Cytogenetic characterization of eight human lymphoblastoid cell lines

Cytogenetic analyses of eight human lymphoblastoid cell lines that were established by a new procedure using B-cell growth factor (BCGF) and interleukin-4 (IL-4) revealed that three cell lines (37.5%) showed structural anomalies in more than 97% of their metaphases, whereas others were predominantly normal diploid. Our results indicate that a) the structural anomalies seen in three cell lines were not induced by culture technique but were constitutional defects present in donors' peripheral blood samples; b) one donor, whose blood gave rise to cell line BCDI, is a constitutional mosaic because both normal diploid and altered metaphases were present; and c) genetic instability should be monitored in regular blood donors because some of them may harbor blood-mediated clastogen or chromosome breakage factor(s) that could induce higher rates of recombinations in somatic cells of some recipients and thus may potentiate such individuals to develop certain types of malignancies.     

Is ribosomal cistron activity increased in human lymphoblastoid cell lines?

The genomic activity of nucleolar organizer regions (NORs) in eight human B-cell lymphoblastoid cell lines was studied following the routinely used Ag-NOR technique. The results demonstrate that (a) Ag-NORs are located in the short arms of D- and G-group chromosomes, (b) two out of eight cell lines have 66.7% and 49.0% of metaphases, respectively, with 9 to 10 active Ag-NORs, and (c) as a whole, Ag-NOR activity is much higher in B-cell lines as compared with conventional 72-hr peripheral blood lymphocyte (T-cell) harvests.     

Influence of altered apoptosis in human lymphoblastoid cell lines on micronucleus frequency

Defects in apoptosis play a decisive role in both tumorigenesis and drug resistance in tumor treatment. The purpose of this study was to investigate the balance between formation of genomic damage and induction of apoptosis upon genotoxic stress. For this, we influenced the apoptotic response and measured the amount of genomic damage expressed as micronucleus formation after treatment with the topoisomerase II inhibitor etoposide. Apoptosis was reduced by the addition of pifithrin (PFT) alpha and enhanced by transient transfection with bcl-2 antisense-oligonucleotide in Bcl-2-overexpressing cells. We used three human lymphoblastoid cell lines with different p53 status (TK6, wild-type p53; WTK1, mutated p53; NH32, p53 double knockout). Under conditions of reduced apoptosis, micronucleus formation was also reduced. When apoptosis was increased, micronucleus formation remained unchanged or was also increased. Overall, we did not find an expected inverse correlation between induction of apoptosis and genomic damage.     

Autophagy is the predominant process induced by arsenite in human lymphoblastoid cell lines

Arsenic is a widespread environmental toxicant with a diverse array of molecular targets and associated diseases, making the identification of the critical mechanisms and pathways of arsenic-induced cytotoxicity a challenge. In a variety of experimental models, over a range of arsenic exposure levels, apoptosis is a commonly identified arsenic-induced cytotoxic pathway. Human lymphoblastoid cell lines (LCL) have been used as a model system in arsenic toxicology for many years, but the exact mechanism of arsenic-induced cytotoxicity in LCL is still unknown. We investigated the cytotoxicity of sodium arsenite in LCL 18564 using a set of complementary markers for cell death pathways. Markers indicative of apoptosis (phosphatidylserine externalization, PARP cleavage, and sensitivity to caspase inhibition) were uniformly negative in arsenite exposed cells. Interestingly, electron microscopy, acidic vesicle fluorescence, and expression of LC3 in LCL 18564 identified autophagy as an arsenite-induced process that was associated with cytotoxicity. Autophagy, a cellular programmed response that is associated with both cellular stress adaptation as well as cell death appears to be the predominant process in LCL cytotoxicity induced by arsenite. It is unclear, however, whether LCL autophagy is an effector mechanism of arsenite cytotoxicity or alternatively a cellular compensatory mechanism. The ability of arsenite to induce autophagy in lymphoblastoid cell lines introduces a potentially novel mechanistic explanation of the well-characterized in vitro and in vivo toxicity of arsenic to lymphoid cells.     

The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines

Formaldehyde (FA), a major industrial chemical and ubiquitous environmental pollutant, has recently been classified by the International Agency for Research on Cancer as a human leukemogen. The major mode of action of FA is thought to be the formation of DNA–protein cross-links (DPCs). Repair of DPCs may be mediated by the Fanconi anemia pathway; however, data supporting the involvement of this pathway are limited, particularly in human hematopoietic cells. Therefore, we assessed the role of FANCD2, a critical component of the Fanconi anemia pathway, in FA-induced toxicity in human lymphoblast cell models of FANCD2 deficiency (PD20 cells) and FANCD2 sufficiency (PD20-D2 cells). After treatment of the cells with 0–150 μM FA for 24 h, DPCs were increased in a dose-dependent manner in both cell lines, with greater increases in FANCD2-deficient PD20 cells. FA also induced cytotoxicity, micronuclei, chromosome aberrations, and apoptosis in a dose-dependent manner in both cell lines, with greater increases in cytotoxicity and apoptosis in PD20 cells. Increased levels of γ-ATR and γ-H2AX in both cell lines suggested the recognition of FA-induced DNA damage; however, the induction of BRCA2 was compromised in FANCD2-deficient PD20 cells, potentially reducing the capacity to repair DPCs. Together, these findings suggest that FANCD2 protein and the Fanconi anemia pathway are essential to protect human lymphoblastoid cells against FA toxicity. Future studies are needed to delineate the role of this pathway in mitigating FA-induced toxicity, particularly in hematopoietic stem cells, the target cells in leukemia.     

Gene expression analysis identifies potential biomarkers of phenanthrene in human hepatocytes (HepG2)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquious in the environment both as natural products and as environmental contaminants. Among PAHs, phenanthrene (PH) that is ubiquitously distributed throughout the environment was subjected in this study. Although environmental distribution and metabolism of PH have been well reported, there are only a few studies examined the expression of mRNA and their functions on PH-induced toxicity. A new paradigm in toxicity screening, toxicogenomic technology represents a useful approach for evaluating the toxic properties of environmental pollutants. In this respect, we elicited the genes which were changed more than 2-fold by analysis of gene expression profiles in human hepatocellular carcinoma (HepG2) cells, exposed to PH by using human oligonucleotide chip. 913 up- and 814 down-regulated genes changed their expression by more than 2-fold and p-values 0.05 through PH exposure. Gene Ontology (GO) analysis on these genes revealed significant enrichments in the several key biological processes related to the hepatotoxicity such as cell migration, wound healing, cytoskeleton organization, microtubule-based process, apoptosis, and cell cycle checkpoint. In conclusion, the present study suggests that PH exerts its toxicity by modulating the mRNA expression in HepG2 cells. we suggest that genes expressed by PH as a molecular signature which can be used more widely implemented in combination with more traditional techniques for assessment and prediction of toxicity by exposure to PH.     

Arsenite exposure in human lymphoblastoid cell lines induces autophagy and coordinated induction of lysosomal genes

Chronic exposure to inorganic arsenic is associated with diverse, complex diseases, making the identification of the mechanism underlying arsenic-induced toxicity a challenge. An increasing body of literature from epidemiological and in vitro studies has demonstrated that arsenic is an immunotoxicant, but the mechanism driving arsenic-induced immunotoxicity is not well established. We have previously demonstrated that in human lymphoblastoid cell lines (LCLs), arsenic-induced cell death is strongly associated with the induction of autophagy. In this study we utilized genome-wide gene expression analysis and functional assays to characterize arsenic-induced effects in seven LCLs that were exposed to an environmentally relevant, minimally cytotoxic, concentration of arsenite (0.75 μM) over an eight-day time course. Arsenic exposure resulted in inhibition of cellular growth and induction of autophagy (measured by expansion of acidic vesicles) over the eight-day exposure duration. Gene expression analysis revealed that arsenic exposure increased global lysosomal gene expression, which was associated with increased functional activity of the lysosome protease, cathepsin D. The arsenic-induced expansion of the lysosomal compartment in LCL represents a novel target that may offer insight into the immunotoxic effects of arsenic.     

Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients

The thiocarbamate drug disulfiram has been used for decades in the treatment of alcohol abuse. Disulfiram induces apoptosis in a number of tumor cell lines and was recently by us proposed to act as a 26S proteasome inhibitor. In this work we characterized disulfiram in vitro with regard to tumor-type specificity, possible mechanisms of action and drug resistance and cell death in human tumor cell lines and in 78 samples of tumor cells from patients using the fluorometric microculture cytotoxicity assay and the automated fluorescence-imaging microscope ArrayScan((R)). Disulfiram induced cytotoxicity in a biphasic pattern in both cell lines and patient tumor cells. Disulfiram induced apoptosis as measured by cell membrane permeability, nuclear fragmentation/condensation and caspase-3/7 activation using high content screening assays. For many of the cell lines tested disulfiram was active in sub-micromolar concentrations. When comparing the logIC(50) patterns with other cytotoxic agents, disulfiram showed low correlation (R<0.5) with all drugs except lactacystin (R=0.69), a known proteasome inhibitor, indicating that the two substances may share mechanistic pathways. Disulfiram was more active in hematological than in solid tumor samples, but substantial activity was observed in carcinomas of the ovary and the breast and in non-small cell lung cancer. Disulfiram also displayed higher cytotoxic effect in cells from chronic lymphocytic leukemia than in normal lymphocytes (p<0.05), which may indicate some tumor selectivity. These results together with large clinical experience and relatively mild side effects encourage clinical studies of disulfiram as an anti-cancer agent.     

Microarray profiling of gene expression in human adipocytes in response to anthocyanins

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and its gene expression in adipocytes. In this study, we have shown the gene expression profile in human adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The human adipocytes were treated with 100 microM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. Based on the gene expression profile, we demonstrated the significant changes of adipocytokine expression (up-regulation of adiponectin and down-regulation of plasminogen activator inhibitor-1 and interleukin-6). Some of lipid metabolism related genes (uncoupling protein2, acylCoA oxidase1 and perilipin) also significantly induced in both common the C3G or Cy treatment groups. These studies have provided an overview of the gene expression profiles in human adipocytes treated with anthocyanins and demonstrated that anthocyanins can regulate adipocytokine gene expression to ameliorate adipocyte function related with obesity and diabetes that merit further investigation.     

多靶点激酶抑制药GNF-7对白血病细胞表达谱的影响

目的探索多靶点激酶抑制药GNF-7对白血病细胞表达谱的影响。方法从基因表达公共数据库下载表达谱数据集GSE49534,用BRB-Array Tools软件包筛选差异表达基因(DEGs),分别对差异基因进行基因本体(GO)功能分析、通路富集分析、基因互作网络分析和通路互作网络分析。结果共筛选出847个差异基因,其中上调表达基因426个,下调表达基因419个。DEGs发挥的分子功能集中在结合、蛋白激酶活性和信号转导因子活性等,主要参与信号转导、小分子代谢和细胞凋亡等生物学过程。DEGs显著富集的通路有核糖体合成、代谢通路、Janus激酶-信号转导和转录激活因子(JAK-STAT)信号通路等。网络分析挖掘出的核心基因有多核糖核苷酸核苷酸转移酶1(PNPT1)、腺苷酸激酶4(AK4)、Janus激酶2(JAK2)、信号转导和转录激活因子2(STAT2)、MYC,核心pathway包括丝裂原活化蛋白激酶(MAPK)信号通路、凋亡、细胞周期和肿瘤通路等。结论 GNF-7通过诱导凋亡和细胞周期阻滞等抑制白血病细胞。     

Differential cytotoxicity of anticancer agents in pre- and post-immortal lymphoblastoid cell lines

We studied the cytotoxic effect of various anticancer agents on lymphoblastoid cell lines transformed by Epstein-Barr virus. Post-immortal N0005 (post-N0005) is an immortalized cell line derived from pre-immortal N0005 (pre-N0005) accompanied by increased telomerase activity, short-telomere, abnormal karyotypes, mutation of p53 gene, down regulation of p16/Rb and the ability to grow in soft agar medium. Compared with pre-N0005 cells, post-N0005 cells were significantly (p<0.001 by the Student t test) more resistant to the killing activity of seven DNA-modifying agents: camptothecin, etoposide, bleomycin, fluorouracil, thioguanine, melphalan and actinomycin D. However, both pre-N0005 and post-N0005 cells showed similar levels of cytotoxicity against four DNA-non-modifying agents: colchicine, paclitaxel, vincristine and methotrexate. DNA-modifying and DNA-non-modifying agents are distinguished by their different sensitivities with pre-N0005 and post-N0005. Based on these results, we propose that pre-N0005 and post-N0005 cell lines be used as a new method to assess and screen anticancer agents.     

MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression

Oral squamous cell carcinoma, the most frequently occurring malignant head and neck tumour, generally exhibits poor prognosis and metastases are the main cause of death. The discovery of reliable prognostic indicators of tumour progression could greatly improve clinical practice. MicroRNAs are involved in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. Since miRNAs have been shown to be abnormally expressed in different tumours their importance as potential cancer prognostic indicators is increasing. To define the role of miRNA in OSCC tumours we investigated the expression profile of 15 OSCC (8 without metastasis and 7 with lymph node metastasis) using microarray analysis. Thirteen miRNA were significantly overexpressed (miR-489, miR-129, miR-23a, miR-214, miR-23b, miR-92, miR-25, miR-210, miR-212, miR-515, miR-146b, miR-21, miR-338) and 6 miRNA were underexpressed (miR-520h, miR-197, miR-378, miR-135b, miR-224, miR-34a) in oral tumours. Underexpression of mir-155, let-7i, mir-146a was found to characterize progression to metastastatic tumours. Further investigations will elucidate whether differentially expressed miRNAs will help to better classify OSCCs, thus improving diagnoses and patient care.     

Epoxide hydrolase expression in human and rodent established cell lines

Four human, four hamster and three mouse established cell lines have been analysed for epoxide hydrolase activity, and these have been compared with activities of human and mouse tissue preparations. Activities expressed by the cell lines, using styrene oxide as substrate, varied between less than 0.005 and 0.44 nmol/min per mg total cellular protein. Human liver and human kidney expressed 12.2 nmol and 2.7 nmol/min per mg total protein respectively. Antibodies prepared against purified human- and mouse-liver epoxide hydrolase enzymes were used to characterize the enzyme in the cell lines. Antihuman antibody was able to inactivate and precipitate enzyme in human cell lines and partially inactivate and precipitate hamster enzymes. It was much less effective against mouse cell lines. Antimouse antibody was able to precipitate mouse enzymes and partially precipitate hamster enzymes but did not precipitate human enzymes. These data may reflect evolutionary divergence of the three species.