SHORT REPORT Inhibition of in vitro proliferation of Epstein Barr Virus infected B cells by an antisense oligodeoxynucleotide targeted against EBV latent membrane protein LMP1 Elena Mattia 1 , Silvia Chichiarelli 1 , Tamas Hickish 3,4 , Aurelia Gaeta 1 , Carlo Mancini 1 , David Cunningham 3 and Jos van Renswoude 2 1 Microbiology Institute, Faculty of Medicine and Surgery; 2 Department of Experimental Medicine and Pathology, Faculty of Medicine and Surgery, University of Rome `La Sapienza', Rome, Italy; 3 Lymphoma Unit, Royal Marsden Hospital, Sutton, Surrey, UK Keywords: EBV; LMP1 expression; antisense oligo- deoxynucleotides Epstein Barr Virus (EBV), an herpesvirus carried by more than 95% of the human population as an asymptomatic infection, is the etiologic agent of infectious mononucleosis (IM) and is clearly asso- ciated with endemic Burkitt's lymphoma (BL), large cell lymphomas that develop in immunosuppressed patients, and with nasopharyngeal carcinoma (NPC) (Miller, 1990). Experimental infection of resting B lymphocytes with EBV leads to permanently virus- transformed lymphoblastoid cell lines (LCLs) that proliferate inde®nitely. LCLs support a predominantly latent viral life cycle in which EBV gene products include 6 nuclear antigens, EBNAs 1, 2, 3a, 3b, 3c, and LP, and three latent membrane proteins, LMP1, 2A and 2B, just as do immunoblastic B cell lymphomas in immunosuppressed patients. In contrast, those tumours in which EBV appears to participate to a complex multi-step pathogenesis, show a more restricted pattern of viral gene expression, limited to EBNA1 only in BL and to EBNA1, LMP1, 2A and 2B in NPC. More recently, EBV has been associated to another lymphoid tumor, Hodgkin's disease (HD), where the presence of EBV genomes of monoclonal origin in the malignant population of Hodgkin and Reed-Sternberg cells (HRS) has been detected in at least 60% of cases (Herbst et al., 1990; Brocksmith et al., 1991). Recent studies aimed at assessing the pattern of latent gene expression in HD have shown that the transcriptional program of the virus in HRS cells involves EBNA1, LMP1 and in most cases LMP2A and LMP2B genes, a pattern similar to that seen in NPC cells (Deacon et al., 1993). In particular, LMP1 transcripts and LMP1 protein were found in virtually all HRS cells in EBV-positive HD cases (Wang et al., 1990; Herbst et al ., 1992; Khan et al ., 1993). Interestingly, LMP1 expression in HRS cells varied according to the histological subtype of HD, being close to 100% of the cases characterized by mixed cellularity (Pallesen et al., 1991; Joske et al., 1992). Several studies have shown that the LMP1 gene has transforming properties. In rodent ®broblast cells, LMP1 reduces serum dependency, contact inhibition and anchorage-dependent growth (Wang et al., 1985). In epithelial cells, LMP1 inhibits dierentiation (Dawson et al., 1990; Wilson et al., 1990) and modulates the phenotype of B lymphoblastoid cells by inducing B-lymphocyte activation markers and cell adhesion molecules (Wang et al., 1990). In addition, LMP1 `protects' B lymphocytes from apoptosis, in part by inducing the expression of the bcl-2 oncogene (Henderson et al., 1991). To further investigate the contribution of LMP1 gene expression to the development of a neoplastic phenotype, we have attempted to down-regulate the gene by devising a synthetic antisense oligodeoxynu- cleotide that, by binding to a target sequence on LMP1 mRNA, would prevent its translation. We have synthesized several, non-chemically modi®ed antisense oligodeoxynucleotides directed to dierent potential target sequences within the translation initiation region of EBV LMP1 mRNA. The eects of the incubation of LMP1 antisense oligodeoxynucleotides were studied on B95.8 cells, EBV transformed marmoset leukocytes (Miller et al., 1972). We have found that one oligomer, denominated AS2, encompassing the predicted transla- tion initiation site of LMP1 mRNA expressed in B95.8 cells (see Table 1), inhibits cell proliferation, leads to a substantial loss of LMP1 mRNA and signi®cantly decreases the intracellular concentration of LMP1 protein as reported below. In order to examine the eect of LMP1 antisense oligodeoxynucleotide AS2 on cellular proliferation, the growth rate of B95.8 cells was studied in the presence of AS2 oligomers. A non-sense LMP1 sequence, termed Sc2 (a randomly scrambled version of the AS2 sequence), was used as a control. Figure 1a shows that LMP1 AS2 oligomers, added to the cell cultures at 24 h intervals to maintain oligomer concentrations at about 20 mM, markedly inhibited the proliferation of B95.8 cells during a 4 day incubation. In contrast, treatment of the cells with the same concentration of Sc2 oligodeoxynucleotides, had little eect on the proliferation of B95.8 cells. Under the conditions used in these experiments, the inhibition of cell growth was maximal at 48 h, the values ranging between 30 and 70% (Figure 1b). When B95.8 cells were treated with LMP1 oligomers in increasing concentrations (in the range between 5 and 60 mM), dose dependent inhibition of cell proliferation by AS2 was observed for antisense Correspondence: E Mattia Received 29 November 1996; revised 26 March 1997; accepted 1 April 1997 Oncogene (1997) 15, 489 ± 493  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00