Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

Cellular gene expression that correlates with EBER expression in Epstein-Barr Virus-infected lymphoblastoid cell lines

Novel Epstein-Barr Virus (EBV) strains with deletion of either EBER1 or EBER2 and corresponding revertant viruses were constructed and used to infect B lymphocytes to make lymphoblastoid cell lines (LCLs). The LCLs were used in microarray expression profiling to identify genes whose expression correlates with the presence of EBER1 or EBER2. Functions of regulated genes identified in the microarray analysis include membrane signaling, regulation of apoptosis, and the interferon/antiviral response. Although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cell gene expression were greater with EBER2 deletion. In this system, deletion of EBER1 or EBER2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting LCLs. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed, but in LCLs almost all of the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not detectably changed by EBER1. The changes in LCL gene expression identified here will provide a basis for identifying the mechanisms of action of EBER RNAs.     

Genetic instability in EBV-transformed lymphoblastoid cell lines

Epstein Barr virus (EBV)-transformed lymphoblastoid cell lines are commonly used to provide an inexhaustible supply of DNA. We examined microsatellite instability in these cell lines in 35 individuals where DNA was available from the original blood samples and from cultured cell lines. Mutations were observed in 0.3% of the analyses, thus providing a quantitative measure of somatic mutation rate.     

Cytogenetic analysis of lymphoblastoid cell lines

Cytogenetic abnormalities were discovered in more than half of 16 lymphoblastoid cell lines established from fragile X individuals and their relatives upon routine cytogenetic analysis of early passage cultures. Subsequently, a second series of lymphoblastoid lines was analyzed to determine if the aneuploidy was a characteristic of lymphoblastoid cell lines derived from fragile X families or a result of the use of cyclosporin A in the establishment of these lines. In the second series of 33 lymphoblastoid cultures, no aneuploid clones were found in the fragile X group, while two were detected in the control cultures, one in a line initiated with cyclosporin A and the other in a line established without cyclosporin A. We conclude that the abnormal clones in our preliminary series were not a characteristic of lines derived from fragile X families and probably not due to the use of cyclosporin A. However, the finding of chromosome abnormalities in a large proportion of lines during the first 3 mo of culture contrasts with previous reports of chromosome stability for the first 12-18 mo of cultivation and indicates that the chromosomes of lymphoblastoid lines should be monitored in any experiment in which a normal diploid complement is critical.     

Erythropoietin gene expression in haemopoietic cell lines

Erythropoietin (Epo) gene expression was studied in a number of different haemopoietic cell lines by in situ hybridization and Northern Blot analysis using a radioisotope-labelled monkey Epo DNA probe. A positive message was expressed by a human cell line, CM-S, derived from a patient with congenital hypoplastic anemia, and by a murine erythro-leukaemic cell line, clone 707, derived from the spleen of Friend virus-infected mice. No message was detected in two megakaryoblastic cell lines, and in a monocytic cell line, derived from a patient with acute monocytic leukaemia. These data may fit with the hypothesis of expression of Epo and other growth factors by haemopoietic cells through a mechanism of so-called autocrine secretion.     

Extreme clonality in lymphoblastoid cell lines with implications for allele specific expression analyses

Lymphoblastoid cell lines (LCL) are being actively and extensively used to examine the expression of specific genes and genome-wide expression profiles, including allele specific expression assays. However, it has recently been shown that approximately 10% of human genes exhibit random patterns of monoallelic expression within single clones of LCLs. Consequently allelic imbalance studies could be significantly compromised if bulk populations of donor cells are clonal, or near clonal. Here, using X chromosome inactivation as a readout, we confirm and quantify widespread near monoclonality in two independent sets of cell lines. Consequently, we recommend where possible the use of bulk, non cell line, ex vivo cells for allele specific expression assays.     

Instability of Mex- phenotype in human lymphoblastoid cell lines

Three lymphoblastoid cell lines (LCLs) had extremely low activities of O6-alkylguanine-DNA alkyltransferase (O6-AGT), and were classified as Mex-. They were highly sensitive to cell killing by 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoure a hydrochloride (ACNU), whereas NMO2, a Mex+ LCL with a high O6-AGT activity, was resistant to the agent. Small fractions of these Mex- LCLs survived the treatment with 10 micrograms/ml of ACNU for 24 h, and the surviving cells were found to be resistant to subsequent treatments with the agent. In addition, they contained O6-AGT activities comparable to that of NMO2 and were therefore regarded as Mex+. These results suggest that the Mex- phenotype in LCLs is unstable.     

Altered gene expression in mouse livers after dichloroacetic acid exposure

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated that DCA exhibits hepatocarcinogenic effects in rodents when administered in drinking water. This chemical does not appear to be highly mutagenic, and the mechanism(s) involved in DCA induction of cancer are not clear. The present work was aimed at identifying changes in gene expression which may indicate critical alterations/pathways involved in this chemical's carcinogenic activities. We used cDNA microarray methods for analyses of gene expression in livers of mice treated with the tumorigenic dose of 2 g/l DCA in drinking water for 4 weeks. Total RNA samples obtained from livers of the control and DCA-treated mice were evaluated for gene expression patterns with Clontech Atlas Mouse 1.2 cDNA and Atlas mouse stress/toxicology arrays, and the data analyzed with AtlasImage 2.01 and one-way ANOVA in JMP4 software. From replicate experiments, we identified 24 genes with altered expression, of which 15 were confirmed by Northern blot analysis. Of the 15 genes, 14 revealed expression suppressed two- to five-fold; they included the following: MHR 23A, cytochrome P450 (CYP) 2C29, CYP 3A11, serum paraoxonase/arylesterase 1 (PON 1), liver carboxylesterase, alpha-1 antitrypsin, ER p72, glutathione S-transferase (GST) Pi 1, angiogenin, vitronectin precursor, cathepsin D (CTSD), plasminogen precursor (contains angiostatin), prothrombin precursor and integrin alpha 3 precursor (ITGA 3). An additional gene, CYP 2A4/5, had a two-fold elevation in expression. Further, in ancillary Northern analyses of total RNA isolated from DCA-induced hepatocellular carcinomas (from earlier reported studies of mice treated with 3.5 g/l DCA for 93 weeks), many of the same genes (11 of 15) noted above showed a similar alteration in expression. In summary, we have identified specific genes involved in the functional categories of cell growth, tissue remodeling, apoptosis, cancer progression and xenobiotic metabolism that have altered levels of expression following exposures to DCA. These findings serve to highlight new pathways in which to further probe DCA effects that may be critical to its tumorigenic activity.     

DNA methylation and chromosome instability in lymphoblastoid cell lines

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus-transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2-rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3-rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.     

Genetic inheritance of gene expression in human cell lines

Combining genetic inheritance information, for both molecular profiles and complex traits, is a promising strategy not only for detecting quantitative trait loci (QTLs) for complex traits but for understanding which genes, pathways, and biological processes are also under the influence of a given QTL. As a primary step in determining the feasibility of such an approach in humans, we present the largest survey to date, to our knowledge, of the heritability of gene-expression traits in segregating human populations. In particular, we measured expression for 23,499 genes in lymphoblastoid cell lines for members of 15 Centre d'Etude du Polymorphisme Humain (CEPH) families. Of the total set of genes, 2,340 were found to be expressed, of which 31% had significant heritability when a false-discovery rate of 0.05 was used. QTLs were detected for 33 genes on the basis of at least one P value <.000005. Of these, 13 genes possessed a QTL within 5 Mb of their physical location. Hierarchical clustering was performed on the basis of both Pearson correlation of gene expression and genetic correlation. Both reflected biologically relevant activity taking place in the lymphoblastoid cell lines, with greater coherency represented in Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathways than in Gene Ontology database pathways. However, more pathway coherence was observed in KEGG pathways when clustering was based on genetic correlation than when clustering was based on Pearson correlation. As more expression data in segregating populations are generated, viewing clusters or networks based on genetic correlation measures and shared QTLs will offer potentially novel insights into the relationship among genes that may underlie complex traits.     

Cancer-testis gene expression profile in human melanoma cell lines

A growing number of evidence indicates that cancer-testis antigens (CTA) can be used as specific targets for immune therapy of malignant melanoma. The aim of this study was to provide a basis for selecting the most suitable CTA by analyzing the mRNA expression profile of genes encoding CTA in melanoma cell lines. We used a real-time quantitative PCR to measure the expression level for the following genes: GAGE1, NY-ESO-1, MAGEA1, PASD, SCP1, SEMG1, SPANXA, SSX1, and PRAME. The objects of study were cell lines mel P, mel Si, mel Mtp, mel Il, mel Hn, mel Ibr, and mel Kor obtained from patients diagnosed with disseminated melanoma. We established that the highest frequency of occurrence and the highest expression level had the following genes: GAGE1, NY-ESO-1, MAGEA1, SCP1, SPANXA, SSX1, and PRAME. Their mRNA translation products can be promising candidates for immunotherapy.