above, this destruction is mediated by a complex of proteins known as the APC, which acts as a cell-cycle regulated ubiquitin-protein ligase. A screen for S. cerevisiae mutants that resulted in excess DNA accumulation (Heichman and Roberts, 1996) resulted in the isolation of two members of the APC, cdc16 and cdc27 (Zachariae et al., 1996). Mutants of these genes arrest in a G2-like state and develop polyploid DNA contents. Morphological evidence suggests that this DNA over-replication occurs within the context of a single cell cycle. The inability of these cells to effectively target and destroy B cyclins explains the presence of high clb2-cdc28 kinase activity in these cells, and indicates that the APC acts downstream of mitotic Cdk activity in preventing re-replication of DNA. The clb2-cdc28 kinase may have a role in activation of the APC, or in targeting replication proteins for APC-mediated destruction.
cdc6p is a S. cerevisiae protein that associates with the origin recognition complex (ORC) and is involved in the assembly of the pre-replication (pre-RC) complex (Liang et al., 1995). Whereas the ORC complex remains bound to chromatin throughout the cell cycle, the MCM proteins cycle on and off during the cycle. A cdc6 mutation has been shown to lead to polyploidization (Liang and Stillman, 1997). Investigation into possible mechanisms revealed that these mutants display constant interaction of the MCM proteins with chromatin for the duration of the cell cycle, possibly allowing for continuous replication competence. Overexpression of the S. pombe homologue, cdc18, also leads to over-replication of DNA (Nishitani and Nurse, 1995). The highly variable DNA contents in these mutant cells suggests that, unlike mutants affecting cdc2, cdc18 overexpression leads to multiple rounds of incomplete DNA replication.
Chromosome segregation mutants
Several yeast mutations display an ability to continue cell cycle progression despite missegregation of chromosomes. The S. cerevisiaeesp1 (Baum et al., 1988) and mps1 (Winey et al., 1991) mutants cause defects in spindle pole body segregation and duplication. The esp1 mutant, although largely lethal, does result in significant polyploidy among surviving cells that is associated with a large reduction in mitotic cdc28 kinase activity (Surana et al., 1993). Mutants of related genes cut1 from S. pombe (Uzawa et al., 1990) and bimB3 from A. nidulans (May et al., 1992) have similar DNA re-replication phenotypes. The unrelated dim1 mutant, which has a suspected role in chromosome segregation during mitosis, also leads to polyploidy (Berry and Gould, 1997).
Mechanisms of polyploidy in Drosophila
Polyploidy is also observed during the various stages of Drosophila embryonic development (reviewed in Edgar and Orr-Weaver, 2001). During the early cycles involving mitotic nuclear divisions, two observations demonstrate that there are marked differences from normal cell cycle control. First, cyclins A and B are present at constant levels. And second, activity of the cdc2 kinase appears to be constitutively high. Several possibilities have been put forth to explain this, including the possibility of localized fluctuations in cyclin levels and cdc2 activity, or of the simple inability to detect oscillations due to the high frequency of these cycles. Whether the later endocycles involve modulation of known cell cycle regulatory elements or some novel mechanism is presently unknown. It is clear, however, that these cycles constitute true endoreduplication–alternating synthesis and gap phases with no detectable entry into mitosis. Under-replication of late-replicating DNA sequences occurs from the first endocycle. Among known cell cycle genes, only cyclin E has been demonstrated to act in both the mitotic and endocycles (Knoblich et al., 1994), triggering S phase entry in each endocycle through its oscillations (Lilly and Spradling, 1996). Activity of the cdc2 kinase is notably absent in this process. In fact, endoreplication appears to be unaffected by mutations not only in the cdc2 gene but also in cyclin A and cyclin B, which are likewise absent during this process (Edgar and Lehner, 1996). A Drosophila homologue of cdk2, known as cdc2c, does appear to be present in the endocycle, forming an active kinase in complex with cyclin E. A different study has shown that a novel gene, fizzy-related (fzr), is required for the transition from mitotic to endocycles, and appears to have a role in the degradation of mitotic cyclins A, B and B3 (Sigrist and Lehner, 1997). Fzr and related proteins may be involved in the regulatory and targeting process of the anaphase promoting complex (APC) (Kallio et al., 1998).
Mechanisms of polyploidy in mammalian cells
Inhibition of mitotic kinases and cyclin B degradation can lead to re-replication in mammalian cells
Treatment of G2 HeLa nuclei with the kinase inhibitor 6-dimethylaminopurine (6-DMAP) allowed a further round of DNA replication without either passage through mitosis or nuclear envelope breakdown (Coverley et al., 1996). 6-DMAP treatment in G2 was shown to induce MCM proteins to re-associate with chromatin, suggesting that a cell cycle-regulated kinase controls MCM binding. In a different study, treatment with the protein kinase inhibitor K-252a was able to induce rat fibroblastic cell lines in culture to polyploidize. Similarly, nocodazole-arrested mouse mammary tumor cells polyploidized when treated with the protein kinase inhibitor staurosporine (Hall et al., 1996). In both cases, at least a partial inhibition of in vivo p34cdc2 kinase activity was associated with kinase inhibition. Cell cycle analysis of a rat choriocarcinoma cell line has shown that high ploidy in these cells is associated with lowered Cdc2 kinase activity and suppression of cyclin B1 expression (MacAuley et al., 1998). Recently, Hixon et al. had shown that in vascular smooth muscle cells polyploidy is associated with reduced cyclin B1 expression and enhanced Cks1, a Cdc2 adapter protein that promotes cyclin B1 degradation, while forced expression of Cks1 increased ploidy. These authors did not examine whether cyclin B was associated with mitotic spindles during a possible early mitosis in these cells. However, based on the above measurements of cyclin B1 in the populations of mitotic and polyploid cells, it was suggested that enhanced degradation of cyclin B1 by Cks1 is responsible for the increase in ploidy (Hixon et al., 2000). As indicated above, cyclin B1 is degraded during anaphase of polyploidizing megakaryocytes (Hixon et al., 2000), and the proteosome pathway responsible for this degradation is enhanced in extracts derived from murine megakaryocytes (Zhang et al., 1998).
Polo family of kinases
The polo kinase was originally identified in Drosophila as a mutant that led to a variety of abnormal mitoses, including polyploid complements of chromosomes. This has led to the discovery of a family of related serine-threonine kinases which has been labeled the polo-like kinases (Golsteyn et al., 1996). The homologues from yeast, Cdc5p (S. cerevisiae) and plo1 (S. pombe), as well as the identified mammalian homologue, polo-like kinase 1 (Plk1), have been shown to function in spindle assembly during mitosis. A study has shown that the yeast Cdc5p may be involved in APC function, both as an activator of proteolysis of particular targets, as well as a substrate (Shirayama et al., 1998). A possible link to the Cdc2 kinase is provided by results showing that the Xenopus homologue, Plx1, is able to phosphorylate and activate Cdc25 in vitro, and microinjection of Plx1 antibody or dominant-negative Plx1 inhibits Cdc25 activation (Qian et al., 1998).
Microtubule inhibitors and the spindle checkpoint
Interruption of the mitotic spindle using microtubule inhibiting drugs leads to M phase arrest. The yeast genes mad2 (Li and Murray, 1991) and bub1 (Hoyt et al., 1991) are components of this checkpoint, and cells harboring mutations in either gene fail to arrest in response to anti-microtubule agents, continuing through multiple rounds of DNA replication. Mammalian cells injected with antibody to the human mad2 homologue, hsMAD2, fail to arrest when treated with nocodazole (Li and Benezra, 1996). Similarly, cells expressing a dominant negative murine Bub1 arrest with a much lower frequency than normal during nocodazole treatment (Taylor and McKeon, 1997). Hence, the true spindle checkpoint genes in mammalian cells, would be those as MAD2 and BUB1, with their inactivation results in bypass of the mitotic arrest (Li et al., 1997; Taylor and McKeon, 1997). Targets of the spindle checkpoint have recently been identified through interactions with Mad proteins in yeast. These targets, the Slp1 gene in fission yeast (Kim et al., 1998) and the Cdc20 gene in budding yeast (Hwang et al., 1998), are important for M phase progression. The spindle and DNA damage checkpoints are, therefore, hypothesized to enforce mitotic arrest through inhibition of these genes. Thus, Cdc20 overexpression is able to overcome the spindle checkpoint to achieve polyploidy, and Slp1 mutants that are unable to interact with Mad proteins bypass the checkpoint in a similar manner. Treatment of mammalian cells with nocodazole or other microtubule-inhibiting agents also activates the spindle checkpoint, leading to mitotic arrest. The tumor suppressor genes p53 and pRb have been implicated in this process (Cross et al., 1995). Mouse embryonic fibroblasts (MEFs) deficient in p53 or pRb fail to arrest when cultured in the presence of these drugs and continue through one or more additional round of DNA replication (Di Leonardo et al., 1997; Khan and Wahl, 1998). Deficiency in the Cdk inhibitor p21 has a similar effect, suggesting that p53 spindle checkpoint function acts through this effector. MEFs lacking p53 or p21 arrested in M phase for the same period of time as wild type MEFs, but were subsequently able to escape the interphase block and continue with another round of replication. Thus p53 could not be labeled a true mitotic checkpoint, since cells lacking the gene behave identically to wild type cells at mitosis (Lanni and Jacks, 1998).
Polyploidy due to forced expression of proto-oncogenes in vivo
Lundgren et al. expressed the proto-oncogene MDM2 in mammary epithelial tissue of transgenic mice. This resulted in polyploidy in approximately one-third of transgenic cells (Lundgren et al., 1997). MDM2 inhibits p53 transcriptional activity by binding the p53 acidic activation domain (Momand et al., 1992; Oliner et al., 1993). Similar results were obtained on a p53−/− background, indicating that the acquisition of polyploidy was not dependent upon the interaction of MDM2 with p53. Overexpression of cyclin D1 in cardiac myocytes in vivo produced polyploid cells, but as in the case of MDM2, it was only in a fraction of the cells, the ploidy level was relatively low and some of the cells were binuclear (Soonpaa et al., 1997). Interestingly, moderate overexpression of cyclin D1 in megakaryocytes of transgenic mice also induced a moderate augmentation in ploidy level (Sun et al., 2001).
Various cells develop low levels of ploidy (4n–16n) during normal development, as a response to stress or as the result of a single mutation. The latter example prevails mainly in yeast cells and frequently involves genes regulating mitosis and/or cytokinesis. Megakaryocytes are unique among mammalian cells in that they attain high ploidy (predominantly 16n and higher) during normal development. Current studies indicate that both the switch from a mitotic to an endomitotic cell cycle in this lineage and the acquisition of high ploidy depend on an orchestrated regulation of several genes. As ploidy in various cell types is achieved by skipping different cell cycle phases (e.g., part of mitosis or all of it and/or cytokinesis), it is expected that somewhat different genetic programs control these shifts to high ploidy. One interesting future pursuit would be to study the common and distinct genetic controls in all of these systems. Studies with megakaryocytes indicate that cells progress through mitosis but skip anaphase B and cytokinesis, thus suggesting that the simultaneous upregulation of G1 phase components, such as cyclin D3, and modified expression of late mitotic and cytokinetic regulators are needed to achieve high ploidy. Several intriguing questions remain regarding the effect of TPO on this process. Although it is not yet clear what are all the components of late mitosis and cytokinesis that are modified by TPO, some studies suggest the involvement of AIM-1 kinase in this process. This cytokine promotes mitotic cell cycle entry and proliferation in precursor cells of the megakaryocytic lineage as well as at one point, the shift to an endomitotic cell cycle. Is this dual role made possible due to TPO-induced changes of gene expression in the proliferating cells, leading the altered cells to further respond to this cytokine by polyploidizing, or do precursor cells rely on an intrinsic oscillator that dictates the number of proliferation cycles prior to endomitotic cycles? It is also not yet clear why the majority of megakaryocytes in the marrow of different species contain a 16n content of DNA. This could reflect a dependency on a programmed oscillator that leads most of the cells to cycle up to 16n, and/or may indicate that the cells with a ploidy higher than 16n undergo rapid fragmentation, apoptosis, and clearance. Exploring these possibilities will enhance our understanding of cell cycle control and would provide a means by which the level of ploidy in the cell may be regulated. Manipulation of such molecular mechanisms would be important under conditions where the degree of ploidy impacts both the level and profile of gene expression and ultimately, the cellular physiology.
We thank Dr. William Vainchenker and Dr. Leah Cataldo for reading portions of this review and for valuable comments. We apologize to those whose studies were not quoted because of limited space.