Spindle Assembly Checkpoint Components Research Articles

Usp7 protects genomic stability by regulating Bub3.

USP7 (Ubiquitin Specific processing Protease-7) is a deubiquitinase which, over the past decade emerged as a critical regulator of cellular processes. Deregulation of USP7 activity has been linked to cancer, making USP7 inhibition an appealing anti-cancer strategy. The identification of novel USP7 substrates and additional USP7-dependent cellular activities will broaden our knowledge towards potential clinical application of USP7 inhibitors. Results presented in this study uncover a novel and pivotal function of USP7 in the maintenance of genomic stability. Upon USP7 depletion we observed prolonged mitosis and mitotic abnormalities including micronuclei accumulation, lagging chromosomes and karyotype instability. Inhibition of USP7 with small molecule inhibitors stabilizes cyclin B and causes mitotic abnormalities. Our results suggest that these USP7-dependent effects are mediated by decreased levels of spindle assembly checkpoint (SAC) component Bub3, which we characterized as an interacting partner and substrate of USP7. In silico analysis across the NCI-60 panels of cell lines supports our results where lower levels of USP7 strongly correlate with genomic instability. In conclusion, we identified a novel role of USP7 as regulator of the SAC component Bub3 and genomic stability.

Phosphorylation Regulates the p31Comet-Mitotic Arrest-deficient 2 (Mad2) Interaction to Promote Spindle Assembly Checkpoint (SAC) Activity

The spindle assembly checkpoint (SAC) ensures the faithful segregation of the genome during mitosis by ensuring that sister chromosomes form bipolar attachments with microtubules of the mitotic spindle. p31(Comet) is an antagonist of the SAC effector Mad2 and promotes silencing of the SAC and mitotic progression. However, p31(Comet) interacts with Mad2 throughout the cell cycle. We show that p31(Comet) binds Mad2 solely in an inhibitory manner. We demonstrate that attenuating the affinity of p31(Comet) for Mad2 by phosphorylation promotes SAC activity in mitosis. Specifically, phosphorylation of Ser-102 weakens p31(Comet)-Mad2 binding and enhances p31(Comet)-mediated bypass of the SAC. Our results provide the first evidence for regulation of p31(Comet) and demonstrate a previously unknown event controlling SAC activity.

Spindle assembly checkpoint protein expression correlates with cellular proliferation and shorter time to recurrence in ovarian cancer.

Ovarian carcinoma (OC) is the most lethal of the gynecological malignancies, often presenting at an advanced stage. Treatment is hampered by high levels of drug resistance. The taxanes are microtubule stabilizing agents, used as first-line agents in the treatment of OC that exert their apoptotic effects through the spindle assembly checkpoint. BUB1-related protein kinase (BUBR1) and mitotic arrest deficient 2 (MAD2), essential spindle assembly checkpoint components, play a key role in response to taxanes. BUBR1, MAD2, and Ki-67 were assessed on an OC tissue microarray platform representing 72 OC tumors of varying histologic subtypes. Sixty-one of these patients received paclitaxel and platinum agents combined; 11 received platinum alone. Overall survival was available for all 72 patients, whereas recurrence-free survival (RFS) was available for 66 patients. Increased BUBR1 expression was seen in serous carcinomas, compared with other histologies (P = .03). Increased BUBR1 was significantly associated with tumors of advanced stage (P = .05). Increased MAD2 and BUBR1 expression also correlated with increased cellular proliferation (P < .0002 and P = .02, respectively). Reduced MAD2 nuclear intensity was associated with a shorter RFS (P = .03), in ovarian tumors of differing histologic subtype (n = 66). In this subgroup, for those women who received paclitaxel and platinum agents combined (n = 57), reduced MAD2 intensity also identified women with a shorter RFS (P < .007). For the entire cohort of patients, irrespective of histologic subtype or treatment, MAD2 nuclear intensity retained independent significance in a multivariate model, with tumors showing reduced nuclear MAD2 intensity identifying patients with a poorer RFS (P = .05).

Non-canonical function of spindle assembly checkpoint proteins after APC activation reduces aneuploidy in mouse oocytes

The spindle assembly checkpoint (SAC) prevents aneuploidy by coupling anaphase onset, through anaphase-promoting complex (APC) activation, with chromosome attachment to spindle microtubules. Here, we examine APC activity in oocytes, noted for their susceptibility to chromosome mis-segregation during the first meiotic division (MI). We find that MI oocytes only contain sub-maximal APC activity, measured through cyclin B1-GFP degradation, because inhibition of SAC proteins when the APC is normally fully active increases cyclin B1 degradation twofold and reduces the length of this division by 2 h. In addition, inhibiting the SAC component Mps1 only when the APC is already active increases aneuploidy rates in the resulting egg by up to 30%. We therefore establish that the activities of SAC proteins and the APC co-exist in oocytes, and such concurrence has a vital role in reducing aneuploidy rates by extending MI, probably by allowing time for numerous erroneous microtubule attachments to be corrected.

NUP98 Fusion Oncoproteins Promote Aneuploidy by Attenuating the Mitotic Spindle Checkpoint

NUP98 is a recurrent fusion partner in chromosome translocations that cause acute myelogenous leukemia. NUP98, a nucleoporin, and its interaction partner Rae1, have been implicated in the control of chromosome segregation, but their mechanistic contributions to tumorigenesis have been unclear. Here, we show that expression of NUP98 fusion oncoproteins causes mitotic spindle defects and chromosome missegregation, correlating with the capability of NUP98 fusions to cause premature securin degradation and slippage from an unsatisfied spindle assembly checkpoint (SAC). NUP98 fusions, unlike wild-type NUP98, were found to physically interact with the anaphase promoting complex/cyclosome (APC/C)(Cdc20) and to displace the BubR1 SAC component, suggesting a possible mechanistic basis for their interference with SAC function. In addition, NUP98 oncoproteins displayed a prolonged half-life in cells. We found that NUP98 stability is controlled by a PEST sequence, absent in NUP98 oncoproteins, whose deletion reproduced the aberrant SAC-interfering activity of NUP98 oncoproteins. Together, our findings suggest that NUP98 oncoproteins predispose myeloid cells to oncogenic transformation or malignant progression by promoting whole chromosome instability.

Selective Inhibition of Pancreatic Ductal Adenocarcinoma Cell Growth by the Mitotic MPS1 Kinase Inhibitor NMS-P715

Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy.

BuGZ Is Required for Bub3 Stability, Bub1 Kinetochore Function, and Chromosome Alignment

During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora B kinase activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologsarehighly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest that BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes.

In Vitro Studies of the Spindle Assembly Checkpoint in Yeast

Although extremely rare, errors in chromosome segregation lead to aneuploidy, which has been linked to cancer and birth defects. Dividing cells rely on a surveillance mechanism called the spindle assembly checkpoint (SAC), which ensures that chromosomes are properly segregated during mitosis. The SAC, comprising several proteins on the mitotic kinetochore emits a diffusible ‘wait’ signal upon detection of improper and/or unattached kinetochores, delaying mitosis. Proper attachment and biorientation silences the ‘wait’ signal allowing progress through mitosis. ‘Wait’ signal silencing is thought of in two possible ways: First, the SAC responds to kinetochore-microtubule tension. Second, the SAC responds to attachment of microtubules to kinetochores, independent of tension. This attachment-tension debate is still unresolved due to the challenge of independently controlling attachment states and levels of tension on kinetochores in vivo. To help shed more light, we purified mitotic kinetochores from budding yeast with core kinetochore components, Mtw1p and Nuf2p, fluorescently labeled. We immobilized purified kinetochores onto a coverslip in our in vitro assay, and using a very sensitive Total Internal Reflection Fluorescence Microscopy system measured an Mtw1p-Nuf2p fluorescence colocalization of 34 ± 2.2%. We performed a similar experiment, immobilizing the kinetochores onto microtubules. We measured a colocalization on microtubules of 81±6.4% indicating that Nuf2p remained bound to the kinetochore upon kinetochore-microtubule attachment in vitro, as expected. We repeated this experiment for kinetochores with fluorescently labeled Mtw1p and Bub1p, a SAC component. We measured a 12 ± 3.4% Mtw1p-Bub1p colocalization when the particles were immobilized onto a coverslip and 50 ± 1.9% on microtubules. These experiments indicate that kinetochore-microtubule attachment in yeast may not be sufficient to release Bub1p. Current efforts are focused on Mad1p, a checkpoint protein that is believed to be released from kinetochores upon microtubule attachment in vivo.

An Overview of the Spindle Assembly Checkpoint Status in Oral Cancer

Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the core components of SAC and its target Cdc20.

The dynamic protein Knl1 - a kinetochore rendezvous.

Knl1 (also known as CASC5, UniProt Q8NG31) is an evolutionarily conserved scaffolding protein that is required for proper kinetochore assembly, spindle assembly checkpoint (SAC) function and chromosome congression. A number of recent reports have confirmed the prominence of Knl1 in these processes and provided molecular details and structural features that dictate Knl1 functions in higher organisms. Knl1 recruits SAC components to the kinetochore and is the substrate of certain protein kinases and phosphatases, the interplay of which ensures the exquisite regulation of the aforementioned processes. In this Commentary, we discuss the overall domain organization of Knl1 and the roles of this protein as a versatile docking platform. We present emerging roles of the protein interaction motifs present in Knl1, including the RVSF, SILK, MELT and KI motifs, and their role in the recruitment and regulation of the SAC proteins Bub1, BubR1, Bub3 and Aurora B. Finally, we explore how the regions of low structural complexity that characterize Knl1 are implicated in the cooperative interactions that mediate binding partner recognition and scaffolding activity by Knl1.

Germline Mutations in the Spindle Assembly Checkpoint Genes BUB1 and BUB3 Are Risk Factors for Colorectal Cancer

The spindle assembly checkpoint controls proper chromosome segregation during mitosis and prevents aneuploidy-an important feature of cancer cells. We performed genome-wide and targeted copy number and mutation analyses of germline DNA from 208 patients with familial or early-onset (40 years of age or younger) colorectal cancer; we identified haploinsufficiency or heterozygous mutations in the spindle assembly checkpoint genes BUB1 and BUB3 in 2.9% of them. Besides colorectal cancer, these patients had variegated aneuploidies in multiple tissues and variable dysmorphic features. These results indicate that mutations in BUB1 and BUB3 cause mosaic variegated aneuploidy and increase the risk of colorectal cancer at a young age.

Abstract 4434: Identification of radiation susceptibility genes in prostate cancer by whole genome RNA interference screening.

Abstract Background: Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in Western world males. PSA testing allows early detection of localized disease. Treatment options for patients with local disease include radiotherapy and surgery. However, local tumor control rates after radiotherapy are highly variable; and ∼40% of treated patients will experience disease recurrence and progression. Advanced prostate cancer has a very poor prognosis, emphasizing the need for more effective early local treatment, preventing advanced disease. Here, we set out to identify molecular targets of radiation susceptibility in PCa cells as a starting-point to increase the therapeutic index of radiotherapy. Methods: Targets were identified by high-throughput screening using a human whole genome siRNA library on the radioresistant PCa cell line PC-3 subjected to 4Gy irradiation. Combined effects of radiation and target gene silencing were quantified using a cell viability assay based on automated cell counting four days after irradiation. Candidate targets were validated using the colony formation assay (CFA) scored 8-10 days after irradiation. Results: We conducted two genome-wide siRNA screens with and without irradiation in PC-3 cells. We used a linear regression model to identify radiation susceptibility genes and selected 45 candidate targets with FDR &amp;lt; 10% for further research. In a conformation screen, multiple independent siRNAs silencing 17 genes induced ≥2-fold more cell death upon 4Gy irradiation compared to a negative control siRNA. Confirmed hits were validated independently using the CFA. The dose modifying factor at 80% clonogenic cell survival (DMF0.8) was calculated for each gene-specific siRNA compared to control siRNA from linear-quadratic survival curves. Sixteen candidate genes were validated with a DMF0.8 ≥ 1.3. This included the mitotic spindle assembly checkpoint component MAD2L2, which was previously reported as a radiosensitizing target in PCa cells (JCI 2011;121:2383-2390), thus validating our screens. Importantly, silencing of 9 genes sensitized PC-3 cells to irradiation more effectively than did silencing of the known radiation susceptibility gene PRKDC. Among these were genes involved in cell cycle control, ubiquitination, cell communication and G-protein coupled receptor signaling. Interestingly, silencing of two druggable genes, i.e. CPNE7 and GPR27, was particularly effective at low dose irradiation, resulting in a very high DMF0.8 of 4.6 and spectacular decreases in surviving fractions after 2Gy irradiation from 70% in controls to 37% and 23%, respectively. Conclusion: We have identified a set of novel candidate targets that could be used to potently enhance the efficacy of radiotherapy in prostate cancer. This could contribute significantly to improving overall success of prostate cancer intervention. Citation Format: Jasmina Hodzic, Ida van der Meulen, Renee de Menezes, Ilse Dingjan, Marielle JP Maas, Winald Gerritsen, Marcel Verheij, Ab A. Geldof, Baukelien van Triest, Victor W. van Beusechem. Identification of radiation susceptibility genes in prostate cancer by whole genome RNA interference screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2013-4434

Hec1-Dependent Cyclin B2 Stabilization Regulates the G2-M Transition and Early Prometaphase in Mouse Oocytes

SummaryThe functions of the Ndc80/Hec1 subunit of the highly conserved Ndc80 kinetochore complex are normally restricted to M phase when it exerts a pivotal kinetochore-based role. Here, we find that in mouse oocytes, depletion of Hec1 severely compromises the G2-M transition because of impaired activation of cyclin-dependent kinase 1 (Cdk1). Unexpectedly, impaired M phase entry is due to instability of the Cdk1-activating subunit, cyclin B2, which cannot be covered by cyclin B1. Hec1 protects cyclin B2 from destruction by the Cdh1-activated anaphase-promoting complex (APCCdh1) and remains important for cyclin B2 stabilization during early M phase, required for the initial stages of acentrosomal spindle assembly. By late M phase, however, Hec1 and cyclin B2 become uncoupled, and although Hec1 remains stable, APCCdc20 triggers cyclin B2 destruction. These data identify another dimension to Hec1 function centered on M phase entry and early prometaphase progression and challenge the view that cyclin B2 is completely dispensable in mammals.

Increased Expression of the Spindle Checkpoint Protein BubR1 is Associated with High Cell Proliferation in Primary Gastrointestinal Diffuse Large B Cell Lymphoma

Primary gastrointestinal diffuse large B cell lymphoma (PGI-DLBCL) is a relatively rare malignancy with limited results on clinical characteristics and carcinogenesis. Chromosome instability (CIN) is a hallmark of cancer cells, including lymphoma. As a key component of spindle assembly checkpoint (SAC), BubR1 plays a crucial role in maintaining genome stability. To elucidate the roles of BubR1 in the pathogenesis of PGI-DLBCL, and its relationship to cell proliferation, Helicobacter pylori infection, and BCL-6 gene translocation, we examined the possible alterations of BubR1 protein expression in PGI-DLBCL patients. Paraffin-embedded cancer tissues from 20 PGI-DLBCL patients were evaluated for BubR1 and H. pylori expression by immunohistochemistry, as well as cell proliferative activity measured by Ki-67 proliferation index (PI). BCL-6 gene rearrangement was assessed by fluorescence in situ hybridization (FISH) and BubR1 expression status was compared with clinicopathological parameters in PGI-DLBCL patients. Overexpression of BubR1 was observed in the majority of PGI-DLBCL patients. The mean expression level of BubR1 was 57.33 ± 23.27% in PGI-DLBCL, which was higher than normal gastrointestinal tissue (10.18 ± 5.65%) and reactive lymph node (26.74 ± 8.60%) (P < 0.01), while it was comparable to nodal lymphoma (54.32 ± 21.28%) (P > 0.05). BubR1 overexpression had a positive correlation with Ki-67 proliferation index (PI) (r = 0.51, P < 0.01) in PGI-DLBCL, but had no relationship to H. pylori infection and BCL-6 gene translocation. In addition, no correlation was found between BubR1 expression levels and overall survivals (P > 0.05). BubR1 overexpression was associated with cell proliferation and may play a role in the carcinogenesis of PGI-DLBCL. Aberrant BubR1 expression may potentially be a biomarker for estimating biologic characteristics of PGI-DLBCL.

Spindle checkpoint deficiency is tolerated by murine epidermal cells but not hair follicle stem cells.

The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by preventing aneuploidy, an event that is detrimental to the fitness and survival of normal cells but oncogenic in tumor cells. Deletion of SAC genes is incompatible with early mouse development, and RNAi-mediated depletion of SAC components in cultured cells results in rapid death. Here we describe the use of a conditional KO of mouse Mad2, an essential component of the SAC signaling cascade, as a means to selectively induce chromosome instability and aneuploidy in the epidermis of the skin. We observe that SAC inactivation is tolerated by interfollicular epidermal cells but results in depletion of hair follicle bulge stem cells. Eventually, a histologically normal epidermis develops within ∼1 mo after birth, albeit without any hair. Mad2-deficient cells in this epidermis exhibited abnormal transcription of metabolic genes, consistent with aneuploid cell state. Hair follicle bulge stem cells were completely absent, despite the continued presence of rudimentary hair follicles. These data demonstrate that different cell lineages within a single tissue respond differently to chromosome instability: some proliferating cell lineages can survive, but stem cells are highly sensitive.

Mitosis-Specific Regulation of Nuclear Transport by the Spindle Assembly Checkpoint Protein Mad1p

Nuclear pore complexes (NPCs) and kinetochores perform distinct tasks, yet their shared ability to bind several proteins suggests their functions are intertwined. Among these shared proteins is Mad1p, a component of the yeast spindle assembly checkpoint (SAC). Here we describe a role for Mad1p in regulating nuclear import that employs its ability to sense a disruption of kinetochore-microtubule interactions during mitosis. We show that kinetochore-microtubule detachment arrests nuclear import mediated by the transport factor Kap121p through a mechanism that requires Mad1p cycling between unattached, metaphase kinetochores and binding sites at the NPC. This signaling pathway requires the Aurora B-like kinase Ipl1p, and the resulting transport changes inhibit the nuclear import of Glc7p, a phosphatase that acts as an Ipl1p antagonist. We propose that a distinct branch of the SAC exists in which Mad1p senses unattached kinetochores and, by altering NPC transport activity, regulates the nuclear environment of the spindle.

Role of the Mad2 Dimerization Interface in the Spindle Assembly Checkpoint Independent of Kinetochores

The spindle assembly checkpoint (SAC) arrests cells when kinetochores are unattached to spindle microtubules. The signaling pathway is initiated at the kinetochores by one SAC component, Mad2, which catalyzes the initial steps of the cascade via the conformational dimerization of its open and closed conformers. Away from kinetochores, the dimerization surface of Mad2 has been proposed, based on data invitro, to either interact with SAC activators or inactivators and thus to contribute to SAC activation or silencing. Here, we analyze its role invivo. To analyze the putative pathway downstream of thekinetochores, we used two complementary approaches: we activated the SAC ectopically and independently from kinetochores, and we separated genetically the kinetochore-dependent and independent pools of Mad2. We found that the dimerization surface is required also downstream of kinetochores to mount a checkpoint response. Our results show that away from kinetochores the dimerization surface is required for stabilizing the end-product of the pathway, the mitotic checkpoint complex. Surprisingly, downstream of kinetochores the surface does not mediate Mad2 dimerization. Instead, our results are consistent with a role of Mad3 as the main interactor of Mad2 via the dimerization surface.

The APC/C Subunit Mnd2/Apc15 Promotes Cdc20 Autoubiquitination and Spindle Assembly Checkpoint Inactivation

The fidelity of chromosome segregation depends onthe spindle assembly checkpoint (SAC). In the presence of unattached kinetochores, anaphase isdelayed when three SAC components (Mad2, Mad3/BubR1, and Bub3) inhibit Cdc20, the activating subunit of the anaphase-promoting complex (APC/C). We analyzed the role of Cdc20 autoubiquitination in the SAC of budding yeast. Reconstitution with purified components revealed that a Mad3-Bub3 complex synergizes with Mad2 to lock Cdc20 on the APC/C and stimulate Cdc20 autoubiquitination, while inhibiting ubiquitination of substrates. SAC-dependent Cdc20 autoubiquitination required the Mnd2/Apc15 subunit of the APC/C. General inhibition of Cdc20 ubiquitination invivo resulted in high Cdc20 levels and a failure to establish a SAC arrest, suggesting that SAC establishment depends on low Cdc20 levels. Specific inhibition of SAC-dependent ubiquitination, by deletion of Mnd2, allowed establishment of a SAC arrest but delayed release from the arrest, suggesting that Cdc20 ubiquitination is also required for SAC inactivation.

Proper Cyclin B3 Dosage Is Important for Precision of Metaphase-to-Anaphase Onset Timing inCaenorhabditis elegans

Cyclin-dependent kinases (CDK) and their compulsory cofactors, the cyclins, are the two key classes of regulatory molecules that determine the eukaryotic cell's progress through the cell cycle by substrate phosphorylation. Cdk1 forms complexes with B-type cyclins and phosphorylates a number of substrates as cells prepare to enter mitosis. CYB-3 (Cyclin B3) is a B-type cyclin that has been recently shown to be required for the timely metaphase-to-anaphase transition, presumably by alleviating a spindle assembly checkpoint (SAC) block. Previously, we have shown that doubling the CYB-3 dosage suppresses sterility in the absence of the essential SAC component MDF-1/Mad1. Here we demonstrate the importance of the Mos1-mediated single-copy insertion method for understanding the effects of gene dosage by generating strains that have more (two or three) copies of the cyb-3 in wild-type and mdf-1(gk2) backgrounds to investigate dosage effect of CYB-3 on mitotic progression as well as development and fertility in the absence and the presence of the MDF-1 checkpoint component. We show that tripling the dosage of CYB-3 results in a significantly variable metaphase-to-anaphase transition, both in wild-type and mdf-1(gk2) mutant backgrounds. Although a majority of embryos initiate anaphase onset normally, a significant number of embryos initiate anaphase with a delay. We also show that tripling the dosage of CYB-3 has no effect on viability in the wild-type background; however, it does reduce the sterility caused by the absence of MDF-1. Together, these data reveal that proper dosage of CYB-3 is important for precision of timely execution of anaphase onset regardless of the presence of the MDF-1 checkpoint component.

Expression of the mitotic-arrest deficiency 2 is associated with chemotherapy resistance in ovarian serous adenocarcinoma

Mitotic-arrest deficiency2 (MAD2) is a key component of spindle assembly checkpoint (SAC) function; SAC mediates spindle microtubule attachment to kinetochores on chromosomes and chromosomal segregation during mitosis. To determine whether MAD2 expression is associated with chemotherapy resistance in ovarian serous adenocarcinoma, we reviewed tumor samples from 41cases of ovarian serous adenocarcinoma at Osaka City University Medical School Hospital (Osaka, Japan), 2000-2007. Of the 41cases, 24 were recurrent and 17 were not recurrent. Expression of MAD2 was investigated in paraffin-embedded sections using a MAD2 antibody. Quantitative analysis of MAD2 expression gave mean weighted scores of 4.3 for the relapsed group and 7.2 for the relapse-free group; the expression was, thus, significantly greater in the relapse-free group compared to the relapsed group (P=0.023). When the 41cases were classified into low- and high-expression, these classifications showed no significant difference in terms of progression-free survival (P=0.0685), however, overall survival for the low-expression group was significantly shorter than that of the high-expression group (P=0.0188). The present study implies that MAD2 expression levels can indicate sensitivity to anticancer agents, and risk for recurrence.