DEPDC1 is a novel cell cycle related gene that regulates mitotic progression

DEPDC1 is a recently identified novel tumor-related gene that is upregulated in several types of cancer and contributes to tumorigenesis. In this study, we have investigated the expression pattern and functional implications of DEPDC1 during cell cycle progression. Expression studies using synchronized cells demonstrated that DEPDC1 is highly expressed in the mitotic phase of the cell cycle. Immunofluorescence assays showed that DEPDC1 is predominantly localized in the nucleus during interphase and is redistributed into the whole cell upon nuclear membrane breakdown in metaphase. Subsequently, siRNA-mediated knockdown of DEPDC1 caused a significant mitotic arrest. Moreover, knockdown of DEPDC1 resulted in remarkable mitotic defects such as abnormal multiple nuclei and multipolar spindle structures accompanied by the upregulation of the A20 gene as well as several cell cycle-related genes such as CCNB1 and CCNB2. Taken together, our current observations strongly suggest that this novel cancerous gene, DEPDC1, plays a pivotal role in the regulation of proper mitotic progression. [BMB Reports 2015; 48(7): 413-418]     

Ubp-M serine 552 phosphorylation by cyclin-dependent kinase 1 regulates cell cycle progression

In eukaryotic cells, genomic DNA is organized into a chromatin structure, which not only serves as the template for DNA-based nuclear processes, but also as a platform integrating intracellular and extracellular signals. Although much effort has been spent to characterize chromatin modifying/remodeling activities, little is known about cell signaling pathways targeting these chromatin modulators. Here, we report that cyclin-dependent kinase 1 (CDK1) phosphorylates the histone H2A deubiquitinase Ubp-M at serine 552 (S552P), and, importantly, this phosphorylation is required for cell cycle progression. Mass spectrometry analysis confirmed Ubp-M is phosphorylated at serine 552, and in vitro and in vivo assays demonstrated that CDK1/cyclin B kinase is responsible for Ubp-M S552P. Interestingly, Ubp-M S552P is not required for Ubp-M tetramer formation, deubiquitination activity, substrate specificity, or regulation of gene expression. However, Ubp-M S552P is required for cell proliferation and cell cycle G₂/M phase progression. Ubp-M S552P reduces Ubp-M interaction with nuclear export protein CRM1 and facilitates Ubp-M nuclear localization. Therefore, these studies confirm that Ubp-M is phosphorylated at S552 and identify CDK1 as the enzyme responsible for the phosphorylation. Importantly, this study specifically links Ubp-M S552P to cell cycle G₂/M phase progression.     

circSVIL regulates bovine myoblast development by inhibiting STAT1 phosphorylation

Science China Life Sciences - Circular RNAs (circRNAs), a novel class of non-coding RNAs with a loop structure, have recently been shown to participate in various pathophysiological processes....     

miR-345-5p regulates proliferation,cell cycle,and apoptosis of acute myeloid leukemia cells by targeting AKT2

Acute myeloid leukemia (AML) is a malignant clonal hematopoietic disease, which is caused by hematopoietic stem cell abnormalities. Epigenetic regulation, especially of microRNAs (miRNAs), mostly results from external or environmental effects and is critical to AML. In this study, for the first time, we report that decreased expression of miR-345-5p facilitates the proliferation of leukemia cells in AML. Further study demonstrated that AKT1/2 was the target of miR-345-5p and was responsible for the dysregulation of leukemia cell proliferation and apoptosis. Inhibition of AKT1/2 ameliorated this malignant effect, which provides new insight into AML diagnosis, treatment, prognosis, and next-step translational investigations.     

miR-377-5p inhibits lung cancer cell proliferation,invasion, and cell cycle progression by targeting AKT1 signaling

Lung carcinoma is the most common type of malignant tumors globally, and its molecular mechanisms remained unclear. With the aim to investigate the effects of microRNA (miR)-377-5p on the cell development, invasion, metastasis, and cycle of lung carcinoma, this study was performed. We evaluated miR-377-5p expression levels in lung cancer tissues and cell models. Cell viability, proliferation, migration, invasion abilities, and cell cycle distribution were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, crystal violet, transwell, and flow cytometry assay. Furthermore, expression levels of protein kinase B α subunit (AKT1) and proteins related to cell cycle and epithelial-mesenchymal transition (EMT) were assessed using Western blot analysis and quantitative real-time polymerase chain reaction. These results suggested that miR-377-5p was downregulated in vivo and in cell models, and miR-377-5p overexpression inhibited cell viability, proliferation, migration, invasion, and induced cell-cycle arrest. In addition, as a target of miR-377-5p, AKT1 alleviated the decreases of cell viability, proliferation, migration, invasion, the S-phase cells, the expression of cyclin D1, fibronectin, and vimentin, as well as the increases of the G0/G1-phase cells, the expression of Foxo1, p27 ^kip1, p21 ^Cip1 and E-cadherin when miR-377-5p overexpressed. In conclusion, miR-377-5p inhibited cell development and regulated cell cycle distribution and EMT by targeting AKT1, which provided a theoretical basis for further study of lung carcinoma therapeutics.     

Dieckol,a natural polyphenolic drug,inhibits the proliferation and migration of colon cancer cells by inhibiting PI3K,AKT, and mTOR phosphorylation

This study investigated that dieckol (DKL), a natural drug, inhibits colon cancer cell proliferation and migration by inhibiting phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) phosphorylation in HCT-116 cells. The cells were treated with DKL in various concentrations (32 and 50 μM) for 24 h and then analyzed for various experiments. MTT (tetrazolium bromide) and crystal violet assay investigated DKL-mediated cytotoxicity. Dichlorodihydrofluorescein diacetate staining was used to assess the reactive oxygen species (ROS) measurement, and apoptotic changes were studied by dual acridine orange and ethidium bromide staining. Protein expression of cell survival, cell cycle, proliferation, and apoptosis protein was evaluated by western blot analysis. Results indicated that DKL produces significant cytotoxicity in HCT-116, and the half-maximal inhibitory concentration was found to be 32 μM for 24-h incubation. Moreover, effective production of ROS and enhanced apoptotic signs were observed upon DKL treatment in HCT-116. DKL induces the expression of phosphorylated PI3K, AKT, and mToR-associated enhanced expression of cyclin-D1, proliferating cell nuclear antigen, cyclin-dependent kinase (CDK)-4, CDK-6, and Bcl-2 in HCT-116. In addition, proapoptotic proteins such as Bax, caspase-9, and caspase-3 were significantly enhanced by DKL treatment in HCT-116. Hence, DKL has been considered a chemotherapeutic drug by impeding the expression of PI3K-, AKT-, and mTOR-mediated inhibition of proliferation and cell cycle-regulating proteins.     

Hsp90 phosphorylation,Wee1 and the cell cycle

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1? yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation.     

Polycystin‐1 regulates cell proliferation and migration through AKT/mTORC2 pathway in a human craniosynostosis cell model

Craniosynostosis is the premature fusion of skull sutures and has a severe pathological impact on childrens’ life. Mechanical forces are capable of triggering biological responses in bone cells and regulate osteoblastogenesis in cranial sutures, leading to premature closure. The mechanosensitive proteins polycystin‐1 (PC1) and polycystin‐2 (PC2) have been documented to play an important role in craniofacial proliferation and development. Herein, we investigated the contribution of PC1 to the pathogenesis of non‐syndromic craniosynostosis and the associated molecular mechanisms. Protein expression of PC1 and PC2 was detected in bone fragments derived from craniosynostosis patients via immunohistochemistry. To explore the modulatory role of PC1 in primary cranial suture cells, we further abrogated the function of PC1 extracellular mechanosensing domain using a specific anti‐PC1 IgPKD1 antibody. Effect of IgPKD1 treatment was evaluated with cell proliferation and migration assays. Activation of PI3K/AKT/mTOR pathway components was further detected via Western blot in primary cranial suture cells following IgPKD1 treatment. PC1 and PC2 are expressed in human tissues of craniosynostosis. PC1 functional inhibition resulted in elevated proliferation and migration of primary cranial suture cells. PC1 inhibition also induced activation of AKT, exhibiting elevated phospho (p)‐AKT (Ser473) levels, but not 4EBP1 or p70S6K activation. Our findings indicate that PC1 may act as a mechanosensing molecule in cranial sutures by modulating osteoblastic cell proliferation and migration through the PC1/AKT/mTORC2 cascade with a potential impact on the development of non‐syndromic craniosynostosis.     

SCAPER, a novel cyclin A-interacting protein that regulates cell cycle progression

Cyclin A/Cdk2 plays an important role during S and G2/M phases of the eukaryotic cell cycle, but the mechanisms by which it regulates cell cycle events are not fully understood. We have biochemically purified and identified SCAPER, a novel protein that specifically interacts with cyclin A/Cdk2 in vivo. Its expression is cell cycle independent, and it associates with cyclin A/Cdk2 at multiple phases of the cell cycle. SCAPER localizes primarily to the endoplasmic reticulum. Ectopic expression of SCAPER sequesters cyclin A from the nucleus and results specifically in an accumulation of cells in M phase of the cell cycle. RNAi-mediated depletion of SCAPER decreases the cytoplasmic pool of cyclin A and delays the G1/S phase transition upon cell cycle re-entry from quiescence. We propose that SCAPER represents a novel cyclin A/Cdk2 regulatory protein that transiently maintains this kinase in the cytoplasm. SCAPER could play a role in distinguishing S phase- from M phase-specific functions of cyclin A/Cdk2.     

Plk1-dependent phosphorylation regulates functions of DNA topoisomerase IIalpha in cell cycle progression

Plk1 (Polo-like kinase 1) has been documented as a critical regulator of many mitotic events. However, increasing evidence supports the notion that Plk1 might also have functions outside of mitosis. Using biochemical fractionation and RNA interference approaches, we found that Plk1 was required for both G(1)/S and G(2)/M phases and that DNA topoisomerase IIalpha (topoIIalpha) was a potential target for Plk1 in both interphase and mitosis. Plk1 phosphorylates Ser(1337) and Ser(1524) of topoIIalpha. Overexpression of an unphosphorylatable topoIIalpha mutant led to S phase arrest, suggesting that Plk1-associated phosphorylation first occurs in S phase. Moreover, overexpression of the unphosphorylatable topoIIalpha mutant activated the ATM/R-dependent DNA damage checkpoint, probably due to reduced catalytic activity of topoIIalpha, and resulted in accumulation of catenated DNA. Finally, we showed that wild type topoIIalpha, but not the unphosphorylatable mutant, was able to rescue topoIIalpha depletion-induced defects in sister chromatid segregation, indicating that Plk1-associated phosphorylation is essential for the functions of topoIIalpha in mitosis.     

Circular RNA circ_001350 regulates glioma cell proliferation,apoptosis, and metastatic properties by acting as a miRNA sponge

Glioma is an aggressive malignancy with increasing incidence and threatens people's health worldwide. Accumulating evidence revealed that circular RNAs (circRNAs) play important functions in cancers. A previous study demonstrated that circ_001350 was elevated in glioma tissue samples than nontumorous tissue specimens screened by high-throughput microarray. The level of circ_001350 in glioma tissue specimens and cell lines was detected by quantitative real-time polymerase chain reaction. The Fisher exact test was carried out to estimate the correlation of circ_001350 level with clinical characteristics. Cell proliferation, apoptosis, and motility abilities were detected using cell counting kit-8, clonogenic, flow cytometry, and transwell experiments, respectively. The potential target of circ_001350 was identified by the luciferase assay. circ_001350 level was significantly enhanced in glioma tissue specimens and cells. Further, elevated expression of circ_001350 was closely linked to patients' clinical severity. Knockdown of circ_001350 could inhibit cell proliferation and metastatic properties and increase apoptotic cells. circ_001350 could directly bind to miR-1236 and regulate its expression to exert oncogenic functions. Collectively, circ_001350 directly sponges miR-1236, thus contributing to malignant progression of glioma.     

CKIP-1 regulates macrophage proliferation by inhibiting TRAF6-mediated Akt activation

Macrophages play pivotal roles in development, homeostasis, tissue repair and immunity. Macrophage proliferation is promoted by macrophage colony-stimulating factor (M-CSF)-induced Akt signaling; yet, how this process is terminated remains unclear. Here, we identify casein kinase 2-interacting protein-1 (CKIP-1) as a novel inhibitor of macrophage proliferation. In resting macrophages, CKIP-1 was phosphorylated at Serine 342 by constitutively active GSK3β, the downstream target of Akt. This phosphorylation triggers the polyubiquitination and proteasomal degradation of CKIP-1. Upon M-CSF stimulation, Akt is activated by CSF-1R-PI3K and then inactivates GSK3β, leading to the stabilization of CKIP-1 and β-catenin proteins. β-catenin promotes the expression of proliferation genes including cyclin D and c-Myc. CKIP-1 interacts with TRAF6, a ubiquitin ligase required for K63-linked ubiquitination and plasma membrane recruitment of Akt, and terminates TRAF6-mediated Akt activation. By this means, CKIP-1 inhibits macrophage proliferation specifically at the late stage after M-CSF stimulation. Furthermore, CKIP-1 deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and CKIP-1^−/− mice spontaneously develop a macrophage-dominated splenomegaly and myeloproliferation. Together, these data demonstrate that CKIP-1 plays a critical role in the regulation of macrophage homeostasis by inhibiting TRAF6-mediated Akt activation.