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Some biological networks exhibit oscillations in their components to convert stimuli to time-dependent responses. The eukaryotic cell cycle is such a case, being governed by waves of cyclin-dependent kinase (cyclin/Cdk) activities that rise and fall with specific timing and guarantee its timely occurrence. Disruption of cyclin/Cdk oscillations could result in dysfunction through reduced cell division. Therefore, it is of interest to capture properties of network designs that exhibit robust oscillations. Here we show that a minimal yeast cell cycle network is able to oscillate autonomously, and that cyclin/Cdk-mediated positive feedback loops (PFLs) and Clb3-centered regulations sustain cyclin/Cdk oscillations, in known and hypothetical network designs. We propose that Clb3-mediated coordination of cyclin/Cdk waves reconciles checkpoint and oscillatory cell cycle models. Considering the evolutionary conservation of the cyclin/Cdk network across eukaryotes, we hypothesize that functional ("healthy") phenotypes require the capacity to oscillate autonomously whereas dysfunctional (potentially "diseased") phenotypes may lack this capacity.

Meiosis with a single round of DNA replication and two successive rounds of chromosome segregation requires specific cyclins associated with cyclin-dependent kinases (CDKs) to ensure its fidelity. But how cyclins control the distinctive meiosis is still largely unknown. In this study, we explored the role of cyclin B3 in female meiosis by generating Ccnb3 mutant mice via CRISPR/Cas9. Ccnb3 mutant oocytes characteristically arrested at metaphase I (MetI) with normal spindle assembly and lacked enough anaphase-promoting complex/cyclosome (APC/C) activity, which is spindle assembly checkpoint (SAC) independent, to initiate anaphase I (AnaI). Securin siRNA or CDK1 inhibitor supplements rescued the MetI arrest. Furthermore, CCNB3 directly interacts with CDK1 to exert kinase function. Besides, the MetI arrest oocytes had normal development after intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA), along with releasing the sister chromatids, which implies that Ccnb3 exclusively functioned in meiosis I, rather than meiosis II. Our study sheds light on the specific cell cycle control of cyclins in meiosis., (© 2019 Li et al.)

Depletion of cyb-1 , a major B-type cyclin expressed during Caenorhabditis elegans spermatogenesis, causes a meiotic division arrest in diakinesis-stage spermatocytes with multiple and mispositioned centrosomes. Association of the two nuclear membrane proteins SUN-1 and ZYG-12 is essential for centrosome-nuclear envelope attachment. We found that depletion of sun-1 causes centrosome defects similar to those caused by cyb-1 depletion in diakinesis-stage spermatocytes. In addition, Ser8 and Ser43 residues in SUN-1 are dephosphorylated in cyb-1 -depleted diakinesis-stage spermatocytes. Nevertheless, dephosphorylation of these residues was not sufficient to reproduce the cyb-1 -related centrosome defects. We then found that the ZYG-12::GFP signal in the nuclear envelope was significantly reduced in the cyb-1 -depleted diakinesis-stage spermatocytes. However, only mispositioned but not multiplied centrosomes were observed in zyg-12 mutant diakinesis-stage spermatocytes, suggesting that zyg-12 is not involved in the centrosome duplication at this stage. Our results suggest that CYB-1 functions to maintain proper positioning of centrosomes during spermatogenesis by regulating phosphorylation of SUN-1, which is possibly crucial for the association between SUN-1 and ZYG-12. This phosphorylation of SUN-1 may also regulate centrosome duplication independently of ZYG-12., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Cyclin B3 is a relatively new member of the cyclin family whose functions are little known. We found that depletion of cyclin B3 inhibited metaphase-anaphase transition as indicated by a well-sustained MI spindle and cyclin B1 expression in meiotic oocytes after extended culture. This effect was independent of spindle assembly checkpoint activity, since both Bub3 and BubR1 signals were not observed at kinetochores in MI-arrested cells. The metaphase I arrest was not rescued by either Mad2 knockdown or cdc20 overexpression, but it was rescued by securin RNAi. We conclude that cyclin B3 controls the metaphase-anaphase transition by activating APC/C(cdc20) in meiotic oocytes, a process that does not rely on SAC activity.

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Chromosome attachment to the mitotic spindle in early mitosis is guarded by an Aurora B kinase-dependent error correction mechanism [1, 2] and by the spindle assembly checkpoint (SAC), which delays cell-cycle progression in response to errors in chromosome attachment [3, 4]. The abrupt loss of sister chromatid cohesion at anaphase creates a type of chromosome attachment that in early mitosis would be recognized as erroneous, would elicit Aurora B-dependent destabilization of kinetochore-microtubule attachment, and would activate the checkpoint [5, 6]. However, in anaphase, none of these responses occurs, which is vital to ensure progression through anaphase and faithful chromosome segregation. The difference has been attributed to the drop in CDK1/cyclin B activity that accompanies anaphase and causes Aurora B translocation away from centromeres [7-12] and to the inactivation of the checkpoint by the time of anaphase [10, 11, 13, 14]. Here, we show that checkpoint inactivation may not be crucial because checkpoint activation by anaphase chromosomes is too slow to take effect on the timescale during which anaphase is executed. In addition, we observe that checkpoint activation can still occur for a considerable time after the anaphase-promoting complex/cyclosome (APC/C) becomes active, raising the question whether the checkpoint is indeed completely inactivated by the time of anaphase under physiologic conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Activation of anaphase-promoting complex/cyclosome (APC/C(Cdc20)) by Cdc20 is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/C(Cdc20) degrades cyclin B and securin, which activates separase [1]. The latter then cleaves cohesin holding sister chromatids together [2]. Because cohesin cleavage also destroys the tension responsible for turning off the SAC, cells must possess a mechanism to prevent SAC reactivation during anaphase, which could be conferred by a dependence of the SAC on Cdk1 [3-5]. To test this, we analyzed mouse oocytes and embryos expressing nondegradable cyclin B together with a Cdk1-resistant form of separase. After biorientation and SAC inactivation, APC/C(Cdc20) activates separase but the resulting loss of (some) cohesion is accompanied by SAC reactivation and APC/C(Cdc20) inhibition, which aborts the process of further securin degradation. Cyclin B is therefore the only APC/C(Cdc20) substrate whose degradation at the onset of anaphase is necessary to prevent SAC reactivation. The mutual activation of tension sensitive SAC and Cdk1 creates a bistable system that ensures complete activation of separase and total downregulation of Cdk1 when all chromosomes have bioriented., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/C(Cdc20)) [2]. Upon satisfaction of both pathways, the APC/C(Cdc20) elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/C(Cdc20) is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/C(Cdc20) couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.

Mcl-1 is one of the major anti-apoptotic members of the Bcl-2 family of apoptotic regulatory proteins. In this study we investigated the role of Mcl-1 in mitotic arrest-induced apoptosis. Vinblastine treatment of KB-3 cells initially resulted in a phosphatase-sensitive mobility shift in Mcl-1 and then subsequent loss of Mcl-1 protein expression which was prevented by MG132, suggesting that phosphorylation triggered proteosome-mediated degradation. Mcl-1 phosphorylation/degradation was a specific response to microtubule inhibition and did not occur in response to lethal concentrations of DNA damaging agents. Vinblastine treatment caused degradation of Mcl-1 in cells in which apoptosis was blocked by Bcl-xL overexpression, indicating that Mcl-1 degradation was not a consequence of apoptosis. A partial reversible phosphorylation of Mcl-1 was observed in synchronized cells traversing mitosis, whereas more extensive phosphorylation and subsequent degradation of Mcl-1 was observed if synchronized cells were treated with vinblastine. Mcl-1 phosphorylation closely paralleled cyclin B expression, and specific cyclin-dependent kinase (Cdk) inhibitors blocked vinblastine-induced Mcl-1 phosphorylation, its subsequent degradation, and improved cell viability after mitotic arrest. Co-immunoprecipitation studies indicated that Mcl-1 was complexed with Bak, but not Bax or Noxa, in untreated cells, and that Bak became activated in concert with loss of Mcl-1 expression. These results suggest that Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis via Mcl-1 phosphorylation, promoting its degradation and subsequently releasing Bak from sequestration., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This short review updates the maturation-inducing hormonal signaling in starfish oocytes. In this system, the activation of cyclin B-Cdc2 kinase (Cdk1) that leads to meiotic resumption does not require new protein synthesis. The key intracellular mediator after hormonal stimulation by 1-methyladenine is the protein kinase Akt/PKB, which in turn directly downregulates Myt1 and upregulates Cdc25 toward the activation of cyclin B-Cdc2. Mitotic kinases including Aurora, Plk1 and Greatwall are activated downstream of cyclin B-Cdc2. The starfish oocyte thus provides a simple model system for the study of meiotic resumption., (Copyright © 2011 Wiley Periodicals, Inc.)

The process of cell division in mammalian cells is orchestrated by cell-cycle-dependent oscillations of cyclin protein levels. Cyclin levels are controlled by redundant transcriptional, post-translational and degradation feedback loops. How each of these separate loops contributes to the regulation of the key cell cycle events and to the connection between the G1-S transition and the subsequent mitotic events is under investigation. Here, we present an integrated computational model of the mammalian cell cycle based on the sequential activation of cyclins. We validate the model against experimental data on liver cells (hepatocytes), which undergo one or two rounds of synchronous circadian-clock gated cell divisions during liver regeneration, after partial hepatectomy (PH). The model exhibits bandpass filter properties that allow the system to ignore strong but transient, or sustained but weak damages after PH. Bifurcation analysis of the model suggests two different threshold mechanisms for the progression of the cell through mitosis. These results are coherent with the notion that the mitotic exit in mammalian cells is bistable, and suggests that Cdc20 homologue 1 (Cdh1) is an important regulator of mitosis. Regulation by Cdh1 also explains the observed G2/M phase prolongation after hepatocyte growth factor (HGF) stimulation during S phase., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.

Scope: Sterigmatocystin (ST), a mycotoxin commonly found in foodstuff and feedstuff, has been shown to be a carcinogenic mycotoxin in animal models. Many studies showed that the high level of ST contamination in grains might be related to the high incidence of gastric carcinoma in rural areas of China. However, up to now, the potential effects of ST on human gastric epithelium cells remain largely unknown. In this study, we explored the effects of ST on cell-cycle distribution and the regulatory mechanism in immortalized human gastric epithelium cells (GES-1)., Methods and Results: The effects of ST on the cell cycle distribution of GES-1 cells were determined with flow cytometric (FCM) analysis, Giemsa staining and immunofluorescence staining, while that on the expression of related gene-Cdc25C, Cdc2, CyclinB1 and the complex of CyclinB1-Cdc2 were studied with Western blot, reverse transcription polymerase chain reaction (RT-PCR) and immunoprecipitation assay respectively. We found that ST induced GES-1 cells arrested at G2 phase by regulating the expression of Cdc25C, Cdc2, CyclinB1 and the formation of CyclinB1-Cdc2 complex. Further study suggested JNK, ERK and PI3K/AKT/mTOR pathways to be involved in the process of G2 arrest induced by ST. The specific inhibitors of JNK and ERK reversed the role of ST, whereas that of PI3K/AKT/mTOR reinforced the effect of ST on cell-cycle distribution., Conclusion: This study demonstrates that JNK, ERK and PI3K/AKT/mTOR pathways participated in the G2 arrest induced by ST through the deregulation of CyclinB1, Cdc2 and Cdc25C. It may play some roles in the gastric carcinogenesis in ST exposure populations., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery. Dosage suppression experiments suggested that the clb5Δ rapamycin hypersensitivity reflects a unique Clb5-associated cyclin-dependent kinase (CDK) function that cannot be performed by mitotic cyclins and that also involves motor proteins, particularly the kinesin-like protein Kip3. Synchronized cell experiments revealed rapamycin-induced defects in pre-anaphase spindle assembly and S phase progression that were more severe in clb5Δ than in wild-type cells but no apparent activation of Rad53-dependent checkpoint pathways. Some rapamycin-treated cells had aberrant spindle morphologies, but rapamycin did not cause gross defects in the microtubule cytoskeleton. We propose a model in which TORC1 and Clb5/CDK act coordinately to promote both spindle assembly via a pathway involving Kip3 and S phase progression.

Cell death by the process of apoptosis plays important roles in development, tissue homeostasis, diseases and drug responses. The cysteine aspartyl protease caspase-9 plays a central role in the mitochondrial or intrinsic apoptotic pathway that is engaged in response to many apoptotic stimuli. Caspase-9 is activated in a large multimeric complex, the apoptosome, which is formed with apoptotic peptidase activating factor 1 (Apaf-1) in response to the release of cytochrome c from mitochondria. Once activated, caspase-9 cleaves and activates the effector caspases 3 and 7 to bring about apoptosis. This pathway is tightly regulated at multiple steps, including apoptosome formation and caspase-9 activation. Recent work has shown that caspase-9 is the direct target for regulatory phosphorylation by multiple protein kinases activated in response to extracellular growth/survival factors, osmotic stress or during mitosis. Here, we review these advances and discuss the possible roles of caspase-9 phosphorylation in the regulation of apoptosis during development and in pathological states, including cancer.

The breast cancer susceptibility gene (BRCA2) is localized mainly in the nucleus where it plays an important role in DNA damage repair. Some BRCA2 protein is also present in the centrosome. Here, we demonstrate that BRCA2 interacts with plectin, a cytoskeletal cross-linker protein, and that this interaction controls the position of the centrosome. Phosphorylation of plectin by cyclin-dependent kinase 1/cyclin B (CDK1/CycB) kinase has been reported to abolish its cross-linking function during mitosis. Here, we induced phosphorylation of plectin in prepared fractions of HeLa cells by adding activated CDK1/CycB kinase. Consequently, there was significant dissociation of the centrosome from the nuclear membrane. Plectin has six homologous ankyrin-like repeat domains (termed PLEC M1-M6). Using a pull-down assay, we found that GST-PLEC M1 and a GST-C-terminal region fusion protein (which comprised PLEC M6, along with an adjacent vimentin site) interacted with BRCA2. Since each PLEC module exhibits high homology to the others, the possibility of all six domains participating in this interaction was indicated. Moreover, when PLEC M1 was overexpressed in HeLa cells, it competed with endogenous plectin and inhibited the BRCA2-plectin interaction. This inhibitory effect resulted in dissociation of the centrosomes from the nucleus and increased the rate of micronuclei formation which may lead to carcinogenesis. In addition, when either BRCA2 or plectin was suppressed by the appropriate siRNA, a similar change in centrosomal positioning was observed. We suggest that the BRCA2-plectin interaction plays an important role in the regulation of centrosome localization and also that displacement of the centrosome may result in genomic instability and cancer development.

CLIP-170, the founding member of microtubule "plus ends tracking" proteins, is involved in many critical microtubule-related functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr(287) in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.

The B-type cyclin Clb5 is involved primarily in control of DNA replication in Saccharomyces cerevisiae. We conducted a synthetic genetic array (SGA) analysis, testing for synthetic lethality between the clb5 deletion and a selected 87 deletions related to diverse aspects of cell cycle control based on GO annotations. Deletion of the spindle checkpoint genes BUB1 and BUB3 caused synthetic lethality with clb5. The spindle checkpoint monitors the attachment of spindles to the kinetochore or spindle tension during early mitosis. However, another spindle checkpoint gene, MAD2, could be deleted without ill effects in the absence of CLB5, suggesting that the bub1/3 clb5 synthetic lethality reflected some function other than the spindle checkpoint of Bub1 and Bub3. To characterize the lethality of bub3 clb5 cells, we constructed a temperature-sensitive clb5 allele. At nonpermissive temperature, bub3 clb5-ts cells showed defects in spindle elongation and cytokinesis. High-copy plasmid suppression of bub3 clb5 lethality identified the C-terminal fragment of BIR1, the yeast homolog of survivin; cytologically, the BIR1 fragment rescued the growth and cytokinesis defects. Bir1 interacts with IplI (Aurora B homolog), and the addition of bub3 clb5-ts significantly enhanced the lethality of the temperature-sensitive ipl1-321. Overall, we conclude that the synthetic lethality between clb5 and bub1 or bub3 is likely related to functions of Bub1/3 unrelated to their spindle checkpoint function. We tested requirements for other B-type cyclins in the absence of spindle checkpoint components. In the absence of the related CLB3 and CLB4 cyclins, the spindle integrity checkpoint becomes essential, since bub3 or mad2 deletion is lethal in a clb3 clb4 background. clb3 clb4 mad2 cells accumulated with unseparated spindle pole bodies. Thus, different B-type cyclins are required for distinct aspects of spindle morphogenesis and function, as revealed by differential genetic interactions with spindle checkpoint components.

Background and Aims: X-linked inhibitor of apoptosis-associated factor 1 (XAF1) was first recognized as an antagonist of X-linked inhibitor of apoptosis in suppressing caspase 3 activity. It has lower expression in cancer cells than normal tissue. Overexpression of XAF1 can inhibit cancer cell growth and sensitize tumor necrosis factor-related apoptosis-inducing ligand- or etoposide-induced apoptosis. The aim of this study is to elucidate the mechanism of XAF1 in regulating cell growth., Methods: Stable transfectants of gastrointestinal (GI) cancer cell lines AGS and SW1116 expressing XAF1 and vector control were generated. Cell growth, apoptosis, mitotic status and cell cycle distribution were assessed. The interaction between XAF1 and G(2)/M checkpoint proteins was evaluated by immunoblotting, kinase assay and co-immunoprecipitation assay. Mitotic catastrophe was identified by occurrence of aberrant nuclei and centrosomal amplification., Results: Our results showed that overexpression of XAF1 suppressed serum-dependent cancer cell growth, induced mitotic catastrophe and G(2)/M cell cycle arrest. Interestingly, XAF1 was predominantly expressed in G(2)/M phase after cell cycle synchronization. XAF1 interacted with and activated checkpoint kinase 1 (Chk1), inactivated Cdc25C and lead to inactivation of Cdc2-cyclin B complex. Suppression of Chk1 abrogated XAF1-induced G(2)/M arrest., Conclusions: Our findings implicate XAF1 as a novel cell cycle modulator that is recruited in G(2)/M phase and thus unravel a novel function pathway of XAF1, suggesting the potential role of XAF1 as the target for the management of GI cancers.

Oncoprotein 18/stathmin (Op18/stathmin) plays a crucial role in maintaining cell biological characteristics by regulating microtubule dynamics, especially entry into mitosis; phosphorylated Op18/stathmin promotes microtubule polymerization to form the mitotic spindle, which is essential for chromosome segregation and cell division. Cdc2 is a critical kinase in starting M phase events in cell-cycle progression and is a positive regulator of the cell cycle. Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncogenic protein that is able to induce carcinogenesis via various signaling pathways. This study focused on regulation by LMP1 of Op18/stathmin signaling in nasopharyngeal carcinoma (NPC) cells and showed that LMP1 regulates Op18/stathmin signaling through cdc2 mediation, LMP1 upregulates cdc2 kinase activity, and Op18/stathmin phosphorylation promotes the interaction of cdc2 with Op18/stathmin and microtubule polymerization during mitosis, and inhibition of LMP1 expression attenuates the interaction of cdc2 and Op18/stathmin and promotes microtubule depolymerization. These results reveal a new pathway via which LMP1 regulates Op18/stathmin signaling by cdc2 mediation; this new signaling pathway not only perfects the LMP1 regulation network but also elucidates the molecular mechanism of LMP1 that leads to carcinogenesis.

The conserved murine gammaherpesvirus 68 ORF20 has recently been demonstrated to induce G2 cell cycle arrest followed by apoptosis in human and mouse cells. Here, we demonstrate that its homologues UL24 in HSV-1, ORF20 in KSHV and UL76 in HCMV are also inducers of cell cycle arrest followed by apoptosis in both human and mouse cells. The mechanism of action is similar to that reported for MHV-68 ORF20, inactivating the mitotic complex cyclinB/Cdc2.

Cell-cycle progression is regulated by cyclin-dependent kinases (CDKs). CDK1 and CDK2 can be also activated by noncyclin proteins named RINGO/Speedy, which were identified as inducers of the G2/M transition in Xenopus oocytes. However, it is unclear how XRINGO triggers M phase entry in oocytes. We show here that XRINGO-activated CDKs can phosphorylate specific residues in the regulatory domain of Myt1, a Wee1 family kinase that plays a key role in the G2 arrest of oocytes. We have identified three Ser that are major phosphoacceptor sites for CDK/XRINGO but are poorly phosphorylated by CDK/cyclin. Phosphorylation of these Ser inhibits Myt1 activity, whereas their mutation makes Myt1 resistant to inhibition by CDK/XRINGO. Our results demonstrate that XRINGO-activated CDKs have different substrate specificity than the CDK/cyclin complexes. We also describe a mechanism of Myt1 regulation based on site-specific phosphorylation, which is likely to mediate the induction of G2/M transition in oocytes by XRINGO.

Testis-specific protein Y-encoded (TSPY) is the putative gene for the gonadoblastoma locus on the Y chromosome (GBY). TSPY and an X-homologue, TSPX, harbor a conserved domain, designated as SET/NAP domain, but differ at their C termini. Ectopic expression of TSPY accelerates cell proliferation by abbreviating the G(2)/M stage, whereas overexpression of TSPX retards cells at the same stage of the cell cycle. Previous studies demonstrated that the SET oncoprotein is capable of binding to cyclin B. Using various protein interaction techniques, we demonstrated that TSPY and TSPX indeed bind competitively to cyclin B at their SET/NAP domains in vitro and in vivo. TSPY colocalizes with cyclin B1 during the cell cycle, particularly on the mitotic spindles at metaphase. TSPY enhances while TSPX represses the cyclin B1-CDK1 phosphorylation activity. The inhibitory effect of TSPX on the cyclin B1-CDK1 complex has been mapped to its carboxyl acidic domain that is absent in TSPY, suggesting that TSPX could serve a normal function in modulating cell-cycle progression at the G(2)/M stage, whereas TSPY has acquired a specialized function in germ cell renewal and differentiation. Epigenetic dysregulation of TSPY in incompatible germ or somatic cells could promote cell proliferation and predispose susceptible cells to tumorigenesis.

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.

S-phase cyclin-dependent kinase Cdc28-Clb5 (CDK-S) and Dbf4-dependent kinase Cdc7-Dbf4 (DDK) are highly conserved kinases well known for their roles in the initiation of DNA replication. CDK-S is also essential for initiation of meiotic recombination because it phosphorylates Ser30 of Mer2, a meiosis-specific double-strand break (DSB) protein. This work shows that the phosphorylation of Mer2 Ser30 by CDK-S primes Mer2 for subsequent phosphorylation by DDK on Ser29, creating a negatively charged "patch" necessary for DSB formation. CDK-S and DDK phosphorylation of Mer2 S30 and S29 can be bypassed by phosphomimetic amino acids, but break formation under these conditions is still dependent on DDK and CDK-S activity. Coordination between premeiotic S and DSB formation may be achieved by using CDK-S and DDK to initiate both processes. Many other proteins important for replication, recombination, repair, and chromosome segregation contain combination DDK/CDK sites, raising the possibility that this is a common regulatory mechanism.

Cell cycle regulation in Eukaryotes is based on common molecular actors and mechanisms. However, the canonical cell cycle is modified in certain cells. Such modifications play a key role in oocyte maturation and embryonic development. They can be achieved either by introduction of new components, pathways, substrates, changed interactions between them, or by elimination of some factors inherited by the cells from previous developmental stages. Here we discuss a particular temporal regulation of the first embryonic M-phase of Xenopus and mouse embryos. These two examples help to understand better the general regulation of M-phase of the cell cycle.

Cyclin-dependent kinase 1 (CDK1) plays a crucial role in establishing metaphase and has also been shown to prevent DNA re-replication. Cyclins B1 and B2 are two known activators of CDK1 operating during mitosis in human cells. Little is known about the specific roles of each of these cyclins in CDK1 activation, but cyclin B2 is thought to play a minor role and to be unable to replace cyclin B1 for mitosis completion. In our study, we found that severe reduction by separate RNA interference of either cyclin B1 or cyclin B2 protein levels results in little or no alteration of the cell cycle and, more specifically, of mitosis progression. In contrast, simultaneous depletion of both B-type cyclins leads to massive accumulation of 4N cells, mitotic failure, premature mitosis exit and DNA re-replication. These defects can be corrected by the ectopic expression of a cyclin B2 resistant to the short hairpin RNA. Altogether, these data show that, in cycling human cells, cyclin B2 can compensate for the downregulation of cyclin B1 during mitosis. They also clearly implicate cyclins B1 and B2 as crucial activators of CDK1 in its biological function of DNA re-replication prevention.

Human gliomas are among the most aggressive tumors, and they respond poorly to treatment. The efficacy of surgical, radiation and chemotherapy treatment of these tumors is limited by the development of resistance. Interventions aimed at altering the response of these tumors to radiation or chemotherapy treatments are needed to improve survival rate and prognosis. Glioblastomas are generally p53 (TP53) functional tumors; however, DNA repair pathways are activated in these tumors instead of the pathways to apoptosis. Thus resistance to treatment is seen in the ability of these tumors to overcome cell death. We present data that demonstrate that U87MG glioblastoma cells transduced with a dominant-negative p53 adenovirus construct become sensitized to radiation-induced mitotic catastrophe through abrogation of G(2)/M checkpoint control and overaccumulation of cyclin B1. These findings suggest that interventions abrogating the G(2)/M checkpoint sensitize these cells to radiation-induced mitotic catastrophe and may represent a novel mechanism to increase the efficacy of radiation in wild-type p53 gliomas that are resistant to apoptosis.

Cyclin B is the key regulatory protein controlling mitosis in all eukaryotes, where it binds cyclin-dependent kinase, cdk1, forming a complex which initiates the mitotic program through phosphorylation of select proteins. Cyclin B regulates the activation, subcellular localization, and substrate specificity of cdk1, and destruction of cyclin B is necessary for mitotic exit. Overexpression of human cyclin B1 has been found in numerous cancers and has been associated with tumor aggressiveness. Here we report the crystal structure of human cyclin B1 to 2.9 A. Comparison of the structure with cyclin A and cyclin E reveals remarkably similar N-terminal cyclin box motifs but significant differences among the C-terminal cyclin box lobes. Divergence in sequence gives rise to unique interaction surfaces at the proposed cyclin B/cdk1 interface as well as the 'RxL' motif substrate binding site on cyclin B. Examination of the structure provides insight into the molecular basis for differential affinities of protein based cyclin/cdk inhibitors such as p27, substrate recognition, and cdk interaction.

DNA replication initiation in S. cerevisiae is promoted by B-type cyclin-dependent kinase (Cdk) activity. In addition, once-per-cell-cycle replication is enforced by cyclin-Cdk-dependent phosphorylation of the prereplicative complex (pre-RC) components Mcm2-7, Cdc6, and Orc1-6. Several of these controls must be simultaneously blocked by mutation to obtain rereplication. We looked for but did not obtain strong evidence for cyclin specificity in the use of different mechanisms to control rereplication: both the S-phase cyclin Clb5 and the mitotic cyclins Clb1-4 were inferred to be capable of imposing ORC-based and MCM-based controls. We found evidence that the S-phase cyclin Clb6 could promote initiation of replication without blocking reinitiation, and this activity was highly toxic when the ability of other cyclins to block reinitiation was prevented by mutation. The failure of Clb6 to regulate reinitiation was due to rapid Clb6 proteolysis, since this toxic activity of Clb6 was lost when Clb6 was stabilized by mutation. Clb6-dependent toxicity is also relieved when early accumulation of mitotic cyclins is allowed to impose rereplication controls. Cell-cycle timing of rereplication control is crucial: sufficient rereplication block activity must be available as soon as firing begins. DNA rereplication induces DNA damage, and when rereplication controls are compromised, the DNA damage checkpoint factors Mre11 and Rad17 provide additional mechanisms that maintain viability and also prevent further rereplication, and this probably contributes to genome stability.

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.

Members of the novel family of proteins that include Drosophila Mip130, Caenorhabditis elegans LIN-9, and mammalian LIN-9 intervene in different cellular functions such as regulation of transcription, differentiation, transformation, and cell cycle progression. Here we demonstrate that LIN-9, designated as Mip/LIN-9, interacts with B-Myb but not with c-Myb or A-Myb. Mip/LIN-9 regulates the expression of B-Myb in a post-transcriptional manner, and its depletion not only decreases the level of the B-Myb protein but also affects the expression of S phase and mitotic genes (i.e. cyclin A, CDK1, and cyclin B). The critical role of Mip/LIN-9 on the expression of S and G(2)/M genes is further supported by the finding that coexpression of Mip/LIN-9 and B-Myb results in the activation of cyclin A and cyclin B promoter-luciferase reporters, and both proteins are detected on the cyclin A and B promoters. Interestingly, although Mip/LIN-9 promoter occupancy peaks earlier than B-Myb, the highest levels of expression of cyclins A and B correlate with the maximum binding of B-Myb to these promoters. These data support the concept that Mip/LIN-9 is required for the expression of B-Myb, and both proteins collaborate in the control of the cell cycle progression via the regulation of S phase and mitotic cyclins.

When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM-independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM-dependent and ATR/ATM-independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38 alpha,beta are not involved in the ATR/ATM-independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM-independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry.

The mammalian bromodomain protein Brd4 interacts with mitotic chromosomes by binding to acetylated histone H3 and H4 and is thought to play a role in epigenetic memory. Mitotic cells are susceptible to antimicrotubule drugs. These drugs activate multiple response pathways and arrest cells at mitosis. We found that Brd4 was rapidly released from chromosomes upon treatment with antimicrotubule drugs, including the reversible agent nocodazole. Yet, when nocodazole was withdrawn, Brd4 was reloaded onto chromosomes, and cells proceeded to complete cell division. However, cells in which a Brd4 allele was disrupted (Brd4+/-), and expressing only half of the normal Brd4 levels, were defective in reloading Brd4 onto chromosomes. Consequently, Brd4+/- cells were impaired in their ability to recover from nocodazole-induced mitotic arrest: a large fraction of +/- cells failed to reach anaphase after drug withdrawal, and those that entered anaphase showed an increased frequency of abnormal chromosomal segregation. The reloading defect observed in Brd4+/- cells coincided with selective hypoacetylation of lysine residues on H3 and H4. The histone deacetylase inhibitor trichostatin A increased global histone acetylation and perturbed nocodazole-induced Brd4 unloading. Brd4 plays an integral part in a cellular response to drug-induced mitotic stress by preserving a properly acetylated chromatin status.

The Rho activator ECT2 functions as a key regulator in cytokinesis. ECT2 is phosphorylated during G2/M phase, but the physiological significance of this event is not well known. In this study, we show that phosphorylation of ECT2 at threonine-341 (T341) affects the autoregulatory mechanism of ECT2. In G2/M phase, ECT2 was phosphorylated at T341 most likely by Cyclin B/Cyclin-dependent kinase 1 (Cdk1), and then dephosphorylated before cytokinesis. Depletion of ECT2 by RNA interference (RNAi) efficiently induced multinucleate cells. Expression of the phospho-deficient mutant of ECT2 at T341 suppressed the multinucleation induced by RNAi to ECT2, indicating that ECT2 is biologically active even when it is not phosphorylated at T341. However, the phospho-mimic mutation at T341 weakly stimulates the catalytic activity of ECT2 as detected by serum response element reporter gene assays. As T341 is located at the hinge region of the N-terminal regulatory domain and C-terminal catalytic domain, phosphorylation of T341 may help accessing downstream signaling molecules to further activate ECT2. We found that the phospho-mimic mutation T341D increases binding with itself or the N-terminal half of ECT2. These results suggest a conformational change of ECT2 upon phosphorylation at T341. Therefore, ECT2 activity might be regulated by the phosphorylation status of T341. We propose that T341 phosphorylation by Cyclin B/Cdk1 could be a trigger for further activation of ECT2.

Previous studies suggested that geminin plays a vital role in both origin assembly and DNA re-replication during S-phase; however, no data to support a role for geminin in G2/M cells have been described. Here it is shown that in G2/M-phase, geminin participates in the promotion of proper cytokinesis. This claim can be supported through a series of observations. First, geminin in G2/M is loaded onto chromatin after it is tyrosine phosphorylated. It is unlike S-phase geminin that resides in the nuclear soluble fraction, where it is exclusively S/T phosphorylated. Secondly, on chromatin, geminin gets S/T phosphorylated in late G1; this modification causes the release of geminin from the chromatin. Cyclins bind and phosphorylate geminin in a sequential, cell cycle-dependent manner. These modifications correlated well with geminin departure from the chromatin. This suggests that cyclin functions to either release geminin from chromatin or at least keep it at bay until late S-phase. Thirdly, depletion of geminin from a diploid mammary epithelial cell line (HME) causes cells to arrest in late G2/M-phase. Massive serine-10 phosphorylated histone H3 staining and survivin localization to mid-body were observed; this suggests that they could be arrested in either mitosis or at cytokinesis. Finally, while in the absence of geminin, cyclin B1, chk1 and cdc7 are all over expressed. This paper will demonstrate that only cdc7 is important in maintaining the cytokinesis arrest in the absence of geminin. Only double depletion of geminin and cdc7 induce apoptosis. Our results taken together show, for the first time, that phosphorylation-induction activates oscillation of geminin between both nuclear soluble and chromatin compartments. Chromatin-bound geminin species functions to initiate or maintain proper cytokineses. In the absence of geminin, cells arrest in cytokinesis; this defines a novel checkpoint, monitored by cdc7, rather than cyclin B1 or chk1.

There are two major problems for the cell to solve in mitosis: how to ensure that each daughter cell receives an equal and identical complement of the genome, and how to prevent cell separation before chromosome segregation. Both these problems are solved by controlling when two specific proteins are destroyed: securin, an inhibitor of chromosome segregation, and cyclin B, which inhibits cell separation (cytokinesis). It has recently become clear that several other proteins are degraded at specific points in mitosis. This review (which is part of the Chromosome Segregation and Aneuploidy series) focuses on how specific proteins are selected for proteolysis at defined points in mitosis and how this contributes to the proper coordination of chromosome segregation and cytokinesis.

Overexpression of the oncogenic serine/threonine kinase Pim-1 has been shown to induce chromosomal missegregation and polyploidy in prostate epithelial cell lines (1). Here we demonstrated that Pim-1-induced polyploidy develops in a passage-dependent manner in culture consistent with a stochastic mode of progression. Induction of chromosomal instability by Pim-1 was not restricted to prostate cells as it was also observed in telomerase-immortalized normal human mammary epithelial cells. Elevated levels of cyclin B1 protein, but not its messenger RNA, were evident in early passage Pim-1 overexpressing cells, suggesting that increased cyclin B1 levels contribute to the development of polyploidy. Furthermore, regulation of cyclin B1 protein and cyclin B1/CDK1 activity after treatment with anti-microtubule agents was impaired. Small interfering RNA targeting cyclin B1 reversed the cytokinesis delay but not the mitotic checkpoint defect in Pim-1 overexpressing cells. These results indicated that chronic Pim-1 overexpression dysregulates cyclin B1 protein expression, which contributes to the development of polyploidy by delaying cytokinesis.

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.

Recent studies have shown that Cyclin B1 is overexpressed in various tumor types but present at low levels in normal tissues. To explore the possibility of employing Cyclin B1 as an anticancer target, we knocked down Cyclin B1 in an HeLa cell line using RNA interference (RNAi). Subsequently, we monitored cell cycle-related molecules by Western blot together with immunofluorescence and determined cell cycle distribution by flow cytometry. XTT and soft agar colony growth experiments were performed to detect cell viability and proliferation. Furthermore, we analyzed cell apoptosis by measuring Bcl-2 and Bax protein level and DNA-ladder assay. After performing Cyclin B1 RNAi, Cyclin B1, Cyclin A and Cdk2 protein levels were found to be markedly downregulated, whereas Cdc2 was almost unaffected; S-phase fraction increased significantly; HeLa cell viability and cell colony forming ability were markedly diminished after the RNAi; Bcl-2 was noticeably attenuated but Bax was hardly changed; and HeLa cells displayed typical DNA ladder. The loss of Cyclin B1 resulted in the downregulation of Cyclin A and Cdk2, S-phase delay and eventually led to cell apoptosis and the decrease of cell viability and proliferation. Our studies suggest that Cyclin B1 may be a promising anticancer target.

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCF(NIPA)) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCF(NIPA). Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCF(NIPA)-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.

In response to DNA damage, the cellular decision of life versus death involves an intricate network of multiple factors that play critical roles in regulation of DNA repair, cell cycle, and cell death. DNA damage checkpoint proteins are crucial for maintaining DNA integrity and normal cellular functions, but they may also reduce the effectiveness of cancer treatment. Here we report the involvement of Cdk5 activator p35-binding protein C53 in regulation of apoptosis induced by genotoxic stress through modulating Cdk1-cyclin B1 function. C53 was originally identified as a Cdk5 activator p35-binding protein and a caspase substrate. Importantly, our results demonstrated that C53 deficiency conferred partial resistance to genotoxic agents such as etoposide and x-ray irradiation, whereas ectopic expression of C53 rendered cells susceptible to multiple genotoxins that usually trigger G(2)/M arrest. Furthermore, we found that Cdk1 activity was required for etoposide-induced apoptosis of HeLa cells. Overexpression of C53 promoted Cdk1 activity and nuclear accumulation of cyclin B1, whereas C53 deficiency led to more cytoplasmic retention of cyclin B1, suggesting that C53 acts as a pivotal player in modulating the G(2)/M DNA damage checkpoint. Finally, C53 and cyclin B1 co-localize and associate in vivo, indicating a direct role of C53 in regulating the Cdk1-cyclin B1 complex. Taken together, our results strongly indicate that in response to genotoxic stress, C53 serves as an important regulatory component of the G(2)/M DNA damage checkpoint. By overriding the G(2)/M checkpoint-mediated inhibition of Cdk1-cyclin B1 function, ectopic expression of C53 may represent a novel approach for chemo- and radio-sensitization of cancer cells.

In mammalian somatic cells, the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation by delaying cell-cycle progression in the face of even a single misaligned chromosome. In contrast, the role of the SAC in unperturbed mammalian oocytes is less well defined as progression through meiosis I is unaltered in mouse oocytes in the presence of one or a few misaligned chromosomes. Furthermore, attempts to disable the function of the SAC protein, Mad2, in mouse oocytes have produced conflicting results. To gain further insight into SAC function during female mammalian meiosis I, we recently utilised a morpholino-based antisense approach to deplete the majority of Mad2 in mouse oocytes. Our results define a clear role for Mad2 in ensuring the proper timing of meiosis I events and ultimately, in ensuring the fidelity of homologue disjunction. We discuss the implications of these results for the regulation of meiosis I in mammalian oocytes and for the genesis of human aneuploidy.

Background and Purpose: To elucidate the role of TP53 on differential effects of topoisomerase I inhibitor topotecan (Hycamtin on radiation sensitivity., Materials and Methods: Cell cycle distribution and protein expression of TP53, p21(WAF1/CIP1) and cyclin B was studied in CCD32 lung fibroblasts, glioblastoma cell lines U118 (mutant TP53), and U87 (wildtype TP53) after treatment with topotecan (0.05 and 1 microM) and/or ionizing radiation (2 Gy)., Results: Cell cycle effects varied with topotecan concentration, resulting in G1 arrest (1 microM), or S/G2/M arrest (0.05 microM), and was modified differentially in fibroblasts and in glioblastoma cells in combination with irradiation. Phosphorylation of TP53 and expression of p21(WAF1/CIP1) was induced by IR and/or topotecan in CCD32 cells, and in U118 cells after topotecan treatment, accompanied by cyclin B degradation. In U87 cells only 1 microM topotecan generated phosphorylation of TP53 and p21(WAF1/CIP1) expression; 0.05 microM caused stabilization of cyclin B., Conclusions: The antagonistic effect of combined topotecan/irradiation treatment in fibroblasts was most likely due to an immediate radiation induced G1 arrest, but was not observed in p53 wildtype glioblastoma cells. Thus, the impact of TP53 on the topotecan response remains indistinct, and is obviously influenced by other genomic alterations acquired by tumor cells.