(a) Volcano plot of changes in apoptosis-related transcripts in EBV-loss BL clones treated with ionomycin and Q-VD.OPh for 48?h 0?h. death-promoting actions of aberrant expression. When EBV infects resting B cells into lymphoblastoid cell lines (LCLs)) and the Latency I EBV gene expression programme (as found in the majority of EBV-positive BL tumours and cell lines derived from these tumours). Latent proteins (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, BHRF1, LMP1 and LMP2A/B) are shown in blue. Non-coding RNAs (EBERs, BHRF1 microRNAs and miR-BARTs) are shown in red, and latent promoters (Cp, Wp, Qp and LMP promoters) are shown in green Crucially, however, most of the Latency III genes are not expressed in established BLs. Instead, in BLs, EBV exhibits more restricted forms of latency characterised by expression of EBNA1, the EBER transcripts and the miR-BARTs (Physique 1). Only a minority of eBLs exhibit a more complex viral gene expression pattern due to a genomic deletion in EBV.9, 10 Cell lines derived from these tumours show marked resistance to apoptosis due to epigenetic silencing of the promoter11 and functional inhibition of BIM, PUMA, BID and BAK by the viral BCL-2 homologue, BHRF1.12 EBV-positive and -negative BLs are genetically distinct, differing in terms of their cellular mutational profiles13, 14 and precise chromosomal translocations.15, 16 It is therefore unsatisfactory to introduce the virus or viral genes into EBV-negative spBL lines to study the role of EBV in eBL. Instead, efforts have focused on wanting to rid EBV-positive eBLs of the virus to assess the contribution of EBV to the growth and survival of BL in an isogenic system. Treatment of EBV-positive BL cells with a dominant-negative form of EBNA1 leads to loss of EBV genomes and widespread apoptosis.17, 18, 19, 20 While implying that EBV is essential for the continued survival of BL cells, this method yielded RG7112 few EBV-loss RG7112 clones for mechanistic studies. Hydroxyurea treatment can also eradicate EBV, but these BL clones do not show a consistent apoptosis predisposition phenotype.21 Additionally, an unusual EBV-positive spBL (Akata-BL) cell line has been reported to spontaneously drop EBV and in a xenograft model of BL. This work has shown unequivocally that EBV in a Latency I contamination can safeguard BL cells from apoptosis mediated by the proapoptotic BH3-only proteins, BIM and PUMA. Results Determining the contribution of EBV to the continued growth of BL cells A panel of EBV-positive BL cell lines (1 spBL and 11 eBLs) were seeded at single-cell dilutions to establish more than 1800 clones. These clones were screened for EBV episome copy number by quantitative, real-time PCR (q-PCR); the results are summarised in Table 1 and Physique 2a. Strikingly, the generation of EBV-loss cells was a rare event, observed in only 61/1800 (3.4%) clones and E1AF seven BL cell lines never yielded EBV-loss clones. These results strongly indicate that while EBV is not essential for continued BL cell growth, there is strong selective pressure to retain EBV. Open in a separate window Physique 2 Genome loads and viral gene expression in BL single-cell clones. (a) Cells from each clonal cell line grown from a single cell by limiting dilution were harvested, lysed and analysed by q-PCR RG7112 to enumerate the average EBV genome copy number per cell. Quantitation was calculated relative to Namalwa-BL cells, which contain two integrated copies of EBV per cell and these data were normalised to the housekeeping gene, EBV-loss clones was 54102 days for Kem-BL, 63113 days for Mutu-BL and 5068 days for Awia-BL (Physique 3a). For direct comparison with previous studies,22, 25 clones of Akata-BL were transplanted by subcutaneous injection into NSG mice at a higher inoculum. The EBV-positive clones gave rise to tumours, whereas the EBV-negative clones were non-tumorigenic (Supplementary Physique 2b). Subsequent analysis confirmed that this Latency I gene expression pattern was retained in all tumours derived from EBV-positive BL cells (Supplementary Physique 2c). Open in a separate window Physique 3 and phenotype of isogenic.