Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment.

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast Schizosaccharomyces pombe. Because cell cycle-regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog Cdc15 controls Cdc15 oligomerization state, localization and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR, with the bulk of phosphorylation being constitutive. In vitro, the casein kinase 1 (CK1) isoforms Hhp1 and Hhp2 can phosphorylate 17 sites, and Cdk1 (also known as Cdc2) can phosphorylate the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 accumulation at the contractile ring (CR). Mutations that eliminate CK1 phosphorylation sites allow CR sliding, and phosphomimetic substitutions at these sites reduce Imp2 protein levels and slow CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive, with milder effects on localization and function. This article has an associated First Person interview with the first author of the paper.

Phosphoregulation of the cytokinetic protein Fic1 contributes to fission yeast growth polarity establishment.

Cellular polarization underlies many facets of cell behavior, including cell growth. The rod-shaped fission yeast Schizosaccharomyces pombe is a well-established, genetically tractable system for studying growth polarity regulation. S. pombe cells elongate at their two cell tips in a cell cycle-controlled manner, transitioning from monopolar to bipolar growth in interphase when new ends established by the most recent cell division begin to extend. We previously identified cytokinesis as a critical regulator of new end growth and demonstrated that Fic1, a cytokinetic factor, is required for normal polarized growth at new ends. Here, we report that Fic1 is phosphorylated on two C-terminal residues, which are each targeted by multiple protein kinases. Endogenously expressed Fic1 phosphomutants cannot support proper bipolar growth, and the resultant defects facilitate the switch into an invasive pseudohyphal state. Thus, phosphoregulation of Fic1 links the completion of cytokinesis to the re-establishment of polarized growth in the next cell cycle. These findings broaden the scope of signaling events that contribute to regulating S. pombe growth polarity, underscoring that cytokinetic factors constitute relevant targets of kinases affecting new end growth.This article has an associated First Person interview with Anthony M. Rossi, joint first author of the paper.

Fission yeast Opy1 is an endogenous PI(4,5)P2 sensor that binds to the phosphatidylinositol 4-phosphate 5-kinase Its3.

Phosphoinositides (PIPs) are a dynamic family of lipids that execute diverse roles in cell biology. PIP levels are regulated by numerous enzymes, but our understanding of how these enzymes are controlled in space and time is incomplete. One role of the PIP phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is to anchor the cytokinetic ring (CR) to the plasma membrane (PM) in Schizosaccharomyces pombe While examining potential PI(4,5)P2-binding proteins for roles in CR anchoring, we identified the dual pleckstrin homology (PH) domain-containing protein Opy1. Although related proteins are implicated in PIP regulation, we found no role for S. pombe Opy1 in CR anchoring, which would be expected if it modulated PM PI(4,5)P2 levels. Our data indicate that although Opy1 senses PM PI(4,5)P2 levels and binds to the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, Opy1 does not regulate Its3 kinase activity or PM PI(4,5)P2 levels, a striking difference from its Saccharomyces cerevisiae homolog. However, overexpression of Opy1 resulted in cytokinesis defects, as might be expected if it sequestered PI(4,5)P2 Our results highlight the evolutionary divergence of dual PH domain-containing proteins and the need for caution when interpreting results based on their overexpression.This article has an associated First Person interview with the first author of the paper.

Fever-range hyperthermia promotes cGAS-STING pathway and synergizes DMXAA-induced antiviral immunity.

Background: Fever-range hyperthermia or fever-range temperature (hereafter FRT) improves survival and shortens disease duration in microbial infections. However, the mechanisms of these beneficial effects still remain elusive. We hypothesized that FRT might enhance cell responsiveness to infections by promoting cGAS-STING signaling to cause enhanced production of IFN-ß. Objective: To investigate the effect fever-range hyperthermia on cGAS-STING pathway. Methods: RAW 264.7 and cGAS-/- RAW 264.7 cells, stimulated with 5µg/ml herring testis DNA (htDNA), were heated to 39.5°C and analyzed for the expression of cGAS, STING, IFN-ß, and the synthesis of cGAMP and IRF3 phosphorylation. In vivo, wild type C57BL/6J mice were subjected to whole body hyperthermia (WBH) at 39.5°C. The mice were then challenged with influenza virus and analyzed for antiviral response in term of IFN-ß expression, body weight and survival. Results: We found that 39.5°C FRT upregulated the expression of cGAS and STING, and induced the synthesis of cGAMP and production of IFN-ß in htDNA-transfected RAW 264.7 cells more potently as compared to 37°C. Moreover, FRT+DMXAA-treated cells were better protected from vesicular stomatitis virus (VSV)-induced cytotoxicity in vitro in contrast to the nonprotected control (no FRT and DMXAA) or DMXAA treatment alone. In vivo, FRT at 39.5°C, co-administered with DMXAA, significantly induced the expression of IFN-ß, showed reduced weight loss mice and exhibited 25% more survival over the course of 14 days as compared to DMXAA treated mice 37°C. Conclusion: We conclude that fever-range hyperthermia promotes cGAS-STING pathway to cause increased expression of IFN-ß and mediate its antiviral effects.

Green and efficient degradation of cefoperazone sodium by Bi4O5Br2 leading to the production of non-toxic products: Performance and degradation pathway.

Photocatalytic process represents a promising approach to overcome the pollution challenge associated with the antibiotics-containing wastewater. This study provides a green, efficient and novel approach to remove cephalosporins, particularly cefoperazone sodium (CFP). Bi4O5Br2 was chosen for the first time to systematically study its degradation for CFP, including the analysis of material structure, degradation performance, the structure and toxicity of the transformation products, etc. The degradation rate results indicated that Bi4O5Br2 had an excellent catalytic activity leading to 78% CFP removal compared with the pure BiOBr (38%) within 120 min of visible light irradiation. In addition, the Bi4O5Br2 presents high stability and good organic carbon removal efficiency. The effects of the solution pH (3.12 - 8.75) on catalytic activity revealed that CFP was mainly photocatalyzed under acidic conditions and hydrolyzed under alkaline conditions. Combined with active species and degradation product identification, the photocatalytic degradation pathways of CFP by Bi4O5Br2 was proposed, including hydrolysis, oxidation, reduction and decarboxylation. Most importantly, the identified products were all hydrolysis rather than oxidation byproducts transformed from the intermediate of ß-lactam bond cleavage in CFP molecule, quite different from the mostly previous studies. Furthermore, the final products were demonstrated to be less toxic through the toxicity analysis. Overall, this study illustrates the detailed mechanism of CFP degradation by Bi4O5Br2 and confirms Bi4O5Br2 to be a promising material for the photodegradation of CFP.

Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site.

Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, the founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.

Thermo-Sensitive PLGA-PEG-PLGA Tri-Block Copolymer Hydrogel as Three-Dimensional Cell Culture Matrix for Ovarian Cancer Cells.

Thermo-sensitive hydrogels which could encapsulate cells and provide a three dimensional (3D) microenvironment have great potential in building new cell culture models in vitro. In this study, a thermal responsive hydrogel based on PLGA-PEG-PLGA tri-block copolymers was developed as matrix for 3D ovarian cancer cell culturing. The gelation of PLGA-PEG-PLGA tri-block copolymer was concentration-dependent. SEM images showed the pores were suitable for the formation of 3D cell structures. Cell morphological results showed that large aggregates of ovarian cancer cells (HO8910) were formed after cultured for 10 days. Therefore, hydrogel based on PLGA-PEG-PLGA tri-block copolymers hold potential as in vitro cell culture matrix for ovarian cancer cells.

A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival.

Deubiquitinating enzymes (DUBs), cysteine or metallo- proteases that cleave ubiquitin chains or protein conjugates, are present in nearly every cellular compartment, with overlapping protein domain structure, localization, and functions. We discovered a cohort of DUBs that are involved in membrane trafficking (ubp4, ubp5, ubp9, ubp15, and sst2) and found that loss of all five of these DUBs but not loss of any combination of four, significantly impacted cell viability in the fission yeast Schizosaccharomyces pombe (1). Here, we delineate the collective and individual functions and activities of these five conserved DUBs using comparative proteomics, biochemistry, and microscopy. We find these five DUBs are degenerate rather than redundant at the levels of cell morphology, substrate selectivity, ubiquitin chain specificity, and cell viability under stress. These studies reveal the complexity of interplay among these enzymes, providing a foundation for understanding DUB biology and providing another example of how cells utilize degeneracy to improve survival.

Effects of Osteoglycin (OGN) on treating senile osteoporosis by regulating MSCs.

BACKGROUND: Significant amount of bone mass is lost during the process of aging due to an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption in bone marrow microenvironment, which leads to net bone loss in the aging population, resulting in the pathogenesis of osteoporosis. METHODS: Firstly, differences in proliferative capacity of adipocyte or adipogenic differentiation in mouse mesenchymal stem cells (MMSCs) and senile mouse model-derived bone marrow mesenchymal stem cells (SMMSCs), as well as mRNA expression of OGN and PPARγ2 were observed. Secondly, osteogenic abilities of MMSCs and SMMSCs treated with rosiglitazone (a PPARγ2 agonist) to induce osteogenic changes were observed, and negative correlation of PPARγ2 with OGN was evaluated. Thirdly, the role of SMMSCs in promoting osteogenesis was examined through enhancing expression of OGN; besides, the related mechanism was investigated by means of expression of related adipocyte and osteoblast specific genes. RESULTS: Forced OGN expression by OGN-infected lentivirus could increase expression of Wnt5b, RUNX2, OCN, ALP and Colla1, as well as bone formation, while decreases expression of adipogenesis marker PPARγ2. It resulted in expression inhibition of adipocyte genes such as adipocytic differentiation related genes adipocyte binding protein 2 (aP2) and osteoclast differentiation factor Rankl in bone marrow, giving rise to increased bone mass. CONCLUSION: OGN may plays a significant role in osteoporosis, which may also provide a potential target for therapeutic intervention of senile osteoporosis characterized by altered differentiation of BMSCs into osteoblasts and adipocytes.

Discovery of genes involved in mitosis, cell division, cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains.

The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini-chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non-essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.

Downregulated lincRNA HOTAIR expression in ovarian cancer stem cells decreases its tumorgeniesis and metastasis by inhibiting epithelial-mesenchymal transition.

BACKGROUND: Emerging evidence indicates that dysregulated long intervening non-coding RNA (lincRNA) HOTAIR correlates highly with tumor invasion and metastasis but a link between the high expression of HOTAIR and the metastatic cascade of cancer stem cells (CSCs) needs to be further studied. The purpose of this study was to investigate the effect of down-regulated HOTAIR expression on tumorgeniesis and metastasis of epithelial ovarian cancer (EOC) CSCs. CD117(+)CD44(+)CSCs were isolated from human EOC SKOV3 cell line by using a magnetic-activated cell sorting system, and were then transfected with the expression vector-based small hairpin RNA targeting HOTAIR; the stably transfected cells were selected for the study. Colony-forming, wound-healing, cellular metastasis and tumorigenicity assays were performed. RESULTS: The results demonstrated that the HOTAIR expression in clinical EOC tissues and SKOV3 CD117(+)CD44(+)CSCs was higher than in SKOV3 tumor tissues and non-CD117(+)CD44(+)CSCs. The CD117(+)CD44(+)-shHOTAIR showed an inhibited HOTAIR expression, reduced cell migration and invasion than CD117(+)CD44(+)- scramble, suggesting the inhibition of an epithelial-mesenchymal transition. Moreover, the downregulated HOTAIR expression in CD117(+)CD44(+) CSCs significantly decreased the tumor growth and lung metastasis in xenograft mice. CONCLUSION: Our findings demonstrated the shHOTAIR-mediated down-regulation of the HOTAIR expression in CD117(+)CD44(+) CSCs can be a promising new opportunity for future clinical trials.

Comprehensive proteomics analysis reveals new substrates and regulators of the fission yeast clp1/cdc14 phosphatase.

The conserved family of Cdc14 phosphatases targets cyclin-dependent kinase substrates in yeast, mediating late mitotic signaling events. To discover substrates and regulators of the Schizosaccharomyces pombe Cdc14 phosphatase Clp1, TAP-tagged Clp1, and a substrate trapping mutant (Clp1-C286S) were purified from asynchronous and mitotic (prometaphase and anaphase) cells and binding partners were identified by 2D-LC-MS/MS. Over 100 Clp1-interacting proteins were consistently identified, over 70 of these were enriched in Clp1-C286S-TAP (potential substrates) and we and others detected Cdk1 phosphorylation sites in over half (44/73) of these potential substrates. According to GO annotations, Clp1-interacting proteins are involved in many essential cellular processes including mitosis, cytokinesis, ribosome biogenesis, transcription, and trafficking among others. We confirmed association and dephosphorylation of multiple candidate substrates, including a key scaffolding component of the septation initiation network called Cdc11, an essential kinase of the conserved morphogenesis-related NDR kinase network named Shk1, and multiple Mlu1-binding factor transcriptional regulators. In addition, we identified Sal3, a nuclear ß-importin, as the sole karyopherin required for Clp1 nucleoplasmic shuttling, a key mode of Cdc14 phosphatase regulation. Finally, a handful of proteins were more abundant in wild type Clp1-TAP versus Clp1-C286S-TAP, suggesting that they may directly regulate Clp1 signaling or serve as scaffolding platforms to localize Clp1 activity.

Dynamics of SIN asymmetry establishment.

Timing of cell division is coordinated by the Septation Initiation Network (SIN) in fission yeast. SIN activation is initiated at the two spindle pole bodies (SPB) of the cell in metaphase, but only one of these SPBs contains an active SIN in anaphase, while SIN is inactivated in the other by the Cdc16-Byr4 GAP complex. Most of the factors that are needed for such asymmetry establishment have been already characterized, but we lack the molecular details that drive such quick asymmetric distribution of molecules at the two SPBs. Here we investigate the problem by computational modeling and, after establishing a minimal system with two antagonists that can drive reliable asymmetry establishment, we incorporate the current knowledge on the basic SIN regulators into an extended model with molecular details of the key regulators. The model can capture several peculiar earlier experimental findings and also predicts the behavior of double and triple SIN mutants. We experimentally tested one prediction, that phosphorylation of the scaffold protein Cdc11 by a SIN kinase and the core cell cycle regulatory Cyclin dependent kinase (Cdk) can compensate for mutations in the SIN inhibitor Cdc16 with different efficiencies. One aspect of the prediction failed, highlighting a potential hole in our current knowledge. Further experimental tests revealed that SIN induced Cdc11 phosphorylation might have two separate effects. We conclude that SIN asymmetry is established by the antagonistic interactions between SIN and its inhibitor Cdc16-Byr4, partially through the regulation of Cdc11 phosphorylation states.

Predictive Value of Impulse Oscillometry Combined with Fractional Expiratory Nitric Oxide Test for Asthma in Preschool Children.

Objective: Prediction of asthma in preschool children is challenging and lacks objective indicators. The aim is to observe and analyze the variances between impulse oscillometry (IOS) and fractional expiratory nitric oxide (FeNO) in preschool children with wheezing, establish a joint prediction model, and explore the diagnostic value of combining IOS with FeNO in diagnosing asthma among preschool children. Patients and methods: This study enrolled children aged 3-6 years with wheezing between June 2021 and June 2022. They were categorized as asthmatic (n=104) or non-asthmatic (n=109) after a 1-year follow-up. Clinical data, along with IOS and FeNO measurements from both groups, underwent univariate regression and multiple regression analyses to identify predictive factors and develop the most accurate model. The prediction model was built using the stepwise (stepAIC) method. The receiver operating characteristic curve (ROC), calibration curve, Hosmer-Lemeshow test, and decision curve analysis (DCA) were employed to validate and assess the model. Results: During univariate analysis, a history of allergic rhinitis, a history of eczema or atopic dermatitis, and measures including FeNO, R5, X5, R20, Fres, and R5-R20 were found to be associated with asthma diagnosis. Subsequent multivariate analysis revealed elevated FeNO, R5, and X5 as independent risk factors. The stepAIC method selected five factors (history of allergic rhinitis, history of eczema or atopic dermatitis, FeNO, R5, X5) and established a prediction model. The combined model achieved an AUROC of 0.94, with a sensitivity of 0.89 and specificity of 0.88, surpassing that of individual factors. Calibration plots and the HL test confirmed satisfactory accuracy. Conclusion: This study has developed a prediction model based on five factors, potentially aiding clinicians in early identification of asthma risk among preschool children.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

The core spindle pole body scaffold Ppc89 links the pericentrin orthologue Pcp1 to the fission yeast spindle pole body via an evolutionarily conserved interface.

Centrosomes and spindle pole bodies (SPBs) are important for mitotic spindle formation and serve as cellular signaling platforms. Although centrosomes and SPBs differ in morphology, many mechanistic insights into centrosome function have been gleaned from SPB studies. In the fission yeast Schizosaccharomyces pombe, the α-helical protein Ppc89, identified based on its interaction with the septation initiation network scaffold Sid4, comprises the SPB core. High-resolution imaging has suggested that SPB proteins assemble on the Ppc89 core during SPB duplication, but such interactions are undefined. Here, we define a connection between Ppc89 and the essential pericentrin Pcp1. Specifically, we found that a predicted third helix within Ppc89 binds the Pcp1 pericentrin-AKAP450 centrosomal targeting (PACT) domain complexed with calmodulin. Ppc89 helix 3 contains similarity to present in the N-terminus of Cep57 (PINC) motifs found in the centrosomal proteins fly SAS-6 and human Cep57 and also to the S. cerevisiae SPB protein Spc42. These motifs bind pericentrin-calmodulin complexes and AlphaFold2 models suggest a homologous complex assembles in all four organisms. Mutational analysis of the S. pombe complex supports the importance of Ppc89-Pcp1 binding interface in vivo. Our studies provide insight into the core architecture of the S. pombe SPB and suggest an evolutionarily conserved mechanism of scaffolding pericentrin-calmodulin complexes for mitotic spindle formation.

Dendritic-cell-targeting virus-like particles as potent mRNA vaccine carriers.

Messenger RNA vaccines lack specificity for dendritic cells (DCs)-the most effective cells at antigen presentation. Here we report the design and performance of a DC-targeting virus-like particle pseudotyped with an engineered Sindbis-virus glycoprotein that recognizes a surface protein on DCs, and packaging mRNA encoding for the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or for the glycoproteins B and D of herpes simplex virus 1. Injection of the DC-targeting SARS-CoV-2 mRNA vaccine in the footpad of mice led to substantially higher and durable antigen-specific immunoglobulin-G titres and cellular immune responses than untargeted virus-like particles and lipid-nanoparticle formulations. The vaccines also protected the mice from infection with SARS-CoV-2 or with herpes simplex virus 1. Virus-like particles with preferential uptake by DCs may facilitate the development of potent prophylactic and therapeutic vaccines.

Evaluation of characteristics of CD44+CD117+ ovarian cancer stem cells in three dimensional basement membrane extract scaffold versus two dimensional monocultures.

BACKGROUND: Cancer stem cells (CSCs) are thought to be capable of surviving conventional chemotherapeutic treatments because the cells have more resistant to anticancer drugs than common cancer cells. Most in vitro studies in experimental cancer cells have been done in a two-dimensional (2D) monocultures, while accumulating evidence suggests that cancer cells behave differently when they are grown within a three-dimensional (3D) culture system. RESULTS: The CD44+CD117+cells isolated from human epithelial ovarian cancer SKOV-3 cell line using magnetic-activated cell sorting were found to grow faster than the SKOV-3 cells in the 3D culture and in the nude mice. Anticancer drugs 5FU, docetaxel, cisplatin, and carboplatin were seen to inhibit growth of the CD44+CD117+ cells by 50% in the 2D culture with IC50 concentration, whereas, in the 3D culture, the four drugs inhibited the cell growth by only 34.4%, 40.8%, 34.8% and 21.9% at 3D one, respectively. Effect of paclitaxel on the CD44+CD117+cell viability indicated that fewer cells underwent apoptosis in 3D culture than that in 2D one. In addition, anticancer drugs markedly increased the expression of ABCG2 and ABCB1 of CD44+CD117+cells in 3D culture. CONCLUSION: Our assay demonstrated that human epithelial ovarian cancer CD44+CD117+cells possessed the properties of CSCs that exhibited more chemoresistance in the 3D culture than that of in 2D one. The 3D culture provides a realistic model for study of the CSC response to anticancer drugs.

Effect of down-regulated transcriptional repressor ZEB1 on the epithelial-mesenchymal transition of ovarian cancer cells.

BACKGROUND: Progress has been made against early events of malignant transformation and drug resistance associated with epithelial ovarian cancer; uncontrolled metastases, however, still accounts for most patient deaths. The molecular mechanism that regulates the process of epithelial ovarian cancer metastases is not yet clearly understood. The purpose of this study was to investigate the effect of down-regulating the transcriptional repressor zinc-finger E-box-binding homeobox 1 (ZEB1) on an epithelial-mesenchymal transition (EMT) of human ovarian cancer SKOV3 cell line in vitro and in vivo. METHODS: The human ovarian cancer cells SKOV3 and HO8910 were transfected with an expression vector-based small hairpin RNA (shRNA) targeting ZEB1 (shZEB1), and the stably transfected cells were selected. Colony-forming, wound-healing, and cellular migration assays were respectively used. The tumorigenicity of shZEB1-SKOV3 was also evaluated in mice. RESULTS: The shZEB1-SKOV3 and shZEB1-HO8910 cells showed a lower level of ZEB1 expression and weaker cell migration than the control cells. Moreover, down-regulating ZEB1 expression with shRNA in the cells enhanced the expression of miR-200c that acted as a tumor suppressor to inhibit the epithelial-mesenchymal transition of shZEB1-SKOV3 cells and to block shZEB1-SKOV3 cell metastasis in vivo. The shRNA-mediated down-regulation ZEB1 in SKOV3 cells significantly decreased the tumor growth in the xenograft mice. CONCLUSION: The shZEB1-mediated down-regulation of the ZEB1 expression in the SKOV3 cells may be considered for future clinical trials.