FSH1 overexpression triggers apoptosis in Saccharomyces cerevisiae.

FSH1 belongs to the family of serine hydrolases in yeast and is homologous to the human ovarian tumor suppressor gene (OVAC2). Our preliminary results showed that cells lacking Fsh1p exhibit an increase in cell growth, and a decrease in the expression of AIF1 and NUC1 (apoptosis responsive genes) when compared to the wild type cells. Growth inhibition of cells overexpressing FSH1 is due to induction of cell death associated with cell death markers typical of mammalian apoptosis namely DNA fragmentation, phosphatidylserine externalization, ROS accumulation, Cytochrome c release, and altered mitochondrial membrane potential. When wild type cells were overexpressed with FSH1 there was up regulation of AIF1 level when compared to control cells suggesting that overexpression of FSH1 regulated cell death in yeast.

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression.

Long noncoding RNAs (lncRNAs) are involved in many aspects of cellular processes, including the antiviral immune response. To identify influenza A virus (IAV)-related lncRNAs, we performed RNA deep sequencing to compare the profiles of lncRNAs in A549 and HEK293T cells with or without IAV infection. We identified an IAV-upregulated lncRNA named lnc-ISG20 because it shares most of its sequence with ISG20. We found that lnc-ISG20 is an interferon-stimulated gene similar to ISG20. Overexpression of lnc-ISG20 inhibited IAV replication, while lnc-ISG20 knockdown favored viral replication, suggesting that lnc-ISG20 is inhibitory to IAV replication. Further study indicated that overexpression of lnc-ISG20 enhances ISG20 protein levels, while knockdown of lnc-ISG20 reduces ISG20 protein levels in A549 cells induced with poly(I·C) and Sendai virus. We demonstrated that lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. As lnc-ISG20 did not affect the mRNA level of ISG20, we postulated that lnc-ISG20 may function as endogenous RNA competing with ISG20 to enhance its translation. Indeed, we identified that microRNA 326 (miR-326) is a mutual microRNA for both ISG20 and lnc-ISG20 that targets the 3' untranslated region of ISG20 mRNA to inhibit its translation. We confirmed that lnc-ISG20 can bind miR-326, which in turn decreased the amount of miR-326 bound to ISG20 mRNA. In conclusion, we identified that the IAV-upregulated lnc-ISG20 is a novel interferon-stimulated gene that elicits its inhibitory effect on IAV replication by enhancing ISG20 expression. We demonstrated that lnc-ISG20 functions as a competitive endogenous RNA to bind miR-326 to reduce its inhibition of ISG20 translation. Our results revealed the mechanism by which lnc-ISG20 inhibits IAV replication.IMPORTANCE The replication of influenza A virus is regulated by host factors. However, the mechanisms by which lncRNAs regulate IAV infection are not well understood. We identified that lnc-ISG20 is upregulated during IAV infection and is also an interferon-stimulated gene. We demonstrated that lnc-ISG20 can enhance ISG20 expression, which in turn inhibits IAV replication. Our studies indicate that lnc-ISG20 functions as a competing endogenous RNA that binds miR-326 and reduces its inhibitory effect on ISG20. Taken together, our findings reveal the mechanistic details of lnc-ISG20 negatively regulating IAV replication. These findings indicate that lnc-ISG20 plays an important role during the host antiviral immune response.

Realizing directional cloning using sticky ends produced by 3'-5' exonuclease of Klenow fragment.

The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5'-3' polymerase activity can extend the 5' overhanging sticky end to the blunt end, and 3'-5' exonuclease activity can cleave the 3' overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3'-5' exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5' overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.

14-3-3σ mediates G2-M arrest produced by 5-aza-2'-deoxycytidine and possesses a tumor suppressor role in endometrial carcinoma cells.

OBJECTIVES: To determine the effect of 5-aza-2'-deoxycytidine (DAC) on human endometrial carcinoma cell (HECC) oncogenicity and demonstrate a molecular mechanism by which DAC modulates HECC oncogenicity. METHODS: The effect of DAC was tested on HECC RL95-2, AN3, Ishikawa and ECC1 cells. The role of 14-3-3σ on HECC oncogenicity in response to DAC treatment was evaluated in RL95-2 and AN3 cells after forced expression or silencing of 14-3-3σ gene expression. RESULTS: Treatment of HECC with DAC produced non-cytotoxic cell growth inhibition and G2/M cell cycle arrest. This effect was strongly correlated with increased expression of p21 and 14-3-3σ. Silencing of 14-3-3σ induced cellular proliferation and reduced the effect of DAC on cell cycle arrest in G2/M phases. Conversely, forced expression of 14-3-3σ showed the opposite effect. Furthermore, forced expression of 14-3-3σ in human endometrial cell lines reduced cell growth and colony formation. CONCLUSIONS: We suggest that 14-3-3σ in HECC suppresses cell proliferation and mediates DAC induced G2/M arrest and inhibition of cell proliferation in HECC.

Aberrant upregulation of 14-3-3Æ¡ expression serves as an inferior prognostic biomarker for gastric cancer.

BACKGROUND: 14-3-3Æ¡ is an intracellular, phosphoserine binding protein and proposed to be involved in tumorigenesis. However, the expression dynamics of 14-3-3Æ¡ and its clinicopathological/prognostic significance in human tumors are still controversial. METHODS: The method of immunohistochemistry (IHC) and Western blot were utilized to examine the protein expression of 14-3-3Æ¡ in gastric cancer and paired normal adjacent gastric mucosal tissues. Receive operating characteristic (ROC) curve analysis was employed to determine a cutoff score for 14-3-3Æ¡ expression in a training set (n = 66). For validation, the ROC-derived cutoff score was subjected to analysis of the association of 14-3-3Æ¡ expression with patient outcome and clinical characteristics in a testing set (n = 86) and overall patients (n = 152). RESULTS: The expression frequency and expression levels of 14-3-3Æ¡ were significantly higher in gastric cancer than in normal gastric mucosal tissues. Correlation analysis demonstrated that high expression of 14-3-3Æ¡ in gastric cancer was significantly correlated with clinical stage and tumor invasion. Furthermore, in the testing set and overall patients, Kaplan-Meier analysis showed that elevated 14-3-3Æ¡ expression predicted poorer overall survival (OS) and progression-free survival (PFS). Importantly, high 14-3-3Æ¡ expression was also associated with shortened survival time in stage III and stage IV gastric cancer patients. Multivariate analyses revealed that 14-3-3Æ¡ expression was an independent prognostic parameter in gastric cancer. CONCLUSIONS: These findings provide evidence that high expression of 14-3-3Æ¡ may be important in the tumor progression and servers as an independent molecular marker for poor prognosis of gastric cancer. Thus, overexpression of 14-3-3Æ¡ identifies patients at high risk and is a novel therapeutic molecular target for this tumor.

Polyinosinic-polycytidylic acid induces the expression of interferon-stimulated gene 20 in mesangial cells.

BACKGROUND/AIMS: Interferon (IFN)-stimulated gene 20 (ISG20) is a 3'-to-5' exonuclease specific for single-stranded RNA and involved in host defense reactions against RNA viruses. The expression and the role of ISG20 in mesangial cells have not been reported. METHODS: Normal human mesangial cells were cultured and treated with polyinosinic-polycytidylic acid (poly (I:C)), an authentic double-stranded RNA which mimics viral infection to cells. The effect of RNA interference of Toll-like receptor 3 (TLR3) or IFN-ß on the ISG20 expression was examined. The effect of a blocking antibody against the receptor for IFN-ß or anti-inflammatory steroid dexamethasone was also examined. RESULTS: Treatment of cells with poly (I:C) induced the expression of ISG20. The poly (I:C)-induced expression of ISG20 was inhibited by knockdown of TLR3, IFN regulatery factor 3 (IRF3) or IFN-ß. Blocking of the receptor for IFN-ß suppressed and overexpression of IFN-ß enhanced ISG20 expression. The poly (I:C)-induced expressions of IFN-ß and ISG20 were inhibited by dexamethasone. Transfection of mesangial cells with poly (I:C) or 5'-triphosphate single-stranded RNA as a complex with cationic lipid also induced the expression of ISG20, and this was inhibited by knockdown of retinoic acid-inducible gene-I (RIG-I). CONCLUSION: Poly (I:C) induces the expression of ISG20 in mesangial cells. ISG20 may be involved in anti-viral reactions in renal mesangial cells. TLR3, IRF3 and de novo synthesized IFN-ß may mediate the poly (I:C)-induced expression of ISG20, and RIG-I may mediate ISG20 expression induced by poly (I:C)/cationic lipid complex.

GNL3L depletion destabilizes MDM2 and induces p53-dependent G2/M arrest.

Guanine nucleotide binding protein-like 3-like (GNL3L) is a nucleolar protein and the vertebrate paralogue of nucleostemin (NS). We previously reported that nucleoplasmic mobilization of NS stabilizes MDM2 (mouse double minute 2). Here, we investigated the role of GNL3L as a novel MDM2 regulator. We found that GNL3L binds MDM2 in vivo and displays the same function as NS in stabilizing MDM2 protein and preventing its ubiquitylation. The interaction between GNL3L and MDM2 also takes place in the nucleoplasm. However, the MDM2 regulatory activity of GNL3L occurs constitutively and does not so much depend on the nucleolar release mechanism as NS does. GNL3L depletion triggers G2/M arrest in the p53-wild-type HCT116 cells more than in the p53-null cells, and upregulates specific p53 targets (that is, Bax, 14-3-3σ and p21) without affecting the ubiquitylation or stability of p53 proteins. The inhibitory activity of GNL3L on p53-mediated transcription correlates with the increased expression of GNL3L and reduced expression of 14-3-3σ and p21 in human gastrointestinal tumors. This work shows that in contrast to most nucleolar proteins that negatively control MDM2, GNL3L and NS are the only two that are designed to stabilize MDM2 protein under basal or induced condition, respectively, and may act as tumor-promoting genes.

Nuclear expression of 14-3-3 sigma is related to prognosis in patients with esophageal squamous cell carcinoma.

BACKGROUND: The 14-3-3 sigma gene is transcriptionally activated by p53 after DNA damage and facilitates DNA repair during G2 arrest. The present study analyzed the clinical significance of 14-3-3 sigma expression in esophageal squamous cell carcinoma. PATIENTS AND METHODS: The relationship between 14-3-3 sigma and p53 expressions was investigated immunohistochemically in surgical specimens of primary tumors from 248 patients with esophageal squamous cell carcinoma. RESULTS: The positive expression rates of cytoplasmic and nuclear 14-3-3 sigma were 61.7% and 41.9%, respectively. There was no correlation between 14-3-3 sigma and p53 expression. Positive expression of nuclear 14-3-3 sigma was significantly correlated with depth of invasion, stage, lymphatic invasion, and poor prognosis. Multivariate analysis indicated that negative expression of nuclear 14-3-3 sigma was an independent prognostic factor. CONCLUSION: Evaluation of the expression of nuclear 14-3-3 sigma proteins may be useful in predicting the outcome in patients with esophageal squamous cell carcinoma.

The novel quinolone CHM-1 induces DNA damage and inhibits DNA repair gene expressions in a human osterogenic sarcoma cell line.

20-Fluoro-6,7-methylenedioxy-2-phenyl-4-quino-lone (CHM-1) has been reported to induce cell cycle arrest and apoptosis in many types of cancer cells. However, there is no available information to show CHM-1 affecting DNA damage and expression of associated repair genes. Herein, we investigated whether or not CHM-1 induced DNA damage and affected DNA repair gene expression in U-2 OS human osterogenic sarcoma cells. The comet assay showed that incubation of U-2 OS cells with 0, 0.75, 1.5, 3 and 6 µM of CHM-1 led to a longer DNA migration smear (comet tail). DNA gel electrophoresis showed that 3 µM of CHM-1 for 24 and 48 h treatment induced DNA fragmentation in U-2 OS cells. Real-time PCR analysis showed that treatment with 3 µM of CHM-1 for 24 h reduced the mRNA expression levels of ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR), breast cancer 1, early onset (BRCA1), 14-3-3sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK) and O(6)-methylguanine-DNA methyltransferase (MGMT) genes in a time-dependent manner. Taken together, the results indicate that CHM-1 caused DNA damage and reduced DNA repair genes in U-2 OS cells, which may be the mechanism for CHM-1-inhibited cell growth and induction of apoptosis.

14-3-3sigma expression as a prognostic marker in undifferentiated nasopharyngeal carcinoma.

In this study, we investigated the expression of 14-3-3sigma tumor suppressor gene in a panel of NPC cell lines, xenografts and primary tumors. Our objective was to determine the correlation between 14-3-3sigma expression and clinical outcome in NPC. We detected reduced 14-3-3sigma expression in 5/6 NPC tumor lines by quantitative RT-PCR and Western blotting. By immunohistochemical staining, significant down-regulation of 14-3-3sigma was also found in 26/72 (36.1%) primary tumors of NPC patients, who were treated with curative radiotherapy. Promoter methylation was confirmed in a subset of primary tumors by methylation-specific PCR analysis. Importantly, we demonstrated that 14-3-3sigma expression is significantly associated with both overall survival (OS) and cancer-specific survival (CSS), but not with the clinical staging of NPC patients. The low 14-3-3sigma expression was associated with improved overall (p=0.029) and cancer-specific survival (p=0.042) on univariate analysis. 14-3-3sigma expression and staging were also independent variables to all the prognostic factors under multivariate analysis. In conclusion, low expression of 14-3-3sigma appears to be a valuable marker for better survival in patient with NPC. These results provide the evidence that 14-3-3sigma expression is a significant prognostic factor for NPC patients.

Identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections.

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-alpha for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-alpha is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.

Emodin, aloe-emodin and rhein induced DNA damage and inhibited DNA repair gene expression in SCC-4 human tongue cancer cells.

In our primary studies, we have shown that emodin, aloe-emodin and rhein induced cytotoxic effects, including cell cycle arrest and apoptosis in SCC-4 human tongue cancer cells. However, details regarding their effects on DNA damage and repair gene expression in SCC-4 cells are not clear. We investigated whether or not emodin, aloe-emodin and rhein induced DNA damage and inhibited DNA repair gene expression in SCC-4 cells. Comet assay (single cell electrophoresis) indicated that incubation of SCC-4 cells with 0, 20, 30 and 40 microM of emodin, 0, 25, 50 and 100 microM of aloe-emodin or rhein led to a longer DNA migration smear (comet tail). This means that all examined agents induced DNA damage in SCC-4 cells and these effects are dose-dependent but emodin is stronger than that of aloe-emodin or rhein. The results from real-time PCR assay demonstrated that 30 microM of emodin or aloe-emodin used for 24 and 48 h treatment in SCC-4 cells significantly inhibited expression of genes associated with DNA damage and repair [ataxia telangiectasia mutated (ATM); ataxia-telangiectasia and Rad3-related (ATR); 14-3-3sigma (14-3-3sigma); breast cancer 1, early onset (BRCA1); and DNA-dependent serine/threonine protein kinase (DNA-PK)]; only rhein suppressed the expression of O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA with 48 h treatment, but had no effect on ATM expression. On 24 h treatment, only aloe-emodin significantly affected ATM expression. These effects may be the vital factors for emodin, aloe-emodin and rhein induction of DNA damage in vitro. In conclusion, these agents induced DNA damage followed by the inhibition of DNA repair-associated gene expressions, including ATM, ATR, 14-3-3sigma, BRCA1, DNA-PK and MGMT in SCC-4 human tongue cancer cells.

Outcome prediction and risk assessment by quantitative pyrosequencing methylation analysis of the SFN gene in advanced stage, high-risk, neuroblastic tumor patients.

The aim of our study was to identify threshold levels of DNA methylation predictive of the outcome to better define the risk group of stage 4 neuroblastic tumor patients. Quantitative pyrosequencing analysis was applied to a training set of 50 stage 4, high risk patients and to a validation cohort of 72 consecutive patients. Stage 4 patients at lower risk and ganglioneuroma patients were included as control groups. Predictive thresholds of methylation were identified by ROC curve analysis. The prognostic end points of the study were the overall and progression-free survival at 60 months. Data were analyzed with the Cox proportional hazard model. In a multivariate model the methylation threshold identified for the SFN gene (14.3.3sigma) distinguished the patients presenting favorable outcome from those with progressing disease, independently from all known predictors (Training set: Overall Survival HR 8.53, p = 0.001; Validation set: HR 4.07, p = 0.008). The level of methylation in the tumors of high-risk patients surviving more than 60 months was comparable to that of tumors derived from lower risk patients and to that of benign ganglioneuroma. Methylation above the threshold level was associated with reduced SFN expression in comparison with samples below the threshold. Quantitative methylation is a promising tool to predict survival in neuroblastic tumor patients. Our results lead to the hypothesis that a subset of patients considered at high risk-but displaying low levels of methylation-could be assigned at a lower risk group.

Aberrant methylation profile of 14-3-3 sigma and its reduced transcription/expression levels in Chinese sporadic female breast carcinogenesis.

To study the relation of 14-3-3 sigma gene promoter hypermethylation and its transcription expression levels in sporadic breast carcinogenesis. Methylation of 14-3-3 sigma gene was detected by sensitive MSP assay in carcinous, non-cancerous, and normal tissue, and its mRNA was also detected by real time PCR based on SYBR Green 1 as well, and protein was detected by west blotting assay. The methylation frequencies of 14-3-3 sigma were 90% in 68 cases of sporadic breast cancer patients. Methylation was presented in portions (2/13, 18%) of hyperplastic samples, and no hypermethylation was presented in normal tissue. The methylation change of 14-3-3 sigma gene was markedly related with various types, grades, and lymph node metastases (P < 0.05), and no significant differences in methylation frequencies were seen between premenopause and postmenopause (P > 0.05). The methylation of 14-3-3 sigma shows reverse relation with its mRNA transcription and expression level (P < 0.05). Only was lymph node metastases strongly associated with poor outcome (P = 0.02). Whether 14-3-3 sigma promoter methylation or not did not affect the 5 years survival rate of sporadic breast cancer patients (P > 0.05). Epigenetics alterations of the 14-3-3 sigma can contribute to reducing or losing expression of 14-3-3 sigma protein, which play an important role in the development of sporadic breast carcinomas and involved in various types, grades, and lymph node metastases. Otherwise, node metastases of breast carcinogenesis patients are strongly associated with poor outcome.

Higher expression levels of 14-3-3sigma in ductal carcinoma in situ of the breast predict poorer outcome.

The protein 14-3-3sigma is involved in the regulation of cellular processes such as apoptosis, cell cycle progression and proliferation. Disruption of protein expression has been implicated in a number of malignancies. Here we examine the expression pattern of 14-3-3sigma in breast cancer and specifically consider whether expression in ductal carcinoma in situ (DCIS) lesions is predictive of disease outcome. We examined 14-3-3sigma protein expression and localization using immunohistochemical staining on a high-density tissue microarray consisting of 157 invasive breast cancer patients. Statistical analyses were used to assess the correlation of 14-3-3sigma expression with clinico-pathological parameters and patient outcome. We observed a statistically significant increase in 14-3-3sigma protein expression in ductal hyperplasia, DCIS, and invasive ductal carcinoma (IDC) as compared normal glandular epithelium. In IDC, lower expression of 14-3-3sigma tended to predicted poorer survival time while in DCIS lesions, there was a stronger correlation between relatively higher levels of 14-3-3sigma predicting shorter survival time. Further, of patients who had concurrent DCIS and IDC lesions, those that exhibited a decrease of 14-3-3sigma expression from DCIS to IDC had significantly shorter survival time. Our findings indicate that 14-3-3sigma expression may be a useful prognostic indicator for survival in patients with breast cancer with an elevated 14-3-3sigma in earlier disease predicting a less favorable disease outcome. To our knowledge this is the first published study associating 14-3-3sigma protein expression with breast cancer survival.

14-3-3sigma gene silencing during melanoma progression and its role in cell cycle control and cellular senescence.

BACKGROUND: The family of 14-3-3 proteins plays an important role in cancer biology by interfering with intracellular signalling pathways and cell cycle checkpoints. The 14-3-3sigma isoform acts as a tumor suppressor and is often inactivated during tumor development. RESULTS: Here, we demonstrate enhanced CpG methylation of the 14-3-3sigma gene in lymph node and cutaneous melanoma metastases compared with primary tumors, associated with dramatically reduced mRNA expression. In line with this, treatment of different metastatic melanoma cell lines with 5-aza-2'-deoxycytidine (5-Aza-CdR), a potent inhibitor of cytosine methylation, significantly induces 14-3-3sigma protein expression. Additional treatment with histone deacetylase inhibitor 4-phenylbutyric acid (Pba) further enhances 14-3-3sigma expression. Induction of 14-3-3sigma expression by 5-Aza-CdR/Pba treatment leads to almost complete inhibition of cell proliferation, with cells predominantly arrested in G2-M. The antiproliferative effect of 5-Aza-CdR/Pba was reversed in 14-3-3sigma knockdown cells. Similarly, melanoma cell lines stably overexpressing 14-3-3sigma show dramatically reduced cell proliferation rates. Moreover, synchronous 14-3-3sigma stably overexpressing cells do not progress through cell cycle, but display a permanent increase in the population of 4n DNA containing cells. Interestingly, overexpression of 14-3-3sigma induces senescence of melanoma cells and is involved in melanoma cell senescence under genotoxic stress. Finally, 14-3-3sigma knockdown supports migratory capacity of melanoma cells in vitro, while 14-3-3sigma overexpression has opposing effects. CONCLUSION: Taken together, the present report indicates that epigenetic silencing of 14-3-3sigma might contribute to tumor progression in malignant melanoma via loss of cell cycle control, impaired cellular senescence program and support of migratory capacity.

14-3-3sigma expression and prognostic value in patients with epithelial ovarian carcinoma: a high throughput tissue microarray analysis.

AIMS: 14-3-3sigma is a potential tumor suppressor gene that when it is silenced by CpG methylation can contribute to cancer development. Previously, we showed that hypermethylation of 14-3-3sigma in human ovarian cancer and ovarian cancer cell lines, and that 14-3-3sigma hypermethylation correlated with loss of its expression by immunohistochemistry. In the present study, our aim is to determine the value of 14-3-3sigma in predicting disease outcome in series of patients with epithelial ovarian cancer. MATERIALS AND METHODS: A tumor microarray (TMA) of 192 patients with a very detailed characteristic and follow-up was performed. The slides were immunostained with 14-3-3sigma antibody and its expression was correlated with age, tumor types, grade, stage, volume of residual tumor, response to therapy, overall survival (OS) and disease-free survival (DFS). RESULTS: A marginal association with the volume of residual tumor after surgery (chi2 p = 0.044, Fischer's exact 0.051) was seen. There was no association between loss of 14-3-3sigma expression and any of age, stage, grade, tumor subtypes, and clinical response to therapy. Survival analysis according to Kaplan-Meier method showed that loss of 14-3-3sigma expression was not associated with OS or DFS (p = 0.702, p = 0.118, respectively). CONCLUSION: Even though 14-3-3sigma is involved in ovarian tumorigenesis, it does not have a prognostic value as a biomarker to predict patients' outcome.

Germ cell specific protein VASA is over-expressed in epithelial ovarian cancer and disrupts DNA damage-induced G2 checkpoint.

OBJECTIVE: Cancer cells have characteristics, such as high telomerase activity and high levels of migration activity and proliferation, which are very similar to those of germ cell lineages. In this study, we examined the expression of VASA, a germ cell lineage specific marker and evaluated its clinical significance in epithelial ovarian cancer (EOC). METHODS: We investigated VASA expression in 75 EOC tissues by immunohistochemistry, correlating results with clinicopathological factors. To clarify the effects of VASA on cellular phenotypes, we compared the protein expression profiles between SKOV-3 cells stably expressing VASA (SKOV-3-VASA) and vector-control cell lines by coupling 2D fingerprinting and identification of proteins by mass spectrometry. RESULTS: VASA expression in tumor cells was found in 21 of 75 cases and was positively correlated with high age and serous histology. Significant down-regulation of 14-3-3sigma was observed in SKOV-3-VASA versus control cells. Over-expression of VASA abrogates the G2 checkpoint, induced by DNA damage, by down-regulating the expression of 14-3-3sigma. CONCLUSIONS: These results suggest that VASA may either play a direct role in the progression of EOC or serve as a valuable marker of tumorigenesis.

Expression of interferon-stimulated gene 20 in vascular endothelial cells.

ISG20 is an ribonuclease specific for single-stranded RNA and considered to play a role in innate immunity against virus infections. We herein show that both poly IC, an authentic double-stranded RNA, and IFN-gamma induced ISG20 expression in cultured HUVEC. Poly IC-induced ISG20 expression was inhibited by LY294002, an inhibitor of PI3K, or by RNA interference against IFN regulatory factor three. ISG20 expression was not induced by IFN-beta, loxoribine or CpG oligonucleotide. These results suggest that ISG20 induction by poly IC may not be dependent on the IRF-3-mediated type I IFN induction pathway in HUVEC. ISG20 may be involved in innate immunity against viral infection in vascular endothelial cells.

Cloning, eukaryotic expression of human ISG20 and preliminary study on the effect of its anti-HBV.

Human ISG20 gene was cloned and the effect of its anti-HBV was primarily studied. The ISG20 gene was amplified from HeLa cells by RT-PCR and recombinant vector expressing ISG20 was constructed by genetic engineering. The overexpression of ISG20 in HepG2 cells was detected by Western blot and the levels of secretion of HBs antigen and HBe antigen tested by ELISA. The results showed that: (1) Sequence of ISG20 cloned was consistent to that published in Genebank; (2) Recombinant vector expressing ISG20 could be expressed in HepG2 cells by transfection; (3) The overexpression of ISG20 protein could reduce the levels of the secretion of HBs antigen and HBe antigen in transfected HepG2 cells. It was suggested that the overexpression of recombinant ISG20 in culture cells could reduce the synthesis of HBV proteins.