Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis¹. LCL have been used to present antigens in a variety of immunologic assays^{2, 3}. In addition, LCL can be used to generate human monoclonal antibodies^{4, 5} and provide a potentially unlimited source when access to primary biologic materials is limited^{6, 7}.A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide⁸, and pokeweed mitogen⁹ to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells^{7, 10-12}.The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23^hiCD58⁺ cells observed as early as three days post-infection indicates a successful outcome.     

Epstein-Barr Viral Antigen in Single Cell Clones of Two Human Leukocytic Lines

A method was devised for obtaining single cell clones of human leukocytic cell lines in the presence of a human placental cell feeder layer. Clones of two lines, LS-B and EB₃, which contain Epstein-Barr viral (EBV) antigen in approximately 1% of cells were tested for EBV antigen. Since all EB₃ clones and LS-B clones contained EBV antigen, it is concluded that in vitro EBV genome is associated with all of the cells.     

Host Genetic Variants and Gene Expression Patterns Associated with Epstein-Barr Virus Copy Number in Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs.     

MiRNA Profiles in Lymphoblastoid Cell Lines of Finnish Prostate Cancer Families

BackgroundHeritable factors are evidently involved in prostate cancer (PrCa) carcinogenesis, but currently, genetic markers are not routinely used in screening or diagnostics of the disease. More precise information is needed for making treatment decisions to distinguish aggressive cases from indolent disease, for which heritable factors could be a useful tool. The genetic makeup of PrCa has only recently begun to be unravelled through large-scale genome-wide association studies (GWAS). The thus far identified Single Nucleotide Polymorphisms (SNPs) explain, however, only a fraction of familial clustering. Moreover, the known risk SNPs are not associated with the clinical outcome of the disease, such as aggressive or metastasised disease, and therefore cannot be used to predict the prognosis. Annotating the SNPs with deep clinical data together with miRNA expression profiles can improve the understanding of the underlying mechanisms of different phenotypes of prostate cancer.ResultsIn this study microRNA (miRNA) profiles were studied as potential biomarkers to predict the disease outcome. The study subjects were from Finnish high risk prostate cancer families. To identify potential biomarkers we combined a novel non-parametrical test with an importance measure provided from a Random Forest classifier. This combination delivered a set of nine miRNAs that was able to separate cases from controls. The detected miRNA expression profiles could predict the development of the disease years before the actual PrCa diagnosis or detect the existence of other cancers in the studied individuals. Furthermore, using an expression Quantitative Trait Loci (eQTL) analysis, regulatory SNPs for miRNA miR-483-3p that were also directly associated with PrCa were found.ConclusionBased on our findings, we suggest that blood-based miRNA expression profiling can be used in the diagnosis and maybe even prognosis of the disease. In the future, miRNA profiling could possibly be used in targeted screening, together with Prostate Specific Antigene (PSA) testing, to identify men with an elevated PrCa risk.     

Genetic Analysis of Human Traits In Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype–phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance—i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.     

Rescue of Epstein-Barr Virus from Somatic Cell Hybrids of Burkitt Lymphoblastoid Cells

A Burkitt lymphoblastoid cell line, P3J-HR-1, was fused and hybridized to a human sternal marrow cell line. The somatic cell hybrids were negative when examined for Epstein-Barr virus (EBV) markers. When the hybrid cells were exposed to 5-iododeoxyuridine, both EBV-specific antigens and virus particles were induced as determined by the immunofluorescence test and by electron microscopy. The data presented suggest that the EBV genome can be transferred from a lymphoblastoid cell to another cell type during cell hybridization, that the EBV genome can persist in the hybrid cells for long periods of time, and that synthesis of the virus can be induced in the heterokaryons.     

The Effect of Freeze-Thaw Cycles on Gene Expression Levels in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.     

47例染色体异常患者永生淋巴细胞株的建立

采用4种不同的血细胞/EB病毒（Epstein-Barr Virus,EBV）比率系列转染冻存血方法建立永生淋巴细胞株,建株成功率达97.87%,并成功地对47例染色体异常患者建立了永生淋巴细胞株,为今后不断进行的深入研究奠定了基础。     

Isolation of Cancer Stem Cells from Three Human Glioblastoma Cell Lines: Characterization of Two Selected Clones

Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca²⁺ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca²⁺-channels but they exhibited increased intracellular Ca²⁺ levels in response to ATP. These Ca²⁺ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca²⁺-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.     

中国10个民族永生细胞系的建立与保存

永久保存我国各少数民族的遗传信息是中国人类基因组计划的重要内容,为此,采用EB病毒(epstein-barr Svirus EBV)上清液及Hepes转化外周血B淋巴细胞,并加入环孢霉素A(cyclosporine A)抑制T淋巴细胞,成功地对中国哈萨克族、满族、朝鲜族、赫哲族、蒙古族、锡伯族、回族、布依族、四川汉族和福建汉族的部分个体建立了永生细胞系。其中哈萨克族64株,朝鲜族58株,赫哲族18株,锡伯族43株,回族63株,布依族67株,满族65株,蒙古族62株,四川汉族51株,福建汉族58株。总计549株。为保存我国各民族遗传资源、分析各少数民族间的遗传学差异及其起源,奠定了材料基础。 Establishment and Preservation of Immortal Lymphoblastoid Cell Lines of the 10 Ethnic Groups in China HUANG Xiao-yi,LIU An,YU Yang,MA Lin-lin,SHI Rong-qian,Lü Fu-qu,JIANG Yan,SUN Wen-jing,XUE Ya-li,FU Song-bin,LI Pu Department of Medical Genetics,Harbin Medical University,Harbin 150086,China Abstract:The immortal lymphoblastoid cell lines were established by EBV transformation of B cells and addition of cyclosporin A to inhibit the activity of T cells.In the present study,549 immortal cell lines of different ethnic groups of Hazak,Manchu,Korea,Hozhe,Mongolia,Sibe,Hui, Puyi,Han in Fujian and Han in Sichuan were established.Through our research,we found it is harmful for B lymphocytes to transform if excessful leucocytes are inoculated.And it is crisis that cyclosporine A shoud be added the last.Our work is an important part of the research of human genome diversity for the exploration of the origin and evolution of different ethnic groups,and it also provides enough research materials for further studies.Moreover,we have sent 50 cell lines of Hozhe,Mongolia,Sibe,Daur,Oroqen to CEPH.Thus it is possible for us to utilize the genetic resources of CEPH freely. Key words：immortal lymphoblastoid cell line;EBV;ethnic groups;reserved in liquid-nitrogen     

非综合征耳聋大家系永生细胞系的建立及几种EB病毒转化外周血淋巴细胞建系方法的探讨

永久保存珍贵的家系材料,是对该家系进行深入研究的基础,为此采用EB病毒（Epstein-Barr virus,EBV）转化淋巴细胞的方法对中国江苏淮阴地区非综合征耳聋大家系行建系工作。该家系患者呈典型的母系遗传特征,且研究发现患者中均具有线粒体DNA 12s RNA A1555G突变,是迄今世界上最大的非综合征耳聋家系之一,在该家系的建系过程中 使用了4种不同的方法。建系结果分别为：微量全血法1株,冻存全血法1株,冻存白细胞法14株及环孢霉素A（CyA）法36株,共计52株。本文就建系工作及这四种转化方法作一简单探讨。     

流行性出血热病毒在人B淋巴母细胞中复制增殖

本文应用细胞培养、免疫荧光及电镜技术研究了B淋巴母细胞对EHF病毒的易感性。结果表明EHF病毒可在该细胞中增殖。感染细胞无明显细胞病变,在形态上与对照组无差别。虽然大部分感染细胞呈现明亮病毒抗原荧光,但在电镜下却难以找到完整的病毒颗粒,仅在扩张的囊泡中发现一些性质待定的微丝样物质。人B淋巴母细胞持续感染的建立,提示患者外周血中大量出现的异型淋巴细胞可能允许EHF病毒在其中复制。     

Amounts of Epstein-Barr Virus DNA in Somatic Cell Hybrids Between Burkitt Lymphoma-Derived Cell Lines

The amounts of Epstein-Barr virus DNA in somatic cell hybrids between human lymphoid cell lines were found to be higher than in the parental cell lines, although the hybrids were not induced to spontaneous virus production.