Hsin-Pai Li

Activation of DNA Methyltransferase 1 by EBV LMP1 Involves c-Jun NH2-Terminal Kinase Signaling

EBV latent membrane protein 1 (LMP1) activates cellular DNA methyltransferases, resulting in hypermethylation and silencing of E-cadherin. However, the underlying mechanism remains to be elucidated. In this study, we show that LMP1 directly induces the dnmt1 promoter activity through its COOH-terminal activation region-2 YYD domain. Using (i) LMP1 mutants, (ii) dominant negative mutants c-jun NH(2)-terminal kinase (JNK)-DN, p38-DN, and constitutive active mutant IkappaB, as well as (iii) dsRNAs targeting c-Jun, JNK, and tumor necrosis factor receptor-associated death domain protein, and (iv) signal transduction inhibitors, we show that LMP1-mediated DNA methyltransferase-1 (DNMT1) activation involves JNK but not nuclear factor kappaB and p38/mitogen-activated protein kinase signaling. In addition, LMP1 is unable to activate dnmt1-P1 promoter with activator protein-1 (AP-1) site mutation. Chromatin immunoprecipitation assay results also confirm that LMP1 activates P1 promoter via the JNK-AP-1 pathway. Furthermore, chromatin immunoprecipitation assay data in LMP1-inducible cells disclose that LMP1 induces formation of a transcriptional repression complex, composed of DNMT1 and histone deacetylase, which locates on E-cadherin gene promoter. Treatment with JNK inhibitor, SP600125, prevents the formation of this repression complex. Statistical analyses of the immunohistochemical staining of 32 nasopharyngeal carcinoma (NPC) biopsies show LMP1 expression (18 of 32, 56.25%), DNMT1 expression (31 of 32, 97%), and phospho-c-Jun (27 of 32, 84.38%), suggesting that overexpression of these proteins is observed in NPC tumor. Overall, these results support a mechanistic link between JNK-AP-1 signaling and DNA methylation induced by the EBV oncogene product LMP1.

Epstein-barr virus latent membrane protein 1: Structure and functions

The Epstein-Barr virus latent membrane protein (LMP) 1 is a versatile protein that has profound effects on target cells through its effect on constitutive cellular proteins, e.g. TRAFs, TRADD, RIP, JAK3, BRAM1, and p85. LMP1 can stimulate or inhibit signaling pathways, resulting in transformation of rodent fibroblast cell lines, blockade of differentiation in epithelial cells, upregulation of anti-apoptotic proteins, production of cytokines, upregulation of cell surface markers, upregulation of DNA methyltransferase activity, and downregulation of cell adhesion molecules and cyclin-dependent kinases. Overall, this results in greater transformation and survival in LMP1-expressing cells. Within nasopharyngeal carcinoma biopsy tissues, a naturally occurring LMP1 variant has been identified as having a 10-amino acid deletion in the C-terminus that seems to confer greater transformation potential than non-deleted LMP1. The role of LMP1 as a viral oncogene and its interaction with cellular factors are discussed.

Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus

Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre‐mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant‐negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective‐interfering RNAs. Similar to the wild‐type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild‐type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA‐dependent RNA synthesis.

Epigenetic changes in virus-associated human cancers

ABSTRACTEpigenetics of human cancer becomes an area of emerging research direction due to a growing understanding of specific epigenetic pathways and rapid development of detection technologies. Aberrant promoter hypermethylation is a prevalent phenonmena in human cancers. Tumor suppressor genes are often hypermethylated due to the increased activity or deregulation of DNMTs. Increasing evidence also reveals that viral genes are one of the key players in regulating DNA methylation. In this review, we will focus on hypermethylation and tumor suppressor gene silencing and the signal pathways that are involved, particularly in cancers closely associated with the hepatitis B virus, simian virus 40 (SV40), and Epstein-Barr virus. In addition, we will discuss current technologies for genome-wide detection of epigenetically regulated targets, which allow for systematic DNA hypermethylation analysis. The study of epigenetic changes should provide a global view of gene profile in cancer, and epigenetic markers could be used for early detection, prognosis, and therapy of cancer.

MCP-1 Promoter Polymorphism at −2518 Is Associated with Metastasis of Nasopharyngeal Carcinoma after Treatment

Purpose: We herein examined whether the single nucleotide polymorphism (SNP) at −2518 of the MCP-1 gene promoter region influences clinical outcomes among nasopharyngeal carcinoma (NPC) patients. Experimental Design: The study population consisted of 411 NPC patients without metastasis at diagnosis. All patients were treated at the Chang Gung Memorial Hospital from March 1994 to November 2004. The MCP-1 SNP−2518 genotype of each patient was determined by TaqMan genotyping kit. Statistical analyses were conducted to compare disease-specific survival (DSS), progression-free survival (PFS), local recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) of patients according to genotype. MCP-1 expression in tumor biopsies was examined by immunohistochemistry. Results: Among 411 NPC patients, carriers of AA and AG genotypes were prone to distant metastasis than that of GG genotype (hazard ratio, 2.21; P = 0.017, and hazard ratio, 2.23; P = 0.005, for AA and AG genotype, respectively) after initial radiotherapy. No genotype-specific significant difference was found in DSS, PFS, and LRFS. Furthermore, immunohistochemistry revealed that MCP-1 expression level was higher in NPC tumor cells from GG carriers compared with those from AA and AG carriers. Conclusions:MCP-1 SNP−2518 may be a valuable genetic marker for assessing the risk of developing distant metastasis after the radiotherapy in NPC patients. Carriers of A allele may require more aggressive chemotherapy implicating a potential marker for personalized medicine. We speculate that a regulatory SNP may be associated with the distant metastasis of NPC. Validation studies are warranted.

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

The cytoplasmic level of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is significantly correlated with the elevated expression of thymidine phosphorylase (TP), and high levels of both proteins are predictive of a poor prognosis in nasopharyngeal carcinoma (NPC). We herein show that TP is highly induced by serum deprivation in NPC cells, and that this is due to an increase in the half-life of the TP mRNA, as shown by nuclear run-on and actinomycin D assays. We further show that the CU-rich element of the TP mRNA directly interacts with hnRNP K, as demonstrated by immunoprecipitation RT–PCR assays, and the nucleus-to-cytoplasm translocation of hnRNP K. Blockade of hnRNP K expression reduces TP expression, suggesting that hnRNP K acts in the upregulation of TP. Mechanistically, both MEK inhibitor and the hnRNP K ERK-phosphoacceptor-site mutant decrease cytoplasmic accumulation of hnRNP K, suggesting that ERK-dependent phosphorylation is critical for TP induction. Furthermore, we found that hnRNP K-mediated TP induction allows NPC cells to resist hypoxia-induced apoptosis. Our results collectively establish the regulation and role of ERK-mediated cytoplasmic accumulation of hnRNP K as an upstream modulator of TP, suggesting that hnRNP K may be an attractive candidate as a future therapeutic target for cancer.

Glutamate receptor, ionotropic, kainate 2 silencing by DNA hypermethylation possesses tumor suppressor function in gastric cancer

Aberrant DNA methylation is considered a major mechanism for silencing tumor suppressor genes in gastric cancer. We used CpG microarray and differential methylation hybridization strategies to identify potential tumor suppressor genes and recovered glutamate receptor, ionotropic, kainate 2 (GRIK2) as a novel epigenetic target in gastric cancer. Additional experiments showed that the promoter region of GRIK2 was hypermethylated in 3 of the 4 tested gastric cancer cell lines, and its expression was restored by treatment of cells with the DNA methylation inhibitor, 5′‐aza‐dC. In clinical samples, the GRIK2 promoter was differentially hypermethylated in tumor tissues compared with adjacent normal tissues (p < 0.001), and this methylation was inversely correlated with the expression level of GRIK2 mRNA (r = −0.44). Functional studies further showed that GRIK2‐expressing gastric cancer cell lines showed decreased colony formation and cell migration. Taken together, these results suggest that GRIK2 may play a tumor‐suppressor role in gastric cancer. Future studies are warranted to examine whether DNA hypermethylation of the GRIK2 promoter can be used as a potential tumor marker for gastric cancer.

ChIPseek, a web-based analysis tool for ChIP data

BackgroundChromatin is a dynamic but highly regulated structure. DNA-binding proteins such as transcription factors, epigenetic and chromatin modifiers are responsible for regulating specific gene expression pattern and may result in different phenotypes. To reveal the identity of the proteins associated with the specific region on DNA, chromatin immunoprecipitation (ChIP) is the most widely used technique. ChIP assay followed by next generation sequencing (ChIP-seq) or microarray (ChIP-chip) is often used to study patterns of protein-binding profiles in different cell types and in cancer samples on a genome-wide scale. However, only a limited number of bioinformatics tools are available for ChIP datasets analysis.ResultsWe present ChIPseek, a web-based tool for ChIP data analysis providing summary statistics in graphs and offering several commonly demanded analyses. ChIPseek can provide statistical summary of the dataset including histogram of peak length distribution, histogram of distances to the nearest transcription start site (TSS), and pie chart (or bar chart) of genomic locations for users to have a comprehensive view on the dataset for further analysis. For examining the potential functions of peaks, ChIPseek provides peak annotation, visualization of peak genomic location, motif identification, sequence extraction, and comparison between datasets. Beyond that, ChIPseek also offers users the flexibility to filter peaks and re-analyze the filtered subset of peaks. ChIPseek supports 20 different genome assemblies for 12 model organisms including human, mouse, rat, worm, fly, frog, zebrafish, chicken, yeast, fission yeast, Arabidopsis, and rice. We use demo datasets to demonstrate the usage and intuitive user interface of ChIPseek.ConclusionsChIPseek provides a user-friendly interface for biologists to analyze large-scale ChIP data without requiring any programing skills. All the results and figures produced by ChIPseek can be downloaded for further analysis. The analysis tools built into ChIPseek, especially the ones for selecting and examine a subset of peaks from ChIP data, provides invaluable helps for exploring the high through-put data from either ChIP-seq or ChIP-chip. ChIPseek is freely available at http://chipseek.cgu.edu.tw.

Plasma epstein‐barr virus DNA concentration and clearance rate as novel prognostic factors for metastatic nasopharyngeal carcinoma

To investigate the pretreatment copy number and the clearance rate of plasma Epstein‐Barr virus (EBV) DNA as novel prognostic outcome markers for metastatic nasopharyngeal carcinoma (NPC).

Thermal aggregation of SARS-CoV membrane protein

Abstract SARS-CoV membrane protein could be detected easily using Western blotting in non-denaturing condition but not regular denaturing treatment. Boiling treatment, causing the aggregation of SARS-CoV membrane protein in the stacking gels, results in the failure to detect the membrane protein in the separating gels. Aggregated membrane proteins could not be dissociated by 1% Triton-X 100, 6M urea, or 2% SDS. The region with amino acid residues from 51 to 170 is responsible for thermal aggregation of SARS-CoV membrane protein. Hydrophobic regions with amino acid residues from 61 to 90, from 91 to 100, from 136 to 170, are essential for this protein aggregation. Thermal aggregation of SARS-CoV membrane protein is not unique among structural proteins of coronaviruses. However, SARS-CoV membrane protein seems to be more sensitive to heat treatment, since the membrane protein of MHV-JHM, another member of the Coronaviridae, would not aggregate after the same treatment. Therefore, if SARS-CoV membrane protein needs to be analyzed using SDS-PAGE, boiling should be avoided. Thermal aggregation of SARS-CoV membrane protein may be one of the reasons for the inactivation of this virus by heat. The unusual property of SARS-CoV membrane protein aggregation induced by heat also provides a model for the study of protein aggregation.