Project description:In this study, we extend array CGH technology by making the accurate detection of segmental aneusomies possible from a single lymphoblast and fibroblast following Phi29 DNA polymerase amplification Array CGH experiments were performed on two segmental aneusomic cell lines derived from patients. As a proof of principle, an interstitial 4q-deletion (46, XX, del(4)(q13.1q22.3)) fibroblast cell line and an unbalanced reciprocal translocation involving chromosomes 14 and X (46, XX, der (X) t(X;14)(q21.3;q23.1)) EBV cell line were used. Array CGH experiments were performed using gDNA from the two cell lines to define the exact size of each rearrangement. For the 4q-deletion cell line, the size of the deleted region was 34 Mb corresponding to 39 clones spotted on the array (from RP11-340A13 to RP11-44P19). For the EBV cell line, the size of the 14q-duplication was 47 Mb corresponding to 63 clones (from RP11-62H20 to CTC-820M16) and the Xq-deletion was 58 Mb corresponding to 70 clones (from RP3-380C13 to RP11-218L14). For each cell line, three single cells were amplified. Following DNA amplification, all cells showed the expected DNA yields (n=6; ï?³ = 2.36 µg ï?± 0.12). Sex-mismatch array CGH experiments were conducted on amplified DNA samples obtained from the 4q-deletion cell line. Using the chromosome specific threshold obtained from our 18 experiments, sex chromosome and autosome ploidy levels were accurately identified with no false negative and no false positive results. Averaging the 39 clones within the chromosome 4q-deleted region enabled the accurate detection of the deletions. For each of the three amplified single-cell DNA of the unbalanced reciprocal translocation involving chromosomes 14 and X, male and female gDNA were used as references. Sex chromosome and autosome ploidy levels were accurately identified. Averaging intensity ratios of the 63 clones within the chromosome 14 duplicated region enabled the accurate detection of the duplications in the three replicate experiments. When, respectively, male or female gDNA were used as a reference, the average log2 mean ratio of the Xq-deleted region was â??0.04 or â??0.93, close to the theoretical expected value of 0 or minus 1. Interestingly, the log2 mean ratios of chromosomes 1 to 22 were highly similar when the same single cell amplified DNA was used for the two experiments using respectively female and male DNA as a reference. Hence, array CGH intensity ratio profiles were very reproducible.
Project description:In this study, we extend array CGH technology by making the accurate detection of chromosomal imbalances possible from a single fibroblast and blastomere following Phi29 DNA polymerase amplification. Firstly, array CGH experiments were performed on four different fibroblast cell lines, derived from patients affected by, respectively, trisomy 13, 18, 21, and monosomy X. For each cell line, three single cells were amplified. Following DNA amplification, all cells showed the expected DNA yields (n=12; 1.87 µg plus:minus 0.39). Sex-mismatch array CGH experiments were conducted on amplified DNA samples obtained from each cell. Sex chromosome ploidy levels, as well as all expected autosomal abnormalities were clearly identified. Secondly, we applied single-cell aneuploidy screening for the detection of chromosomal imbalances in preimplantation embryos. DNA from blastomeres from three 7-8 cell-stage embryos was amplified by ï¦29 DNA polymerase. Following DNA amplification, all cells yielded the expected amount of DNA (n=16; 2.45 µg plus:minus 0.41). Chromosomal aneuploidies were accurately detected using a simple and rapid array CGH protocol.
Project description:Nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia and southern China. The primary treatment for NPC is radiotherapy. Despite of the encouraging results of radiotherapy, local recurrence or distant metastases of NPC after the initial therapy is frequently found due to radioresistance. Therefore, there is an urgent need to identify genes that control radiosensitivty, aiming to reduce disease recurrence. Epstein-Barr virus (EBV) infection was closely associated with undifferentiated NPC. EBV-encoded microRNAs (miRNAs) played crucial roles in the pathogenesis of NPC. Ebv-miR-BART7 belongs to the 44 EBV BART miRNAs and was found to be up-regulated in NPC tissues and plasma. Forced expression of ebv-miR-BART7 enhanced the radiosensitivity of NPC cells. However, the mechanisms underlying the sensitizing effect of ebv-miR-BART7 on radiation remain largely unknown. Given that miRNA exerts biological functions by regulating its targets, we used microarray to identify the targets of ebv-miR-BART7 that regulate radiosensitivity of NPC cells.
Project description:We proposed that the frequency of EBV reactivation may be crucial for its pathogenic role and highly recurrent EBV reactivations exert a profound influence on genome instability. Recurrent reactivations were induced in the EBV-latently infected NPC cells (NA cells) by treating with 12-o-tetradecanoylphorbol-13-acetate (TPA) and sodium n-butyrate (SB) once per passage periodically. Genome-wild oligoarray-based comparative genomic hybridization (CGH) technology was applied to investigate the copy-number aberrations in response for different frequencies of EBV reactivation. The results showed that the NR15 cells, NA cells with the highest frequency (15 times) of reactivation, exhibit extensive genomic copy-number alterations (CNAs) mostly involving chromosome 3p, 3q, 8p, 8q, 9p and 9q, whereas limited number of CNAs were observed both in NR1 cells where only the initial reactivation take place and NP15 cells which were culture in parallel with NR15 cells without EBV-reactivation. We concluded that it is the highly recurrent reactivations of EBV, but neither just a primary reactivation nor EBV-latent infection, may intimately involve in carcinogenesis of nasopharyngeal epithelial cells with the progressive genome instabilities and the accumulation of genetic mutations. Experiment Overall Design: Samples of four sources were analyzed. The NP1 cells denoted the EBV-latently infected nasopharyngeal carcinoma cell lines, NA cells, with low-passage-number and mock-treated. The NR1 denoted the NA cells harboring one time of EBV reactivation. The NR15 cells denoted the NA cells harboring fifteen times of EBV reactivation. The NP15 cells denoted the NA cells cultured in parallel with NR15 (experienced 15 times of passages) without EBV-reactivation. Genomic DNAs extracted from NP15, NR1 and NR15 cells were used as experimental samples and subjected to Cy5-labeling reactions. Genomic DNAs extracted from NP1 cells were used as the common reference samples subjected to Cy3-labeling reactions. All three experiments were verified by dye-swap design where the targets labeling conditions were exchanged, NP1 labeled with Cy5, and NP15, NR1 or NR15 labeled with Cy3.
Project description:EBV-positive cell lines were assayed for expression of EBV miRNAs. The names of the miRNAs are from miRBase from Fall 2007. Microarray probes are tandem complements of the mature miRNA sequence. We assayed Burkitt's lymphoma (BL), Nasopharyngeal carcinoma, post-transplant lymphoproliferative disease (PTLD), primary effusion lymphoma, and lymphoblastoid cell lines. We also assayed primary B cells that were infected with the B95-8 strain of EBV, which was found to express EBV miRNAs as early as 20 hours post infection. We have found PTLD and BLs from HIV-positive donors both express EBV miRNAs. These types of cell lines have not previously been found to express viral miRNAs. We have found that cells that support type I and type II latency express only the BART miRNAs, whereas cells that support type III latency express BART and BHRF1 miRNAs. Furthermore, BL cell lines that spontaneously lose EBV express levels of the viral miRNAs that are at least 5-fold lower than cell lines that do not lose EBV. In total, 48 samples have been assayed and included in this study. EBV-negative control samples are not included in this data set, but raw and processed data may be requested from the contributors. These EBV-negative cell lines include the Burkitt's lymphoma cell lines, BJAB and Akata-negative, the gastric carcinoma cell line, AGS, and uninfected primary B cells. Of the 48 samples, we have assayed 22 different EBV-positive cell lines and 4 different time points after infection of primary B cells with EBV. Replicates of the majority of cell lines is included in this data set. Replicates are from independent RNA isolations that were then hybridized to individual microarrays.
Project description:Differential gene expression in RNA isolated from stably-transfected EBERs-negative versus EBERs-positive HK1 cell lines We established stable transfection of EBERs in the EBV-negative NPC cell line, HK1,to elucidate the role of the EBERs in NPC pathogenesis. Microarray gene expression profiling was carried out to investigate candidate genes which expression correlates with the expression of the EBERs.
Project description:Latent infection with Epstein-Barr virus (EBV) is recognised as a factor in the pathogenesis of nasopharyngeal carcinoma (NPC). We found that EBV encoded Latent membrane protein 2A (LMP2A) enhances lipid accumulation significantly in NPC cells. We used microarrays to identify differential genes regulated by LMP2A in NPC cell lines.
Project description:Nasopharyngeal carcinoma (NPC) is a unique malignancy derived from the epithelium of the nasopharynx. Despite great advances in the development of radiotherapy and chemotherapy, relapse and metastasis in NPC patients remain major causes of mortality. Evidence accumulated over recent years indicates that Epstein-Barr virus (EBV) lytic replication plays an important role in the pathogenesis of NPC and inhibition of EBV reactivation is now being considered as a goal for the therapy of EBV-associated cancers. With this in mind, a panel of dietary compounds was screened and emodin was found to have potential anti-EBV activity. Through Western blotting, immunofluorescence, and flow cytometric analysis, we show that emodin inhibits the expression of EBV lytic proteins and blocks virion production in EBV- positive epithelial cell lines. In investigating the underlying mechanism, reporter assays indicated that emodin represses Zta promoter (Zp) and Rta promoter (Rp) activities, triggered by various inducers. Mapping of the Zp construct reveals that the SP1 binding region is important for emodin-triggered repression and emodin is shown to be able to inhibit SP1 expression, suggesting that it likely inhibits EBV reactivation by suppression of SP1 expression. Moreover, we also show that emodin inhibits the tumorigenic properties induced by repeated EBV reactivation, including micronucleus formation, cell proliferation, migration, and matrigel invasiveness. Emodin administration also represses the tumor growth in mice which is induced by EBV activation. Taken together, our results provide a potential chemopreventive agent in restricting EBV reactivation and NPC recurrence.