Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

Abstract

Background: The Epstein-Barr virus (EBV) is associated with lymphoid malignancies, including Burkitt's lymphoma (BL), and can transform human B cells in vitro. EBV-harboring cell lines are widely used to investigate lymphocyte transformation and oncogenesis. Qualitative EBV gene expression has been extensively described, but knowledge of quantitative transcription is lacking. We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines.

Results: To compare quantitative gene transcription in the BL cell lines Namalwa, Raji, Akata, Jijoye, and P3HR1, we developed an oligonucleotide microarray chip, including 17 housekeeping genes, six latent EBV genes (EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2), and four lytic EBV genes (BZLF1, BXLF2, BKRF2, BZLF2), and used the cell line B95.8 as a reference for EBV gene transcription. Quantitative polymerase chain reaction assays were used to validate microarray results. We found that transcription levels of housekeeping genes differed considerably among BL cell lines. Using a selection of housekeeping genes with similar quantitative transcription in the tested cell lines to normalize EBV gene transcription data, we showed that transcription levels of EBNA1 were quite similar in very different BL cell lines, in contrast to transcription levels of other EBV genes. As demonstrated with Akata cells, the chip allowed us to accurately measure EBV gene transcription changes triggered by treatment interventions.

Conclusion: Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology.

Figure 1

Selection of microarray probes. The specificity of EBV gene probes was tested in the reference cell line B95.8 as positive control (A) and in the EBV-negative cell line BJAB (B). The P3HR1 strain of EBV is characterized by a large deletion in the region coding for EBNA2 and was used to validate the specificity of EBNA2 probes (C). Black bars represent probes considered specific and selected for the final version of the chip. Mean ± SEM values (with background subtracted) were normalized to the set of eight housekeeping genes. Robust signals were measured for most latent and lytic EBV genes in the reference cell line B95.8. White bars indicate probes that were not selected.

Figure 2

Quantitative analysis of EBV gene transcription in cultured BL cell lines at steady state. EBV gene transcription levels in exponentially growing cultured cells were determined by competitive hybridization to the reference cell line B95.8. Shown are mean ± SD values of dye-swap microarray experiments expressed as transcription ratio to B95.8. Dotted lines indicate the range of mean transcription values. Stars represent "not detected."

Figure 3

Validation of microarray results by qPCR analysis. EBV gene transcription levels in exponentially growing cultured cells were determined by competitive hybridization to the reference cell line B95.8 and by qPCR. Shown are mean ± SD values from dye-swap microarray experiments (open squares) and for three independent qPCR experiments normalized over B95.8 (closed triangles). Stars represent "not detected."

Figure 4

Effect of induction of lytic EBV infection on Zta and EBV gene transcription. (A) Western blot showing protein transcription levels of Zta in IgG cross-linking induced (+) and non-treated (-) Akata cells. (B) Transcription of EBV genes was quantified by microarray analysis in Akata cells upon induction of lytic infection. Treated and non-treated cells were harvested at different times after induction of lytic infection by BCR cross-linking. Competitive hybridization of labeled samples from treated cells was performed against non-treated cells. Shown are mean values of two dye-swap experiments. (C) EBV gene transcription was quantified during induction of lytic EBV infection in Akata cells by BCR cross-linking. For each time point, treated and non-treated cells were harvested and subjected to qPCR. Each point represents the difference between induced and non-induced cells, normalized to the HMBS housekeeping gene. Results are from at least two biological replicates and are given as: ΔΔC_T = (C_T(EBV gene)-C_T(HMBS)}_treated-{C_T(EBV gene)-C_T(HMBS))_{not treated.}