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Regulation of cdc2 transcription

Multiple species of G1 cyclins and cyclin-dependent kinases are induced sequentially during G1 phase; the expression of cyclin A and cdc2 genes is subsequently induced at the G1/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5'-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, which contains the E box, SpI, and E2F motifs. The expression of 5' sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells reveals the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses reveal that the enhancer sequence, AAGTTACAAATA, located from -276 to -265, confers strong inducibility on the basal promoter at the G1/S boundary. The base substitutions introduced into the motifs of transcription factors indicate that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G1/S boundary. Electrophoretic mobility shift assays and DNase I footprinting show that a factor which interacts with the enhancer element is induced late in G1 phase (Shimizu, 1997).

In quiescent cells, cdc2 mRNA is almost undetectable. Stimulation of cells to reenter the cell cycle results in induction of cdc2 expression, beginning at the G1-to-S transition and reaching maximum levels during late S and G2 phases. To investigate cdc2 transcriptional regulation throughout cell cycle progression, protein-DNA interactions were monitored by in vivo footprinting along 800 bp of the human cdc2 promoter in quiescent fibroblasts and at different time points following serum stimulation. There are 11 in vivo protein-binding sites, but no protein binding is observed at a high-affinity E2F site that has been implicated in cdc2 regulation. Nine of the identified in vivo binding sites (among them were two inverted CCAAT boxes, two Sp1 sites, and one ets-2 site) bind transcription factors constitutively throughout the cell cycle. However, at two elements located at positions -60 and -20 relative to the transcription start site, the binding pattern changes significantly as the cells are entering S phase. A G0- and G1-specific protein complex disappears at the -20 element at the beginning of S phase. This sequence deviates at one base position from known E2F consensus binding sites. The major E2F activity in human fibroblasts contains E2F-4 and p130. The -20 element of the cdc2 gene specifically interacts with a subset of E2F-4-p130 complexes present in G0 cells but does not interact with S-phase-specific E2F complexes. Transient-transfection experiments with wild-type and mutant cdc2 promoter constructs indicate that the -20 element is involved in suppressing cdc2 activity in quiescent cells. It is suggested that the presence of the p130-E2F-4 complex in G0/G1 blocks access of components of the basal transcription machinery or prevents transaction by the constitutively bound upstream activator proteins (Tommasi, 1995).

The S/G2-specific transcription of the human cdc25C gene is due to the periodic occupation of a repressor element, the cell cycle-dependent element (CDE), located in the region of the basal promoter. Protein binding to the major groove of the CDE in G0 and G1 results in a phase-specific repression of activated transcription. CDE-mediated repression is also the major principle underlying the periodic transcription of the human cyclin A and cdc2 genes. A single point mutation within the CDE results in a 10- to 20-fold deregulation in G0 and an almost complete loss of cell cycle regulation of all three genes. The cdc25C, cyclin A and cdc2 genes share an identical 5 bp region known as the cell cycle genes homology region (CHR), starting at an identical position, six nucleotides 3' to the CDE. Strikingly, mutation of the CHR region in each of the three promoters produces the same phenotype as the mutation of the CDE, i.e. a dramatic deregulation in G0. In agreement with these results, in vivo DMS footprinting showed the periodic occupation of the cyclin A CDE in the major groove, and of the CHR in the minor groove. All three genes bear conspicuous similarities in their upstream activating sequences (UAS). This applies in particular to the presence of NF-Y and Sp1 binding sites which, in the cdc25C gene, have been shown to be the targets of repression through the CDE (Zwicker, 1995).

The cdc25C , cdc2 and cyclin A promoters are controlled by transcriptional repression through two contiguous protein binding sites, termed the CDE and CHR. In the present study CDF-1 has been identified as the factor that interacts with the cdc25C CDE-CHR module. CDF-1 binds to the CDE in the major groove and to the CHR in the minor grove in a cooperative fashion in vitro, in a manner similar to that seen by genomic footprinting. In agreement with in vivo binding data and its putative function as a periodic repressor, DNA binding by CDF-1 in nuclear extracts is down-regulated during cell cycle progression. CDF-1 also binds avidly to the CDE-CHR modules of the cdc2 and cyclin A promoters, but not to the E2F site in the B-myb (see Drosophila Myb oncogene-like) promoter. Conversely, E2F complexes do not recognize the cdc25C CDE-CHR and CDF-1 is immunologically unrelated to all known E2F and DP family members. This indicates that E2F- and CDF-mediated repression is controlled by different factors acting at different stages during the cell cycle. While E2F-mediated repression seems to be associated with genes such as B-myb that are up-regulated early (around mid G1), CDE-CHR-controlled genes (such as cdc25C, cdc2 and cyclin A), become derepressed later. The fractionation of native nuclear extracts on glycerol gradients leads to separation of CDF-1 from both E2F complexes and pocket proteins of the pRb family. This emphasizes the conclusion that CDF-1 is not an E2F family member and points to profound differences in the cell cycle regulation of CDF-1 and E2F (N. Liu, 1997).

The c-myb proto-oncogene is preferentially expressed in hematopoietic cells and is required for cell cycle progression at the G1/S boundary. Because c-myb encodes a transcriptional activator that functions via DNA binding, it is likely that c-myb exerts its biological activity by regulating the transcription of genes required for DNA synthesis and cell cycle progression. One such gene, cdc2, encodes a 34-kDa serine-threonine kinase that appears to be required for G1/S transition in normal human T-lymphocytes. To determine whether c-myb is a transcriptional regulator of cdc2 expression, a segment of a cdc2 gene containing extensive 5'-flanking sequences and part of the first exon was cloned. Sequence analysis reveals the presence of two closely spaced Myb binding sites that interact with bacterially synthesized Myb protein within a region extending from nucleotides -410 to -392 upstream of the transcription initiation site. A 465-base pair segment of 5'-flanking sequence containing these sites is linked to the CAT reporter gene and was shown to have promoter activity in rodent fibroblasts. Cotransfection of this construct with a full-length human c-myb cDNA driven by the early simian virus 40 promoter results in a 6-8-fold enhancement of CAT activity, which is abrogated by mutations in the Myb binding sites. These data suggest that c-myb participates in the regulation of cell cycle progression by activating the expression of the cdc2 gene (Ku, 1993).

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, an E2f3 mutant mouse strain was generated. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, the properties of embryonic fibroblasts derived from E2f3 mutant mice were analyzed. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. A number of genes, including B-myb, cyclin A, cdc2, cdc6, and DHFR, could be identified whose expression is dependent on the presence of E2F3 but not E2F1. A critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G1/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. It is concluded that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells (Humbert, 2000).

Previous work has provided evidence for E2F-dependent transcription control of both G1/S- and G2/M-regulated genes. Analysis of the G2-regulated cdc2 and cyclin B1 genes reveals the presence of both positive- and negative-acting E2F promoter elements. Additional elements provide both positive (CCAAT and Myb) and negative (CHR) control. Chromatin immunoprecipitation assays identify multiple interactions of E2F proteins that include those previously shown to activate and repress transcription. E2F1, E2F2, and E2F3 were found to bind to the positive-acting E2F site in the cdc2 promoter, whereas E2F4 binds to the negative-acting site. Binding of an activator E2F is dependent on an adjacent CCAAT site that is bound by the NF-Y transcription factor and binding of a repressor E2F is dependent on an adjacent CHR element, suggesting a role for cooperative interactions in determining both activation and repression. Finally, the kinetics of B-Myb interaction with the G2-regulated promoters coincides with the activation of the genes, and RNAi-mediated reduction of B-Myb inhibits expression of cyclin B1 and cdc2. The ability of B-Myb to interact with the cdc2 promoter is dependent on an intact E2F binding site. These results thus point to a role for E2Fs, together with B-Myb, which is an E2F-regulated gene expressed at G1/S, in linking the regulation of genes at G1/S and G2/M (Zhu, 2004).

To understand cell cycle control mechanisms in early development and how they change during differentiation, embryonic stem cells were used to model embryonic events. The results demonstrate that as pluripotent cells differentiate, the length of G(1) phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb-E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the down-regulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F-pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F-pRb pathway (White, 2005).

Regulation of cdc2 protein level

In the fission yeast Schizosaccharomyces pombe, p34(cdc2) plays a central role controlling the cell cycle. A new gene has been isolated, named srw1(+), that encodes a WD repeat protein, as a multicopy suppressor of hyperactivated p34(cdc2). Cells lacking srw1(+) are sterile and defective in cell cycle controls. When starved for a nitrogen source, they fail to effectively arrest in G1 and die of accelerated mitotic catastrophe when regulation of p34(cdc2)/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34(cdc2). srw1(+) shares functional similarity with rum1(+), having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. It is concluded that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34(cdc2) and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments (Yamaguchi, 1997).

Mitosis requires activity of the cyclin B cyclin-dependent kinase 1 (cdc2) heterodimer. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B. Cdk2 is also active during mitosis. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2. Cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. These findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit (D'Angiolella, 2001).

The cell-cycle oscillator includes an essential negative-feedback loop: Cdc2 activates the anaphase-promoting complex (APC), which leads to cyclin destruction and Cdc2 inactivation. Under some circumstances, a negative-feedback loop is sufficient to generate sustained oscillations. However, the Cdc2/APC system also includes positive-feedback loops, whose functional importance was assessed in this study. Short-circuiting positive feedback makes the oscillations in Cdc2 activity faster, less temporally abrupt, and damped. This compromises the activation of cyclin destruction and interferes with mitotic exit and DNA replication. This work demonstrates a systems-level role for positive-feedback loops in the embryonic cell cycle and provides an example of how oscillations can emerge out of combinations of subcircuits whose individual behaviors are not oscillatory. This work also underscores the fundamental similarity of cell-cycle oscillations in embryos to repetitive action potentials in pacemaker neurons, with both systems relying on a combination of negative and positive-feedback loops (Pomerening, 2005).

Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. This study reports the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 binds to the core APC, and this binding is disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 is antagonized by PP2A, which can bind to Emi2 and promote Emi2-APC interactions. It is concluded that constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest (Wu, 2007)

Developmental biology of cdc2

In mammalian cells the Cdc25 family of dual-specificity phosphatases has three distinct isoforms, termed A, B, and C, which are thought to play discrete roles in cell-cycle control. Xenopus Cdc25A exhibits developmental regulation and demonstrates a key role in embryonic cell-cycle control. Northern and Western blot analyses show that Cdc25A is absent in oocytes, and synthesis begins within 30 min after fertilization. The protein product is localized in the nucleus during interphase and accumulates continuously until the midblastula transition (MBT), after which it is degraded. Upon injection into newly fertilized eggs, wild-type Cdc25A shortens the cell cycle and accelerates the timing of cleavage, whereas embryos injected with phosphatase-dead Cdc25A display a dose-dependent increase in the length of the cell cycle and a slower rate of cleavage. In contrast, injection of the phosphatase-dead Cdc25C isoform has no effect. Western blotting with an antibody specific for phosphorylated tyr15 in Cdc2/Cdk2 reveals a cycle of phosphorylation/dephosphorylation in each cell cycle in control embryos, and in embryos injected with phosphatase-dead Cdc25A there is a twofold increase in the level of phospho-tyrosine in Cdc2/Cdk2. Consistent with this, the levels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity are both reduced by approximately 50% after phosphatase-dead Cdc25A injection. The phosphatase-dead Cdc25A can be recovered in a complex with both Cdks, suggesting that it acts in a dominant-negative fashion. These results indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15 occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 by Cdc25A controls at least in part the length of the cell cycle and the timing of cleavage in pre-MBT embryos. The disappearance of Cdc25A after the MBT may underlie in part the lengthening of the cell cycle at that time (Kim, 1999).

After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukaryotes remain postmitotic during their entire lifespan. This requires that a very stringent control be exerted on the cell division apparatus, whose molecular mechanisms remain quite elusive. Quail neuroretina was used as a model to study the control of cell division in the developing CNS. In vertebrates, embryonic neuroretinal cells (NR cells) stop their proliferation at different times depending on the cell type. Most NR cells in the quail embryo become postmitotic between E7 and E8. To acquire a better understanding of the molecular events leading to quiescence in NR cells, the expression of cdc2 as well as cyclin A and cyclin B2 [two activators of p34(cdc2)] were analyzed in the developing neuroretina. These three proteins are downregulated between E7 and E9, suggesting that a common mechanism could block their transcription in differentiating neurons. p34(cdc2) downregulation is correlated with the appearance of the microtubule-associated protein tau. These results strongly suggest that inhibition of cdc2 gene expression is closely linked to the achievement of terminal differentiation in neurons. However, postmitotic ganglion cell precursors begin to synthesize the early neuronal differentiation marker beta3-tubulin while p34(cdc2) is still detectable in these cells, suggesting that p34(cdc2) or a closely related kinase could play a role in some "young" postmitotic neurons (Espanel, 1997).

Cyclin B-dependent CDC2 kinase activity has a key role in triggering the G2/M-phase transition during the mitotic and meiotic cell cycles. The Hsp70-2 gene is expressed only in spermatogenic cells at a significant level. In Hsp70-2 gene knock-out (Hsp70-2[-/-]) mice, primary spermatocytes fail to complete meiosis I, suggesting a link between HSP70-2 heat-shock protein and CDC2 kinase activity during this phase of spermatogenesis. Members of the HSP70 protein family are molecular chaperones that mediate protein de novo folding, translocation and multimer assembly. This study used immunoprecipitation-coupled western blot and in vitro reconstitution experiments to show that HSP70-2 (1) interacts with CDC2 in the mouse testis; (2) appears to be a molecular chaperone for CDC2, and (3) is required for CDC2/cyclin B1 complex formation. Previous studies reported that most CDC2 kinase activity in the mouse testis is present in pachytene spermatocytes. Although CDC2 kinase activity for histone H1 is present in the testis of wild-type mice, it is nearly absent from the testis of Hsp70-2(-/-) mice, probably due to defective CDC2/cyclin B1 complex formation. The addition of HSP70-2 to freshly prepared extracts of testis from Hsp70-2(-/-) mice not only restores CDC2/cyclin B1 complex formation but also reconstitutes CDC2 kinase activity in vitro. It appears that one cause of the failure to complete meiosis I during spermatogenesis in Hsp70-2(-/-) mice is disruption of CDC2/cyclin B1 assembly in pachytene spermatocytes, thereby preventing development of the CDC2 kinase activity required to trigger G2/M-phase transition. These studies provide novel in vivo evidence for a link between an HSP70 molecular chaperone and CDC2 kinase activity essential for the meiotic cell cycle in spermatogenesis (Zhu, 1997).

Major developmental events in early Xenopus embryogenesis coincide with changes in the length and composition of the cell cycle. These changes are mediated in part through the regulation of CyclinB/Cdc2 and they occur at the first mitotic cell cycle, the mid-blastula transition (MBT) and at gastrulation. The contribution has been investigated of maternal Wee1, a kinase inhibitor of CyclinB/Cdc2, to these crucial developmental transitions. By depleting Wee1 protein levels using antisense morpholino oligonucleotides, it is shown that Wee1 regulates M-phase entry and Cdc2 tyrosine phosphorylation in early gastrula embryos. Moreover, Wee1 is required for key morphogenetic movements involved in gastrulation, but is not needed for the induction of zygotic transcription. In addition, Wee1 is positively regulated by tyrosine autophosphorylation in early gastrula embryos and this upregulation of Wee1 activity is required for normal gastrulation. Overexpression of Cdc25C, a phosphatase that activates the CyclinB/Cdc2 complex, induces gastrulation defects that can be rescued by Wee1, providing additional evidence that cell cycle inhibition is crucial for the gastrulation process. Together, these findings further elucidate the developmental function of Wee1 and demonstrate the importance of cell cycle regulation in vertebrate morphogenesis (Murakami, 2004).

Modulation of the cell cycle appears to play an important role in both Xenopus and Drosophila embryogenesis. Prior to gastrulation, both organisms undergo a burst of rapid cell divisions followed by a gradual expansion of the cell cycle. Zygotic cell cycle components are synthesized after the MBT and previous studies have indicated that zygotic proteins do play a role in regulating Cdc2 activity during gastrulation. In Drosophila, cell cycle inhibition is observed at the ventral furrow, a region somewhat analogous to the Xenopus blastopore, and this inhibition is achieved by the removal of a zygotic activator of Cdc2. Specifically, the spatially restricted expression of the Tribbles protein results in the degradation of the String/Cdc25C phosphatase in cells surrounding the ventral furrow. In Xenopus, the zone of non-mitotic cells in the mid-late gastrula is identical to the area of zygotic Wee1B/Wee2 RNA expression, suggesting that zygotic expression of a Cdc2 inhibitor, Wee1B/Wee2, might play an analogous role in frog embryogenesis. Interestingly, the expansion of the cell cycle after the MBT (and during gastrulation) is regulated by zygotic components in Drosophila, but is regulated by maternally derived components in Xenopus. In Xenopus, the maternally regulated program of cell cycle expansion has been implicated in the onset of zygotic transcription, cytoplasmic blebbing and pseudopod formation (at the MBT), but has not been previously implicated in the coordinated tissue morphogenesis that takes place during gastrulation. This study demonstrates that the maternal Wee1 protein contributes to the cell cycle downregulation that occurs during Xenopus gastrulation. The findings also indicate that the maternally directed program of cell cycle control, rather than simply facilitating the transcription of zygotic components, plays a direct role in morphogenesis (Murakami, 2004).

The requirement of cell cycle regulation for the coordinated cell movements of gastrulation is another shared feature of Drosophila and Xenopus embryogenesis. In flies, the Tribbles-mediated degradation of Cdc25C permits the invagination of mesodermal cells at the ventral furrow, one of the earliest events of gastrulation. Similarly, in this study, cell cycle inhibition mediated by Wee1 was found to be important for epiboly, involution and convergent-extension, all of which are major morphogenetic processes that contribute to normal Xenopus gastrulation. Thus, although flies and frogs may use different molecular components to regulate the embryonic cell cycle, it appears that in both organisms the inhibition of cell division is essential for the complex morphogenetic movements required for gastrulation (Murakami, 2004).

Wee1 is upregulated by tyrosine autophosphorylation following the MBT and at gastrulation. This upregulation appears to be required for Wee1 function in early gastrula embryos given that neither kinase-inactive Wee1 or a Wee1 protein containing mutations in the tyrosine phosphorylation sites are able to rescue the defects produced by MO-Wee1-depletion. These findings are consistent with previous observations that upregulation of Wee1 activity by tyrosine autophosphorylation is critical for Wee1 function in the first mitotic cell cycle. Taken together, these studies indicate that the maternal Wee1 protein functions at distinct developmental points to coordinate cell cycle progression with events that control the organization of the embryonic body plan. Moreover, this work contributes to a growing body of evidence that cell cycle regulation is likely to be crucial for a wide variety of morphogenetic processes. Wee1 is a primary cell cycle target of the budding morphogenesis checkpoint in S. cerevisiae, and in mammalian cells, there is evidence that inhibition of cell proliferation is necessary for cell migration. Collectively, these studies suggest that 'morphogenesis' checkpoints, which coordinate cell shape changes and movement with cell proliferation, will be crucial for normal development and organogenesis, and may also play an important role in the balance between deregulated cell proliferation and metastasis (Murakami, 2004).

The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit

Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. This study introduced a live-cell sensor for CDK2 activity in mammalian cultured cells. It was unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity (Spencer, 2013).

The restriction point was originally defined as a point in late G1 after which cells would continue through the cell cycle even if mitogens were withdrawn. It has also been proposed that commitment to the next cell cycle is made at the end of the preceding cycle. The current study argues that these apparently contradictory models can be explained by taking into account the two types of cell behaviors that this study has observed within the same cell population. The results argue that cells integrate mitogenic and potentially other inputs during a restriction window at the end of the previous cell cycle (R1) to regulate the bifurcation into the intermediate level of CDK2 (CDK2inc) or or low level of CDK2 (CDK2low) state upon completion of mitosis. Only the cells in the CDK2low state experience a second restriction window (R2) in which mitogens are needed to re-enter the cell cycle and build up CDK2 activity. Given the different fractions of CDK2low cells that were found in the cell types that were tested, the relative importance of R1 versus R2 will vary depending on the cell type, strength of mitogen stimuli and other conditions. Although the two restriction windows R1 and R2 cover different phases of the cell cycle, they reflect an underlying principle that cells require the continued presence of mitogens for several hours before they commit to building up CDK2 activity. Finally, the relationship between CDK2 activity and cell-cycle commitment that is described in this study may be integral to other cell fate decisions such as the senescence of somatic cells, the differentiation of stem cells, or the progression of cancer cells (Spencer, 2013).

Cdc2 and tumorogenesis

The cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1 are induced in response to anti-proliferative stimuli and block G1/S-phase progression through the inhibition of CDK2. Although the cyclin E-CDK2 pathway is often deregulated in tumors, the relative contribution of p21Cip1 and p27Kip1 to tumorigenesis is still unclear. The MYC transcription factor is an important regulator of the G1/S transition and its expression is frequently altered in tumors. It has been suggested that p27Kip1 is a crucial G1 target of MYC. In mice, deficiency for p27Kip1 but not p21Cip1 results in decreased survival to retrovirally-induced lymphomagenesis. Importantly, in such p27Kip1 deficient lymphomas an increased frequency of Myc activation is observed. p27Kip1 deficiency also collaborates with MYC overexpression in transgenic lymphoma models. Thus, in vivo, the capacity of MYC to promote tumor growth is fully retained and even enhanced upon p27Kip1 loss. In lymphocytes, MYC overexpression and p27Kip1 deficiency independently stimulate CDK2 activity and augment the fraction of cells in S phase, in support of their distinct roles in tumorigenesis (Martins, 2002).

Doxorubicin (DOX) is a DNA topoisomerase II inhibitor widely used in anticancer treatment, however, it can lead to irreversible cardiac damage with severe debilitation. TBP-binding associated factor 1 (TAF1) is increased in DOX damaged hearts in vivo and in cardiomyocytes in vitro. To identify the functional role for TAF1 in DOX-treated heart wild type and mutant TAF1 was overexpressed in H9c2 cells. Overexpression of wild-type TAF1, but not N-terminal kinase domain mutants, increased tolerance to DOX in confluent cells. DOX treatment can cause prolonged G1 arrest. Increased cdk2 activity coupled to increased cyclin E protein and decreased p21(waf1Cip1) and p27(Kip1) protein was found to correlate only with increased DOX tolerance and wild-type TAF1. DOX sensitivity was restored when the cdk2-inhibitor Roscovitine was co-administered with DOX. Overexpression of cdk2-alone increased resistance to DOX. Thus, TAF1 induced DOX tolerance in confluent cells through an increase in cdk2 activity is directed by the TAF1 N-terminal domain. These studies suggest new avenues for myocardial protection against DOX toxicity and suggest a role for cdk2 in chemorefractory cells (Servent, 2004)

Table of contents

cdc2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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