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.     

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.     

含HTNV S基因重组痘苗病毒在HFRS患者B淋巴母细胞系中的表达

采用Ficoll密度梯度离心法(淋巴细胞分离液)分离肾综合征出血热(HFRS)患者外周血单个核细胞（PBMC),并用EB病毒(EBV)感染B淋巴细胞,建立永生化的B淋巴母细胞系(B—LCL)。然后,用含汉滩病毒(Hantaan virus,HTNV)S基因的重组痘苗病毒感染B—LCL,应用问接免疫荧光检测核衣壳蛋白的表达。结果表明,B淋巴细胞经EBV感染4周左右,可形成永生化B—LCL。成功转化后的B—LCL,体积增大,且增殖的淋巴细胞积聚成团。汉滩病毒S基因在B—LCL中能有效表达核衣壳蛋白。含S基因的重组痘苗病毒感染的B—LCL可用作HTNV核衣壳蛋白特异性CTL活性研究的靶细胞。     

含HTNV S基因重组痘苗病毒在HFRS患者B淋巴母细胞系中的表达

采用Ficoll密度梯度离心法(淋巴细胞分离液)分离肾综合征出血热(HFRS)患者外周血单个核细胞(PBMC),并用EB病毒(EBV)感染B淋巴细胞,建立永生化的B淋巴母细胞系(B-LCL).然后,用含汉滩病毒(Hantaan virus ,HTNV) S基因的重组痘苗病毒感染B-LCL,应用间接免疫荧光检测核衣壳蛋白的表达.结果表明,B淋巴细胞经EBV感染4周左右,可形成永生化B-LCL.成功转化后的B-LCL,体积增大,且增殖的淋巴细胞积聚成团.汉滩病毒S基因在B-LCL中能有效表达核衣壳蛋白.含S基因的重组痘苗病毒感染的B-LCL可用作HTNV核衣壳蛋白特异性CTL活性研究的靶细胞.     

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.     

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.     

Epstein-Barr Virus-associated Antigens in Non-producing Clones of Human Lymphoblastoid Cell Lines

NUCLEIC acid hybridization suggests that the Epstein-Barr virus (EBV) genome may be present in human lymphoblastoid cell lines that are free of detectable EBV^1,2. We describe here a plentiful appearance of EBV-associated early antigens (EA) and the viral capsid antigen (VCA) in non-producing Raji and NC-37 cell lines when exposed to 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine (IUdR). These antigens were synthesized in all the Raji and NC-37 clones exposed to BUdR or IUdR, strongly suggesting that a complete, but unexpressed, EBV genome exists in the cells of these non-producing lines.     

Effect of Freeze-Thaw Cycles on Bacterial Communities of Arctic Tundra Soil

The effect of freeze-thaw (FT) cycles on Arctic tundra soil bacterial community was studied in laboratory microcosms. FT-induced changes to the bacterial community were followed over a 60-day period by terminal restriction fragment length polymorphism (T-RFLP) profiles of amplified 16S rRNA genes and reverse transcribed 16S rRNA. The main phylotypes of the active, RNA-derived bacterial community were identified using clone analysis. Non-metric multidimensional scaling ordination of the T-RFLP profiles indicated some shifts in the bacterial communities after three to five FT cycles at −2, −5, and −10°C as analyzed both from the DNA and rRNA. The dominating T-RFLP peaks remained the same, however, and only slight variation was generally detected in the relative abundance of the main T-RF sizes of either DNA or rRNA. T-RFLP analysis coupled to clone analysis of reverse transcribed 16S rRNA indicated that the initial soil was dominated by members of Bacteroidetes, Acidobacteria, Alpha-, Beta-, and Gammaproteobacteria. The most notable change in the rRNA-derived bacterial community was a decrease in the relative abundance of a Betaproteobacteria-related phylotype after the FT cycles. This phylotype decreased, however, also in the control soil incubated at constant +5°C suggesting that the decrease was not directly related to FT sensitivity. The results indicate that FT caused only minor changes in the bacterial community structure.     

人TNFAIP1 基因在常见细胞系中的表达

TNFAIP1蛋白是第一个被鉴定受肿瘤坏死因子α诱导的蛋白,TNFAIP1 蛋白可能参与DNA复制、合成、细胞凋亡以及人类各种疾病的发生等重要过程,但对该基因确切功能和作用机制研究不多.该文利用实时定量PCR的方法检测该基因在几种常见细胞系的表达情况,发现在COS7以及NIH3T3细胞系中高表达,而在HeLa、HepG2、SW480、PanC1、MCF7等癌细胞系中低表达.由此推测TNFAIP1可能在癌症的发生中起到一定的作用.如果把TNFAIP1 基因克隆到真核表达载体pCMV-Myc中,转染到HeLa细胞系中,发现过表达TNFAIP1 可促进HeLa细胞的凋亡.     

AFP的亚细胞定位及对肿瘤细胞生长的影响

目的观察甲胎蛋白（AFP）在不同肿瘤细胞中的亚细胞定位及对肿瘤细胞生长的影响。方法运用免疫荧光的方法观察内源性AFP在HeI。a细胞、QGY-7703细胞、MCF-7细胞中的亚细胞定位。将构建的表达AFP的质粒pcDNA3-AFP及AFP腺病毒siRNA干涉载体Adv—AFPsiRNA作用于QGY-7703细胞,MCF-7细胞,运用M1Tr,集落形成实验检测细胞增殖状况。结果免疫荧光显示,内源性的AFP在HeLa细胞、QGY-7703细胞、MCF-7细胞均只在细胞质中表达。pcDNA3-AFP使QGY-7703的细胞活性增加了2l％（P〈0．05）及集落形成能力增加了32％（P〈0．01）,MCF-7实验组比对照组细胞活性降低了30％（P〈0．01）．克隆形成能力降低82％（P〈0．01）。Adv—AFPsiRNA使QGY-7703的细胞活性降低了22％（P〈0．05）,平均克隆形成能力降低52％（P〈0．01）,MCF-7细胞活性提高了24．5％（P〈0．05）,克隆形成能力提高了89％（P〈O．01）。结论内源性的AFP只在细胞质中表达。AFP能促进QGY-7703细胞的增殖及克隆形成能力,而在MCF-7细胞中发挥相反的作用。腺病毒介导的内源性的AFP表达的下调能降低QGY-7703的增殖,却增加了MCF-7的细胞活性及克隆形成能力。     

在肾癌细胞系GRC-1细胞周期不同阶段Wnt-5A基因的表达情况

Wnt基因所编码的蛋白质与许多生长因子一样具有分泌型生长因子的结构特点,其家族成员Wnt-5A是许多恶性肿瘤的自分泌生长因子,在肾细胞癌中表达显著升高.为研究在细胞周期的不同阶段生长因子Wnt-5A在转录水平的表达情况,我们采用胸腺嘧啶双阻断及高压笑气处理的方法,使肾细胞癌细胞系GRC-1细胞同步化.用半定量反转录多聚酶链反应对处于细胞周期不同阶段的细胞cDNA进行扩增,S期与G1,M期Wnt-5A mRNA表达存在差异显著(P＜0.05）.结果提示生长因子Wnt-5A在肾细胞癌的发生中具有潜在的作用,在S期作用可能尤为显著.     

URI基因稳定表达对肝癌SMMC-7721细胞增殖能力的影响

目的:瞬转以及筛选出能够稳定表达URI(RPB5-mediating protein)基因的SMMC-7721细胞株,以其为模型研究URI基因对人肝癌SMMC-7721细胞增殖的影响.方法:首先,抽提URI的重组质粒并转染到SMMC-7721细胞中,在G418药物的筛选下选出能够稳定表达URI基因的细胞株,RT-PCR法和酶切检测该稳定细胞中URI基因的表达效率以确认URI是否稳定表达,MTT法和克隆形成实验检测URI基因对SMMC-7721细胞增殖的影响.结果:成功建立URI基因过表达的稳定细胞株.与SMMC-7721细胞对照组比较,其URI mRNA表达水平显著上调,能稳定表达URI细胞株的增殖,克隆形成率明显升高.结论:pFLAG-CMV-4-URI重组质粒能使URI在肝癌SMMC-7721细胞内稳定表达,过表达URI基因有可能帮助细胞通过G2/M期检验点来提高肝癌细胞SMMC-7721的增殖能力.     

Characterization of MTAP Gene Expression in Breast Cancer Patients and Cell Lines

MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors´ MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNBC.     

野生型P53、P16基因协同对肺腺癌细胞系生长抑制作用的研究

为了探讨野生型P53基因及P16基因在恶性肿瘤基因治疗中的作用,用腺病毒为载体将野生型P53基因转入高、低转移的肺腺癌细胞系Anip973、AGZY83-a和经野生型P16基因质粒转染的高、低转移肺腺癌细胞系Anip973（Anip973P16）、AGZY83-a（AGZY83-aP16）。对各组转染细胞进行生长曲线、MTT生长抑制率、原位末端标记、Western-blotting等技术检测分析。结果发现（1）野生型P53蛋白的过表达对上述肺腺癌细胞系均呈现出较强的生长抑制作用。（2）野生型P53蛋白的过表达对高转移肺癌细胞系Anip973的抑制作用明显高于低转移细胞系AGZY83-a。（3）野生型p53蛋白的过表达对经野生型P16基因转染的高、低转移的肺癌细胞Anip973、AGZY83-a抑制作用明显高于未经P16基因转染的细胞。野生型P53基因可以作为肺腺癌基因治疗的候选基因。肿瘤抑制基因P53、P16的联合转染可能是对肺腺癌进行基因治疗的有效手段。 Abstract：To investigate the suppression effect of tumor suppressor genes in lung adenocarcinoma cell lines,we transferred a pair of lung adenocarcinoma cell lines with different metastasis potential,Anip973(High-metastasis potential cell line) and AGZY83-a (Low-metastasis potential cell line)and this pair of cell lines transfected with P16 gene:AGZY83-a P16 and Anip973 P16 with wild type P53 gene with adenovirus vector.The suppression effects of P53 gene were evaluated by cell growth curve,MTT,western-blotting analysis and TUNEL technique.Overexpression of wild-type P53 gene in AGZY83-a,Anip973,Anip973 P16 and AGZY83-a P16 inhibited the growth of these four kinds of lung cancer cells and induced apoptosis of the cells.The suppression effect of P53 gene in Anip973 and Anip973 P16 was higher than AGZY83-a and AGZY83-a P16 while co-expression of P53 and　P16 in this pair of cell lines inhibited the cells more efficiently comparing with the expression of P53 alone.Wild-type P53 gene might act as a candidate gene in lung adenocarcinoma gene therapy while co-transfection of P53 and P16 genes was a more effective method.     

The Effect of Proteases on Gene Expression and Cell Differentiation in Dictyostelium discoideum

We have examined the effects of chymotrypsin or pronase on the differentiation of monolayers of Dictyostelium discoideum amoebae developing in the presence of 1–5 mM cyclic AMP. Using sporogenous mutants, which are capable of forming both spores and stalk cells under these conditions, we have observed that low concentrations of either protease selectively inhibit a late step of spore formation. Higher levels of the proteases act at an earlier time and by a distinct mechanism to reduce the accumulation of the prespore cell specific enzyme UDP galactose polysaccharide transferase while not affecting the appearance of glycogen phosphorylase. The latter is present in both prestalk and prespore cells.     

DNA甲基化抑制鼻咽癌细胞系膜联蛋白A1基因表达

为了研究不同分化程度和转移潜能鼻咽癌(NPC)细胞系膜联蛋白A1(ANXA1)mRNA和蛋白质表达情况及其与基因甲基化的关系．培养NPC细胞系CNE1、CNE2、5-8F、6-10B和永生化非癌性人鼻咽黏膜上皮细胞NP69细胞用于实验,用甲基化特异性聚合酶链反应(MSP)方法检测ANXA1基因甲基化状态,同时利用逆转录-聚合酶链反应(RT-PCR)方法检测ANXA1基因的mRNA表达水平．然后用不同浓度的5-杂氮-2′-脱氧胞苷(5-aza-2dC)对NPC细胞进行去甲基化处理,MSP和RT-PCR方法检测处理组和对照组细胞ANXA1基因甲基化状况和mRNA表达水平,并用Western-blotting方法检测ANXA1基因蛋白质表达水平．结果发现,NP69细胞ANXA1基因无甲基化,4株NPC细胞系ANXA1基因都存在不同程度的甲基化,甲基化程度与细胞的分化程度和转移潜能相关．NPC细胞ANXA1基因mRNA表达水平降低,低于NP69细胞,其降低的程度与基因的甲基化程度相关．5-aza-2dC能够剂量依赖性地引起ANXA1基因去甲基化,经去甲基化处理后,NPC细胞系ANXA1基因的mRNA和蛋白质的表达水平相应提高．研究证明,NPC细胞系ANXA1基因的mRNA和蛋白质表达水平出现下调,甲基化是导致表达下调的主要原因,5-aza-2dC去甲基化处理能够恢复ANXA1基因的表达水平．     

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。