Integrated bioinformatics analysis reveals CDK1 and PLK1 as potential therapeutic targets of lung adenocarcinoma.

ABSTRACT: This study is to identify potential biomarkers and therapeutic targets for lung adenocarcinoma (LUAD).GSE6044 and GSE118370 raw data from the Gene Expression Omnibus database were normalized with Robust Multichip Average. After merging these two datasets, the combat function of sva packages was used to eliminate batch effects. Then, limma packages were used to filtrate differentially expressed genes. We constructed protein-protein interaction relationships using STRING database and hub genes were identified based on connectivity degrees. The cBioportal database was used to explore the alterations of the hub genes. The promoter methylation of cyclin dependent kinase 1 (CDK1) and polo-like Kinase 1 (PLK1) and their association with tumor immune infiltration in patients with LUAD were investigated using DiseaseMeth version 2.0 and TIMER databases. The Cancer Genome Atlas-LUAD dataset was used to perform gene set enrichment analysis.We identified 10 hub genes, which were upregulated in LUAD, among which 8 were successfully verified in the Cancer Genome Atlas and Oncomine databases. Kaplan-Meier analysis indicated that the expressions of CDK1 and PLK1 in LUAD patients were associated with overall survival and disease-free survival. The methylation levels in the promoter regions of these 2 genes in LUAD patients were lower than those in normal lung tissues. Their expressions in LUAD were associated with tumor stages and relative abundance of tumor infiltrating immune cells, such as B cells, CD4+ T cells, and macrophages. Moreover, cell cycle, DNA replication, homologous recombination, mismatch repair, P53 signaling pathway, and small cell lung cancer signaling were significantly enriched in CDK1 and PLK1 high expression phenotype.CDK1 and PLK1 may be used as potential biomarkers and therapeutic targets for LUAD.

DNMT3A Mutation-Induced CDK1 Overexpression Promotes Leukemogenesis by Modulating the Interaction between EZH2 and DNMT3A.

DNMT3A mutations are frequently identified in acute myeloid leukemia (AML) and indicate poor prognosis. Previously, we found that the hotspot mutation DNMT3A R882H could upregulate CDK1 and induce AML in conditional knock-in mice. However, the mechanism by which CDK1 is involved in leukemogenesis of DNMT3A mutation-related AML, and whether CDK1 could be a therapeutic target, remains unclear. In this study, using fluorescence resonance energy transfer and immunoprecipitation analysis, we discovered that increased CDK1 could compete with EZH2 to bind to the PHD-like motif of DNMT3A, which may disturb the protein interaction between EZH2 and DNMT3A. Knockdown of CDK1 in OCI-AML3 cells with DNMT3A mutation markedly inhibited proliferation and induced apoptosis. CDK1 selective inhibitor CGP74514A (CGP) and the pan-CDK inhibitor flavopiridol (FLA) arrested OCI-AML3 cells in the G2/M phase, and induced cell apoptosis. CGP significantly increased CD163-positive cells. Moreover, the combined application of CDK1 inhibitor and traditional chemotherapy drugs synergistically inhibited proliferation and induced apoptosis of OCI-AML3 cells. In conclusion, this study highlights CDK1 overexpression as a pathogenic factor and a potential therapeutic target for DNMT3A mutation-related AML.

Upregulation of ATP Binding Cassette Subfamily C Member 5 facilitates Prostate Cancer progression and Enzalutamide resistance via the CDK1-mediated AR Ser81 Phosphorylation Pathway.

The treatment of advanced prostate cancer, castration-resistant prostate cancer, remains challenging. The mechanisms of action of ATP binding cassette subfamily C member 5 (ABCC5) in prostate cancer and its relationship with drug resistance are still unclear. Expression and prognostic analyses of ABCC5 were performed through bioinformatic methods and immunohistochemistry analyses in multiple public databases as well as in our own prostate cancer cohort. The biological function of ABCC5 in prostate cancer cells was evaluated by in vitro and in vivo cell proliferation and migration and invasion assays. The regulation of CDK1 by ABCC5 was determined via RT-qPCR, western blots, and immunofluorescence. ABCC5 was significantly overexpressed in prostate cancer and positively associated with unfavorable clinicopathological features and prognosis. Upregulation of ABCC5 could enhance the cell proliferation, migration, and invasion of prostate cancer in vitro and in vivo. Mechanistically, ABCC5 exerts a protumor effect by binding to and inhibiting the protein degradation of CDK1, which promotes the phosphorylation of AR at Ser81 by CDK1 and activates the transcriptional activity of AR on target genes. Moreover, the addition of a CDK1 inhibitor or knockdown of CDK1 significantly improved the efficacy of enzalutamide on prostate cancer cells. The ABCC5-CDK1-AR regulatory pathway could be a potential therapeutic target for advanced prostate cancer, especially castration-resistant prostate cancer (CRPC), to enhance the therapeutic effect of enzalutamide.

CDKN2A, CDK1, and CCNE1 overexpression in sebaceous gland carcinoma of eyelid.

PURPOSE: To investigate the overexpression of genes in sebaceous gland carcinoma (SGC) of the eyelid compared to sebaceous adenoma of the eyelid in order to elucidate the molecular mechanism underlying pathogenesis. METHODS: We performed histopathological examination of eyelid tissues surgically removed from four patients diagnosed with SGC (cases 1-3) and sebaceous adenoma (case 4) of the eyelid. Next, we performed global gene expression analysis of surgical tissue samples using a GeneChip® system and the Ingenuity Pathways Knowledge Base. The results of the GeneChip® analysis were explored with quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RESULTS: In the SGC samples, we found that 211, 199, and 199 genes, respectively, showed ≥ 2.0-fold higher expression than those in the sebaceous adenoma sample (case 4); 194 genes were common to all three SGC samples. For the 194 genes with upregulated expression, functional category analysis showed that SGC of the eyelid employed a unique gene network, including cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase 1 (CDK1), and cyclin E1 (CCNE1), which are related to cell cycle progression, incidence of tumor, and cell viability. Furthermore, qRT-PCR analysis showed that the expression levels of CDKN2A, CDK1, and CCNE1 were significantly upregulated in all SGC cases compared to those in the sebaceous adenoma case. These data were similar to the results of microarray analysis. CONCLUSION: Overexpression of cell cycle-related genes CDKN2A, CDK1, CCNE1, and their gene network may help elucidate the pathogenic pathway of SGC of the eyelid at the molecular level.

Involvement of CaMKII in regulating the release of diplotene-arrested mouse oocytes by pAkt1 (Ser473).

Calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) had been reported to play a role in the process of fertilization. However, the role of CaMKII in the release of diplotene-arrested oocytes is poorly understood. In this study, we explored the potential effect of CaMKII on Akt1 and the relationship among CaMKII, Akt1 and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) during the meiotic resumption of mouse oocytes. We found that inhibition of CaMKII aggravated diplotene arrest. We detected the expression and distribution of pCaMKII (Thr286), pAkt1 (Ser473), Cdc25B and pCdc2 (Tyr15) when oocytes were treated with KN-93, SH-6, LY294002 or PIP3, respectively. Our data showed that down-regulated CaMKII by KN-93 decreased the levels of pAkt1 (Ser473) and rearranged the distribution of pAkt1 (Ser473). Meanwhile, down-regulated pAkt1 (Ser473) by SH-6 also decreased the levels of pCaMKII (Thr286), Cdc25B and pCdc2 (Tyr15) significantly and rearranged the distributions of pCaMKII (Thr286). Furthermore, our data showed that exogenous PIP3 up-regulated GVBD rates significantly and increased the levels of pCaMKII (Thr286) and pAkt1 (Ser473). On the contrary, down-regulation of PIP3 by LY294002 decreased GVBD rates and the levels of pCaMKII (Thr286) and pAkt1 (Ser473), respectively. Our results showed that Akt1 and CaMKII regulated each other, and PIP3 may be involved in these regulations during the release of mouse oocytes from diplotene arrest.

LncRNA CASC11 promotes the development of lung cancer through targeting microRNA-302/CDK1 axis.

OBJECTIVE: To elucidate whether long non-coding RNA cancer susceptibility candidate 11 (lncRNA CASC11) could participate in the development of lung cancer through targeting microRNA-302/CDK1 axis. PATIENTS AND METHODS: Expression levels of CASC11, microRNA-302 and CDK1 in lung cancer tissues and paracancerous tissues were determined by quantitative real-time polymerase chain reaction (qRT-PCR). CASC11 expression in lung cancer cell lines was also determined. The regulatory effect of CASC11 on proliferative potential of lung cancer cells was accessed by cell counting kit-8 (CCK-8) and colony formation assay. The binding condition between microRNA-302 to CASC11 and CDK1 was evaluated by dual-luciferase reporter gene assay. CDK1 expression in lung cancer cells with CASC11 or microRNA-302 knockdown was detected by Western blot. The proliferation of lung cancer cells was determined after transfection of microRNA-302 inhibitor or co-transfection of microRNA-302 inhibitor and si-CASC11. RESULTS: CASC11 and CDK1 were highly expressed, whereas microRNA-302 was lowly expressed in lung cancer tissues. Identically, CASC11 was highly expressed in lung cancer cell lines (A547, H157 and SPC-A-1) than controls as well. CASC11 knockdown attenuated proliferative capacity of lung cancer cells. The opposite trend was observed by microRNA-302 knockdown. Dual-luciferase reporter gene assay verified that CASC11 could bind to microRNA-302 and microRNA-302 could bind to CDK1. CDK1 expression in lung cancer cells was negatively regulated by CASC11. MicroRNA-302 knockdown reversed the inhibitory effect of CASC11 on CDK1 expression. The proliferation of lung cancer cells co-transfected with microRNA-302 inhibitor and si-CASC11 decreased compared with those transfected with microRNA-302 inhibitor. CONCLUSIONS: High expression of CASC11 promotes the development of lung cancer through upregulating CDK1 expression by binding to microRNA-302.

High expression of CDK1 and BUB1 predicts poor prognosis of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related death. Increasing evidence suggests that cell cycle dysregulation is one of the hallmarks of cancer. In this study, by using the GEO database, we predicted the cell cycle-related protein CDK1 and BUB1 to be significantly overexpressed in PDAC tissues. Thus, this study aimed to investigate the clinical pathological significance of CDK1 and BUB1 in PDAC. METHODS: To explore the role of CDK1 and BUB1 in PDAC progression and evaluate their prognostic value, we investigated the expression patterns of CDK1 and BUB1 by using immunohistochemical staining in 99 PDAC and 71 normal pancreatic tissues with complete pathological parameters and survival data. RESULTS: CDK1 and BUB1 were significantly overexpressed in PDAC tissues. The expression of CDK1 was correlated with tumor size and histological grade, and the expression of BUB1 was correlated with the tumor size of PDAC. With regard to survival, a high expression of either CDK1 or BUB1 was correlated with a short survival of PDAC patients. Additionally, PDAC patients with a concurrent high expression of CDK1 and BUB1 showed the shortest survival. CONCLUSIONS: Our study demonstrated that CDK1 and BUB1 may play a role in PDAC progression and could be prognostic biomarkers for PDAC patients.

MiR-31-5p acts as a tumor suppressor in renal cell carcinoma by targeting cyclin-dependent kinase 1 (CDK1).

BACKGROUND: Renal cell carcinoma (RCC) accounts for more than 90% of cancers in the kidney. RCC is often asymptomatic, as a result people with RCC generally have advanced disease by the time it is discovered and has a poor prognosis compared to other cancers. Therefore, it is necessary to explore its pathogenesis and identify some reliable prognostic biomarker of RCC. miRNAs are emerging as important players in the development and progression of RCC. miR-31-5p has been reported to act as a tumor suppressor in hepatocellular carcinoma (HCC). The aim of this study is to determine the detailed molecular mechanism of miR-31-5p in the progression of RCC and to investigate its potential clinical value. METHODS: In this study, RT-qPCR, EdU assay, CCK-8 assay, wound scratch assay, transwell assay, flow cytometry assay and cell cycle assay were performed to detect miR-31-5p expression and its functions in RCC. Moreover, 42 formalin-fixed paraffin-embedded (FFPE) RCC samples were used to analyze the relationship between miR-31-5p expression and patients' overall survival. Finally, luciferase reporter assay, RT-qPCR assay and western blot were used to explore the association between miR-31-5p and its potential targets. RESULTS: miR-31-5p was significantly down-regulated in RCC tissues and RCC cell lines compared with paired adjacent normal tissues and normal cell lines. miR-31-5p downregulation was associated with poor prognosis in RCC patients. Overexpression of miR-31-5p inhibited RCC cell proliferation, migration and invasion and cell cycle. Conversely, down-regulation of miR-31-5p promoted cell proliferation, migration and invasion. Furthermore, cyclin-dependent kinasec1 (CDK1), a key player in cell cycle regulation, was identified as a functional target of miR-31-5p. CONCLUSIONS: Our results suggest that miR-31-5p serves as a tumor suppressor in RCC and is expected to be a molecular biomarker for poor prognosis of RCC.

Nucleolar and Spindle Associated Protein 1 (NUSAP1) Inhibits Cell Proliferation and Enhances Susceptibility to Epirubicin In Invasive Breast Cancer Cells by Regulating Cyclin D Kinase (CDK1) and DLGAP5 Expression.

BACKGROUND Differentially expressed genes (DEGs) of IBC were selected from the Gene Expression Omnibus (GEO) chip data: GSE21422 and GSE21974. Network analysis of the DEGs and IBC-related genes was performed in STRING database to find the core gene. Thus, this study aimed to determine the role of NUSAP1 in invasive breast cancer (IBC) and to investigate its effect on drug susceptibility to epirubicin (E-ADM). MATERIAL AND METHODS The mRNA expression of NUSAP1 was determined by quantitative polymerase chain reaction (q-PCR). The protein expression was detected by Western blotting. Cell growth and growth cycle were detected by MTT assay and flow cytometry, respectively. Cell migration and invasion were tested by Transwell assay. RESULTS Through use of gene network analysis, we found that NUSAP1 interacts with IBC-related genes. NUSAP1 presented high expression in IBC tissue samples and MCF-7 cells. NUSAP1 overexpression promoted the growth, migration, and invasion of MCF-7 cells. While NUSAP1 gene silencing downregulated the expression of genes associated with cell cycle progression in G2/M phase, cyclin D kinase (CDK1) and DLGAP5 arrested cells in G2/M phase and significantly inhibited the growth, migration, and invasion of MCF-7 cells. si-NUSAP1 increased the susceptibility of MCF-7 cells to E-ADM-induced apoptosis. CONCLUSIONS Our study provides evidence that downregulation of NUSAP1 can inhibit the proliferation, migration, and invasion of IBC cells by regulating CDK1 and DLGAP5 expression and enhances the drug susceptibility to E-ADM.

Translatome analysis at the egg-to-embryo transition in sea urchin.

Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis.

Cyclin-dependent kinase 1 targeting improves sensitivity to radiation in BRAF V600E colorectal carcinoma cells.

OBJECTIVES: Preoperative chemoradiation is currently the standard of care in locally advanced rectal carcinoma, even though a subset of rectal tumors does not achieve major clinically meaningful responses upon neoadjuvant chemoradiation. At present, no molecular biomarkers are available to predict response to neoadjuvant chemoradiation and select resistant tumors willing more intense therapeutic strategies. Thus, BRAF mutational status was investigated for its role in favoring resistance to radiation in colorectal carcinoma cell lines and cyclin-dependent kinase 1 as a target to improve radiosensitivity in BRAF V600E colorectal tumor cells. METHODS: Colony-forming assay and apoptotic rates were evaluated to compare the sensitivity of different colon carcinoma cell lines to ionizing radiation and their radiosensitivity upon exposure to BRAF and/or cyclin-dependent kinase 1 inhibitory/silencing strategies. Cyclin-dependent kinase 1 expression/subcellular distribution was studied by immunoblot analysis. RESULTS: Colon carcinoma BRAF V600E HT29 cells exhibited poor response to radiation compared to BRAF wild-type COLO320 and HCT116 cells. Interestingly, neither radiosensitizing doses of 5-fluoruracil nor BRAF inhibition/silencing significantly improved radiosensitivity in HT29 cells. Of note, poor response to radiation correlated with upregulation/relocation of cyclin-dependent kinase 1 in mitochondria. Consistently, cyclin-dependent kinase 1 inhibition/silencing as well as its targeting, through inhibition of HSP90 quality control pathway, significantly inhibited the clonogenic ability and increased apoptotic rates in HT29 cells upon exposure to radiation. CONCLUSION: These data suggest that BRAF V600E colorectal carcinoma cells are poorly responsive to radiation, and cyclin-dependent kinase 1 represents a target to improve radiosensitivity in BRAF V600E colorectal tumor cells.

The switch from cAMP-independent to cAMP-dependent arrest of meiotic prophase is associated with coordinated GPR3 and CDK1 expression in mouse oocytes.

Mammalian oocytes are arrested in meiotic prophase from around the time of birth until just before ovulation. Following an extended period of growth, they are stimulated to mature to the metaphase II stage by a preovulatory luteinizing hormone (LH) surge that occurs with each reproductive cycle. Small, growing oocytes are not competent to mature into fertilizable eggs because they do not possess adequate amounts of cell cycle regulatory proteins, particularly cyclin-dependent kinase 1 (CDK1). As oocytes grow, they synthesize CDK1 and acquire the ability to mature. After oocytes achieve meiotic competence, meiotic arrest at the prophase stage is dependent on high levels of cAMP that are generated in the oocyte under the control of the constitutively active Gs-coupled receptor, GPR3. In this study, we examined the switch between GPR3-independent and GPR3-dependent meiotic arrest. We found that the ability of oocytes to mature, as well as oocyte CDK1 levels, were dependent on follicle size, but CDK1 expression in oocytes from preantral follicles was not acutely altered by the activity of follicle stimulating hormone (FSH). Gpr3 was expressed and active in incompetent oocytes within early stage follicles, well before cAMP is required to maintain meiotic arrest. Oocytes from Gpr3-/- mice were less competent to mature than oocytes from Gpr3+/+ mice, as assessed by the time course of germinal vesicle breakdown. Correspondingly, Gpr3-/- oocytes contained significantly lower CDK1 levels than their Gpr3+/+ counterparts that were at the same stage of follicle development. These results demonstrate that GPR3 potentiates meiotic competence, most likely by raising cAMP.

Platelet rich plasma releasate promotes proliferation of skeletal muscle cells in association with upregulation of PCNA, cyclins and cyclin dependent kinases.

Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The purpose of this study is to investigate the effect and molecular mechanism of PRP releasate on proliferation of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP releasate. Cell proliferation was evaluated by 3-[4,5-Dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and immunocytochemistry with Ki-67 stain. Flow cytometric analysis was used to evaluate the cell cycle progression. Western blot analysis was used to evaluate the protein expressions of PCNA, cyclin E1, cyclin A2, cyclin B1, cyclin dependent kinase (cdk)1 and cdk2. The results revealed that PRP releasate enhanced proliferation of skeletal muscle cells by shifting cells from G1 phase to S phase and G2/M phases. Ki-67 stain revealed the increase of proliferative capability after PRP releasate treatment. Protein expressions including cyclin A2, cyclin B1, cdk1, cdk2 and PCNA were up-regulated by PRP releasate in a dose-dependent manner. It was concluded that PRP releasate promoted proliferation of skeletal muscle cells in association with the up-regulated protein expressions of PCNA, cyclin A2, cyclin B1, cdk1 and cdk2.

Elevated E2F7 expression predicts poor prognosis in human patients with gliomas.

E2F transcription factors have been studied extensively in a broad range of organisms as major regulators of cell cycle, apoptosis, and differentiation. The E2F family includes the atypical member E2F7, which has been rarely studied in gliomas. The aim of this study is to determine the expression status of E2F7 in gliomas, its relationship to clinicopathological features, and patients' outcome. The mRNA levels of E2F7 in the human brain and different grades of gliomas were analysed using datasets from the publically available Oncomine database. One of the most significant co-expression factors, CDK1, together with E2F7, was further validated by immunohistochemistry in 90 different grades of gliomas. Furthermore, univariate and multivariate analyses were performed to identify prognostic variables relative to patient and tumour characteristics and treatment modalities. E2F7 mRNA expression was found to be elevated in gliomas by Oncomine-database analysis. Immunohistochemistry showed an increase in E2F7 labelling index in high- versus low-grade gliomas (62.1±11.8% vs. 18.9±10.2%, p<0.0001). There was a positive correlation between E2F7 and CDK1 immunoreactivity (Spearman r=0.446, p=0.037). Clinicopathological evaluation suggested that E2F7 expression was associated with tumour grade (p<0.0001) and recurrence (p=0.025). In Cox multivariate analysis, pathological classification and recurrence were independent prognostic factors of gliomas, and E2F7 was significantly related to progression-free survival (p=0.011), but not overall survival (p=0.062). Our findings suggested that E2F7 might act as an independent prognostic factor of gliomas and might constitute a potential therapeutic target for this disease.

Chelidonine induces mitotic slippage and apoptotic-like death in SGC-7901 human gastric carcinoma cells.

The aim of the present study was to investigate the effect of chelidonine on mitotic slippage and apoptotic-like death in SGC-7901 human gastric cancer cells. The MTT assay was performed to detect the antiproliferative effect of chelidonine. Following treatment with chelidonine (10 µmol/l), the ultrastructure changes in SGC-7901, MCF-7 and HepG2 cells were observed by transmission electron microscopy. The effects of chelidonine on G2/M phase arrest and apoptosis of SGC-7901 cells were determined by flow cytometry. Indirect immunofluorescence assay and laser scanning confocal microscopy (LSCM) were used to detect the phosphorylation level of histone H3 (Ser10) and microtubule formation was detected using LSCM following immunofluorescent labeling. Subsequent to treatment with chelidonine (10 µmol/l), expression levels of mitotic slippage-associated proteins, including BUB1 mitotic checkpoint serine/threonine kinase B (BubR1), cyclin-dependent kinase 1 (Cdk1) and cyclin B1, and apoptosis-associated protein, caspase-3 were examined by western blotting at 24, 48 and 72 h. The half maximal inhibitory concentration of chelidonine was 23.13 µmol/l over 48 h and chelidonine induced G2/M phase arrest of cells. The phosphorylation of histone H3 at Ser10 was significantly increased following treatment with chelidonine for 24 h, indicating that chelidonine arrested the SGC-7901 cells in the M phase. Chelidonine inhibited microtubule polymerization, destroyed microtubule structures and induced cell cycle arrest in the M phase. Giant cells were observed with multiple micronuclei of varying sizes, which indicated that following a prolonged arrest in the M phase, the cells underwent mitotic catastrophe. Western blotting demonstrated that the protein expression levels of BubR1, cyclin B1 and Cdk1 decreased significantly between 48 and 72 h. Low expression levels of BubR1 and inactivation of the cyclin B1-Cdk1 complex results in the cells being arrested at mitosis and leads to mitotic slippage. In addition, apoptotic morphological changes in multinucleated cells were observed, the apoptosis rates increased gradually with administration of chelidonine in a time-dependent manner and the protein levels of caspase-3 increased significantly between 24 and 72 h. Thus, chelidonine induces mitotic slippage, and apoptotic-like death occurs in SGC-7901 cells undergoing mitotic catastrophe. Gastric cancer is a common malignancy, and ranks second in overall cancer-associated mortalities worldwide. The present study demonstrated that chelidonine induces M phase arrest and mitotic slippage of SGC-7901 human gastric carcinoma cells via downregulating the expression of BubR1, Cdk1 and cyclin B1 proteins. With the prolongation of chelidonine treatment, the giant cells with multiple micronuclei underwent mitotic slippage and were maintained in the G1 phase and did not survive. A number of multinucleated cells underwent apoptosis via a caspase-dependent signaling pathway. The current study proposes that chelidonine induces mitotic slippage and apoptotic-like death of SGC-7901 cells.

Ndd1 turnover by SCF(Grr1) is inhibited by the DNA damage checkpoint in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage. Here, we show that Ndd1 is also subject to regulation by protein turnover during the mitotic cell cycle: Ndd1 is unstable during an unperturbed cell cycle, but is strongly stabilized in response to DNA damage. We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCF(Grr1). In response to DNA damage, Ndd1 stabilization requires the checkpoint kinases Mec1/Tel1 and Swe1, the S. cerevisiae homolog of the Wee1 kinase. In both humans and yeast, the checkpoint promotes Wee1-dependent inhibitory phosphorylation of Cdk1 following exposure to DNA damage. While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood. We propose that the DNA damage checkpoint stabilizes Ndd1 by inhibiting Cdk1, which we show is required for targeting Ndd1 for destruction.

A genetic interactome of the let-7 microRNA in C. elegans.

The heterochronic pathway controls temporal patterning during Caenorhabditis elegans larval development. The highly conserved let-7 microRNA (miRNA) plays a key role in this pathway, directing the larval-to-adult (L/A) transition. Hence, knowledge of the genetic interactome of let-7 has the potential to provide insight into both control of temporal cell fates and mechanisms of regulation and function of miRNAs. Here, we report the results of a genome-wide, RNAi-based screen for suppressors of let-7 mutant vulval bursting. The 201 genetic interaction partners of let-7 thus identified include genes that promote target silencing activity of let-7, seam cell differentiation, or both. We illustrate the suitability of our approach by uncovering the mitotic cyclin-dependent kinase CDK-1 as a downstream effector of let-7 that affects both seam cell proliferation and differentiation, and by identifying a core set of candidate modulators of let-7 activity, which includes all subunits of the condensin II complex. We propose that the genes identified in our screen thus constitute a valuable resource for studies of the heterochronic pathway and miRNAs.

The clinical significance of CDK1 expression in oral squamous cell carcinoma.

OBJECTIVES: To evaluate the clinical significance of cyclin-dependent kinase 1 (CDK1) in 77 oral squamous cell carcinomas (OSCC) using immunohistochemical methods. STUDY DESIGN: Immunohistochemical expression of CDK1 was compared with various clinicopathological features in 77 OSCC and 60 controlled epithelia adjacent to the tumours. In addition, correlation of CDK1 expression and prognostic and the 5-year accumulative survival rate of OSCC were investigated. RESULTS: The CDK1 protein was expressed in 52 cases of 77 tumor tissues (67.5%), compared with 21 cases of 60 controlled (35.0%). The expression of CDK1 was significantly correlated with the histological grade of OSCC (P<0.05). The CDK1 protein was over-expressed in recurrent tumors or in those with lymph node metastasis. Statistical analysis showed a significant reduction in the 5-year accumulative survival rate in CDK1 positive cases compared with CDK1 negative cases (P<0.05). Namely, the CDK1 positive patients had poor prognosis. CONCLUSIONS: The expression of CDK1 might serve as malignant degree and prognostic markers for the survival of OSCC.

Inhibition of CDK2 promotes inducible regulatory T-cell differentiation through TGFß-Smad3 signaling pathway.

Inducible regulatory T-cells (iTReg) can be generated from CD4(+)Foxp3(-) naïve conventional T-cells by a combination of TGF-ß and T-cell receptor (TCR) signaling. It is of enormous clinical importance to identify agents that can promote the generation and differentiation of functional iTreg cells. We have established a phenotypic screening platform to identify new compounds that can promote the TGFß-mediated iTreg differentiation. We have found Kenpaullone, a potent CDK1, CDK2 and CDK5 inhibitor, as new enhancer for iTreg cell differentiation. Kenpaullone promotes iTreg cell differentiation through increased and prolonged transcription of foxp3 gene by enhancing TGFß-Smad3 signaling pathway. Thus, we have demonstrated that CDK2 is the biological target of Kenpaullone and proven that CDK2 is a novel negative regulator of iTreg cell differentiation.

Targeting glutamine transport to suppress melanoma cell growth.

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF(WT) (C8161 and WM852) and BRAF(V600E) mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF(WT) and BRAF(V600E) melanoma.