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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., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

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.

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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.

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.

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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.

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.

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.

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 G s -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., (Published by Elsevier Inc.)

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.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2014 UICC.)

Inducible regulatory T-cells (iTReg) can be generated from CD4(+)Foxp3(-) naïve conventional T-cells by a combination of TGF-β 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 TGFβ-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 TGFβ-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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin-dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that isoproterenol (ISO)-induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a nonselective β-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. Quantitative PCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO-responsive element was mapped to the proximal -100-bp sequence of the CDK1 promoter region using various 5'-flanking sequence deletion constructs. Sequence analysis of the -100-bp CDK1 minimal promoter region revealed two putative nuclear factor-Y (NF-Y) binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of the -100-bp CDK1 promoter construct but not the ISO-induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site-directed mutagenesis of the distal NF-Y binding site in the -100-bp CDK1 promoter region completely abolished both basal and ISO-induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.

Breast cancer is one of the most common malignancies in women. Approximately 15% of the patients belong to the triple-negative breast cancer (TNBC) group, and have the disadvantage of not benefiting from currently available receptor-targeted systemic therapies. Some cancers in the TNBC group harbor defects in DNA double-strand break repair by homologous recombination (HR), such as BRCA1 dysfunction, and are hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition. However, only a small fraction of the tumors are BRCA-deficient, and this restricts the therapeutic utility of the PARP inhibitor monotherapy. Cyclin-dependent kinase 1 (CDK1) is necessary not only for BRCA1-mediated S phase checkpoint activation, but also for HR, because it phosphorylates BRCA1 for the efficient formation of BRCA1 foci. In this study, we showed that the combined inhibition of CDK1 and PARP in BRCA-proficient MDA-MB-231 breast cancer cells resulted in dramatically reduced cell growth compared to PARP inhibition alone. Mechanistic investigations revealed that this sensitivity appears to be mediated by sustained DNA damage and inefficient DNA repair triggering mitochondrial-mediated apoptosis as well as autophagy. Our results suggest that CDK1 inhibition represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA‑proficient breast cancers.

Ginsenosides, extracted from the traditional Chinese herb ginseng, are a series of novel natural anticancer products known for their favorable safety and efficacy profiles. The present study aimed to investigate the cytotoxicity of ginsenoside Rf to human osteosarcoma cells and to explore the anticancer molecular mechanisms of ginsenoside Rf. Five human osteosarcoma cell lines (MG-63, OS732, U-2OS, HOS and SAOS-2) were employed to investigate the cytotoxicity of ginsenoside Rf by MTT and colony forming assays. After treatment with ginsenoside Rf, MG-63 cells which were the most sensitive to ginsenoside Rf, were subjected to flow cytometry to detect cell cycle distribution and apoptosis, and nuclear morphological changes were visualized by Hoechst 33258 staining. Caspase-3, -8 and -9 activities were also evaluated. The expression of cell cycle markers including cyclin B1 and Cdk1 was detected by RT-PCR and western blotting. The expression of apoptotic genes Bcl-2 and Bax and the release of cytochrome c were also examined by western blotting. Change in the mitochondrial membrane potential was observed by JC-1 staining in situ. Our results demonstrated that the cytotoxicity of ginsenoside Rf to these human osteosarcoma cell lines was dose-dependent, and the MG-63 cells were the most sensitive to exposure to ginsenoside Rf. Additionally, ginsenoside Rf induced G2/M phase cell cycle arrest and apoptosis in MG-63 cells. Furthermore, we observed upregulation of Bax and downregulation of Bcl-2, Cdk1 and cyclin B1, the activation of caspase-3 and -9 and the release of cytochrome c in MG-63 cells following treatment with ginsenoside Rf. Our findings demonstrated that ginsenoside Rf induces G2/M phase cell cycle arrest and apoptosis in human osteosarcoma MG-63 cells through the mitochondrial pathway, suggesting that ginsenoside Rf, as an effective natural product, may have a therapeutic effect on human osteosarcoma.

This study showed that silencing BMP4 expression significantly activated caspase-2, 3, and 9, while decreasing Matrigel colony formation in Cytarabine (Ara-C)-treated leukemia HL-60 cells. In contrast, Ara-C significantly upregulated Atg5 and Beclin-1 expression, the ratio of LC3-II/LC3-I, and CDK1 and cyclin B1 expression in leukemia cells expressing BMP4. BafA significantly sensitized the apoptotic effect of Ara-C in leukemia cells. Injection of Ara-C significantly inhibited tumor growth in mice inoculated with leukemia cells with BMP4 silenced. In conclusion, BMP4 plays a crucial role in the chemoresistance of leukemia cells through the activation of autophagy and subsequent inhibition of apoptosis.

Tribbles-related protein 3 (TRB3) has been shown to be a crucial modulator of tumorigenesis. However, the precise role and the functional morphology of TRB3 are not clearly understood. To elucidate these enigmas we established the cell line, M2TRB3, by introducing the human TRB3 gene and protein in Cl66M2 (M2) mouse mammary tumor cells. This cell line stably expressed the TRB3 gene and protein. After 72 h of cell culture, there was a 34% increase in the growth of M2TRB3 cells compared to the control M2 mock cells. The mean volume of the tumors originating from the M2TRB3 cells was significantly increased by 38% when compared to the mean volume of the M2 mock tumors, and the proliferating cell nuclear antigen (PCNA) labeling index in the M2TRB3 tumors was higher when compared to that of the M2 and M2 mock cells. In the tumor tissue samples, the mean diameter of nuclei in the M2TRB3 tumor cells (9.4±0.3 µm) showed a significant increase compared to that of the M2 mock tumor cells (7.0±0.2 µm). M2TRB3 cells also showed a marked increase in the population of tetraploid or octaploid nuclei compared to M2 mock cells bearing mainly either diploid or tetraploid nuclei. Western blot analysis revealed the overexpression of cyclin B1 and cyclin D1 in M2TRB3 cells when compared to that in the M2 mock cells. These novel findings provide further evidence that TRB3 promotes cell proliferation and chromosomal instability by causing polyploidization during development.

Incidence of hepatocellular carcinoma (HCC) is dramatically increasing and is the third cause of cancer death worldwide. One key approach to control HCC is chemoprevention by naturally occurring agents. This study aims at investigating the antitumor effect of oleanolic acid (OA) and the molecular mechanisms. BALB/c mice were injected subcutaneously with HepG2 cells to establish transplanted tumors. Apoptosis and cell cycle arrest-related markers and signaling cascades were determined by western blot, immunofluorescence, reverse transcriptase-polymerase chain reaction and flow cytometric analysis. OA exhibited inhibitory effect on HCC through induction of apoptosis and cell cycle arrest both in transplanted tumors and in HepG2 cells. OA induced apoptosis through mitochondrial pathway, evidenced by inhibition of Akt/mammalian target of rapamycin pathway, mitochondrial dysfunction, transient increase of adenosine triphosphate, increase of Bax/Bcl-2 ratio, increased release of cytochrome c and activation of caspase/poly (ADP-ribose) polymerase. Activation of mitochondrial apoptotic pathway may be due to reactive oxygen species generated by mitochondrial fatty acid oxidation, resulted from enhancement of lipolysis regulated by cyclic adenosine 3',5'-monophosphate response element-binding protein-hormone-sensitive lipase/peroxisome proliferator-activated receptor γ signaling. OA induced G2/M cell cycle arrest through p21-mediated downregulation of cyclin B1/cdc2. Cyclooxygenase-2 (COX-2) and p53 were involved in OA-exerted effect, and extracellular signal-regulated kinase-p53 signaling played a central role in OA-activated cascades responsible for apoptosis and cell cycle arrest. OA demonstrated significant antitumor activities in HCC in vivo and in vitro models. These data provide new insights into the mechanisms underlying the antitumor effect of OA.

Objectives: Lycoris is aurea agglutinin (LAA) has attracted rising attention due to its remarkable bioactivities. Here, we aimed at investigating its anti-tumor activities., Material and Methods: In vitro methods including MTT, cellular morphology observation, FCM and immunoblotting were performed. In vivo methods like detection of tumor volume, body weight and survival ratio, as well as TUNEL staining were performed., Results and Conclusion: LAA triggers G2 /M phase cell cycle arrest via up-regulating p21expression as well as down-regulating cdk-1cyclinA singling pathway, and induces apoptotic cell death through inhibiting PI3K-Akt survival pathway in human lung adenocarcinoma A549 cells. While LAA has no significant cytotoxic effect toward normal human embryonic lung fibroblast HELF cells, and moreover, LAA could amplify the antineoplastic effects of cisplatin toward A549 cells. Lastly LAA also bears anti-cancer and apoptosis-inducing effects in vivo, and it could decrease the volume and weight of subcutaneous tumor mass obviously as well as expand lifespan of mice. These findings may provide a new perspective for elucidating the complicated molecular mechanisms of LAA-induced cancer cell growth-inhibition and death, providing a new opportunity of LAA as a potential candidate anti-neoplastic drug for future cancer therapeutics., (© 2013 Blackwell Publishing Ltd.)

In the present study, in order to investigate the anticancer effects of O-desmethylangolensin (O-DMA) on human hepatocellular carcinoma Hep3B cells, we first examined the antiproliferative effect of O-DMA. When Hep3B cells were treated with O-DMA at various concentrations (5-200 µM) for 24, 48 or 72 h, cell proliferation decreased significantly in a dose- and time-dependent manner. Moreover, O-DMA exposure at the IC50 concentration for 72 h arrested cells at the G2/M phase, which was accompanied by a reduction in CDK1, and an increase in cyclin A and B. Under the same conditions, O-DMA significantly increased the number of sub-G1 phase cells. Additionally, an Annexin V assay revealed that exposure to O-DMA affected the rate of cell apoptosis. O-DMA caused the downregulation of Bcl-2 and upregulation of Bax, which led to cytochrome c release from the mitochondria and activation of caspase-3. Taken together, these data suggest that O-DMA exhibits anticancer activity by arresting the cell cycle at G2/M phase and causing mitochondrial-dependent apoptosis in Hep3B cells.

Objectives: A number of factors involved in the control of energy balance and metabolism act as modulators of gonadal axis. Ghrelin, a peptide secreted from the stomach and hypothalamus, has emerged as an orexigenic food intake controlling signal acting upon hypothalamus. Recently, the potential reproductive role of ghrelin has received great attention. This study was designed to investigate the influence of food restriction and consequent metabolic hormone (ghrelin) on the level and gene expression of female reproductive hormones in adult rats., Materials and Methods: To study the effect of chronic food restriction on ghrelin level in adult female rats and its relation to female reproductive hormones, 32 adult female Sprague Dawley rats divided into 4 groups: Group I (control group) comprised 8 rats fed ad libitum for 30 days, Group II, III and IV (food-restricted groups for 10, 20 and 30 days respectively) each consisted of 8 rats fed 50% of ad libitum intake determined by the amount of food consumed by the control group., Results: Mean body weight of food restricted rats was observed to decrease during the period of the experiment. Food restriction produced significant increase of serum ghrelin with significant decrease of both gastric and hypothalamic ghrelin accompanied with significant increase in its gene expression in stomach and hypothalamus. Estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels showed significant decrease correlated with down-regulation of gonadotropins, cyclin-dependent kinase (cdc2), cyclin B and kisspeptin (Kiss1) genes in food restricted rats compared with control group., Conclusions: Ghrelin could be one of the hormones responsible for the suppression of female reproductive axis in case of negative energy balance. Thus, ghrelin may operate as an autocrine/paracrine regulator of ovarian function. Overall, ghrelin may represent an additional link between body weight homeostasis and reproductive function.

The duration of S phase in early embryos is often short, and then increases as development proceeds because of the appearance of late-replicating regions of the genome. In the April 1, 2012, issue of Genes & Development, Farrell and colleagues (pp. 714-725) demonstrate that the down-regulation of cyclin-dependent kinase 1 (Cdk1) activity triggers the onset of late-replicating DNA and an increase in S-phase length in Drosophila embryos, revealing an unexpected role for Cdk1 in replication control during development.

Nearly half of human cancers harbor p53 mutations, which can promote cancerous growth, metastasis, and resistance to therapy. The gain of function of mutant p53 is partly mediated by its ability to form a complex with NF-Y or p63/p73. Here, we demonstrate that TopBP1 mediates these activities in cancer, and we provide both in vitro and in vivo evidence to support its role. We show that TopBP1 interacts with p53 hot spot mutants and NF-YA and promotes mutant p53 and p300 recruitment to NF-Y target gene promoters. TopBP1 also facilitates mutant p53 interaction with and inhibition of the transcriptional activities of p63/p73. Depletion of TopBP1 in mutant p53 cancer cells leads to downregulation of NF-Y target genes cyclin A and Cdk1 and upregulation of p63/p73 target genes such as Bax and Noxa. Mutant p53-mediated resistance to chemotherapeutic agents depends on TopBP1. The growth-promoting activity of mutant p53 in a xenograft model also requires TopBP1. Thus, TopBP1 mediates mutant p53 gain of function in cancer. Since TopBP1 is often overexpressed in cancer cells and is recruited to cooperate with mutant p53 for tumor progression, TopBP1/mutant p53 interaction may be a new therapeutic target in cancer.

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti-cancer treatment have been sought from natural resources. Here, we have investigated anti-proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells., Material and Methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate levels were analysed by HPLC., Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G(2) /M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21(waf1/cip1) was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate in myriocin-treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells., Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21(waf1/cip1) , followed by inhibition of cyclin B1 and cdc2, resulting in G(2) /M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism-based therapy for this type of skin cancer., (© 2011 Blackwell Publishing Ltd.)

Ethnopharmacological Relevance: Tian-Xian liquid (TXL), a commercially available Chinese medicine decoction, has been used as an anticancer dietary agent for more than 10 years without reported side effects., Aim of the Study: The safety and quality consistency of TXL and its mechanisms of action on antiproliferation, antimetastasis, and reversion of multidrug resistance (MDR) regimens were explored., Materials and Methods: In this study, an atomic absorption spectrophotometer and reversed phase high performance liquid chromatography with photodiode array detection (HPLC-DAD) were used to evaluate the main toxic elements and the quality consistency among different batches of TXL extracts, respectively. HT29 human colon cancer cell line and tumor-bearing nude mice were used. TXL was provided by China-Japan Feida Union Company Limited. The effect of TXL on in vitro proliferation of HT29 human colon cancer cell line was examined. The percentages of treated cells distributed in different phases of the cell cycles were analyzed by flow cytometry. Antiproliferative effect after treatment with TXL was assessed by determination of the protein levels of p21, cyclinD1, PCNA, and cdk-2, which are the key regulators for cell cycle progression. Meanwhile, the protein levels of MMP-1 and MDR-1 (multidrug resistance protein-1) were also determined to assess the effect of TXL on antimetastasis and reversion of MDR regimen, respectively., Results: The contents of main toxic elements were lower in TXL extract compared with the standard set by the Department of Health of the Government of Hong Kong Special Administrative Region (SAR). Our HPLC results showed that the relative standard deviations of the amount of the 5 standards were less than 5% in different batches of TXL. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of cyclinD1, PCNA, and cdk-2 in the TXL-treated in vitro models, thereby, impeding cell progression from G1/S phase. Results obtained from the in vivo study also demonstrated that TXL upregulated the protein level of p21 and downregulated the protein levels of MMP-1 and MDR-1., Conclusions: Results obtained from the present investigation not only demonstrate the safety and quality of TXL extract but also demonstrate that TXL possesses antiproliferative and antimetastatic activities and brings about reversion of MDR on HT29 cell and on xenografted tissue in tumor-implanted nude mice., (© The Author(s) 2011)

Curcuma aromatica is a common Chinese herb for treating diseases with blood stasis and has been regarded as an anticancer herb in modern clinical practice. However, the anticancer effects and related molecular mechanisms of Curcuma aromatica remain unclear. In the present study, human colon carcinoma LS-174-T cell line with wild-type p53 was used as a model cell to evaluate the anticancer effects of aqueous extract of Curcuma aromatica (AECA). AECA inhibits LS-174-T cell proliferation in a dose- and time-dependent manner and colony formation in a dose-dependent manner. AECA treatment induces apoptosis accompanied by caspase-8, -9, and -3 activation in LS-174-T cells. Moreover, blocking the activities of these caspases with a specific inhibitor significantly protected LS-174-T cells from AECA-induced apoptosis. AECA treatment also induces G2/M phase arrest in LS-174-T cells. Expression of p53 was unchanged after AECA treatment; specific silence of p53 did not influence AECA-induced apoptosis and G2/M phase arrest. Further, the expression of cyclin B1 and CDK1 was reduced by AECA. This study suggests that AECA might be effective as an antiproliferative herb for colon carcinoma, the antitumor activity of AECA may involve both extrinsic and intrinsic apoptosis, and AECA induces G2/M phase arrest via downregulation of cyclin B1 and CDK1 and without the participation of p53.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.

Background: Hepatocellular carcinoma (HCC) still is a big burden for China. In recent years, the third-generation platinum compounds have been proposed as potential active agents for HCC. However, more experimental and clinical data are warranted to support the proposal. In the present study, the effect of lobaplatin was assessed in five HCC cell lines and the underlying molecular mechanisms in terms of cell cycle kinetics were explored., Methods: Cytotoxicity of lobaplatin to human HCC cell lines was examined using MTT cell proliferation assay. Cell cycle distribution was determined by flow cytometry. Expression of cell cycle-regulated genes was examined at both the mRNA (RT-PCR) and protein (Western blot) levels. The phosphorylation status of cyclin-dependent kinases (CDKs) and retinoblastoma (Rb) protein was also examined using Western blot analysis., Results: Lobaplatin inhibited proliferation of human HCC cells in a dose-dependent manner. For the most sensitive SMMC-7721 cells, lobaplatin arrested cell cycle progression in G1 and G2/M phases time-dependently which might be associated with the down-regulation of cyclin B, CDK1, CDC25C, phosphorylated CDK1 (pCDK1), pCDK4, Rb, E2F, and pRb, and the up-regulation of p53, p21, and p27., Conclusion: Cytotoxicity of lobaplatin in human HCC cells might be due to its ability to arrest cell cycle progression which would contribute to the potential use of lobaplatin for the management of HCC.

Objectives: To determine if SAHA, a histone deacetylase inhibitor, decreases ovarian cancer cell viability when combined with paclitaxel in vitro, and to explore molecular alterations of combined paclitaxel+SAHA treatment., Methods: SKOV3 and Hey ovarian cancer cell lines were treated for 24 h with paclitaxel, then re-treated with SAHA or paclitaxel for an additional 48 h. Protein extracts were prepared at 48 h for western blot analysis. Cell viability was assessed at 72 h using the ApoAlert Annexin V Apoptosis Kit., Results: SAHA causes G1 and G2 cell cycle arrest in ovarian cancer cell lines. Cell viability was significantly reduced by combined paclitaxel+SAHA treatment. In Hey cells, viability was reduced to 67% with paclitaxel, and to 48% with paclitaxel+SAHA (p<0.001). In the SKOV3 cell line, viability was reduced to 70% with continuous paclitaxel treatment, and was further reduced to 57% in the combined treatment group (p<0.05). Increased PARP cleavage was noted in the paclitaxel+SAHA groups. SAHA increased expression of p21cip1/waf1 and p27Kip1, down regulated cyclins A and B, and suppressed CDK1. Paclitaxel induced expression of survivin, an inhibitor of apoptosis protein, was reduced to baseline control levels with the addition of SAHA. The pro-apoptotic protein, Bad, was also increased with SAHA., Conclusions: Paclitaxel+SAHA reduces cell viability in excess of either agent alone in ovarian cancer cell lines. Cell death is mediated via several mechanisms including G1/G2 arrest from CDK1 downregulation, inhibition of paclitaxel-induced survivin accumulation, and from increased Bad expression.

Here we review some of our work over the last decade on Xenopus oocyte maturation, a cell fate switch, and the Xenopus embryonic cell cycle, a highly dynamical process. Our approach has been to start with wiring diagrams for the regulatory networks that underpin the processes; carry out quantitative experiments to describe the response functions for individual legs of the networks; and then construct simple analytical models based on chemical kinetic theory and the graphical rate-balance formalism. These studies support the view that the all-or-none, irreversible nature of oocyte maturation arises from a saddle-node bifurcation in the regulatory system that drives the process, and that the clock-like oscillations of the embryo are built upon a hysteretic switch with two saddle-node bifurcations. We believe that this type of reductionistic systems biology holds great promise for understanding complicated biochemical processes in simpler terms.

Background and Purpose: Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death., Experimental Approach: Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry., Key Results: Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G(1) arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis., Conclusion and Implications: The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G(1) phase cell cycle arrest, coupled with induction of apoptosis.

Tocotrienols, a subgroup within the vitamin E family of compounds, have shown antiproliferative and anticancer properties, however, the molecular basis of these effects remains to be elucidated. In this study, the effect of 3-tocotrienol on cell cycle arrest was assessed by studying the retinoblastoma protein (Rb) levels and phosphorylation status, levels of E2F (a transcription factor critically involved in the G1/S-phase transition of the mammalian cell cycle; originally identified as a DNA-binding protein essential for early region 1A-dependent activation of the adenovirus promoter designated E2), and other cell cycle controlling proteins in estrogen receptor-negative MDA-MB-231 breast cancer cells. The cell growth assay demonstrated that exposure of the MDA-MB-231 cells to 6-tocotrienol (1-20 microM) resulted in a dose- and time-dependent inhibition of cell growth as compared with vehicle treated cells and the magnitude of growth inhibition was higher at 10 and 20 microM treatment for 48 and 72 h. The phosphorylation status of Rb plays a central role in the control of the cell cycle at the G0/G1-phase. delta-Tocotrienol treatment reduced the total Rb and its phosphorylation at the Ser780, Ser795, Ser 807/811 and Thr826 positions in a dose- and time-dependent fashion. The site-specific inhibition of the phosphorylation of Rb by delta-tocotrienol was tightly associated with a marked reduction in the expression of cyclin D1 and its regulatory partner cyclin-dependant kinase 4 (CDK4), which is responsible for the phosphorylation of Rb at Ser780, Ser795, Ser 807/811 and Thr826. In addition, delta-tocotrienol also reduced the expression of E2F that occurred simultaneously with the loss of Rb phosphorylation and inhibition of cell cycle progression. Interestingly, delta-tocotrienol also caused a marked reduction in the expression of G2/M regulatory proteins including cyclin B1 and CDK1. To the best of our knowledge, this study was the first to reveal that the target of cell proliferative inhibitory action of delta-tocotrienol in a model estrogen receptor-negative human breast cancer cell line MDA-MB-231 is mediated by the loss of cyclin D1 and associated suppression of site-specific Rb phosphorylation, suggesting its future development and use as an anticancer agent.

Suppression of Cdc2 activity is essential for DNA damage-induced G2 arrest. In the present study, we elucidated regulatory mechanism of Cdc2 activity during radiation-induced transient G2 arrest. Exposure of the cells to gamma-radiation (4 Gy) led to a transient increase of cells in G2 at 12 h rather than M phase and then the cells resumed cell cycle progression from the G2 arrest. However, the levels of cyclin B1 and Cdc2 activity were increased in the whole cell extracts at 12 h. Despite cyclin B1 induction and increased level of Cdc2 activity after irradiation the activities in the nuclear fractions were transiently decreased at 12 h and returned to control levels by 24-48 h, demonstrating transient inhibition of nuclear translocation of cyclin B1 in response to radiation. Moreover, inhibitory phosphorylation of the Cdc2 on Tyr15 and the Cdc25C on Ser216 were increased concomitant with transient G2 arrest. The level of phosphorylated Wee1 and its activity were also markedly increased at 12 h after irradiation. In addition, radiation caused nuclear accumulation of p21(CIP1/WAF1) at 12 h, resulting in increased-binding of p21(CIP1/WAF1) to Cdc2. Nuclear p21(CIP1/WAF1) protein level and its binding to Cdc2 gradually returned to control level when the cells resumed cell cycle progression. However, total protein level of p21(CIP1/WAF1) continued to increase until 48 h after irradiation. Collectively, these results indicate that the suppression of nuclear import of cyclin B1, the induction of Wee1 kinase activity, and the transient nuclear accumulation of p21(CIP1/WAF1) may play important roles in the transient cell cycle delay in response to ionizing radiation.

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotion factor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism of oocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greater at previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during early embryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and early embryogenesis except for a decline at early vitellogenesis. In situ hybridization analysis indicated that CB1, CB2 and Cdc2 transcripts were present in oocytes of different developmental stages as well as in all spermatogenic cells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein was absent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2 and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins were present throughout spermatogenesis, whereas CB1 protein was only detected in spermatogonia and spermatocytes, but not in spermatids. Thus, it appears that CB1, CB2 and Cdc2 transcripts have similar expression patterns during oogenesis and spermatogenesis, but CB1 protein varies in amount during these processes. These data suggest that CB1 may have a leading role in the regulation of meiotic maturation of oocytes and spermotocytes.

Norcantharidin (NCTD), a demethylated form of cantharidin, is currently used as an anticancer drug in China, but five newly synthesized derivatives have not been tested for antitumor efficacy. In this study, we investigated the in-vitro and in-vivo activity of five derivatives on Bel-7402, HeLa and PC-3M1E8 cell lines on a sulfarhodamine B assay. All of the derivatives showed significant antiproliferative activity, hence we elected to study further one of them, NCTD-Nd3II, in an in-vivo mouse model, and to examine its effects on cell cycle and protein expression. NCTD-Nd3II inhibited H22 tumors in mice in a dose-dependent manner with low toxicity. Flow cytometry results showed that apoptosis and G2/M cell cycle arrest contributed to the cytotoxic and cytostatic effects of NCTD-Nd3II. Further studies showed that Bax and p21 protein expression was upregulated, whereas cyclin B1, Cdc-2 and Bcl-2 protein expression was downregulated. Our findings show that NCTD-Nd3II might be a promising chemotherapeutic agent for hepatomas.

After reports on regression of cancer in humans and animals infected with microbial pathogens date back more than 100 years, much effort has been spent over the years in developing wild type or attenuated bacterial and purified bacterial proteins for the treatment of cancer. Pseudomonas aeruginosa exotoxin A (ETA) is known to inhibit cell growth and trigger significant cell death in various cancer cells. Although ETA induces apoptosis of cancer cells, its exact mechanism of action is not yet known. Four different assays were performed in this study: morphological assessment of apoptotic cells, cell cytotoxicity, cell cycle analysis and Western blot analysis. The proliferation and survival in the cells treated with ETA was decreased. In addition, percentages of apoptotic HeLa S(3) cells treated with ETA were increased. ETA-induced apoptosis rates were confirmed to have increased in a dose-dependent manner through annexin V binding assay. Flow cytometric analysis was examined to ascertain whether ETA could regulate cell cycle in HeLa S(3) cells. ETA treatment demonstrated that the expression of 14-3-3delta proteins was increased, while expression of cdc and cyclin B proteins was decreased, suggesting that ETA induces cell cycle arrest and then progresses to apoptosis. Therefore, these results suggest that P. aeruginosa ETA induced apoptosis in HeLa S(3) cells.

Background: The epidermal growth factor receptor (EGFR) is frequently overexpressed in uterine cervix carcinoma. The role of the pre-treatment EGFR expression levels and the changes of expression induced by ionizing radiation (IR) have not been conclusively defined., Patients and Methods: The staining intensity (SI) and labeling index (LI) of EGFR were determined in 38 patients by immunohistochemistry (IHC). Biopsies were taken before after 1 week of RT. EGFR expression was correlated with cell cycle, apoptosis and angiogenesis., Results: Before RT, 87% and after 1 week of RT, 95% of samples were positive for EGFR (p=0.2). Two patterns were observed, either increasing or decreasing expression after initiating RT. An increase of the EGFR SI was seen in 63% of patients from a mean of 57 SI (SD+/-60) before RT to 142 SI (SD+/-80.8) (p=0.001) during RT. In 32% of cases, EGFR decreased from 165 SI before (SD+/-83.0) to 75 SI (SD+/-73.0) (p< or =0.001) during RT. Two of five (5%) patients negative for EGFR before RT remained negative. An increase of the RT-induced EGFR LI was associated with reduced microvessel density (MVD) (p=0.02). Changes of the EGFR LI did neither correlate with cell cycle arrest nor apoptosis., Conclusions: EGFR expression changes unpredictably during RT. The implications of changing EGFR during RT remain to defined. Repeated biopsies and EGFR reassessment during RT may help to better define EGFR-targeted treatment.

Schwann cell migration facilitates peripheral nerve regeneration after injury. We have recently found increased activation of Cdc2 kinase in regenerating sciatic nerves. Here we show that Cdc2 phosphorylation of caldesmon regulates Schwann cell migration and nerve regeneration. A robust but transient increase in Cdc2 expression was found in cultured Schwann cells prepared from the sciatic nerve in rats that had undergone crush injury for 7 days. These ;injury-preconditioned' Schwann cells exhibited enhanced migration compared with non-preconditioned control cells and treatment with the cdk inhibitor roscovitine prevented cell migration. After transduction with recombinant Cdc2 DNA adenoviral vectors, Schwann cells were implanted into sciatic nerves; those expressing wild-type Cdc2 migrated further in the distal direction than those expressing dominant-negative Cdc2. We identified caldesmon as a downstream substrate of Cdc2 in Schwann cells and its phosphorylation by Cdc2 changed its subcellular localization. Overexpression of dominant-negative caldesmon significantly counteracted the migration effect caused by Cdc2. Finally, neurite outgrowth of cultured DRG sensory neurons, facilitated by co-culture with injury-preconditioned Schwann cells, was suppressed by roscovitine treatment. The results indicate that activation of the Cdc2-caldesmon pathway is necessary for Schwann cell migration and suggest a role for this pathway in peripheral axonal growth.

The cyclin-dependent kinase (Cdk)-associated protein phosphatase KAP is a dual-specificity phosphatase of which the only known function is to dephosphorylate Cdk2 and inhibit cell cycle progression. Paradoxically, we find increased KAP mRNA expression in malignant astrocytomas, which correlates with increasing histologic grade and decreased patient survival. We have resolved this apparent paradox with the discovery of aberrant KAP splicing in malignant astrocytomas that leads to increased expression of KAP-related transcripts but decreased KAP protein expression. In addition, the aberrant splicing generates a dominant negative KAP variant that increases proliferation. We provide the first evidence that KAP not only regulates proliferation but also inhibits migration by decreasing cdc2 mRNA and protein expression. The effect of KAP on cdc2 expression requires its phosphatase activity but does not involve direct dephosphorylation of cdc2. Thus, KAP regulates both cdc2-dependent migration and Cdk2-dependent proliferation, and its loss due to aberrant splicing increases malignancy in human gliomas.

The cell cycle-regulatory transcription factor E2F1 induces apoptosis of postmitotic neurons in developmental and pathological situations. E2F1 transcriptionally activates many proapoptotic genes including the cyclin-dependent protein kinase cell division cycle 2 (Cdc2). Necdin is a potent mitotic suppressor expressed predominantly in postmitotic neurons and interacts with E2F1 to suppress E2F1-mediated gene transcription. The necdin gene NDN is maternally imprinted and expressed only from the paternal allele. Deletion of the paternal NDN is implicated in the pathogenesis of Prader-Willi syndrome, a genomic imprinting-associated neurodevelopmental disorder. Here, we show that paternally expressed necdin represses E2F1-dependent cdc2 gene transcription and attenuates apoptosis of postmitotic neurons. Necdin was abundantly expressed in differentiated cerebellar granule neurons (CGNs). Neuronal activity deprivation elevated the expression of both E2F1 and Cdc2 in primary CGNs prepared from mice at postnatal day 6, whereas the necdin levels remained unchanged. In chromatin immunoprecipitation analysis, endogenous necdin was associated with the cdc2 promoter containing an E2F-binding site in activity-deprived CGNs. After activity deprivation, CGNs underwent apoptosis, which was augmented in those prepared from mice defective in the paternal Ndn allele (Ndn(+m/-p)). The levels of cdc2 mRNA, protein, and kinase activity were significantly higher in Ndn(+m/-p) CGNs than in wild-type CGNs under activity-deprived conditions. Furthermore, the populations of Cdc2-immunoreactive and apoptotic cells were increased in the cerebellum in vivo of Ndn(+m/-p) mice. These results suggest that endogenous necdin attenuates neuronal apoptosis by suppressing the E2F1-Cdc2 system.

Objective: To investigate the inhibitory effect of recombinant polypeptide CH50 of fibronectin on invasion and angiogenesis of tumors, and analyze the possible molecular mechanism of the therapeutic effect of polypeptide CH50 on tumors., Methods: The tumor model was established by inoculation of H22 hepatocarcinoma cells in mice. The tumor gene therapy was performed by in vivo gene transfection with a method based on hydrodynamics to express polypeptide CH50. After treatment, the inhibitory effect on tumor invasion and angiogenesis was observed by histotology with HE staining of tumor tissues. The expresison of MMP-9 mRNA and protein at the edge of tumor tissue was evaluated by RT-PCR and gelatin zymography, respectively. RT-PCR was used to detect the expression of the related genes in H22 cells treated with polypeptide CH50. Cell adhesion assay was used to analyze the influence of polypeptide CH50 on the binding of cells to fibrinogen., Results: (1) Eukaryotic expression plasmid pCH510 was expressed in vivo in a non-targeting manner and produced a significant inhibitory effect on tumor growth. The therapy with polypeptide CH50 resulted in pronounced necrosis of tumor cells in pCH510 group, compared with that in control groups at histological level. (2) Polypeptide CH50 could inhibit the growth, invasion and angiogenesis of the tumor, and interfere the formation of new collateral circulation in the tumor. (3) The expression level of MMP-9 protein at the edge of tumor tissue was significantly decreased after treatment, especially the activation of pro-MMP-9 was inhibited significantly, whereas the expression level of MMP-9 mRNA was not influenced. (4) The expression of alphav, 33 and cdc2 mRNAs in H22 cells treated with polypeptide CH50 was down-regulated. (5) Cell adhesion assay manifested that polypeptide CH50 can affect the adhesion ability of H22 cells., Conclusion: Polypeptide CH50 can inhibit tumor growth and angiogenesis by suppressing the functions of MMP-9 and integrin alphavbeta3.

It has been proposed that cannabinoids are involved in the control of cell fate. Thus, these compounds can modulate proliferation, differentiation, and survival in different manners depending on the cell type and its physiopathologic context. However, little is known about the effect of cannabinoids on the cell cycle, the main process controlling cell fate. Here, we show that Delta(9)-tetrahydrocannabinol (THC), through activation of CB(2) cannabinoid receptors, reduces human breast cancer cell proliferation by blocking the progression of the cell cycle and by inducing apoptosis. In particular, THC arrests cells in G(2)-M via down-regulation of Cdc2, as suggested by the decreased sensitivity to THC acquired by Cdc2-overexpressing cells. Of interest, the proliferation pattern of normal human mammary epithelial cells was much less affected by THC. We also analyzed by real-time quantitative PCR the expression of CB(1) and CB(2) cannabinoid receptors in a series of human breast tumor and nontumor samples. We found a correlation between CB(2) expression and histologic grade of the tumors. There was also an association between CB(2) expression and other markers of prognostic and predictive value, such as estrogen receptor, progesterone receptor, and ERBB2/HER-2 oncogene. Importantly, no significant CB(2) expression was detected in nontumor breast tissue. Taken together, these data might set the bases for a cannabinoid therapy for the management of breast cancer.