Endoreplication Cell Cycles
2001; Cell Press; Volume: 105; Issue: 3 Linguagem: Inglês
10.1016/s0092-8674(01)00334-8
ISSN1097-4172
AutoresBruce A. Edgar, Terry L. Orr‐Weaver,
Tópico(s)Plant Molecular Biology Research
ResumoMolecular studies from the past decade have revealed striking conservation in the mechanisms of eukaryotic cell cycle control. Yet before the advent of molecular genetics, it was clear that eukaryotes possessed many different cell cycle variations, and thus that there must be diversity in mechanisms of control. One common cell cycle variant is the endoreplication cycle, in which cells increase their genomic DNA content without dividing. Although endocycles are sometimes dismissed as an evolutionary peculiarity, they are widespread in protists, plants, and many animals including arthropods, mollusks, and mammals. Endocycling cells can become incredibly polyploid, with chromatin values (C values denote DNA content as a multiple of the normal haploid genome) as high as 24,000 reported in some plant endosperms (Traas et al. 1998Traas J. Hülskamp M. Gendreau E. Höfte H. Endoreplication and development rule without dividing?.Curr. Opin. Plant Biol. 1998; 1: 498-503Crossref PubMed Scopus (164) Google Scholar). Because cell size for a given cell type is generally proportional to the amount of nuclear DNA, endoreplication constitutes an effective strategy of cell growth, and it is often found in differentiated cells that are large or highly metabolically active (see Figure 1 for a classic example). Recent studies show that endocycles utilize much of the same G1/S regulatory machinery as mitotic cycles, with some informative, and some perplexing, alterations. Here we review what is known about the different endocycle types found in nature and how they are regulated. We also offer some ideas about why endocycles are so commonly used. Endoreplication, also known as endoreduplication, gives rise to cells with extra copies of the genomic DNA. In many cases the chromosome number is increased in multiples of N (the normal haploid chromosome number). Such cells are referred to as polyploid or endopolyploid (Figure 2A). Endoreplicated cells in which the sister chromatids remain closely associated are referred to as polytene (Figure 2B). Thus whereas sperm nuclei are 1N, 1C, diploid cells in G1 are 2N, 2C, and diploid cells in G2 are 2N, 4C, a polyploid cell might be 32N, 32C or 32N, 64C. Polytene cells are normally descended from diploid progenitors, but since homologs are paired, the equivalent polytene cell would be 1N, 64C (White 1973White M.J.D. Animal Cytology and Evolution. Third Edition. Cambridge University Press, Cambridge1973Google Scholar). The best known example of polyteny is found in the giant salivary gland chromosomes of Drosophila, which have up to 2048 copies of the euchromatic genome neatly aligned in parallel arrays (see Urata et al. 1995Urata Y. Parmelee S.J. Agard D.A. Sedat J.W. A three-dimensional structural dissection of Drosophila polytene chromosomes.J. Cell Biol. 1995; 131: 279-295Crossref PubMed Scopus (30) Google Scholar), but polytene chromosomes greater than 16,000C have been noted in another insect, Chironomus. The distinction between polyteny and polyploidy is not absolute, and various intermediate DNA configurations can occur, differing primarily in the degree of association between duplicated chromatids (Figure 2D, Hammond and Laird 1985Hammond M.P. Laird C.D. Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster.Chromosoma. 1985; 91: 267-278Crossref PubMed Scopus (80) Google Scholar). For this reason the term polyploid is often used to refer to endoreplicated cells with virtually any chromosomal configuration. In endoreplication cell cycles, or endocycles, S phases alternate with distinct gap phases that lack DNA replication, but there is no cell division (Figure 3). Few, if any, cases of polypoidy resulting from continuous DNA replication have been reported. Some endocycling cell types retain hallmarks of mitosis, but many examples lack all vestiges of mitosis, including chromosome condensation, nuclear envelope breakdown, and the reorganization of microtubules that builds the spindle. The term endomitosis initially referred to a rare cell cycle in which mitosis occurred without nuclear envelope breakdown or cytokinesis (for review see Nagl 1978Nagl W. Endopolyploidy and Polyteny in Differentiation and Evolution. North-Holland Publishing Company, Amsterdam1978Google Scholar). However, now this term is more generally used to describe cycles that proceed through anaphase but lack nuclear division and cytokinesis. In yet another cell cycle variant, nuclear division occurs without cytokinesis, giving rise to multinucleate cells. Such cycles are seen in mammalian hepatocytes and osteoclasts, and also in syncytial slime molds like Physarum and early insect embryos. The regulation of these cycles is thought to be similar to that found in normal mitotic cells, and is not covered here. In proliferating cells, phosphorylation events triggered by cyclin-dependent kinases (CDKs) control the onset of both mitosis and S phase. Mitotic control is mediated by A and B type cyclins complexed with Cdk1, and also by Cdc25 type phosphatases that serve to activate Cdk1. S phase inititation and progression are mediated by Cyclin E/Cdk2 and Cyclin A/Cdk2 complexes, whereas earlier cell cycle events including G0→G1 transitions involve the D type cyclins complexed with Cdks 4 and 6. Endoreplicating cells appear to have simplified this regulatory machinery by eliminating the expression of components that are no longer required. For instance, some endoreplicating cell types that bypass mitosis do not express Cdk1 or its activators, Cyclin B, Cyclin A, and Cdc25C. Cell types in which mitosis is partially traversed but incomplete have been reported to have lowered levels of these G2/M regulators. This suggests that levels of mitotic CDK activity may be responsible for determining the extent of mitotic functions retained in an endocycling cell. However, some studies have found that endocycling cells can also lose structural components required for mitosis, such as centrosomes (Mahowald et al. 1979Mahowald A.P. Caulton J.H. Edwards M.K. Floyd A.D. Loss of centrioles and polyploidization in follicle cells of Drosophila melanogaster.Exp. Cell Res. 1979; 118: 404-410Crossref PubMed Scopus (48) Google Scholar). Although the loss of structural mitotic components is a plausible mechanism for initiating endocycling, it could also be a secondary event with little functional relevance. What enables endocycling cells to replicate their DNA many times within a single interphase, and thus escape the mechanisms that block rereplication in mitotic cells? This is not entirely clear yet, but work in diverse mitotic systems does suggest what sort of mechanisms to expect. Many recent studies indicate that after completion of an S phase, cyclin/CDK activity must drop to low levels before rereplication is possible. A window of low CDK activity is initiated by destruction of the G2 cyclins during late metaphase by the anaphase promoting complex (APC), which remains active through early G1. Low CDK activity during early G1 allows the assembly of prereplication complexes (preRCs) containing ORC proteins, Cdc6, Cdt1/Dup1, and MCMs onto DNA replication origins, thus “licensing” the DNA for another round of replication. Cdc6 and the MCM proteins are removed from the DNA during and after S phase, and their ability to rebind and relicense the DNA for replication is inhibited during the S and G2 phases by high levels of Cdk2 and Cdk1 activity. It is only after levels of Cdk activity drop during the M→G1 transition that relicensing becomes possible again. In this way only a single round of DNA replication is allowed in each mitotic cycle (see Donaldson and Blow 1999Donaldson A.D. Blow J.J. The regulation of replication origin activation.Curr. Opin. Genet. Dev. 1999; 9: 62-68Crossref PubMed Scopus (74) Google Scholar, for review). Other switches that are built into this CDK↔APC tug-of-war may further ensure against inappropriate rereplication. For instance CDK inhibitors such as Sic1 in yeast and p27Kip1 and p57Kip2 in vertebrates can be targeted for turnover by CDK-dependent phosphorylation, and Geminin, a protein that sequesters Cdt1/Dup1 and thus inhibits replication licensing, is periodically degraded at anaphase, probably by the APC (Tada et al. 2001Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Repression of origin assembly in metaphase depends on inhibition of RLF- B/Cdt1 by geminin.Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (385) Google Scholar, Wohlschlegel et al. 2000Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Inhibition of eukaryotic DNA replication by geminin binding to cdt1.Science. 2000; 290: 2309-2312Crossref PubMed Scopus (555) Google Scholar). In endoreplicating cells, these mechanisms could in principle confer intrinsic oscillatory capability upon the S phase kinase, CycE/Cdk2, and thus also upon the machinery that first assembles and then “fires” (activates) preRCs. Oscillations in Cyclin E activity and MCM localization (Su and O'Farrell 1998Su T.T. O'Farrell P.H. Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.J. Cell Biol. 1998; 140: 451-460Crossref PubMed Scopus (49) Google Scholar) have been reported in endocycles, and the Geminin target Cdt1/Dup1 also appears to be used (Whittaker et al. 2000Whittaker A.J. Royzman I. Orr-Weaver T.L. Drosophila double parked a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts.Genes Dev. 2000; 14: 1765-1776PubMed Google Scholar). Moreover, the fact that all endocycles exhibit gap phases suggests that, as in mitotic cycles, CDK activity must periodically cease to allow reassembly of preRCs and renewed DNA replication. Indeed, enforcing continuous CycE/Cdk2 activity causes endocycles to stall in Drosophila (Follette et al. 1998Follette P.J. Duronio R.J. O'Farrell P.H. Fluctuations in cyclin E levels are required for multiple rounds of endocycle S phase in Drosophila.Curr. Biol. 1998; 8: 235-238Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Weiss et al. 1998Weiss A. Herzig A. Jacobs H. Lehner C.F. Continuous Cyclin E expression inhibits progression through endoreduplication cycles in Drosophila.Curr. Biol. 1998; 8: 239-242Abstract Full Text Full Text PDF PubMed Google Scholar). Below we describe the different types of endocycles characterized in the literature and note how each fits with these general notions of S phase control. Megakaryocytes are a blood cell type specialized to produce platelets. As part of their differentiation program, megakaryocytes become polyploid up to 128N (for review, see Zimmet and Ravid 2000Zimmet J. Ravid K. Polyploidy occurrence in nature, mechanisms, and significance for the megakaryocyte-platelet system.Exp. Hematol. 2000; 28: 3-16Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). This conversion to polyploidy is achieved via endomitosis, and is triggered by the secreted signal thrombopoietin. The large increase in megakaryocyte size that results from polyploidy is correlated with their ability to bud off adequate numbers of platelets. Cyclin D3 protein is upregulated in response to thrombopoietin in these cells, and Cyclin D3 overexpression in the megakaryocyte lineage has been found to increase megakaryocyte ploidy in transgenic mice (Zimmet et al. 1997Zimmet J.M. Ladd D. Jackson C.W. Stenberg P.E. Ravid K. A role for cyclin D3 in the endomitotic cell cycle.Mol. Cell. Biol. 1997; 17: 7248-7259Crossref PubMed Scopus (101) Google Scholar). Antisense experiments with megakaryocyte cell lines show that decreasing the levels of Cyclin D3 blocks polyploidization, and thus Cyclin D3 appears to be critical for driving thrombopoietin-induced endocycles (Wang et al. 1995Wang Z. Zhang Y. Kamen D. Lees E. Ravid K. Cyclin D3 is essential for megakaryocytopoiesis.Blood. 1995; 86: 3783-3788Crossref PubMed Google Scholar). Studies of Drosophila Cyclin D/Cdk4 show that this complex can also increase ploidy in endocycling tissues, and suggest that it does this by potentiating cellular growth, rather than via direct effects on cell cycle regulators such as RBF, a fly retinoblastoma homolog (Datar et al. 2000Datar S.A. Jacobs H.W. Lehner C.F. Edgar B.A. The Drosophila cyclinD/Cdk4 complex promotes cellular growth.EMBO J. 2000; 19: 4543-4554Crossref PubMed Scopus (223) Google Scholar). Knockout studies in mice are also consistent with the idea that Cdk4 and the D-type cyclins are used to regulate growth, but this possibility has not yet been tested extensively. In the case of megakaryocyte endocycling, it is not yet known what the critical targets of the Cyclin D3 kinase complex are. Endomitosis in megakaryocytes involves nuclear envelope breakdown and the appearance of condensed chromosomes and multipolar spindles (Nagata et al. 1997Nagata Y. Muro Y. Todokoro K. Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.J. Cell Biol. 1997; 139: 449-457Crossref PubMed Scopus (117) Google Scholar). Sister chromatids have been observed to separate in anaphase A, but anaphase B does not occur. As a consequence, replicated copies of the chromosomes are incorporated into the same nucleus when the nuclear envelope reforms at a stage equivalent to telophase. Cytokinesis appears to be bypassed completely. Megakaryocyte endomitosis is reported to occur at subnormal levels of the mitotic regulatory kinase, Cyclin B/Cdk1 (reviewed in Zimmet and Ravid 2000Zimmet J. Ravid K. Polyploidy occurrence in nature, mechanisms, and significance for the megakaryocyte-platelet system.Exp. Hematol. 2000; 28: 3-16Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), and the observation has been made that degradation of Cyclin B is relatively enhanced (Zhang et al. 1998Zhang Y. Wang Z. Liu D.X. Pagano M. Ravid K. Ubiquitin-dependent degradation of cyclin B is accelerated in polyploid megakaryocytes.J. Biol. Chem. 1998; 273: 1387-1392Crossref PubMed Scopus (67) Google Scholar). Thus, premature or excess degradation of Cyclin B might account for the exit from mitosis prior to anaphase B, nuclear division, and cytokinesis. Mammalian trophoblasts, which contribute to the placenta, also become polyploid via endocycles. These cells increase their DNA content to >1000C, possibly to facilitate the high metabolic activity required of them (reviewed in Zybina and Zybina 1996Zybina E.V. Zybina T.G. Polytene chromosomes in mammalian cells.Int. Rev. Cytol. 1996; 165: 53-119Crossref PubMed Google Scholar). The chromosomes of trophoblast giant cells have regions in which replicated copies are tightly associated (Varmuza et al. 1988Varmuza S. Prideaux V. Kothary R. Rossant J. Polytene chromosomes in mouse trophoblast giant cells.Development. 1988; 102: 127-134Crossref PubMed Google Scholar). Thus their chromosomes are polytene, although they are not aligned along their entire lengths to produce the intricate banding patterns seen in insect polytene cells. DNA replication in trophoblasts is cyclic, and aspects of mitosis are present. The chromosomes bundle in what has been termed “endoprophase,” and then decondense and dissociate in “endointerphase” when DNA replication occurs. Changes in cell cycle regulators were investigated in the trophoblast cell line, Rcho1 (MacAuley et al. 1998MacAuley A. Cross J.C. Werb Z. Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells.Mol. Biol. Cell. 1998; 9: 795-807Crossref PubMed Scopus (161) Google Scholar). During Rcho1 polytenization, Cyclin D1 is induced, Cyclins E and A remain high, and levels of Cyclin B are reduced. The CDK inhibitor (CKI) p57Kip2 is upregulated as Rcho1 cells differentiate and become polytene (Hattori et al. 2000Hattori N. Davies T.C. Anson-Cartwright L. Cross J.C. Periodic expression of the cyclin-dependent kinase inhibitor p57(Kip2) in trophoblast giant cells defines a G2-like gap phase of the endocycle.Mol. Biol. Cell. 2000; 11: 1037-1045Crossref PubMed Scopus (92) Google Scholar). p57 protein accumulates at the end of each S phase and disappears several hours before each subsequent S phase. Furthermore, a mutation that stabilizes p57 blocks S phase entry, suggesting that this inhibitor may mediate fluctuations in Cyclin E/Cdk2 activity and thus allow periodic replication origin licensing and firing. Interestingly, the mutation used to stabilize p57 encompasses a putative CDK phosphorylation site. This suggests that there may be feedback between CDK activity and p57 stability, and so provides one of the switches required to build a biphasic oscillator. As described below, a CDK/CKI-based oscillator may also be a plausible explanation for how some endocycles are controlled in Drosophila. An interesting parallel exists between the control of differentiation and polytenization of trophoblasts and the maintenance of diploidy during Drosophila development. The Drosophila Escargot protein, a member of the snail family of transcription factors, is required to prevent diploid abdominal histoblasts from becoming polyploid during larval development, when the majority of the tissues are in the endocycle (Fuse et al. 1994Fuse N. Hirose S. Hayashi S. Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot.Genes Dev. 1994; 8: 2270-2281Crossref PubMed Scopus (109) Google Scholar, Hayashi 1996Hayashi S. A cdc2 dependent checkpoint maintains diploidy in drosophila.Development. 1996; 122: 1051-1058PubMed Google Scholar, Hayashi et al. 1993Hayashi S. Hirose S. Metcalfe T. Shirras A.D. Control of imaginal cell development by the escargot gene of Drosophila.Development. 1993; 118: 105-115PubMed Google Scholar). A mouse gene (mSna) that is most homologous to Escargot is downregulated during trophoblast differentiation and polytenization (Nakayama et al. 1998Nakayama H. Scott I.C. Cross J.C. The transition to endoreduplication in trophoblast giant cells is regulated by the mSNA zinc finger transcription factor.Dev. Biol. 1998; 199: 150-163Crossref PubMed Scopus (90) Google Scholar). Overexpression of mSna in rat Rcho1 cells reduced the frequency with which these cells differentiated to form giant polytene trophoblast cells, but did not affect the kinetics of endocycles in trophoblasts that had already differentiated. Both the Drosophila Escargot and mSna proteins are expressed in a variety of diploid tissues. These observations have led to the proposal that the Escargot and mSna transcription factors affect differentiation decisions that preclude endoreplication. Many plants contain tissues that become highly polyploid during development. A survey of Arabidopsis revealed polyploidy in hair trichomes, leaf epidermal cells, root tip cells, and cells in the hypocotyl (Galbraith et al. 1991Galbraith D.W. Harkins K.R. Knapp S. Systemic endopolyploidy in Arabidopsis thaliana.Plant Physiol. 1991; 96: 985-989Crossref PubMed Scopus (367) Google Scholar, Melaragno et al. 1993Melaragno J.E. Mehrotra B. Coleman A.W. Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis.Plant Cell. 1993; 5: 1661-1668PubMed Google Scholar). In some cell types the extent of endoreplication appears to be intrinsically controlled by the differentiation program, but environmental influences such as light can also affect endoreplication (see Traas et al. 1998Traas J. Hülskamp M. Gendreau E. Höfte H. Endoreplication and development rule without dividing?.Curr. Opin. Plant Biol. 1998; 1: 498-503Crossref PubMed Scopus (164) Google Scholar for review). Examples of somatic polyploidy are particularly prevalent in plants with small genome sizes, raising the possibility that high ploidy may be needed for certain aspects of plant growth or function (De Rocher et al. 1990De Rocher E.J. Harkins K.R. Galbraith D.W. Bohnert H.J. Developmentally regulated endopolyploidy in succulents with small genomes.Science. 1990; 250: 99-101Crossref PubMed Scopus (149) Google Scholar, Nagl 1976Nagl W. DNA endoreduplication and polyteny understood as evolutionary strategies.Nature. 1976; 261: 614-615Crossref PubMed Scopus (187) Google Scholar). Proposals include a requirement for increased cell size to achieve a particular morphology, the need to change nuclear to organelle ratio, and the use of increased gene copy number to cope with environmental damage. Polyploidization in the maize endosperm has been proposed to increase its metabolic capability. Cell cycle regulators have been analyzed in Arabidopsis, maize, and alfalfa endocycles. Regulators that control the G1-S transition in mammalian cells, particularly the Rb-Cyclin D pathway, are conserved in plants (for review see den Boer and Murray 2000den Boer B.G. Murray J.A. Triggering the cell cycle in plants.Trends Cell Biol. 2000; 10: 245-250Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). During polyploidization of maize endosperm, mitotic cyclin/Cdk activity has been reported to decrease, whereas S phase cyclin/Cdk is maintained. In this case, however, the specific cyclins and CDKs involved have not been defined (Grafi et al. 1996Grafi G. Burnett R.J. Helentjaris T. Larkins B.A. DeCaprio J.A. Sellers W.R. Kaelin Jr., W.G. A maize cDNA encoding a member of the retinoblastoma protein family involvement in endoreduplication.Proc. Natl. Acad. Sci. USA. 1996; 93: 8962-8967Crossref PubMed Scopus (197) Google Scholar, Grafi and Larkins 1995Grafi G. Larkins B.A. Endoreduplication in maize endosperm—involvement of m-phase-promoting factor inhibition and induction of s-phase-related kinases.Science. 1995; 269: 1262-1264Crossref PubMed Scopus (171) Google Scholar). Investigation of polyploidization in alfalfa demonstrated a novel role for proteolysis in the endocycle (Cebolla et al. 1999Cebolla A. Vinardell J.M. Kiss E. Olah B. Roudier F. Kondorosi A. Kondorosi E. The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants.EMBO J. 1999; 18: 4476-4484Crossref PubMed Scopus (257) Google Scholar). An ortholog to the Cdh1/Fzr-related family of proteins, which activate the anaphase promoting complex and promote G2 cyclin degradation in other species, was isolated from alfalfa and called ccs52. Strikingly, the ccs52 gene is expressed in endocycling cells, and when antisense RNA was used to reduce the level of ccs52 transcripts, ploidy in these cells was reduced. There was no effect on diploid cells. This suggests that APC-mediated proteolysis may be a key event in generating polyploidy. As described above, periodic APC activation is one mechanism that might permit preRC assembly and licensing between S phases. It remains unclear which specific APC targets might be involved in endocycling in alfalfa, or whether APC-mediated proteolysis plays a role in other endocycles. Investigations in this area may shed considerable light on the mechanisms of endocycle progression. Endoreplication is widespread in arthropods (White 1973White M.J.D. Animal Cytology and Evolution. Third Edition. Cambridge University Press, Cambridge1973Google Scholar) and has been extensively characterized in the tiny but highly polyploid fruit fly, D. melanogaster. Following the cell-proliferative phase of Drosophila embryogenesis, many tissues initiate endocycles that lack all visible aspects of mitosis (Smith and Orr-Weaver 1991Smith A.V. Orr-Weaver T.L. The regulation of the cell cycle during Drosophila embryogenesis—the transition to polyteny.Development. 1991; 112: 997-1008Crossref PubMed Google Scholar). These tissues, which include the gut, epidermis, fat body (liver), malipighian tubules (kidney), trachea, and salivary glands, continue to endocycle during larval development long after they are fully differentiated. Some adult tissues, including ovarian follicle and nurse cells, and sensory neurons in the wing, also employ endocycles. Endoreplication parallels larval growth, and experimental inhibition of endocycle progression using DNA replication inhibitors, or mutations in genes essential for DNA replication, shows that increases in C values are required for both cell and organismal growth (Figure 4). Conversely, conditions that arrest growth invariably block endocycle progression, suggesting a tight regulatory linkage between these two processes (Galloni and Edgar 1999Galloni M. Edgar B.A. Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster.Development. 1999; 126: 2365-2375PubMed Google Scholar, Zhang et al. 2000Zhang H. Stallock J.P. Ng J.C. Reinhard C. Neufeld T.P. Regulation of cellular growth by the Drosophila target of rapamycin dTOR.Genes Dev. 2000; 14: 2712-2724Crossref PubMed Scopus (485) Google Scholar). Final DNA levels in the larval cells appear to be developmentally programmed, ranging from 16C to 2048C depending on cell type. In switching from mitotic to endo cycles, cells in the Drosophila embryo terminate the expression of mRNAs encoding the mitotic regulators Cdk1, Cyclin A, Cyclin B, Cyclin B3, and Cdc25/string (Sauer et al. 1995Sauer K. Knoblich J.A. Richardson H. Lehner C.F. Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis.Genes Dev. 1995; 9: 1327-1339Crossref PubMed Scopus (199) Google Scholar). How transcription of these genes is shut off at this juncture is not known, but developmentally programmed signals are presumed to be important. In addition to preprogrammed transcriptional controls, embryonic cells switching to the endocycle downregulate their mitotic machinery at the protein level. Studies of Fzr/Cdh1, a regulator of the APC, show that APC activity is required for the switch from mitotic to endocycles. fzr mutant embryos show inappropriate accumulation of G2 cyclins in G1 cells that are poised to initiate endocycling, and these cells never initiate their first endocycle S phases (Sigrist and Lehner 1997Sigrist S.J. Lehner C.F. Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles.Cell. 1997; 90: 671-681Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). This is presumed to be due to the inhibitory action of persistent APC targets such as Cyclin A, Cyclin B, or perhaps Geminin, on preRC formation. Consistent with this idea, experimental inhibition of Cdk1 activity in fission yeast, budding yeast, and Drosophila can force cells that are normally mitotic to become endoreplicative (Hayashi 1996Hayashi S. A cdc2 dependent checkpoint maintains diploidy in drosophila.Development. 1996; 122: 1051-1058PubMed Google Scholar). The possibility that APC-mediated proteolysis might be generally used in endocycle progression is suggested by the observation in alfalfa noted above (Cebolla et al. 1999Cebolla A. Vinardell J.M. Kiss E. Olah B. Roudier F. Kondorosi A. Kondorosi E. The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants.EMBO J. 1999; 18: 4476-4484Crossref PubMed Scopus (257) Google Scholar), but it is still unclear whether Fzr or the APC functions during successive Drosophila endocycles. The expression pattern of Fzr suggests that the Fzr/APC may be required only for the initial switch from mitotic to endocycles, and that it is irrelevant thereafter. Analysis of the Drosphila mutant morula, however, suggests that this may not be the case in all cell types (Reed and Orr-Weaver 1997Reed B.H. Orr-Weaver T.L. The Drosophila gene morula inhibits mitotic functions in the endo cell cycle and the mitotic cell cycle.Development. 1997; 124: 3543-3553PubMed Google Scholar). Weak alleles of morula exhibit the striking phenotype that polyploid nurse cells exit the endocycle and resume mitosis. This is accompanied by reappearance of Cyclin B protein via a posttranscriptional mechanism, suggesting that APC function may be compromised. Of the examples described here, Drosophila's endocycles are the most distinct from the archetypal cell cycle. Not only do these cycles lack mitosis, but genomic replication during each S phase is incomplete. In most polytene tissues heterochromatin, which makes up approximately 30% of the genome, is underreplicated (Figure 2, Figure 3). This includes centromeric heterochromatin, intercalary (or interband) heterochromatin, and telomereic sequences. In addition, certain euchromatic regions, such as the histone genes, can be underreplicated (Laird 1980Laird C.D. Structural paradox of polytene chromosomes.Cell. 1980; 22: 869-874Abstract Full Text PDF PubMed Scopus (47) Google Scholar, Hammond and Laird 1985Hammond M.P. Laird C.D. Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster.Chromosoma. 1985; 91: 267-278Crossref PubMed Scopus (80) Google Scholar). Heterochromatic replication is affected by levels of Cyclin E; in weak cyclin E mutants heterochromatin in the ovarian nurse cells is replicated (Lilly and Spradling 1996Lilly M.A. Spradling A.C. The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion.Genes Dev. 1996; 10: 2514-2526Crossref PubMed Scopus (215) Google Scholar). Although the mechanism by which Cyclin E affects heterochromatic DNA replication is unclear, the idea most consistent with available data is that lowered levels of Cyclin E allow S phase to continue for longer, and that this in turn allows heterochromatin (which is normally late replicating even in mitotic cells) to replicate (Lilly and Spradling 1996Lilly M.A. Spradling A.C. The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion.Genes Dev. 1996; 10: 25
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