Cdc2 Protein Research Articles

Ginsenoside induces apoptosis, autophagy and cell cycle arrest in gastric cancer cells by regulation of reactive oxygen species and activation of MAPK pathway

Purpose: To study the influence of ginsenoside on apoptosis, cell cycle and autophagy in gastric carcinoma (GC), and its effect on reactive oxygen species (ROS) levels and the mitogen-activated protein kinase (MAPK) pathway. 
 Methods: Human gastric cancer cell line BGC-823 was randomly divided into the following groups: control, 100 μM ginsenoside (Rg5), 150 μM Rg5, and 200 μM Rg5 groups. Western blot assay was used to determine the expressions of autophagy-associated protein 12 (Atg12), Beclin-1, lc3b II, cycle-related protein, phosphated mitotic cyclin 25 homologous protein C (p-cdc25c), cyclin B1, and MAPK signaling pathway-related proteins. 
 Results: There was significantly higher apoptosis in Rg5-treated BGC-823 cells than in untreated cells. Relative protein levels of Beclin-1, Atg5, Atg12, and lc3b II in BGC-823 cells in Rg5 groups were significantly and concentration-dependently up-regulated, relative to the corresponding expression levels in untreated cells. There were markedly up-regulated proteins of p-cdc25c, cyclin B1 and p-cdc2 in Rg5-exposed BGC-823 cells than in untreated cells, while CDC2 protein expression was significantly and concentration-dependently lower than that of control group (p < 0.05). Rg5 treatment resulted in marked and concentration-dependent increases in ROS levels in BGC-823 cells, relative to control cells (p < 0.05), whereas the expression levels of p-p38, p-JNK and p-ERK were significantly higher in Rg5-exposed cells than in unexposed cells (p < 0.05). 
 Conclusion: Ginsenoside induces apoptosis, autophagy and cycle interruption in GC cells by regulating ROS production and activating MAPK pathway. Therefore, ginsenoside may be a promising agent for the management of gastric cancer. However, there is a need to conduct in vivo studies on the compound.

Ochratoxin A induces cytotoxicity through ROS-mediated endoplasmic reticulum stress pathway in human gastric epithelium cells

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium species that greatly threatens human health. We previously showed that OTA induced cycle arrest, apoptosis and autophagy in human gastric epithelium cells (GES-1). However, the mechanism underlying these effects is still unclear. Here, we showed that OTA exposure increased the expression of endoplasmic reticulum (ER) stress indicators (GRP78, PERK, ATF6, eIF2α, and CHOP), suggesting the activation of the unfolded protein response pathway. 4-phenylbutyric acid (4-PBA), an ER stress-specific inhibitor, attenuated OTA-induced loss of cell viability and apoptosis in GES-1 cells. It also attenuated the G2 phase arrest and autophagy induced by OTA, as evidenced by upregulated G2 phase-related proteins (Cdc2, Cdc25C, and cyclinB1) and downregulated autophagy markers (LC3B and Beclin-1). Moreover, OTA was found to increase ROS generation, and the inhibition of ROS formation by N-acetylcysteine (NAC), an ROS inhibitor, attenuated OTA-induced ER stress and subsequent apoptosis, cell cycle arrest, and autophagy. Collectively, these results suggest that the ROS-mediated ER stress pathway contributes to the OTA toxin-induced cytotoxicity in GES-1 cells. This study offers new insights into the molecular mechanisms underlying OTA toxicity in gastric cells.

Impaired Cdc20 signaling promotes senescence in normal cells and apoptosis in non–small cell lung cancer cells

Cellular senescence is a form of irreversible growth arrest that cancer cells evade. The cell division cycle protein 20 homolog (Cdc20) is a positive regulator of cell division, but how its dysregulation may relate to senescence is unclear. Here, we find that Cdc20 mRNA and protein expression are downregulated in stress-induced premature senescent lung fibroblasts in a p53-dependent manner. Either Cdc20 downregulation or inhibition of anaphase-promoting complex/cyclosome (APC/C) is sufficient to induce premature senescence in lung fibroblasts, while APC/C activation inhibits stress-induced premature senescence. Mechanistically, we show both Cdc20 downregulation and APC/C inhibition induce premature senescence through glycogen synthase kinase (GSK)-3β–mediated phosphorylation and downregulation of securin expression. Interestingly, we determined Cdc20 expression is upregulated in human lung adenocarcinoma. We find that downregulation of Cdc20 in non–small cell lung cancer (NSCLC) cells is sufficient to inhibit cell proliferation and growth in soft agar and to promote apoptosis, but not senescence, in a manner dependent on downregulation of securin following GSK-3β-mediated securin phosphorylation. Similarly, we demonstrate securin expression is downregulated and cell viability is inhibited in NSCLC cells following inhibition of APC/C. Furthermore, we show chemotherapeutic drugs downregulate both Cdc20 and securin protein expression in NSCLC cells. Either Cdc20 downregulation by siRNA or APC/C inhibition sensitize, while securin overexpression inhibits, chemotherapeutic drug-induced NSCLC cell death. Together, our findings provide evidence that Cdc20/APC/C/securin-dependent signaling is a key regulator of cell survival, and its disruption promotes premature senescence in normal lung cells and induces apoptosis in lung cancer cells that have bypassed the senescence barrier.

Enhanced reparative dentinogenesis of biphasic calcium phosphate ceramics containing calcium-deficient hydroxyapatite (CDHA) and strontium-incorporated CDHA in direct pulp capping

Despite that dental materials and therapeutic strategies have made progress, reserving dental pulp vitality remains a great challenge during clinical practices of dentistry. Biphasic calcium phosphate (BCP) ceramic is a promising biomaterial for bone regeneration and dentin restoration, whose biological activities need to be further improved to meet the needs of regenerative medicine. Calcium-deficient hydroxyapatite (CDHA), which is more similar in composition to natural bone and tooth minerals, imparts superior bioactivity to BCP ceramics. Besides, owing to the positive roles of strontium in stimulating bone formation, the strontium-incorporated CDHA has excellent biocompatibility and further promotes bone regeneration. However, the effect of CDHA and strontium-incorporated CDHA in dental pulp capping has been rarely investigated. Herein, we presented an effective method to integrate CDHA and strontium-incorporated CDHA into BCP ceramics and investigated their biological properties as a potential strategy for dental pulp-capping and dentin regeneration. The characterization of phase composition and crystal structure confirmed that novel BCP ceramics with high CDHA content (BCP-A) and strontium-incorporated CDHA (BCP-A/Sr) have been successfully synthesized. In vitro, BCP-C (conventional BCP), BCP-A, and BCP-A/Sr provided an appropriate microenvironment and significantly promoted the proliferation of human dental pulp cells (hDPCs). BCP-A did not affect the migration of hDPCs, yet BCP-C and BCP-A/Sr displayed a suppressing trend. In vivo, BCP-A significantly reduced the inflammation response and improved the formation of reparative dentin bridges. Compared with mineral trioxide aggregate (MTA), a clinical direct pulp capping material, the texture of reparative dentin bridges in BCP-A showed more uniformity. Moreover, RNA-seq analysis further clarified that direct co-culture of hDPCs with BCP-A mainly up-regulated the cell cycle signaling pathway and CDC20 protein. These findings indicate that the novel BCP-A is a promising biomaterial for direct dental pulp capping due to its unique intrinsic biological activities, which warrants further investigation.

A De Novo Transcriptome Analysis Identifies Cold-Responsive Genes in the Seeds of Taxillus chinensis (DC.) Danser.

Taxillus chinensis (DC.) Danser, a parasitic plant of the Loranthaceae family, grows by attacking other plants. It has a long history of being used in Chinese medicine to treat multiple chronic diseases. We previously observed that T. chinensis seeds are sensitive to cold. In this study, we performed transcriptome sequencing for T. chinensis seeds treated by cold (0°C) for 0 h, 12 h, 24 h, and 36 h. TRINITY assembled 257,870 transcripts from 223,512 genes. The GC content and N50 were calculated as 42.29% and 1,368, respectively. Then, we identified 42,183 CDSs and 35,268 likely proteins in the assembled transcriptome, which contained 1,622 signal peptides and 6,795 transmembrane domains. Next, we identified 17,217 genes (FPKM > 5) and 2,333 differentially expressed genes (DEGs) in T. chinensis seeds under cold stress. The MAPK pathway, as an early cold response, was significantly enriched by the DEGs in the T. chinensis seeds after 24 h of cold treatment. Known cold-responsive genes encoding abscisic acid-associated, aquaporin, C-repeat binding factor (CBF), cold-regulated protein, heat shock protein, protein kinase, ribosomal protein, transcription factor (TF), zinc finger protein, and ubiquitin were deregulated in the T. chinensis seeds under cold stress. Notably, the upregulation of CBF gene might be the consequences of the downregulation of MYB and GATA TFs. Additionally, we identified that genes encoding CDC20, YLS9, EXORDIUM, and AUX1 and wound-responsive family protein might be related to novel mechanisms of T. chinensis seeds exposed to cold. This study is first to report the differential transcriptional induction in T. chinensis seeds under cold stress. It will improve our understanding of parasitic plants in response to cold and provide a valuable resource for future studies.

Up-regulation of cell division cycle 20 expression alters the morphology of neuronal dendritic spines in the nucleus accumbens by promoting FMRP ubiquitination.

The nucleus accumbens (NAc) is the key area of the reward circuit, but its heterogeneity has been poorly studied. Using single-cell RNA sequencing, we revealed a subcluster of GABAergic neurons characterized by cell division cycle 20 (Cdc20) mRNA expression in the NAc of adult rats. We studied the coexpression of Cdc20 and Gad1 mRNA in the NAc neurons of adult rats and assessed Cdc20 protein expression in the NAc during rat development. Moreover, we microinjected AAV2/9-hSyn-Cdc20 with or without the dual-AAV system into the bilateral NAc for sparse labeling to observe changes in the synaptic morphology of mature neurons and assessed rat behaviors in open field and elevated plus maze tests. Furthermore, we performed the experiments with a Cdc20 inhibitor, Cdc20 over-expression AAV vector, and Cdc20 conditional knockout primary striatal neurons to understand the ubiquitination-dependent degradation of fragile X mental retardation protein (FMRP) in vitro and in vivo. We confirmed the mRNA expression of Cdc20 in the NAc GABAergic neurons of adult rats, and its protein level was decreased significantly 3 weeks post-birth. Up-regulated Cdc20 expression in the bilateral NAc decreased the dendritic spine density in mature neurons and induced anxiety-like behavior in rats. Cdc20-APC triggered FMRP degradation through K48-linked polyubiquitination in Neuro-2a cells and primary striatal neurons and down-regulated FMRP expression in the NAc of adult rats. These data revealed that up-regulation of Cdc20 in the bilateral NAc reduced dendritic spine density and led to anxiety-like behaviors, possibly by enhancing FMRP degradation via K48-linked polyubiquitination.

Norcantharidin promotes cancer radiosensitization through Cullin1 neddylation-mediated CDC6 protein degradation.

Radiotherapy (RT) is a conventional cancer therapeutic modality. However, cancer cells tend to develop radioresistance after a period of treatment. Diagnostic markers and therapeutic targets for radiosensitivity are severely lacking. Our recently published studies demonstrated that the cell division cycle (CDC6) is a critical molecule contributing to radioresistance, and maybe a potential therapeutic target to overcome radioresistance. In the present study, we for the first time reported that Norcantharidin (NCTD), a demethylated form of cantharidin, re-sensitized radioresistant cancer cells to overcome radioresistance, and synergistically promoted irradiation (IR)-induced cell killing and apoptosis by inducing CDC6 protein degradation. Mechanistically, NCTD induced CDC6 protein degradation through the ubiquitin-proteasome pathways. By using small interfering RNA (siRNA) interference or small compound inhibitors, we further determined that NCTD induced CDC6 protein degradation through a neddylation-dependent pathway, but not through Huwe1, Cyclin F, and APC/C-mediated ubiquitin-proteasome pathways. We screened the six most relevant Cullin subunits (CUL1, 2, 3, 4A, 4B, and 5) using siRNAs. The knockdown of Cullin1 but not the other five cullins remarkably elevated CDC6 protein levels. NCTD promoted the binding of Cullin1 to CDC6, thereby promoting CDC6 protein degradation through a Cullin1 neddylation-mediated ubiquitin-proteasome pathway. NCTD can be used in combination with radiotherapy to achieve better anticancer efficacy, or work as a radiosensitizer to overcome cancer radioresistance.

Crude extract of Desmodium gangeticum process anticancer activity via arresting cell cycle in G1 and modulating cell cycle-related protein expression in A549 human lung carcinoma cells.

BackgroundDesmodium gangeticum (L.)DC., which belongs to the Leguminosae family, has been used in Taiwan and other subtropical countries as an external medicine to remove blood stasis, activate blood circulation, and reduce inflammation. It has been reported to have antioxidant effects and improve inflammatory responses in rats stimulated by pro-inflammatory agents and induced gastric ulcers in experimental animals over the past few decades. This plant has also been used to treat parasitic infections, but there are no reports regarding its effects on lung cancer. Therefore, this study attempted to investigate its water crude extract (in abbreviation DG) on lung cancer cells.MethodsA549 human lung cancer cells were tested for survival using MTT, trypan blue, and propidium iodide. The effects of various concentrations of the crude extract of D. gangeticum (DG) (0.125~1 mg/ml) on the cell cycle and apoptosis of A549 cells were analyzed by flow cytometry and Western blotting methods.ResultsDG can inhibit the growth of A549 human lung cancer cells in a concentration- and time-dependent manner. DG arrested A549 cells in the G1 phase by increasing the proteins expression of p21, p27, cyclin D1, and cyclin E. Additionally, DG decreased the expression of cyclin A, B1, and Cdc 2 (CDK1) proteins.ConclusionsDG demonstrated the anti-lung cancer activity by arresting the cell cycle in G1 via increasing the p21, p27, cyclin D1, cyclin E, and decreasing Cdc2, cyclin A, and B1 proteins expression in A549 human lung cancer cells.

Identification of a seven-cell cycle signature predicting overall survival for gastric cancer.

While genetic alterations in several regulators of the cell cycle have a significant impact on the gastric carcinogenesis process, the prognostic role of them remains to be further elucidated. The TCGA-STAD training set were downloaded and the mRNA expression matrix of cell cycle genes was extracted and corrected for further analysis after taking the intersection with GSE84437 dataset. Differentially expressed mRNAs were identified between tumor and normal tissue samples in TCGA-STAD. Univariate Cox regression analysis and lasso Cox regression model established a novel seven-gene cell cycle signature (including GADD45B, TFDP1, CDC6, CDC25A, CDC7, SMC1A and MCM3) for GC prognosis prediction. Patients in the high-risk group shown significantly poorer survival than patients in the low-risk group. The signature was found to be an independent prognostic factor for GC survival. Nomogram including the signature shown some clinical net benefit for overall survival prediction. The signature was further validated in the GSE84437 dataset. In tissue microarray, CDC6 and MCM3 protein expression were significant differences by the immunohistochemistry-based H-score between tumor tissues and adjacent tissues, and CDC6 is an independent prognostic factor for GC. Interestingly, our GSEA revealed that low-risk patients were more related to cell cycle pathways and might benefit more from therapies targeting cell cycle. Our study identified a novel robust seven-gene cell cycle signature for GC prognosis prediction that may serve as a beneficial complement to clinicopathological staging. The signature might provide potential biomarkers for the application of cell cycle regulators to therapies and treatment response prediction.

G9a Responds to KRAS‐Mediated Replication Stress by Increased Origin Licensing

Pancreatic Ductal Adenocarcinoma (PDAC) remains as one of the leading causes of cancer death in the US. KRAS mutations are recognized as the leading oncogenic driver of PDAC growth. Activation of KRAS causes increased proliferation that requires a level of adaptation during processes, such as DNA replication. This rapid duplication of the genome and epigenome generates havoc in the timing and coordination of replication origin firing, triggering a response known as replication stress (RS). The epigenomic regulator G9a is responsible for catalyzing histone H3 lysine 9 mono‐ and di‐methylation (H3K9me1 and H3K9me2). G9a is abundantly present in many forms of cancer, including PDAC, and because of its interaction with replication proteins, such as RPA and PCNA, it is known to have an important role during DNA replication. DNA replication happens at the S phase of the cell cycle, which requires that replication origins are recognized at late G1 phase by the origin recognition complex (ORC) followed by assembly of the pre‐replication complex (pre‐RC), comprised of CDC6, cdt1 and mini chromosome maintenance (MCM) proteins, to allow origin licensing and subsequent firing. We have shown that the G9a complex increases in response to KRAS activation and is critical to the growth‐promoting effects of this oncogene in PDAC. Though it is known that KRAS reduces distances between replication origins, there has been a paucity of knowledge regarding the role of G9a during replication origin licensing and whether it is directly involved with the pre‐RC. In the current study, we aim to better understand whether G9a interacts with the replication origins and the pre‐RC to increase origin licensing under conditions of KRAS‐mediated RS. To simulate the early events in oncogene‐induced RS to study compensatory mechanisms, we generated an inducible KRAS model (iKRAS‐HPNE) in an hTERT‐immortalized human non‐cancerous pancreatic ductal cell line, hTERT‐HPNE E6/E7. We found that KRAS induction for 48h in this iKRAS‐HPNE model activated the ATR RS‐response pathway, indicated by increased levels of P‐T1989‐ATR, P‐S345‐CHK1 and P‐S33‐RPA32 by western blot and immunofluorescence‐based microscopy. Concurrently, members of the G9a epigenomic complex (G9a, GLP and WIZ) significantly increased along with two subunits of the ORC (ORC1 and ORC2) and several other pre‐RC proteins (Cdt1, MCM2, MCM5 and MCM7). Co‐immunoprecipitation of G9a in the iKRAS‐HPNE cells revealed that G9a interacts with ORC2 protein under conditions of oncogene‐induced RS. Pharmacological inhibition of G9a with UNC0642 during KRAS‐induced RS resulted in a decrease of chromatin‐bound ORC1 and ORC2 as well as pre‐RC proteins in subcellular fractionation experiments, suggesting that functional G9a is critical for enhanced origin licensing. In summary, our results demonstrate that G9a directly interacts with replication origins and the pre‐RC during origin licensing under conditions of RS. Our study reveals one potential mechanism by which cancerous cells with KRAS mutation meet the demands for rapid cellular proliferation during RS and reinforces the role of G9a as a promising therapeutic target for PDAC.

Localization of Cdc14 Isoforms to Ciliary Structures in Tetrahymena thermophila

Cdc14 protein phosphatase is highly conserved across the eukaryotic kingdom, from single‐celled yeast and protozoa to multicellular organisms including mammals. In budding yeast, where it was first studied, Cdc14 is required for mitotic exit; however, this function is not widely conserved. In humans and mice, Cdc14A mutations cause deficiencies in hearing and male fertility due to defects in cilia formation and maintenance. There is evidence for Cdc14 localization to the basal bodies at the proximal end of cilia. The large number of cilia in the free‐living protozoan Tetrahymena thermophila make it an attractive model for molecular studies of cilia structure and function. Our goal is to determine the localization of Cdc14 isoforms to gain some insight into their cellular function and to test if Cdc14 cilia localization is broadly conserved. Interestingly, T. thermophilahas a larger number of Cdc14 isoforms than most organisms. Thus far, genes encoding three of the seven T. thermophilaCdc14 isoforms have been used to create C‐terminal gene fusions with YFP that are expressed in vivo. The constructs were inserted into the T. thermophilamacronuclear genome adjacent to the RPL29 gene and cell lines were selected due to the resulting conversion to cycloheximide resistance. Expression of the transgenes was regulated by a metallothionine promoter. Cells were examined via fluorescence microscopy in the presence and absence of cadmium. All three isoforms localize along ciliary rows and in the oral apparatus after induction during vegetative growth, consistent with basal body localization. No differences in localization have been noted between the three isoforms thus far. The presence of multiple isoforms with the same localization raises questions regarding the redundancy and/or functional specialization of Cdc14 in this highly ciliated organism.

Interphase chromosome condensation in nutrient-starved conditions requires Cdc14 and Hmo1, but not condensin, in yeast

When asynchronously growing cells suffer from nutrient depletion and inactivation of target of rapamycin complex 1 (TORC1) protein kinase, the rDNA (rRNA gene) region is condensed in budding yeast Saccharomyces cerevisiae, which is executed by condensin and Cdc14 protein phosphatase. However, it is unknown whether these mitotic factors can condense the rDNA region in nutrient-starved interphase cells. Here, we show that condensin is not involved in TORC1 inactivation-induced rDNA condensation in G1 cells. Instead, the high-mobility group protein Hmo1 drove this process. The histone deacetylase Rpd3 and Cdc14, which repress rRNA transcription, were both required for the interphase rDNA condensation. Furthermore, interphase rDNA condensation necessitated CLIP and cohibin that tether rDNA to inner nuclear membranes. Finally, we showed that Hmo1, CLIP, Rpd3, and Cdc14 were required for survival in nutrient-starved G1 cells. Thus, this study disclosed novel features of interphase chromosome condensation.

Hinokiflavone induces apoptosis, cell cycle arrest and autophagy in chronic myeloid leukemia cells through MAPK/NF-\u03baB signaling pathway

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative tumor originating from hematopoietic stem cells, and resistance to tyrosine kinase inhibitors (TKI) has become a major cause of treatment failure. Alternative drug therapy is one of the important ways to overcome TKI resistance. Hinokiflavone (HF) is a C-O-C type biflavonoid with low toxicity and antitumor activity. This study investigated the antitumor effect and possible mechanisms of HF in CML cells.MethodsCell viability was measured by CCK-8 assay. Cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blotting was used to assess protein expression levels.ResultsOur results showed that HF significantly inhibited the viability of K562 cells in a concentration- and time-dependent manner and induced G2/M phase arrest by up-regulating p21 and down-regulating Cdc2 protein. Furthermore, HF induced caspase-dependent apoptosis by activating JNK/p38 MAPK signaling pathway and inhibiting NF-κB activity. In addition, HF induced autophagy by increasing LC3-II expression and p62 degradation. Pretreatment with CQ, a late autophagy inhibitor, significantly increased the levels of LC3-II and p62 proteins and promoted cell survival.ConclusionHF shows a good anti-leukemia effect and is expected to become a potential therapeutic drug for CML.

A noncoding regulatory RNA Gm31932 induces cell cycle arrest and differentiation in melanoma via the miR-344d-3-5p/Prc1 (and Nuf2) axis

Emerging evidence has shown that long non-coding RNAs (lncRNAs) play an important role in inhibiting tumor cell proliferation and inducing differentiation. In this study, integrative analysis of whole transcriptome sequencing data demonstrated that lncRNA-Gm31932 is significantly decreased in all-trans retinoic acid (ATRA)-induced and sodium 4-phenylbutanoate (PB-4)-induced mouse melanoma B16 cells. Silencing lncRNA-Gm31932 could inhibit B16 cell proliferation, with cell cycle arrest at the G0/G1 phase and obvious differentiation characteristics, e.g., increased cell volume, melanin content and tyrosinase (Tyr) activity. Furthermore, a series of experiments (luciferase reporter assay, RNA pull-down assay, and western blotting) showed that lncRNA-Gm3932 down-regulated Prc1 and Nuf2 by competitively sponging miR-344d-3-5p, which subsequently reduced the expression of cell cycle-related proteins CDK2, CDC2, and Cyclin B1, and increased the expression of P21 and P27. Moreover, silencing lncRNA-Gm31932 could significantly inhibit tumor growth in B16 melanoma-bearing mice. Taken together, these results indicate that as a possible signaling pathway for ATRA and PB-4, lncRNA-Gm31932 can induce cell cycle arrest and differentiation via miR-344d-3-5p/Prc1 (and Nuf2) axis.

Role of ubiquitin-protein ligase UBR5 in the disassembly of mitotic checkpoint complexes

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation in mitosis by preventing the onset of anaphase until correct bipolar attachment of sister chromosomes to the mitotic spindle is attained. It acts by promoting the assembly of a mitotic checkpoint complex (MCC), composed of mitotic checkpoint proteins BubR1, Bub3, Mad2, and Cdc20. MCC binds to and inhibits the action of ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), which targets for degradation regulators of anaphase initiation. When the checkpoint system is satisfied, MCCs are disassembled, allowing the recovery of APC/C activity and initiation of anaphase. Many of the pathways of the disassembly of the different MCCs have been elucidated, but the mode of their regulation remained unknown. We find that UBR5 (ubiquitin-protein ligase N-recognin 5) is associated with the APC/C*MCC complex immunopurified from extracts of nocodazole-arrested HeLa cells. UBR5 binds to mitotic checkpoint proteins BubR1, Bub3, and Cdc20 and promotes their polyubiquitylation invitro. The dissociation of a Bub3*BubR1 subcomplex of MCC is stimulated by UBR5-dependent ubiquitylation, as suggested by observations that this process in mitotic extracts requires UBR5 and α-β bond hydrolysis of adenosine triphosphate. Furthermore, a system reconstituted from purified recombinant components carries out UBR5- and ubiquitylation-dependent dissociation of Bub3*BubR1. Immunodepletion of UBR5 from mitotic extracts slows down the release of MCC components from APC/C and prolongs the lag period in the recovery of APC/C activity in the exit from mitotic checkpoint arrest. We suggest that UBR5 may be involved in the regulation of the inactivation of the mitotic checkpoint.

The telomeric 5' end nucleotide is regulated in the budding yeast Naumovozyma castellii.

The junction between the double-stranded and single-stranded telomeric DNA (ds–ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5′ end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5′ end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5′ end, indicating that not all ds‐ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds–ss junction ensures the protection of both 5′ ends and 3′ overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.

The effect of paclitaxel on apoptosis, autophagy and mitotic catastrophe in AGS cells

Paclitaxel is an anti-microtubule agent that has been shown to induce cell death in gastric cancer. However, the detailed mechanism of action is unclear. In this study, we reveal that the paclitaxel-induced cell death mechanism involves mitotic catastrophe, autophagy and apoptosis in AGS cells. Paclitaxel induced intrinsic apoptosis by activating caspase-3, caspase-9 and PARP. In addition, the significant increase in autophagy marker LC3B-II, together with Atg5, class III PI3K and Beclin-1, and the down-regulation of p62 following paclitaxel treatment verified that paclitaxel induced autophagy. Further experiments showed that paclitaxel caused mitotic catastrophe, cell cycle arrest of the accumulated multinucleated giant cells at the G2/M phase and induction of cell death in 24 h. Within 48 h, the arrested multinucleated cells escaped mitosis by decreasing cell division regulatory proteins and triggered cell death. Cells treated with paclitaxel for 48 h were grown in fresh medium for 24 h and checked for CDC2, CDC25C and lamin B1 protein expressions. These proteins had decreased significantly, indicating that the remaining cells became senescent. In conclusion, it is suggested that paclitaxel-induced mitotic catastrophe is an integral part of the cell death mechanism, in addition to apoptosis and autophagy, in AGS cells.

NF2 Gene Participates in Regulation of the Cell Cycle of Meningiomas by Restoring Spindle Assembly Checkpoint Function and Inhibiting the Binding of Cdc20 Protein to Anaphase Promoting Complex/Cyclosome

The neurofibromatosis type 2 (NF2) gene mutation is the leading genetic event in meningiomas, usually accompanied by malignant features. Dysfunction of the spindle assembly checkpoint (SAC) induces tumorigenesis. However, the crosstalk between NF2 and SAC in meningiomas remains unclear. Cell proliferation, invasion, apoptosis, and cell cycle of meningiomas were determined by cell counting kit-8 assay, transwell assay, and flow cytometry, respectively. The expression of SAC in meningioma cells was detected by quantitative real-time polymerase chain reaction and Western blot. The interaction between anaphase promoting complex/cyclosome (APC/C) and cell division cycle 20 (Cdc20) protein in meningioma cells was further explored by co-immunoprecipitation. We found that the expression of NF2/merlin was low or absent in malignant meningiomas. Overexpression of NF2 suppressed the proliferation and invasion of meningioma cells, prolonged the G2/M phase, and elevated the expression of SAC proteins at posttranscription. Furthermore, the interaction between APC/C and Cdc20 was inhibited by NF2. Our findings suggested that NF2 might restore SAC function by impairing the binding of APC/C and Cdc20, thereby limiting the mitotic rate and inhibiting proliferation of meningiomas.

Phosphorylation of luminal region of the SUN-domain protein Mps3 promotes nuclear envelope localization during meiosis.

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.

Peptide Nanoparticle-Mediated Combinatorial Delivery of Cancer-Related siRNAs for Synergistic Anti-Proliferative Activity in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is one of the deadliest types of cancer for women of different age groups. Frequently this cancer does not respond to conservative treatment. Combinatorial RNAi can be suggested as an advanced approach to TNBC therapy. Due to the fact that TNBC cells overexpress chemokine receptor 4 we used modular L1 peptide-based nanoparticles modified with CXCR4 ligand for combinatorial delivery of siRNAs suppressing major transduction pathways. TNBC cell line MDA-MB-231 was used as a cellular model. Genes encoding the AQP3, CDC20, and COL4A2 proteins responsible for proliferative activity in TNBC cells were selected as RNAi targets. The siRNA binding ability of the carrier was studied at different charge ratios. The silencing specificity was demonstrated for all siRNAs studied. Alamar Blue proliferation assay has shown significant reduction in the anti-proliferative activity after combinatorial siRNA transfection compared to single siRNA delivery. The most significant synergistic effect has been demonstrated for combinatorial transfection of anti-COL4A2 and anti-CDC20 siRNAs what resulted in 1.5–2 fold inhibition of proliferation and migration of TNBC cells. Based on our findings, we have concluded that combinatorial treatment by CXCR4-ligand modified L1-polyplexes formed with AQP3, CDC20, and COL4A2 siRNAs effectively inhibits proliferation of TNBC cells and can be suggested as useful tool for RNAi-mediated cancer therapy.