Original articleCopy number increase of 1p36.33 and mitochondrial genome amplification in Epstein–Barr virus-transformed lymphoblastoid cell lines
Introduction
Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) have been widely used as genomic resources for a variety of human genetic studies; however, the genomic and biological characteristics of LCLs that differ from primary B-cells have not been well characterized. Although EBV is typically maintained as an episome in infected cells, rarely integrated into host chromosomes, many reports have described viral integrations in EBV-infected cells [1], [2], [3], [4], [5], [6], [7]. Chromosomal instability is increased by viral integration, which causes deletion or duplication of host and viral genome sequences flanking the integration junction sites [8], [9]. LCLs frequently acquire chromosomal aberrations during the long-term culture required for immortalization, up to a population doubling level of 160–180, at which the LCL is considered to be terminally immortalized [10]. EBV-infected B-cells are transformed into LCL cells, which are polygonal at very early population doubling levels.
Array CGH is effective in detecting chromosomal aberrations in genetic diseases and cancer on a whole-genome scale [11], [12]. For example, the frequencies of 1:8 for segmental deletion and of 1:50 for segmental duplications were estimated to generate large-scale copy number polymorphisms in human newborns [13]. Recently, large-scale copy number polymorphisms (CNPs) or large-scale copy number variations (LCVs) were identified in phenotypically normal individuals by oligo- or bacterial artificial chromosome (BAC) array CGH approaches [14], [15]. Array CGH has been successfully performed using various DNA sources from whole blood cells, LCLs, and formalin-fixed, paraffin-embedded tissues [16], [17]. In our study, BAC array CGH was applied to investigate whether EBV infection induces a chromosomal change during EBV-transformed B-cell immortalization. Here we also report segmental copy number polymorphisms of Korean normal individuals.
Given that LCLs are used as major common genome resource for genomic and genetic studies in human, we attempted to detect genomic differences in array CGH profiles between primary B-cells and their corresponding EBV-transformed LCLs from the same donor. The copy number change of the 1p36.33 segment corresponding to the mtDNA increase was the only copy number change in chromosomal segments between EBV-transformed LCLs and primary B-cells. This feature of 1p36.33 should be taken into account in analyses of array CGH profiles of diseases. Additionally, our array CGH of LCLs revealed potential copy number polymorphisms of chromosomal segments among Korean populations.
Section snippets
Cells
Peripheral blood was obtained from healthy donors with informed consent. Ficoll-Hypaque gradient centrifugation was performed to isolate peripheral blood mononuclear cells. EBV stock was prepared from an EBV-transformed B95-8 marmoset cell line. EBV-infection of the mononuclear cells was performed to generate LCLs, as described elsewhere [18]. EBV-infected cells were incubated in RPMI-1640 supplemented with cyclosporin A (0.5 μg/mL), 10% fetal bovine serum (FBS), and penicillin–streptomycin
Array CGH analysis
To detect the copy number changes of chromosomal segments in the EBV-transformed B-cell immortalization, BAC array CGH was first performed with dye-swap experiments to analyze the genomic profile of four different LCLs compared to pooled whole human blood (Fig. 2). Each LCL showed a relatively different genomic profile of chromosomal copy number variations of which two segments, 1p36.33 and 2p11.2, were commonly increased or decreased in all four LCLs tested in this study, respectively (Table 1
Discussion
Here we report the increase in mtDNA copy numbers of LCLs in EBV-mediated B-cell transformation, as well as copy number polymorphisms of chromosomal segments among Korean subjects. Comparison of array CGH profiles of four LCLs using pooled reference DNA from multiple donors showed that copy number changes in the 1p36.33 segment and two immunoglobulin (Ig) loci (2p11.2 and 14q32.33) were common in all LCLs tested in this study. The copy number changes of Ig light chain κ and Ig heavy chain loci
Acknowledgments
This work was supported by an intramural grant of the National Institute of Health, Korea.
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