EBV-transformed Lymphoblastoid Cell Lines Research Articles

Association between Single Nucleotide Polymorphisms in ERCC4 and Risk of Squamous Cell Carcinoma of the Head and Neck

BackgroundExcision repair cross-complementation group 4 gene (ERCC4/XPF) plays an important role in nucleotide excision repair and participates in removal of DNA interstrand cross-links and DNA double-strand breaks. Single nucleotide polymorphisms (SNPs) in ERCC4 may impact repair capacity and affect cancer susceptibility.Methodology/Principal FindingsIn this case-control study, we evaluated associations of four selected potentially functional SNPs in ERCC4 with risk of squamous cell carcinoma of the head and neck (SCCHN) in 1,040 non-Hispanic white patients with SCCHN and 1,046 cancer-free matched controls. We found that the variant GG genotype of rs2276466 was significantly associated with a decreased risk of SCCHN (OR = 0.69, 95% CI 0.50–0.96), and that the variant TT genotype of rs3136038 showed a borderline significant decreased risk with SCCHN (OR = 0.76, 95% CI: 0.58–1.01) in the recessive model. Such protective effects were more evident in oropharyngeal cancer (OR = 0.61, 95% CI: 0.40–0.92 for rs2276466; OR = 0.69, 95% CI: 0.48–0.98 for rs3136038). No significant associations were found for the other two SNPs (rs1800067 and rs1799798). In addition, individuals with the rs2276466 GG or with the rs3136038 TT genotypes had higher levels of ERCC4 mRNA expression than those with the corresponding wild-type genotypes in 90 Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Caucasians.ConclusionsThese results suggest that these two SNPs (rs2276466 and rs3136038) in ERCC4 may be functional and contribute to SCCHN susceptibility. However, our findings need to be replicated in further large epidemiological and functional studies.

Correlation between Base-Excision Repair Gene Polymorphisms and Levels of In-Vitro BPDE–Induced DNA Adducts in Cultured Peripheral Blood Lymphocytes

In vitro benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in cultured peripheral lymphocytes have been shown to be a phenotypic biomarker of individual’s DNA repair phenotype that is associated with cancer risk. In this study, we explored associations between genotypes of base-excision repair genes (PARP1 Val762Ala, APEX1 Asp148Glu, and XRCC1 Arg399Gln) and in vitro BPDE-induced DNA adducts in cultured peripheral blood lymphocytes in 706 cancer-free non-Hispanic white subjects. We found that levels of BPDE-induced DNA adducts were significantly higher in ever smokers than in never smokers and that individuals with the Glu variant genotypes (i.e., Asp/Glu and Glu/Glu) exhibited lower levels of BPDE-induced DNA adducts than did individuals with the common Asp/Asp homozygous genotype (median RAL levels: 32.0 for Asp/Asp, 27.0 for Asp/Glu, and 17.0 for Glu/Glu, respectively; P trend = 0.030). Further stratified analysis showed that compared with individuals with the common APEX1-148 homozygous Asp/Asp genotype, individuals with the APEX1-148Asp/Glu genotype or the Glu/Glu genotype had a lower risk of having higher-level adducts (adjusted OR = 0.60, 95% CI: 0.36–0.98 and adjusted OR = 0.47, 95% CI: 0.26–0.86, respectively; P trend = 0.012) among smokers. Such an effect was not observed in non-smokers. However, there was no significant interaction between the APEX1 Asp148Glu polymorphism and smoking exposure in this study population (P = 0.512). Additional genotype-phenotype analysis found that the APEX1-148Glu allele had significantly increased expression of APEX1 mRNA in 270 Epstein-Barr virus-transformed lymphoblastoid cell lines, which is likely associated with more active repair activity. Our findings suggest that the functional APEX1-148Glu allele is associated with reduced risk of having high levels of BPDE-induced DNA adducts mediated with high levels of mRNA expression.

Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia.

Abnormal replication timing has been observed in cancer but no study has comprehensively evaluated this misregulation. We generated genome-wide replication-timing profiles for pediatric leukemias from 17 patients and three cell lines, as well as normal B and T cells. Nonleukemic EBV-transformed lymphoblastoid cell lines displayed highly stable replication-timing profiles that were more similar to normal T cells than to leukemias. Leukemias were more similar to each other than to B and T cells but were considerably more heterogeneous than nonleukemic controls. Some differences were patient specific, while others were found in all leukemic samples, potentially representing early epigenetic events. Differences encompassed large segments of chromosomes and included genes implicated in other types of cancer. Remarkably, differences that distinguished leukemias aligned in register to the boundaries of developmentally regulated replication-timing domains that distinguish normal cell types. Most changes did not coincide with copy-number variation or translocations. However, many of the changes that were associated with translocations in some leukemias were also shared between all leukemic samples independent of the genetic lesion, suggesting that they precede and possibly predispose chromosomes to the translocation. Altogether, our results identify sites of abnormal developmental control of DNA replication in cancer that reveal the significance of replication-timing boundaries to chromosome structure and function and support the replication domain model of replication-timing regulation. They also open new avenues of investigation into the chromosomal basis of cancer and provide a potential novel source of epigenetic cancer biomarkers.

Ectopically Expressed Human Tumor Biomarker MutS Homologue 2 Is a Novel Endogenous Ligand That Is Recognized by Human γδ T Cells to Induce Innate Anti-tumor/Virus Immunity

Human (h) MutS homologue 2, a nuclear protein, is a critical element of the DNA mismatch repair system. Our previous studies suggest that hMSH2 might be a protein ligand for TCRγδ. Here, we show that hMSH2 is ectopically expressed on a large panel of epithelial tumor cells. We found that hMSH2 interacts with both TCRγδ and NKG2D and contributes to Vδ2 T cell-mediated cytolysis of tumor cells. Moreover, recombinant human MSH2 protein stimulates the proliferation and IFN-γ secretion of Vδ2 T cells in vitro. Finally, hMSH2 expression is induced on the cell surface of Epstein-Barr virus-transformed lymphoblastoid cell lines, and the induction increases the sensitivity of these lymphoblastoid cell lines to γδ T cell-mediated cytolysis. Our data suggest that hMSH2 functions as a tumor-associated or virus infection-related antigen recognized by both Vδ2 TCR and NKG2D, and it plays a role in eliciting the immune responses of γδ T cells against tumor- and virus-infected cells. The recognition of ectopic surface-expressing endogenous antigen by TCRγδ and NKG2D may be an important mechanism of innate immune response to carcinogenesis and viral infection.

Constitutive AP-1 Activity and EBV Infection Induce PD-L1 in Hodgkin Lymphomas and Posttransplant Lymphoproliferative Disorders: Implications for Targeted Therapy

Programmed cell death ligand 1 (PD-L1) is a molecule expressed on antigen-presenting cells that engages the PD-1 receptor on T cells and inhibits T-cell receptor signaling. The PD-1 axis can be exploited by tumor cells to dampen host antitumor immune responses and foster tumor cell survival. PD-1 blockade has shown promise in multiple malignancies but should be directed toward patients in whom it will be most effective. In recent studies, we found that the chromosome 9p24.1 amplification increased the gene dosage of PD-L1 and its induction by JAK2 in a subset of patients with classical Hodgkin lymphoma (cHL). However, cHLs with normal 9p24.1 copy numbers also expressed detectable PD-L1, prompting analyses of additional PD-L1 regulatory mechanisms. Herein, we utilized immunohistochemical, genomic, and functional analyses to define alternative mechanisms of PD-L1 activation in cHL and additional EBV(+) lymphoproliferative disorders. We identified an AP-1-responsive enhancer in the PD-L1 gene. In cHL Reed-Sternberg cells, which exhibit constitutive AP-1 activation, the PD-L1 enhancer binds AP-1 components and increases PD-L1 promoter activity. In addition, we defined Epstein-Barr virus (EBV) infection as an alternative mechanism for PD-L1 induction in cHLs with diploid 9p24.1. PD-L1 was also expressed by EBV-transformed lymphoblastoid cell lines as a result of latent membrane protein 1-mediated, JAK/STAT-dependent promoter and AP-1-associated enhancer activity. In addition, more than 70% of EBV(+) posttransplant lymphoproliferative disorders expressed detectable PD-L1. AP-1 signaling and EBV infection represent alternative mechanisms of PD-L1 induction and extend the spectrum of tumors in which to consider PD-1 blockade.

The extract of Chrysanthemum indicum Linne inhibits EBV LMP1-induced NF-κB activation and the viability of EBV-transformed lymphoblastoid cell lines

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-κB activation is essential for LCL survival. To identify a novel inhibitor candidate for LMP1-induced NF-κB activation, crude ethanol extracts of medicinal plants were screened for the potential NF-κB inhibitory activity. Seventy percent ethanol extract of Chrysanthemum indicum Linne extract (CIE) strongly reduced LMP1-induced NF-κB activation. In addition, CIE inhibited LMP1-induced IKKα or IKKβ activation. Interestingly, CIE treatment rapidly reduced LCL viability without exhibiting any adverse effects on the viability of human foreskin fibroblasts (HFF), EBV negative Burkitt’s lymphoma cell lines (BL41) or HeLa cells.Taken together, CIE has potent inhibitory effect on EBV LMP1-induced NF-κB activation and EBV-transformed LCL viability.

Understanding Disease Susceptibility through Population Genomics

Genetic epidemiology studies have established that the natural variation of gene expression profiles is heritable and has genetic bases. A number of proximal and remote DNA variations, known as expression quantitative trait loci (eQTLs), that are associated with the expression phenotypes have been identified, first in Epstein-Barr virus-transformed lymphoblastoid cell lines and later expanded to other cell and tissue types. Integration of the eQTL information and the network analysis of transcription modules may lead to a better understanding of gene expression regulation. As these network modules have relevance to biological or disease pathways, these findings may be useful in predicting disease susceptibility.

Longitudinal analysis of frequency and reactivity of epstein–barr virus‐specific T lymphocytes and their association with intermittent viral reactivation

Persistent Epstein-Barr virus (EBV) infection is controlled tightly by virus-specific T cells. EBV infection is reactivated intermittently over time, even in apparently healthy carriers. Changes in frequency and reactivity of memory T cells, particularly of CD8(+) origin, have not been assessed in this context. It is hypothesized that viral reactivation is facilitated by diminished EBV-specific T-cell immunity. To this end, blood samples from 14 healthy donors were collected at irregular time intervals for a period of about 1 year. Samples were screened for both EBV plasma viremia and increases in viral load in PBMCs as parameters of EBV reactivation. PBMCs were subject to IFN-γ ELISPOT analysis using the autologous EBV-transformed lymphoblastoid cell line (EBV-LCL) or appropriate HLA class I-restricted EBV peptides as stimulators. Frequencies of epitope-specific CD8(+) T cells were monitored further using HLA tetramers and flow cytometry. Twelve of 14 donors exhibited signs of asymptomatic EBV reactivation. Viral reactivation was accompanied by either substantially decreased IFN-γ responses against autologous EBV-LCL (eight of 12 study participants) and/or increased responses against particular EBV peptides (six of 12 donors). In seven persons with HLA-A2 and/or -B8 alleles numbers of HLA tetramer-positive CD8(+) T cells also varied over time, but showed no correlation to episodes of detectable viral activity. In summary, IFN-γ reactivity of EBV-specific T cells is not constant. Viral reactivation is detected preferably at times of diminished EBV-LCL-specific cellular immunity. However, increased reactivity of single immunodominant CD8(+) EBV-specific T-cell clones may occur in response to virus replication.

C-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

LMP1-mediated activation of nuclear factor of kappaB (NF-κB) is critical for the ligand independent proliferation and cell survival of in vitro EBV-transformed lymphoblastoid cell lines (LCLs). Previous experiments revealed that a majority of LMP1-dependent responses are regulated by NF-κB. However, the extent that individual NF-κB family members are required for these responses, in particular, c-Rel, whose expression is restricted to mature hematopoietic cells, remains unclear. Here we report that low c-Rel expression in LCLs derived from a patient with hyper-IgM syndrome (Pt1), resulted in defects in proliferation and cell survival. In contrast to studies that associated loss of NF-κB with increased apoptosis, Pt1 LCLs failed to initiate apoptosis and alternatively underwent autophagy and necrotic cell death. Whereas the proliferation defect appeared linked to a c-Rel-associated decrease in c-myc expression, identified pro-survival and pro-apoptotic targets were expressed at or near control levels consistent with the absence of apoptosis. Ultrastructural examination of Pt1 LCLs revealed a high level of cellular and ER stress that was further supported by gene expression profiling showing the upregulation of several genes involved in stress and inflammation. Apoptosis-independent cell death was accompanied by increased expression of the inflammatory marker, caspase-4. Using gene overexpression and siRNA knockdown we demonstrated that levels of c-Rel directly modulated expression of caspase-4 as well as other ER stress genes. Overall, these findings reveal the importance of c-Rel in maintaining LCL viability and that decreased expression results in ER stress and a default response leading to necrotic cell death.

Optimized Internal Control and Gene Expression Analysis in Epstein-Barr Virus-Transformed Lymphoblastoid Cell Lines

The Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) is one of the major genomic resources for human genetics and immunological studies. Use of LCLs is currently extended to pharmacogenetic studies to investigate variations in human gene expression as well as drug responses between individuals. We evaluated four common internal controls for gene expression analysis of selected hematopoietic transcriptional regulatory genes between B cells and LCLs. In this study, the expression pattern analyses showed that TBP (TATA box-binding protein) is a suitable internal control for normalization, whereas GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is not a good internal control for gene expression analyses of hematopoiesis-related genes between B cells and LCLs at different subculture passages. Using the TBP normalizer, we found significant gene expression changes in selected hematopoietic transcriptional regulatory genes (downregulation of RUNX1, RUNX3, CBFB, TLE1, and NOTCH2 ; upregulation of MSC and PLAGL2) between B cells and LCLs at different passage numbers. These results suggest that these hematopoietic transcriptional regulatory genes are potential cellular targets of EBV infection, contributing to EBV-mediated B-cell transformation and LCL immortalization.

CYP17A1 Intron Mutation Causing Cryptic Splicing in 17α-Hydroxylase Deficiency

17α-hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

The Use of Genome-Wide eQTL Associations in Lymphoblastoid Cell Lines to Identify Novel Genetic Pathways Involved in Complex Traits

The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097–16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future.

Establishment of the first WHO international genetic reference panel for Prader Willi and Angelman syndromes

Prader Willi and Angelman syndromes are clinically distinct genetic disorders both mapping to chromosome region 15q11-q13, which are caused by a loss of function of paternally or maternally inherited genes in the region, respectively. With clinical diagnosis often being difficult, particularly in infancy, confirmatory genetic diagnosis is essential to enable clinical intervention. However, the latter is challenged by the complex genetics behind both disorders and the unmet need for characterised reference materials to aid accurate molecular diagnosis. With this in mind, a panel of six genotyping reference materials for Prader Willi and Angelman syndromes was developed, which should be stable for many years and available to all diagnostic laboratories. The panel comprises three Prader Willi syndrome materials (two with different paternal deletions, and one with maternal uniparental disomy (UPD)) and three Angelman syndrome materials (one with a maternal deletion, one with paternal UPD or an epigenetic imprinting centre defect, and one with a UBE3A point mutation). Genomic DNA was bulk-extracted from Epstein-Barr virus-transformed lymphoblastoid cell lines established from consenting patients, and freeze-dried as aliquots in glass ampoules. In total, 37 laboratories from 26 countries participated in a collaborative study to assess the suitability of the panel. Participants evaluated the blinded, triplicate materials using their routine diagnostic methods against in-house controls or externally sourced uncertified reference materials. The panel was established by the Expert Committee on Biological Standardization of the World Health Organization as the first International Genetic Reference Panel for Prader Willi and Angelman syndromes.

Granulocyte-Macrophage Colony Stimulating Factor Auto-Antibodies Are Produced by Lamina Propria Mononuclear Cells Isolated From Ileal Strictures of Pediatric Patients With Crohn Disease Undergoing Ileo-Cecal Resection

G A A b st ra ct s that the STAT3 risk allele would be associated with increased cellular responsiveness to IL6 stimulation as measured by STAT3 tyrosine phosphorylation. Methods: B cells were isolated by magnetic bead columns from peripheral blood samples obtained from 69 pediatric IBD patients and transfected with EBV to create immortalized EBV-transformed lymphoblastoid cell lines (EBLs). Patients were genotyped for the STAT3 rs744166 and JAK2 rs10758669 risk alleles. EBLs selected for experimentation included STAT3 AA or GG (A=risk) while including only those which were also JAK2 AA (homozygote for the non-risk allele). EBLs were cultured overnight in serum-free media with 1x106 cells/mL and then stimulated with 100 ng/mL IL-6 for 30 minutes prior to analysis. Analyses included flow cytometry for cell surface IL-6 receptor and Glycoprotein130 (GP130) and intracellular pSTAT3 and pSTAT1; Real Time Polymerase Chain Reaction (RT-PCR) for STAT3, JAK2, and GP130; and western blot for nuclear pSTAT3 and pSTAT1, cytosolic STAT3, STAT1, JAK2, GP130, and IL6 receptor, and membrane fraction JAK2. Protein bands were quantified by normalized chemoluminescent arbitrary units (AU) via LAS Image Reader and MultiGauge Software (Fujifilm®). Differences were tested using unpaired and paired t-tests in GraphPad Prism Software®. Results: Pediatric IBD patient demographics did not vary by STAT3 genotype (age 15.6 vs 14.3 years, percent male 50 vs 40, CD patients 2 vs 2, UC patients 8 vs 6). Neither cell surface IL-6 receptor or GP130; cytosolic abundance of STAT3, STAT1, JAK2, GP130, IL-6 receptor; nor messenger RNA expression of STAT3, JAK2, or GP130 varied by STAT3 genotype. The mean nuclear protein pSTAT3 AU following IL-6 stimulation was 31.5 AU in EBLs from patients homozygous for the STAT3 risk allele compared to 19.1 AU in those lacking the STAT3 risk allele (p=0.04). The mean nuclear protein pSTAT1 AU following IL-6 stimulation was inversely 2.8 AU in EBLs from patients homozygous for the STAT3 risk allele compared to 4.3 AU in those lacking the STAT3 risk allele (p=0.001). The mean membrane fraction JAK2 AU was 80.2 AU in EBLs from patients homozygous for the STAT3 risk allele compared to 24.9 AU in those lacking the STAT3 risk allele (p= 0.008). Conclusions: The STAT3 genetic variant which increases risk for IBD is associated with increased JAK2 membrane fraction abundance and increased IL-6 dependent STAT3 tyrosine phosophorylation in EBV-transformed lymphocytes. These differences in cell signaling may promote IBD risk via STAT3 dependent effects upon T-lymphocyte differentiation and survival.

Variants in ZNF365 isoform D are associated with Crohn's disease

ObjectiveGenome-wide association studies have identified multiple Crohn's disease (CD) susceptibility loci, including association with non-coding intergenic single-nucleotide polymorphisms (SNPs) at 10q21.DesignTo fine-map the 10q21 locus, the authors genotyped 86 SNPs...

The NP9 protein encoded by the human endogenous retrovirus HERV‐K(HML‐2) negatively regulates gene activation of the Epstein‐Barr virus nuclear antigen 2 (EBNA2)

Epstein-Barr virus (EBV) is a human tumour virus that efficiently growth-transforms primary human B-lymphocytes in vitro. The viral nuclear antigen 2 (EBNA2) is essential for immortalisation of B-cells and stimulates viral and cellular gene expression through interaction with DNA-bound transcription factors. Like its cellular homologue Notch, it associates with the DNA-bound repressor RBPJκ (CSL/CBF1) thereby converting RBPJκ into the active state. For instance, both EBNA2 and Notch activate the cellular HES1 promoter. In EBV-transformed lymphocytes, the RNA of the NP9 protein encoded by human endogenous retrovirus HERV-K(HML-2) Type 1 is strongly up-regulated. The NP9 protein is detectable both in EBV-positive Raji cells, a Burkitt's lymphoma cell line, and in IB4, an EBV-transformed human lymphoblastoid cell line. NP9 binds to LNX that forms a complex with the Notch regulator Numb. Therefore, the function of NP9 vis-à-vis Notch and EBNA2 was analysed. Here, we show that NP9 binds to EBNA2 and negatively affects the EBNA2-mediated activation of the viral C- and LMP2A promoters. In contrast, NP9 did neither interfere in the activation of the HES1 promoter by Notch nor the induction of the viral LMP1 promoter by EBNA2. In an electrophoretic mobility shift analysis, NP9 reduced the binding of EBNA2 to DNA-bound RBPJκ by about 50%. The down-regulation of EBNA2-activity by NP9 might represent a cellular defence mechanism against viral infection or could, alternatively, represent an adaptation of the virus to prevent excessive viral protein production that might otherwise be harmful for the infected cell.

MicroRNA signatures associated with immortalization of EBV-transformed lymphoblastoid cell lines and their clinical traits

MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) and associated clinical traits. Hence, we performed miRNA microarray approach with early- (p6) and late-passage (p161) LCLs. Microarray data showed that nine miRNAs (miR-20b*, miR-28-5p, miR-99a, miR-125b, miR-151-3p, miR-151:9.1, miR-216a, miR-223* and miR-1296) were differentially expressed in most LCLs during long-term culture. In particular, miR-125b was up-regulated in all the tested late-passage LCLs. miR-99a, miR-125b, miR-216a and miR-1296 were putative negative regulators of RASGRP3, GPR160, PRKCH and XAF1, respectively, which were found to be differentially expressed in LCLs during long-term culture in a previous study. Linear regression analysis showed that miR-200a and miR-296-3p correlated with triglyceride and HbA1C levels, respectively, suggesting that miRNA signatures of LCLs could provide information on the donor's health. In conclusion, our study suggests that expression changes of specific miRNAs may be required for terminal immortalization of LCLs. Thus, differentially expressed miRNAs would be a potential marker for completion of cell immortalization during EBV-mediated tumorigenesis.

Population Differences in the Rate of Proliferation of International HapMap Cell Lines

The International HapMap Project is a resource for researchers containing genotype, sequencing, and expression information for EBV-transformed lymphoblastoid cell lines derived from populations across the world. The expansion of the HapMap beyond the four initial populations of Phase 2, referred to as Phase 3, has increased the sample number and ethnic diversity available for investigation. However, differences in the rate of cellular proliferation between the populations can serve as confounders in phenotype-genotype studies using these cell lines. Within the Phase 2 populations, the JPT and CHB cell lines grow faster (p < 0.0001) than the CEU or YRI cell lines. Phase 3 YRI cell lines grow significantly slower than Phase 2 YRI lines (p < 0.0001), with no widespread genetic differences based on common SNPs. In addition, we found significant growth differences between the cell lines in the Phase 2 ASN populations and the Han Chinese from the Denver metropolitan area panel in Phase 3 (p < 0.0001). Therefore, studies that separate HapMap panels into discovery and replication sets must take this into consideration.

Curcumin Down-Regulates Cytokine-Mediated Tissue Factor and Plasminogen Activator Type 1 Expression In Human Endothelial Cells

AbstractAbstract 3333Curcumin (diferuloyl methane), an active component of the spice turmeric, has been shown to exhibit anti-inflammatory and antioxidant activities in addition to an anticartinogenic activity in vitro and in vivo. Recent studies reported that curcumin acts as a non-stressful and non-cytotoxic inducer of the cytoprotective heme oxigenase-1 (HO-1). HO-1 is an inducible enzyme which degrades oxidative heme into biliverdin, free iron, and carbon monoxide. The first human case of HO-1 deficiency was described from Japan by Yachie, et al (J Clin Invest 1999; 103:129). This HO-1 deficient child exhibited severe persistent endothelial damage and extremely hyper-coagulable state. These findings prompted us to study roles of HO-1 in the regulation of coagulability. Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) derived from the HO-1 deficient patient was not able to produce HO-1 production at all. In comparison to control LCLs, the HO-1 deficient LCLs expressed markedly higher levels of tissue factor (TF) mRNA and protein (2-fold, p<0.05) as well as of plasminogen activator inhibitor type 1 (PAI-1) (2-fold, p<0.05), upon exposure to oxidative stress such as hemin, suggested important roles of HO-1 or HO-1 products in the regulating of coagulation and fibrinolytic system. We then hypothesized that endothelial HO-1 induced by curcumin may ameliorate hyper-coagulable state associated with inflammation or oxidative stress. The exposure of human umbilical vein endothelial cells (HUVECs) to curcumin (15 mM) for 6 hr resulted in a significant increase in the level of both HO-1 mRNA (15-fold) and protein. The treatment of HUVECs with 15 mM curcumin for 3hr before stimulation with tumor necrosis factor-α (TNF-α, 10 ng/ml) for additional 5hr markedly inhibited TNF-a-induced expression of TF (approximately 90 % suppression) and PAI-1 (approximately 70 % suppression) (Figure1). The inhibitory effect of curcumin on the TF expression was partially abrogated by tin protoporphyrin IX (SnPP) (20 mM), an inhibitor of heme oxigenase activity, indicating that the curcumin-induced suppression of TF is partly mediated by HO-1 (Figure 1). Moreover, curcumin inhibited TNF-a-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK), indicating the inhibition of these signal pathways may also be responsible for the suppression of TF/PAI-1 expressions in HUVECs by curcumin. In conclusion, these results indicate that curcumin exerts anti-thrombotic effects directly and indirectly through the induction of HO-1. Curcumin may serve as a novel anti-thrombotic agent for the treatment of hyper-coagulable state or thrombus formation associated with inflammation.Figure 1. Effects of curcumin on TNF-α-induced TF and PAI-1 protein (A) and mRNA (B) expression in HUVECs. The columns represent the mean ± SD (n=3) percentages of the control culture. *p<0.05 versus TNF-α alone. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Resveratrol, a Natural Antioxidant From Grapes and Red Wines, Prevents EBV-Associated Lymphoproliferation and Transformation through Inducing Apoptosis

AbstractAbstract 2543Epstein-Barr virus (EBV) infection and reactivation is an increasing complication in immune deficient patients, particularly after allogeneic hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). Whereas tapering of immunosuppression, donor lymphocyte infusion, and rituximab may be effective for this complication after HSCT, severe adverse events such as fatal GVHD and infections could develop thereafter. Resveratrol, a natural antioxidant compound found in grapes and red wines, possesses various biological activities including antitumor, anti-inflammatory, and antimicrobial efficacies “(Nature Reviews Drug Discovery 5, 493–506, 2006). We report here anti-EBV effects of resveratrol in eliminating EBV infected cells and preventing EBV-associated B-cell transformation. Resveratrol, at nontoxic concentrations to normal cells, effectively prevented the transformation of primary B cells derived from a panel of 16 healthy donors as well as cord blood mononuclear cells derived from 10 different donors. In addition, resveratrol induced dose-dependent apoptosis, assessed by FACS-based detection of Annexin V, in 7 different established EBV-transformed lymphoblastoid cell lines (LCLs) and in the Burkitt lymphoma EBV+ Raji and Daudi cell lines. These effects were consistently associated with the activation of caspase-8 and a 60 % down-regulation of the anti-apoptotic protein BCL-2. The addition of the caspase-8 inhibitor c-FLIPL to the cell culture vigorously inhibited the resveratrol induced apoptosis. Moreover, the resveratrol treatment decreased by 80% the EBV nuclear antigen 1 (EBNA1) expression, a critical protein required for the persistency of EBV infection. These results suggest that resveratrol may have potential roles in preventing EBV-associated lymphoproliferative diseases in immune compromised patients. Since resveratrol is a nontoxic, inexpensive and clinically available agent, it may be useful in preventing EBV- infection and reactivation after HSCT and SOT. Disclosures:No relevant conflicts of interest to declare.