Mitotic Checkpoint Protein Mad2 Research Articles

Regulation of mitotic checkpoint protein MAD2 by its interacting partners

Regulation of mitotic checkpoint protein MAD2 by its interacting partners

Abstract PR007: The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers

Abstract Many diverse, common, and lethal cancer types have inactivation of the retinoblastoma (RB1, Rb) tumor suppressor pathway including cancers caused by the human papillomavirus (HPV), which causes >5% of all cancers worldwide. Despite therapeutic advances, advanced Rb-deficient cancers are still lethal with therapy-related adverse effects. Moreover, there are no therapies that uniquely target HPV-driven or Rb-deficient cancers. Therefore, biomarker-selected, targeted therapy for Rb-deficient cancers represents both a substantial unmet need and a significant translational knowledge gap. Rb-deficient cancer cells depend on Aurora A kinases to bypass the spindle assembly checkpoint (SAC) and survive due to overexpression of the mitotic checkpoint protein Mad2. Thyroid hormone receptor interactor 13 (TRIP13) catalyzes the ATP-dependent conversion of Mad2 from its active to inactive form, which turns off the SAC. Our lab discovered that the combination of Aurora A inhibition with alisertib plus TRIP13 depletion induced extensive apoptosis selectively in Rb-deficient cancer cells. Moreover, our results demonstrated that the synthetic lethality between Aurora A and TRIP13 reflects mitotic catastrophe. The combination led to highly abnormal mitoses in Rb-deficient cancer cells, evidenced by unaligned, dispersed chromosomes, mitotic spindle defects, and G2/M arrest. Moreover, live cell imaging showed that alisertib treatment increased the duration of first mitoses and induced mitotic cell death, which was even more pronounced in second mitotic events. The combination resulted in both prolonged mitotic arrest and enhanced lethality, indicated by increased mitotic cell death after the first mitoses and interphase cell death following the first mitotic arrest. Furthermore, using degron tagging to rapidly degrade TRIP13, we showed that TRIP13 depletion led to transient mitotic arrest in cells that were in late G2, supporting the role of TRIP13 in regulating the SAC and mitotic progression. Since prolonged mitotic arrest is known to cause DNA damage and both Aurora A and TRIP13 are known to be involved in the DNA damage response, we also tested the effect of the combination treatment on DNA damage. Treating Rb-deficient cancer cells with the combination resulted in robust increases in gamma-H2AX, pTIF1 beta (KAP1, TRIM28), and DNA PKcs. The lethal effects of the combination required mitotic entry as concurrent CDK1 inhibition with RO-3306 prevented apoptosis and DNA damage induced by the combination. The selective effect of the combination in Rb-deficient cancer cells may have a wide therapeutic index resulting in less toxic and more effective therapy. Additionally, targeting mitosis may contribute to a cancer-specific effect due to cancer cells’ high proliferation rate. Our study suggests that targeting TRIP13 and Aurora A led to DNA damage and death specifically during mitosis in Rb-deficient cells. This work has potential to establish a foundation for future clinical trials and may change current clinical practice paradigms. Citation Format: Lacin Yapindi, Soma Ghosh, Tuhina Mazumdar, Faye M. Johnson. The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR007.

FOXM1 is critical for the fitness recovery of chromosomally unstable cells

Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein MAD2, leading to mitotic arrest and cell death. However, some tumor cells are capable of exiting mitosis and consequently increasing CIN. How cells escape the mitotic arrest induced by MAD2 and proliferate with CIN is not well understood. Here, we explored loss-of-function screens and drug sensitivity tests associated with MAD2 levels in aneuploid cells and identified that aneuploid cells with high MAD2 levels are more sensitive to FOXM1 depletion. Inhibition of FOXM1 promotes MAD2-mediated mitotic arrest and exacerbates CIN. Conversely, elevating FOXM1 expression in MAD2-overexpressing human cell lines reverts prolonged mitosis and rescues mitotic errors, cell death and proliferative disadvantages. Mechanistically, we found that FOXM1 facilitates mitotic exit by inhibiting the spindle assembly checkpoint (SAC) and the expression of Cyclin B. Notably, we observed that FOXM1 is upregulated upon aneuploid induction in cells with dysfunctional SAC and error-prone mitosis, and these cells are sensitive to FOXM1 knockdown, indicating a novel vulnerability of aneuploid cells.

Abstract 1858: Rb deficient HPV+ HNSCC experienced enhanced sensitivity to aurora kinase inhibitors by altering the balance of MAD2 and TRIP13 levels

Abstract Human papilloma virus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) is common in young patients (<60 years). All patients experience either permanent toxicity from standard therapy or death from recurrent disease. To address this unmet need for more effective and less toxic therapy, we performed a high throughput drug screen comparing drug efficacy in HPV+ and HPV- cell lines. We tested the drug sensitivity of 864 unique drugs (0-3.1 µM) divided into 51 classes based on their targets in 33 squamous cancer cell lines using a parameter that is independent of cell division. Only one class of drugs, Aurora kinase inhibitors, was more effective in HPV+ than HPV- cell lines. To validate our screening results, we tested two Aurora kinase inhibitors (Alisertib, Barasertib) and observed significant cell death in HPV+ cells, but not in HPV- cells. Alisertib reduced tumor volume in HPV+ PDX mouse models without toxicity. To investigate the underlying mechanism, we compared the expression of ~300 proteins, using RPPA, to drug efficacy and validated the results using immunoblotting. The expression of Rb, pRb(S807/811), and p16 correlated with drug sensitivity. To establish role for Rb, we used a bidirectional approach to manipulate RB1. Reduction in RB1 levels using either shRNA or stable E7 overexpression in HPV- cells resulted in increased Alisertib-induced apoptosis. We also used siRNA to knock down E7 expression in two HPV+ cell lines which resulted in elevated levels of Rb, thereby rescuing the cells from Alisertib-induced apoptosis. Studies have shown Rb-deficient cells have elevated levels of the mitotic-checkpoint protein MAD2, that may impact cell fitness. To test the role of MAD2, we depleted the levels of MAD2 and observed diminishing levels of cyclin B1 accumulation and reduced Alisertib-induced apoptosis. Additionally, we altered the activated levels of MAD2 protein by overexpression of TRIP13 that resulted in partially rescuing cells from undergoing Alisertib-induced apoptosis. These results indicate that Rb- deficient MAD2 overexpressing cells require a finely tuned balance of MAD2 and TRIP13 levels for their mitotic exit. Furthermore, we observed combined inhibition of TRIP13 with Aurora A led to more apoptosis in Rb-deficient cells than the single agents. Our results demonstrate that Rb-deficient cancers are sensitive to Aurora A inhibition due to an imbalance between MAD2 and TRIP13. Based on the above finding, our present study shows that TRIP13 inhibition in combination with Aurora kinase inhibitors leads to more apoptosis and can pave a path for novel therapies in the management of HPV+ tumors. Citation Format: Soma Ghosh, Tuhina Mazumdar, Wei Xu, Reid T. Powell, Clifford Stephan, Li Shen, Curtis R. Pickering, Jing Wang, Faye M. Johnson. Rb deficient HPV+ HNSCC experienced enhanced sensitivity to aurora kinase inhibitors by altering the balance of MAD2 and TRIP13 levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1858.

Indibulin dampens microtubule dynamics and produces synergistic antiproliferative effect with vinblastine in MCF-7 cells: Implications in cancer chemotherapy

Indibulin, a synthetic inhibitor of tubulin assembly, has shown promising anticancer activity with a minimal neurotoxicity in preclinical animal studies and in Phase I clinical trials for cancer chemotherapy. Using time-lapse confocal microscopy, we show that indibulin dampens the dynamic instability of individual microtubules in live breast cancer cells. Indibulin treatment also perturbed the localization of end-binding proteins at the growing microtubule ends in MCF-7 cells. Indibulin reduced inter-kinetochoric tension, produced aberrant spindles, activated mitotic checkpoint proteins Mad2 and BubR1, and induced mitotic arrest in MCF-7 cells. Indibulin-treated MCF-7 cells underwent apoptosis-mediated cell death. Further, the combination of indibulin with an anticancer drug vinblastine was found to exert synergistic cytotoxic effects on MCF-7 cells. Interestingly, indibulin displayed a stronger effect on the undifferentiated neuroblastoma (SH-SY5Y) cells than the differentiated neuronal cells. Unlike indibulin, vinblastine and colchicine produced similar depolymerizing effects on microtubules in both differentiated and undifferentiated SH-SY5Y cells. The data indicated a possibility that indibulin may reduce chemotherapy-induced peripheral neuropathy in cancer patients.

Negative Selection and Chromosome Instability Induced by Mad2 Overexpression Delay Breast Cancer but Facilitate Oncogene-Independent Outgrowth.

SummaryChromosome instability (CIN) is associated with poor survival and therapeutic outcome in a number of malignancies. Despite this correlation, CIN can also lead to growth disadvantages. Here, we show that simultaneous overexpression of the mitotic checkpoint protein Mad2 with KrasG12D or Her2 in mammary glands of adult mice results in mitotic checkpoint overactivation and a delay in tumor onset. Time-lapse imaging of organotypic cultures and pathologic analysis prior to tumor establishment reveals error-prone mitosis, mitotic arrest, and cell death. Nonetheless, Mad2 expression persists and increases karyotype complexity in Kras tumors. Faced with the selective pressure of oncogene withdrawal, Mad2-positive tumors have a higher frequency of developing persistent subclones that avoid remission and continue to grow.

Abstract 3784: Regulation of chromatin condensation by mitotic checkpoint protein MAD2

Abstract The process of cell division is stringently regulated by spindle and mitotic checkpoint components mainly MAD2, MAD1, CDC20, BUBR1, BUB3 and MAD3. These checkpoint proteins detect unattached or improperly attached kinetochore-microtubule binding sites and generate an anaphase-wait signal by inhibiting the anaphase-promoting complex (APC/C). Activation of checkpoint signaling arrests the cells at metaphase until the attachment errors are rectified and all the chromosomes are properly bi-oriented at the metaphase plate. Together with these checkpoint proteins, the expression of checkpoint-regulatory factors such as p31 (comet) and recently identified, WT1 can hypo- or hyper-activate the signaling pathway, respectively and regulate the timing of mitotic exit. Recently we found that WT1 can interact with MAD2 and regulate the mitotic checkpoint function. Furthermore our studies show that MAD2 and WT1 can also influence the chromatin condensation process required for mitotic progression. We analyzed the effect of MAD2 and WT1 perturbations on the epigenetic mark (phosphorylation of histone H3) associated with condensed chromatin state. Our results show that MAD2 regulates the recruitment of chromatin modifying enzyme Aurora kinase B during mitosis. We further show that MAD2 is an important factor required for synchronization of key events beyond the checkpoint signaling sites to ensure the fidelity of chromosome segregation. Citation Format: Jayasha Shandilya, Stefan GE Roberts. Regulation of chromatin condensation by mitotic checkpoint protein MAD2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3784. doi:10.1158/1538-7445.AM2015-3784

MiR-28-5p Promotes Chromosomal Instability in VHL-Associated Cancers by Inhibiting Mad2 Translation

Chromosomal instability enables tumor development, enabled in part by aberrant expression of the mitotic checkpoint protein Mad2. Here we identify a novel regulatory mechanism for Mad2 expression involving miR-28-5p-mediated inhibition of Mad2 translation, and we demonstrate that this mechanism is triggered by inactivation of the tumor suppressor VHL, the most common event in clear cell renal cell carcinoma (ccRCC). In VHL-positive cancer cells, enhanced expression of miR-28-5p diminished Mad2 levels and promoted checkpoint weakness and chromosomal instability. Conversely, in checkpoint-deficient VHL-negative renal carcinoma cells, inhibition of miR-28-5p function restored Mad2 levels, mitotic checkpoint proficiency, and chromosomal stability. Notably, chromosome missegregation errors and aneuploidy that were produced in a mouse model of acute renal injury (as a result of kidney-specific ablation of pVHL function) were reverted in vivo also by genetic inhibition of miR-28-5p. Finally, bioinformatic analyses in human ccRCC associated loss of VHL with increased miR-28-5p expression and chromosomal instability. Together, our results defined miR-28-5p as a critical regulator of Mad2 translation and mitotic checkpoint function. By identifying a potential mediator of chromosomal instability in VHL-associated cancers, our work also suggests a novel microRNA-based therapeutic strategy to target aneuploid cells in VHL-associated cancers.

Elevated Expression Of CKS1B Inhibits Senescence Thorough Enhanced Degradation Of p21 In Multiple Myeloma

BackgroundHigh expression of CKS1B gene as a result of amplification of region 1q21 identifies a subset of patients with poor prognosis in multiple myeloma (MM). We have previously shown that over-expression of CKS1B enhances degradation of p27 and activates both MEK/ERK and JAK/STAT3 signaling pathways, promotes MM cell drug-resistance. However, other mechanisms of drug resistance induced by high expression of CKS1B remain to be identified. Materials and methodsCKS1B was over-expressed or knocked-down by shRNA in OCI-MY5 and KMS28PE myeloma cell lines. Cell viability was detected by PrestoBlue assay; Annexin-V assay was employed to detect cell apoptosis. Colony formation was assessed in soft agar. The direct dye efflux to assess multidrug resistance was performed using eFluxx-ID Multidrug resistance assay. Senescence was detected by SA-β-galactosidase (β-Gal) staining. Expression of SCF-Skp2 substrates was evaluated by western blots. ResultsWe demonstrated that overexpression of CKS1B significant decreased sensitivity to bortezomib and increased the clonogenic capacity compared to cells transfected with empty vector in the presence of bortezomib (p < 0.05). A prominent mechanism of drug resistance involved upregulation of multidrug resistance associated proteins (MRPs). We investigated whether the level of MDR1, BCRP, and MRP1, mitotic checkpoint protein MAD2 and ABC transporter family members was increased in cells with elevated CKS1B. However, none of drug pump proteins was up-regulated or stabilized by over-expression of CKS1B in OCI-MY5 and KMS28PE. Accordingly, over-expression of CKS1B did not promote efflux of the hydrophilic eFluxx-ID™ gold fluorescent dye from MM cells any greater than control cells, suggesting alternative mechanisms. We therefore tested whether CKS1B can inhibit drug-induced senescence in MM cells after treatment with bortezomib by quantifying the percentage of cells expressing senescence-associated β-Gal. Interestingly, MM cells with elevated CKS1B were significantly less β-Gal positive following treatment with bortezomib compared to control cells (p < 0.05). To explore molecular mechanism underlying CKS1B induced anti-senescence, expression of SCF-Skp2 substrates involved in senescence pathway was examined as CKS1B is an essential cofactor for SCF-Skp2 complex. Over-expression of CKS1B significantly reduced expression of p21, p27, and p57 while knock-down of CKS1B stabilized these proteins. Notably, cells with high expression of CKS1B failed to accumulate p21 protein in response to bortezomib. ConclusionOur findings suggest that anti-senescence is a novel mechanism in CKS1B-induced drug resistance in MM and the turnover of p21 regulated by CKS1B could have important implications not only for bortezomib therapy but also to develop therapies aimed at enhancing p21 function independent of p53. Disclosures:No relevant conflicts of interest to declare.

Chromosomal Instability: Mad2 beyond the Spindle Checkpoint

What specific defects can cause chromosomal instability in cancer cells? Overexpression of the mitotic checkpoint protein Mad2 triggers chromosome missegregation but, surprisingly, Mad2 exerts this effect through a previously unknown effect on microtubule dynamics.

The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4.

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.

Mad2 inhibits the mitotic kinesin MKlp2

We identified the mitotic kinesin-like protein 2 (MKlp2), a kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis, as a target of the mitotic checkpoint protein Mad2. MKlp2 possesses a consensus Mad2-binding motif required for Mad2 binding. Mad2 prevents MKlp2 from loading onto the mitotic spindle, a prerequisite step for its function as a mitotic kinesin. Furthermore, Mad2 inhibits the ability of MKlp2 to relocate the CPC from centromeres, an essential step to promote cytokinesis. An MKlp2 mutant that is refractory to Mad2-mediated inhibition prematurely translocates to the mitotic spindle and mislocalizes the CPC component Aurora B from the midbody of dividing cells. This correlates with an increased incidence of cytokinesis failure. Together, these findings reveal that MKlp2 is a novel mitotic target of Mad2 necessary for proper mitotic progression and cytokinesis.

Nek2 targets the mitotic checkpoint proteins Mad2 and Cdc20: A mechanism for aneuploidy in cancer

In mitosis, the duplicated chromosomes are separated and equally distributed to progeny cells under the guidance of the spindle, a dynamic microtubule network. Previous studies revealed a mitotic checkpoint that prevents segregation of the chromosomes until all of the chromosomes are properly attached to microtubules through the kinetochores. A variety of kinetochore-localized proteins, including Mad2 and Cdc20, have been implicated in controlling the mitotic checkpoint. Here we report that both Mad2 and Cdc20 can physically associate with Nek2, a serine/threonine kinase implicated in centrosome functions. We show that, similar to Nek2, the endogenous Cdc20 protein can be detected in the centrosome and the spindle poles. Both Cdc20 and Mad2 can be phosphorylated by Nek2. Moreover, our studies demonstrate that overexpression of Nek2 enhances the ability of Mad2 to induce a delay in mitosis. These observations indicate that Nek2 may act upon the Mad2–Cdc20 protein complex and play a critical role in regulating the mitotic checkpoint protein complex. We propose that overexpression of Nek2 may promote aneuploidy by disrupting the control of the mitotic checkpoint.

The basal body gene, RPGRIP1L, is a candidate tumour suppressor gene in human hepatocellular carcinoma

Loss of heterozygosity (LOH) on chromosome 16q is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC). Our previous data showed that the smallest common deleted region was between D16S415 and D16S419, encompassed approximately by a 0.75 cM region on 16q12.2, suggesting that the putative tumour suppressor genes (TSGs) at this locus might be involved in the development of HCC. Of the four genes ( CHD9, RBL2, AKTIP and RPGRIP1L) located in this region, only RPGRIP1L was downregulated in HCCs. Downregulation of RPGRIP1L was found in 91% (10/11) HCC cell lines and in 35% (14/40) HCCs, respectively. To investigate the role of RPGRIP1L in HCCs, we used the overexpression of RPGRIP1L in four HCC cell lines (HepG2, Huh6, Huh7 and Hep3B). Overexpression of RPGRIP1L suppressed colony formation of tumour cells. Conversely, expression of RPGRIP1L M (dominant negative form) in HCC cells enhanced colony formation. Furthermore, knockdown RPGRIP1L by RNA interference in SK-HepI cells promoted colony formation. Taken together, these data strongly suggest that RPGRIP1L might be the putative TSG in HCC. Moreover, we showed that Mad2, Survivin and Securin were elevated in RPGRIP1L M- HepG2 transfectants and RPGRIP1L-shRNA-SK-HepI transfectants. After knockdown of MAD2 in RPGRIP1L-shRNA-SK-HepI transfectants partly reverse cellular colony formation capability. These data suggest that RPGRIP1L suppresses anchorage-independent growth partly through the mitotic checkpoint protein Mad2.

Inhibition of protein kinase C ζ blocks the attachment of stable microtubules to kinetochores leading to abnormal chromosome alignment

The attachment of spindle microtubules to kinetochores is crucial for accurate segregation of chromosomes to daughter cells during mitosis. While a growing number of proteins involving this step are being identified, its molecular mechanisms are still not clear. Here we show that protein kinase C ζ (PKCζ) is localized at the mitotic spindle during mitosis and plays a role in stable kinetochore-microtubule attachment. Striking staining for PKCζ was observed at the mitotic spindle and spindle poles in cells at prometaphase and metaphase. PKCζ molecules at these stages were phosphorylated at Thr-410, as detected by a phosphospecific antibody. PKCζ was also detected at the spindle midzone and the midbody during anaphase and telophase, respectively, and PKCζ at these stages was no longer phosphorylated at Thr-410. The polarity determinants Par3 and Par6, which are known to associate with PKCζ, were also localized to the spindles and spindle poles at prometaphase and metaphase. Knockdown of PKCζ by RNA interference affected normal chromosome alignment leading to generation of cells with aberrant nuclei. A specific PKCζ inhibitor strongly blocked the formation of cold-sensitive stable kinetochore microtubules, and thus prevented microtubule-kinetochore attachment. Treatment of cells with the PKCζ inhibitor also dislocated the minus-end directed motor protein dynein from kinetochores, but not the mitotic checkpoint proteins Mad2 and CENP-E. Prolonged exposure to the PKCζ inhibitor eventually resulted in cell death. These results suggest a critical role of PKCζ in spindle microtubule-kinetochore attachment and subsequent chromosomal separation.

Inactivation of Human MAD2B in Nasopharyngeal Carcinoma Cells Leads to Chemosensitization to DNA-Damaging Agents

Rev7p has been suggested to play an important role in regulating DNA damage response in yeast, and recently, the human homologue (i.e., MAD2B) has been identified, which shares significant homology to the mitotic checkpoint protein MAD2. In this study, we investigated whether MAD2B played a key role in cellular sensitivity to DNA-damaging anticancer drugs by suppressing its expression using RNA interference in nasopharyngeal carcinoma cells. Using colony formation assay, we found that suppression of MAD2B conferred hypersensitivity to a range of DNA-damaging agents, especially DNA cross-linkers, such as cisplatin, and gamma-irradiation. This effect was associated with reduced frequencies of spontaneous and drug-induced mutations, elevated phosphorylation of histone H2AX, and markedly increased chromosomal aberrations in response to DNA damage. In addition, there was also a significant decrease in cisplatin-induced sister chromatid exchange rate, a marker for homologous recombination-mediated post-replication repair in MAD2B-depleted cells. These results indicate that MAD2B may be a key factor in regulating cellular response to DNA damage in cancer cells. Our findings reveal a novel strategy for cancer therapy, in which cancer cells are sensitized to DNA-damaging anticancer drugs through inactivation of the MAD2B gene.

Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control.

Advanced human cancers are invariably aneuploid, in that they harbour cells with abnormal chromosome numbers. However, the molecular defects underlying this trait, and whether they are a cause or a consequence of the malignant phenotype, are not clear. Mutations that disable the retinoblastoma (Rb) pathway are also common in human cancers. These mutations promote tumour development by deregulating the E2F family of transcription factors leading to uncontrolled cell cycle progression. We show that the mitotic checkpoint protein Mad2 is a direct E2F target and, as a consequence, is aberrantly expressed in cells with Rb pathway defects. Concordantly, Mad2 is overexpressed in several tumour types, where it correlates with high E2F activity and poor patient prognosis. Generation of Rb pathway lesions in normal and transformed cells produces aberrant Mad2 expression and mitotic defects leading to aneuploidy, such that elevated Mad2 contributes directly to these defects. These results demonstrate how chromosome instability can arise as a by-product of defects in cell cycle control that compromise the accuracy of mitosis, and suggest a new model to explain the frequent appearance of aneuploidy in human cancer.

Metaphase I Arrest upon Activation of the Mad2-Dependent Spindle Checkpoint in Mouse Oocytes

Background: The importance of mitotic spindle checkpoint control has been well established during somatic cell divisions. The metaphase-to-anaphase transition takes place only when all sister chromatids have been properly attached to the bipolar spindle and are aligned at the metaphase plate. Failure of this checkpoint may lead to unequal separation of sister chromatids. On the contrary, the existence of such a checkpoint during the first meiotic division in mammalian oocytes when homologous chromosomes are segregated has remained controversial.Results: Here, we show that mouse oocytes respond to spindle damage by a transient and reversible cell cycle arrest in metaphase I with high Maturation Promoting Factor (MPF) activity. Furthermore, the mitotic checkpoint protein Mad2 is present throughout meiotic maturation and is recruited to unattached kinetochores. Overexpression of Mad2 in meiosis I leads to a cell cycle arrest in metaphase I. Expression of a dominant-negative Mad2 protein interferes with proper spindle checkpoint arrest.Conclusions: Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.

Differences in Spindle Association of the Mitotic Checkpoint Protein Mad2 in Mammalian Spermatogenesis and Oogenesis

We have investigated expression and subcellular localization of the spindle checkpoint protein Mad2 during rat and mouse spermatogenesis and in superovulated mouse oocytes. Our immunofluorescence studies demonstrate substantial differences in the localization patterns of kinetochore-associated Mad2 in these meiotic systems compared with previous studies of mitosis. In addition, the association of Mad2 with second-division-metaphase kinetochores differed significantly in male versus female meiosis. In spermatogenesis, Mad2 remained at most kinetochores throughout the entire first meiotic division and was lost only at metaphase of the second meiotic division. This result indicates that loss of kinetochore-associated Mad2 is not essential for the metaphase-to-anaphase transition during the first meiotic division. Disruption of the male meiotic spindles with the microtubule depolymerizing agent nocodazole resulted in the appearance of Mad2 at nearly all kinetochores. In contrast, the microtubule stabilizer taxol induced the loss of Mad2 from the majority of the first-division-metaphase kinetochores in which it was normally present in untreated cells. In contrast to the situation in spermatogenesis, Mad2 persisted at the kinetochores of normal, second-division oocytes at metaphase. These findings suggest that the role of the kinetochore in signaling in the spindle checkpoint may differ markedly between mammalian mitosis and meiosis, between the two meiotic divisions, and between male and female meiosis.

Chromosome Missegregation and Apoptosis in Mice Lacking the Mitotic Checkpoint Protein Mad2

The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome–microtubule attachment during metaphase. To determine the function of the mitotic checkpoint protein Mad2 during normal cell division and when mitosis goes awry, we have knocked out Mad2 in mice. We find that E5.5 embryonic cells lacking Mad2, like mad2 yeast, grow normally but are unable to arrest in response to spindle disruption. At E6.5, the cells of the epiblast begin rapid cell division and the absence of a checkpoint results in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survive without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells, and for embryonic viability, even in the absence of spindle damage.