Mitotic Arrest Deficient Protein Research Articles

Direct Observation and Real-Time Tracking of an Extraordinarily Stable Folding Intermediate in Mitotic Arrest Deficient Protein 2 Folding by Single-Molecule Fluorescence Resonance Energy Transfer.

Although ensemble experiments have suggested that mitotic arrest deficient protein 2 (Mad2), a metamorphic protein, has folding intermediates, direct evidence and characterization are not available. It remains an outstanding challenge to capture the folding intermediates in real time, which is crucial to elucidate the folding mechanism, but the folding intermediates are normally unstable and only exist transiently. By combining confocal-microscopy-based and total internal reflection fluorescence (TIRF)-microscopy-based single-molecule Förster resonance energy transfer (sm-FRET) techniques, we have investigated the folding/unfolding process of Mad2 and captured its folding intermediate at the single-molecule level. This provides direct evidence for the existence of an intermediate along the folding pathway of Mad2. The folding intermediate proved to be extraordinarily stable, with an extremely long average dwell time of 2.3 s under the conditions of 3 M GdmCl at ambient temperature. The folding trajectories obtained from TIRF experiments further suggest that the intermediate is on-pathway to native Mad2.

MAD2B Blunts Chronic Unpredictable Stress and Corticosterone Stimulation-Induced Depression-Like Behaviors in Mice.

Depression is a prevalent and recurrent psychiatric disorder. Aberrant neural structure and activity play fundamental roles in the occurrence of depression. Mitotic arrest deficient protein (MAD2B) is highly expressed in neurons and may be implicated in synaptic plasticity in the central nervous system. However, the effect of MAD2B in depression, as well as the related molecular mechanism, is uncertain. Here, we employed mouse models of depression induced by chronic unpredictable stress exposure or corticosterone (CORT) stimulation. Depression-like behaviors in mice were evaluated by sucrose preference, forced swimming, and tail suspension tests. Hippocampal MAD2B overexpression was mediated by adeno-associated virus 8 containing enhanced green fluorescent protein. In vitro primary neuronal cells were obtained from the hippocampus of rat embryos and were treated with CORT, and MAD2B overexpression was performed using lentivirus. MAD2B and glutamate metabotropic receptor 4 (GRM4) levels were evaluated by western blots and quantitative PCR. Primary neuronal miR-29b-3p expression was detected by quantitative PCR. MAD2B expression was reduced in the hippocampus in mice exhibiting depressive-like behaviors. However, hippocampal MAD2B overexpression protected mice from developing either chronic unpredictable stress- or CORT-induced depression-like behaviors, an effect associated with reduced expression of GRM4, a presynaptic receptor involved in depression. Moreover, MAD2B overexpression in primary neuronal cells also decreased GRM4 expression while enhancing the level of miR-29b-3p; this phenomenon was also observed under CORT stimulation. Our results suggest an important role of neuronal MAD2B in the pathogenesis of depression via the miR-29b-3p/GRM4 signaling pathway. MAD2B could be a potential therapeutic target for depressive disorders.

Conditional deletion of MAD2B in forebrain neurons enhances hippocampus-dependent learning and memory in mice.

Mitotic arrest deficient 2-like protein 2 (MAD2B) is not only a DNA damage repair agent but also a cell cycle regulator that is widely expressed in the hippocampus and the cerebral cortex. However, the functions of MAD2B in hippocampal and cerebral cortical neurons are poorly understood. In this study, we crossed MAD2Bflox/flox and calcium/calmodulin-dependent protein kinase II alpha (Camk2a)-Cre mice to conditionally knock out MAD2B in the forebrain pyramidal neurons by the Cre/loxP recombinase system. First, RNA sequencing suggested that the differentially expressed genes in the hippocampus and the cerebral cortex between the WT and the MAD2B cKO mice were related to learning and memory. Then, the results of behavioral tests, including the Morris water maze test, the novel object recognition test, and the contextual fear conditioning experiment, suggested that the learning and memory abilities of the MAD2B cKO mice had improved. Moreover, conditional knockout of MAD2B increased the number of neurons without affecting the number of glial cells in the hippocampal CA1 and the cerebral cortex. At the same time, the number of doublecortin-positive (DCX+) cells was increased in the dentate gyrus (DG) of the MAD2B cKO mice. In addition, as shown by Golgi staining, the MAD2B cKO mice had more mushroom-like and long-like spines than the WT mice. Transmission electron microscopy (TEM) revealed that spine synapses increased and shaft synapses decreased in the CA1 of the MAD2B cKO mice. Taken together, our findings indicated that MAD2B plays an essential role in regulating learning and memory.

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy.

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.

MAD2L1 Functions As a Novel Diagnostic and Predictive Biomarker in Cholangiocarcinoma.

Background: Most cholangiocarcinoma (CCA) patients are diagnosed at an advanced stage of disease, and the postoperational recurrence rates are high in those undergoing resection. The lack of satisfying biomarkers for early diagnoses and effective targeting of driver pathways is the leading reason for therapeutic failures. The goal of this study was to find a biomarker for making improved diagnoses with enhanced prognostic capabilities for CCA. Materials and Methods: Our study used bioinformatic analyses of microarray data from the Gene Expression Omnibus (GEO) database and investigated mitotic arrest deficient 2-like protein 1 (MAD2L1) expression in tumor and adjacent non-neoplastic biliary ducts through immunocytochemistry in 42 surgically removed primary CCAs from a single institute. In vitro and in vivo models were used to explore the function of MAD2L1. Results: In total, 297 high probability differentially expressed genes (DEGs) were obtained from overlapping the DEGs from the three individual data sets. Through enrichment assays and protein-protein interaction networks analyses, seven hub genes were identified. MAD2L1 was picked up as a novel biomarker based on hierarchical cluster analyses and Kaplan-Meier survival analyses. MAD2L1 was expressed in cancer tissues but not in the surrounding normal tissue, with 31 (73.81%) of 42 CCAs MAD2L1 positive by immunohistochemistry (IHC). MAD2L1 expression levels were significantly correlated with tumor size, pathological grade, and clinical stage. A Kaplan-Meier survival analysis demonstrated an inverse correlation with MAD2L1 expression. Real-time polymerase chain reaction and immunoblotting results further confirmed the results of IHC and bioinformatic analyses. In vitro and in vivo models demonstrated decreasing MAD2L1 could significantly suppress tumor growth, whereas increasing MAD2L1 could promote tumor growth. Conclusion: MAD2L1 could be used as a biomarker to predict prognosis and potential therapeutic target in CCA. Clinical Trial Registration Number: [2020]KY157-01.

Inhibition of MAD2B alleviates venous neointimal formation by suppressing VSMCs proliferation and migration

The proliferation and migration of vascular smooth muscle cells (VSMCs) are essential events in venous neointimal hyperplasia (VNH), a culprit of arteriovenous fistula (AVF) malfunction. Mitotic arrest-deficient protein 2B (MAD2B) is a critical regulator of cell proliferation and differentiation in many scenarios. To address the role of MAD2B in VSMCs proliferation and migration during VNH, AVFs from patients with end-stage renal disease (ESRD) and chronic kidney disease (CKD) mice were used to evaluate MAD2B expression. In cultured VSMCs treated with platelet-derived growth factor-BB (PDGF-BB), the effect of MAD2B on VSMCs proliferation and migration was detected by cell counting kit-8 (CCK8) assay, immunofluorescence, wound-healing scratch and transwell assays. Besides, we exploited different small interfering RNAs (siRNAs) to explore the potential mechanisms in the issue. Furthermore, rapamycin was applied to reveal whether MAD2B-associated pathways were involved in its inhibitory effect on VSMCs proliferation and migration. Accordingly, we found that MAD2B expression was enhanced in AVFs from patients with ESRD, CKD mice and VSMCs stimulated by PDGF-BB. Meanwhile, inhibition of MAD2B alleviated VSMCs proliferation and migration while the number of ski-related novel gene (SnoN)-positive VSMCs was also increased in vivo and in vitro. Moreover, gene deletion of MAD2B decreased the level of SnoN protein in PDGF-BB-stimulated VSMCs. Furthermore, rapamycin suppressed the increased expressions of MAD2B and SnoN induced by PDGF-BB. Thus, our study demonstrates that inhibition of MAD2B suppresses the proliferation and migration of VSMCs during VNH via reducing SnoN expression. Moreover, rapamycin exerts an inhibitory effect on intimal hyperplasia, possibly via the MAD2B-SnoN axis.

MAD2B-mediated cell cycle reentry of podocytes is involved in the pathogenesis of FSGS.

Rationale: Focal segmental glomerulosclerosis (FSGS) is characterized by the dysfunction of “post-mitotic” podocytes. The reentry of podocytes in the cell cycle will ultimately result in cell death. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of anaphase-promoting complex (APC)/cyclosome, precisely controls the metaphase to anaphase transition and ordered cell cycle progression. However, the role of MAD2B in FSGS podocyte injury remains unknown.Methods: To explore MAD2B function in podocyte cell cycle reentry, we used conditional mutant mice lacking MAD2B selectively in podocytes in ADR-induced FSGS murine model. Additionally, KU-55933, a specific inhibitor of ataxia-telangiectasia mutated (ATM) was utilized in vivo and in vitro to explore the role of ATM in regulating MAD2B.Results: The expression of MAD2B in podocytes was dramatically increased in patients with FSGS and ADR-treated mice along with podocyte cell cycle reentry. Podocyte-specific knockout of MAD2B effectively attenuated proteinuria, podocyte injury, and prevented the aberrant cell cycle reentry. By bioinformatics analysis we revealed that ATM kinase is a key upstream regulator of MAD2B. Furthermore, inhibition of ATM kinase abolished MAD2B-driven cell cycle reentry and alleviated podocyte impairment in FSGS murine model. In vitro studies by site-directed mutagenesis and immunoprecipitation we revealed ATM phosphorylated MAD2B and consequently hampered the ubiquitination of MAD2B in a phosphorylation-dependent manner.Conclusions: ATM kinase-MAD2B axis importantly contributes to the cell cycle reentry of podocytes, which is a novel pathogenic mechanism of FSGS, and may shed light on the development of its therapeutic approaches.

SAT-159 MAD2B Promotes Podocyte Injury through Regulating Numb-Dependent Notch1 Pathway in Diabetic Nephropathy

The pathological features of diabetic nephropathy（DN）include glomerulosclerosis, thickening of the glomerular basement membrane, glomerular hypertrophy, mesangial cell expansion, podocyte loss, renal-cell hypertrophy and tubulointerstitial fibrosis. Recent studies have demonstrated the loss of podocyte is a critical event in DN. Many studies suggested that DN podocyte injury was induced by the association of multiple factors, including mechanical stress, inflammatory reaction, oxidative stress, TGF-β1 induction, renin angiotensin aldosterone system (RAAS) activation, and AGEs accumulation. Previously, our group have found the mitotic arrest deficient protein MAD2B is implicated in high glucose (HG)-induced podocyte injury by regulating cell cycle. However, the exact mechanism of MAD2B in podocyte injury is lacking. Numb was originally discovered as an intrinsic cell fate determinant in Drosophila by antagonizing Notch signaling which may play a pivotal pathogenic role in podocyte injury. In vivo study, DN model was constructed on C57BL/6 mice by a single intra-peritoneal injection of STZ. Our group also successfully constructed gene knockout mice with podocyte-specific depletion of MAD2B (NMKO). In vitro study, immortalized human podocyte cell line was employed, and exposed to different treatments after differentiation. The expression of MAD2B and Numb was suppressed by recombinant lentivirus infection，while Numb was elevated by adenovirus infection. Our latest finding revealed that HG exposure downregulated Numb expression in podocytes and DN mice resulting in increase of NICD and Hes-1 and mitotic catastrophe of podocyte. In addition, the overexpression of Numb could alleviate podocyte injury through decreasing the expression of Desmin and increasing autophagy. Previously we have demonstrated the enhancement of MAD2B in DN mice and HG treated podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes when exposed to HG. Accordingly, the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased. Afterwards we identified Numb as a novel MAD2B binding protein in human kidney through screens using a yeast two-hybrid interaction trap. Through confocal laser scanning microscopy we confirmed the co-localization of MAD2B and Numb in podocyte in vivo and in vitro. Subsequent endogenous co-immunoprecipitation established the direct interaction between MAD2B and Numb. The mechanisms of interaction between them still remains to be established. Upregulated MAD2B expression contributes to Numb depletion and its downstream protein accumulation such as NICD and Hes-1 in HG condition，which ultimately leads to podocyte mitosis abnormality and cell polarity disorder. However, inhibiting MAD2B expression could reverse podocyte injury by maintaining normal cell cycle and increasing autophagy.

Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80.

Objective: Mitotic arrest-deficient protein 1 (MAD1) is a kinetochore protein essential for the mitotic spindle checkpoint. Proteomic studies have indicated that MAD1 is a component of the DNA damage response (DDR) pathway. However, whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods: We ectopically expressed the wild type, or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects. We used the comet assay, colony formation assay, immunofluorescence staining, and flow cytometry to assess the DDR, radiosensitivity, and the G2/M checkpoint. We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins. Data were analyzed using the unpaired Student’s t-test.Results: We showed that MAD1 was required for an optimal DDR, as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation (IR). We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated (ATM) kinase-dependent. Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR. Using mass spectrometry, we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR. Among them, we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage. Finally, we showed that MAD1 interaction with KU80 required serine 214 phosphorylation, and it was essential for activation of DNA protein kinases catalytic subunit (DNA-PKcs).Conclusions: MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.

A Study of the Mechanism of Binding between Neratinib and MAD2L1 Based on Molecular Simulation and Multi-spectroscopy Methods.

Nilatinib is an irreversible tyrosine kinase inhibitor, which is used in the treatment of some kinds of cancer. To study the interaction between Neratinib and MAD2L1, a potential tumor target, is of guiding significance for enriching the medicinal value of Neratinib. The binding mechanism between Mitotic arrest deficient 2-like protein 1 (MAD2L1) and Neratinib under simulative physiological conditions was investigated by molecule simulation and multi-spectroscopy approaches. Molecular docking showed the most possible binding mode of Neratinib-MAD2L1 and the potential binding sites and interaction forces of the interaction between MAD2L1 and Neratinib. Fluorescence spectroscopy experiments manifested that Neratinib could interact with MAD2L1 and form a complex by hydrogen bond and van der Waals interaction. These results were consistent with the conclusions obtained from molecular docking. In addition, according to Synchronous fluorescence and three-dimensional fluorescence results, Neratinib might lead to the conformational change of MAD2L1, which may affect the biological functions of MAD2L1. This study indicated that Neratinib could interact with MAD2L1 and lead to the conformational change of MAD2L1. These works provide helpful insights for the further study of biological function of MAD2L1 and novel pharmacological utility of Neratinib.

Mitotic Arrest-Deficient Protein 2B Overexpressed in Lung Cancer Promotes Proliferation, EMT, and Metastasis.

As the noncatalytic subunit of mammalian DNA polymerase, mitotic arrest-deficient protein 2B (MAD2B) has been reported to play a role in cell cycle regulation, DNA damage tolerance, gene expression, and carcinogenesis. Although its expression is known to be associated with poor prognosis in several types of human cancers, the significance of MAD2B expression in lung malignancies is still unclear. Our study showed that MAD2B expression significantly increased in lung cancer, especially in the metastatic tissues. We also found that knockdown of MAD2B inhibited the migration, invasion, and epithelial–mesenchymal transition of lung cancer cells in vitro and the metastasis in vivo, while overexpression of MAD2B had the opposite effect. Microarray and Western blotting data indicated that slug might be its downstream target since knockdown of MAD2B inhibited, while overexpression increased, the expression of slug. Moreover, the expression of MAD2B was found to be positively correlated with slug in lung cancer tissues as well. Collectively, these findings indicate an oncogenic role of MAD2B in lung cancer, and slug might be involved in the process.

Abstract 3370: Mad2l2 inhibits colorectal cancer growth by promoting ncoa3 ubiquitination and degradation

Abstract Nuclear receptor coactivator 3 (NCOA3) is a transcriptional coactivator that has elevated expression in multiple tumor types, including colorectal cancer (CRC). However, the molecular mechanisms that regulate the tumorigenic functions of NCOA3 in CRC remain largely unknown. In this study, we aimed to discover and identify the novel regulatory proteins of NCOA3 and explore their mechanisms of action. Immunoprecipitation coupled with mass spectrometry analysis was used to detect, identify and verify the proteins that interacted with NCOA3 in CRC cells. The biological functions of the candidate proteins and the underlying molecular mechanism were investigated in CRC cell lines and a mouse model in vitro and in vivo. The clinical significance of NCOA3 and its interaction partner protein in CRC patients was also studied. We discovered and identified mitotic arrest deficient 2-like protein 2 (MAD2L2, also known as MAD2B or REV7) as an interaction partner of NCOA3 by immunoprecipitation coupled with mass spectrometry analysis. Overexpression of MAD2L2 suppressed the cell proliferation, migration and clonogenecity in CRC cell lines and a mouse model by inducing the degradation of NCOA3. We also found that increased MAD2L2 expression in CRC cells activated p38, which was required for the phosphorylation of NCOA3 that led to its ubiquitination and degradation by the proteasome. Moreover, we demonstrated that MAD2L2 predicted favorable prognosis in CRC patients. These results indicate that MAD2L2 inhibits colorectal cancer growth by promoting NCOA3 ubiquitination and degradation. Our study have discovered a novel role of MAD2L2 in the regulation of NCOA3 degradation, and proposed that MAD2L2 serves as a potential tumor suppressor in CRC. Citation Format: yixin li, ge qin, qian long, changlin zhang, dingbo shi, miao chen, wuguo deng. Mad2l2 inhibits colorectal cancer growth by promoting ncoa3 ubiquitination and degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3370.

BRCA1 and MAD2 Are Coexpressed and Are Prognostic Indicators in Tubo-ovarian High-Grade Serous Carcinoma.

The aim of this study was to investigate the relationship between BRCA1 and mitotic arrest deficiency protein 2 (MAD2) protein expression, as determined by immunohistochemistry, and clinical outcomes in epithelial ovarian carcinoma (EOC). A tissue microarray consisting of 94 formalin-fixed paraffin-embedded EOC with fully matched clinicopathological data were immunohistochemically stained with anti-BRCA1 and anti-MAD2 antibodies. The cores were scored in a semiquantitative manner evaluating nuclear staining intensity and extent. Coexpression of BRCA1 and MAD2 was evaluated, and patient survival analyses were undertaken. Coexpression of BRCA1 and MAD2 was assessed in 94 EOC samples, and survival analysis was performed on 65 high-grade serous carcinomas (HGSCs). There was a significant positive correlation between BRCA1 and MAD2 expression in this patient cohort (P < 0.0001). Both low BRCA1 and low MAD2 are independently associated with overall survival because of HGSC. Low coexpression of BRCA1 and MAD2 was also significantly associated with overall survival and was driven by BRCA1 expression. BRCA1 and MAD2 expressions are strongly correlated in EOC, but BRCA1 expression remains the stronger prognostic factor in HGSC.

Dynamic feature of mitotic arrest deficient 2–like protein 2 (MAD2L2) and structural basis for its interaction with chromosome alignment–maintaining phosphoprotein (CAMP)

Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis.

MAD2B acts as a negative regulatory partner of TCF4 on proliferation in human dermal papilla cells

Dermal papilla cells (DPCs) are important components of hair follicles and play a critical role in hair follicle development. However, the mechanisms by which DPCs induce hair follicle development remain unclear. In the present study, we identified the mitotic arrest deficient protein MAD2B as a modifier of DPCs. Overexpression of MAD2B inhibited DPC aggregative growth and proliferation induced by the Wnt signaling activator T cell factor 4 (TCF4), and decreased TCF4-induced expression and the release of hair growth-related cytokines, including hepatocyte growth factor, insulin-like growth factor-1, and vascular endothelial growth factor in DPCs. In contrast, knockdown of MAD2B promoted TCF4-induced DPC proliferation, but did not affect the expression and secretion of cytokines by TCF4-induced DPCs. These results suggest a functional antagonism between MAD2B and TCF4 in DPC-induced hair follicle development. Mechanistically, MAD2B physically interacted with TCF4 to repress TCF4 transcriptional activity via β-catenin mediation, leading to reduced β-catenin/TCF4-dependent transactivation and Wnt signaling activity. These results demonstrate, for the first time, that MAD2B plays a negative role in TCF4-induced DPC growth and proliferation.

The association between MAD2 and prognosis in cancer: a systematic review and meta-analyses.

This systematic review and meta-analyses investigates the expression of the cell checkpoint regulator, mitotic arrest deficiency protein 2 (MAD2) in cancerous tissue and examines whether an association exists between MAD2 levels and cancer survival and recurrence. Studies investigating MAD2 expression in cancer tissue utilising immunohistochemistry (IHC) were identified by systematic literature searches of Medline, Embase and Web of Science databases by October 2015. Random effects meta-analyses were performed to generate pooled hazard ratios (HRs) with 95% confidence intervals (CIs) of overall and progression-free survival according to MAD2 expression. Forty-three studies were included in the overall review. In 33 studies investigating MAD2 expression by IHC in cancer tissue, a wide range of expression positivity (11–100%) was reported. Higher MAD2 expression was not associated with an increased risk of all-cause mortality in a range of cancers (pooled HR 1.35, 95% CI 0.97–1.87; P = 0.077, n = 15). However, when ovarian cancer studies were removed, a significant pooled HR of 1.59 for risk of all-cause mortality in other cancer patients with higher expressing MAD2 tumours was evident (95% CI, 1.17–2.17; P = 0.003, n = 12). In contrast, higher MAD2 expression was associated with significant decreased risk of all-cause mortality in ovarian cancer patients (pooled HR = 0.50, 95% CI, 0.25–0.97; P = 0.04, n = 3). In conclusion, with the exception of ovarian cancer, increased MAD2 expression is associated with increased risk of all-cause mortality and recurrence in cancer. For ovarian cancer, reduced levels of MAD2 are associated with poorer outcome. Further studies are critical to assess the clinical utility of a MAD2 IHC biomarker.

Maturation promoting factor destabilization facilitates postovulatory aging-mediated abortive spontaneous egg activation in rat.

The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging-mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee-1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging-mediated abortive SEA in rats eggs aged invivo. For this purpose, sexually immature female rats were given a single injection (20IU IM) of pregnant mare serum gonadotropin for 48h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21h post-hCG surge to induce postovulatory aging invivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr-14/Tyr-15 and dephosphorylation of Thr-161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging-mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging-mediated abortive SEA in rat eggs.

Involvement of Cyclin-Dependent Kinase 1 during Postovulatory Aging-Mediated Abortive Spontaneous Egg Activation in Rat Eggs Cultured In Vitro.

Freshly ovulated rat eggs do not remain arrested at metaphase II (MII) and undergo exit from MII arrest with initiation of extrusion of the second polar body (PBII), a characteristic feature of abortive spontaneous egg activation (SEA). The biochemical and molecular changes during postovulatory aging-mediated abortive SEA remain poorly understood. We investigated the morphological, cellular, and molecular changes during postovulatory aging-mediated abortive SEA in eggs cultured in vitro. Our results suggest that postovulatory egg aging in vitro induced initiation of PBII extrusion in a time-dependent manner. Postovulatory aging increased Wee1 kinase and Thr-14/Tyr-15 phosphorylated cyclin-dependent kinase 1 (Cdk1) levels, whereas Thr-161 phosphorylated Cdk1 and cyclin B1 levels were significantly decreased in eggs cultured in vitro. The early mitotic inhibitor 2 (Emi2) level was significantly reduced, but anaphase promoting complex/cyclosome (APC/C) and mitotic arrest deficient protein (MAD2) levels were increased initially and then reduced during a later period of in vitro culture. These results suggest that an increased Wee1 kinase level modulated the specific phosphorylation status of Cdk1, increased Cdk1 activity, and decreased the cyclin B1 level. Furthermore, the decreased Emi2 level was associated with an increased level of APC/C and decreased level of cyclin B1, which resulted in maturation promoting factor (MPF) destabilization and finally led to postovulatory aging-mediated abortive SEA in rat eggs cultured in vitro.

Mad2 overexpression is associated with high cell proliferation and reduced disease-free survival in primary gastrointestinal diffuse large B-cell lymphoma

Objectives: Primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL) is a rare hematological malignancy with limited results on carcinogenesis and clinical characteristics. The aims of the current study were to examine mitotic arrest deficiency protein 2 (Mad2) expressions in PGI-DLBCL, and assess its association with Ki-67 expression, Helicobacter pylori (H. pylori) infection, BCL-6 gene rearrangement, and clinicopathological variables.Methods: Cancer tissues from 38 PGI-DLBCL patients were examined for Mad2, Ki-67, and H. pylori expression by immunohistochemistry, using normal gastrointestinal tissues and nodal DLBCL as controls. BCL-6 gene translocation was analyzed by fluorescence in situ hybridization (FISH), and Mad2 expression status was evaluated along with clinicopathological characteristics.Results: Mad2 expression was increased in PGI-DLBCL patients when compared with controls. The expression of Mad2 was 51.55 ± 22.88% in PGI-DLBCL, which was higher than reactive lymph node (28.77 ± 10.89%) and lymphoid nodule in normal gastrointestinal tissue (26.41 ± 11.30%) (P = 0.002), while it was comparable to nodal DLBCL (57.23 ± 20.79%) (P = 0.358). Mad2 overexpression had a positive correlation with Ki-67 proliferation index (r = 0.55, P = 0.01) in PGI-DLBCL, and patients with BCL-6 gene rearrangement had lower Mad2 expression (P = 0.032) than patients with intact BCL-6, while no relation was found between Mad2 expression and H. pylori infection. PGI-DLBCL patients with higher Mad2 expression had lower estimated disease-free survival (DFS) (17.10% vs. 53.00%) (P = 0.049). However, no correlation was found between Mad2 expression levels and overall survival (OS) (P = 0.443).Conclusions: Aberrant Mad2 expression was associated with cell proliferation and genetic instability, which may contribute to the carcinogenesis of PGI-DLBCL. Mad2 overexpression indicated a poor DFS and may be a potential biomarker for estimating prognosis for PGI-DLBCL patients.

Metformin Protects Neurons against Oxygen-Glucose Deprivation/Reoxygenation -Induced Injury by Down-Regulating MAD2B

Background/Aims: Metformin, the common medication for type II diabetes, has protective effects on cerebral ischemia. However, the molecular mechanisms are far from clear. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of the anaphase-promoting complex (APC), is widely expressed in hippocampal and cortical neurons and plays an important role in mediating high glucose-induced neurotoxicity. The present study investigated whether metformin modifies the expression of MAD2B and to exert its neuroprotective effects in primary cultured cortical neurons during oxygen-glucose deprivation/reoxygenation (OGD/R), a widely used in vitro model of ischemia/reperfusion. Methods: Primary cortical neurons were cultured, deprived of oxygen-glucose for 1 h, and then recovered with oxygen-glucose for 12 h and 24 h. Cell viability was measured by detecting the levels of lactate dehydrogenase (LDH) in culture medium. The levels of MAD2B, cyclin B and p-histone 3 were measured by Western blot. Results: Cell viability of neurons was reduced under oxygen-glucose deprivation/reoxygenation (OGD/R). The expression of MAD2B was increased under OGD/R. The levels of cyclin B1, which is a substrate of APC, were also increased. Moreover, OGD/R up-regulated the phosphorylation levels of histone 3, which is the induction of aberrant re-entry of post-mitotic neurons. However, pretreatment of neurons with metformin alleviated OGD/R-induced injury. Metformin further decreased the expression of MAD2B, cyclin B1 and phosphorylation levels of histone 3. Conclusion: Metformin exerts its neuroprotective effect through regulating the expression of MAD2B in neurons under OGD/R.