P11: Proteoglycans expression correlates with the phenotype of malignant and non-malignant EBV-positive B-cell lines

Abstract

In vitro Epstein-Barr virus (EBV) infects B-cells resulting in immortalization, In vivo the virus resides latent in resting B-cells. Rarely the EBV-host cell interaction may contribute to development of malignant lymphoma. It is well known that both c-myc translocation and the viral infection are observed in patients with EBV+ Burkitt’s lymphoma (BL). Proteoglycans (PGs) are complex glycosylated proteins. They are key components of ECM and play a critical role in cell–cell and cell–matrix interactions. Disruptions of such interactions will affect B cell interaction with surrounding stroma and may thus perturb the cell phenotypes. The purpose of this study was to investigate expressinon of proteoglycans in EBV+ cell lines with different origins and phenotypes. We analysed the expression of 12 of the main proteoglycans in primary B cells, lymphoblastoid cell lines (LCLs) generated by EBV infection of normal human B cells in vitro and EBV-positive BL cell lines. An EBV-negative BL cell line was used for reference. According to RT-PCR analysis, primary B-lymphocytes expressed different PGs, mainly serglycin, CD44, perlecan and syndecan-1. The high expression of PGs in normal B cells probably reflects interactions of these cells with the neighbouring cells and microenvironment. B cell lines which carry EBV, in general, showed lower levels of PGs. The PGs expression pattern was similar in LCLs and in primary B cells, however, distinguished by high levels of perlecan and serglycin and low expression of CD44 in LCLs. BL cells showed the most significant down-regulation of PGs compared to primary B cells. There was a correlation between the type of EBV latency program, and PGs expression. Serglycin was expressed at a low levels in BL-cells with EBV latency III-program compared to LCLs, while in EBV latency I BL cells both serglycin and perlecan were down-regulated. Cells with latency I-program show general hypermethylation of the cellular genome in contrast to cells with latency III-program. Thus we explored the possibility of epigenetic regulation of the PG-coding genes by treating cells with 5′-deoxyazacytidine (5-AzaC, a demethylating agent) and Trichostatin A (TSA, a chromatin structure modulator). There was no significant change in PGs expression upon this treatment in LCLs or in EBV latency III BL cells, while EBV latency I BL cells showed up-regulation of several PGs. This suggests that PGs expression is at least partly regulated by epigenetic mechanisms. Interestingly EBV latency is also partly regulated at the epigenetic level. Similar trends were observed for the key ECM components (collagen 1A1, fibronectin and elastin). Normal B lymphocytes expressed collagen, fibronectin and elastin, whereas LCLs and BL cells showed no expression of these. Treatment of these cells with 5-AzaC or TSA resulted in similar changes in PGs expression patterns. Up-regulation of ECM components was detected only in EBV latency I BL cells. Taken together, our data show that proteoglycans are expressed in primary B lymphocytes whereas they are not or only partly expressed in EBV-carrying cell lines, depending on their latency program. Expression of PGs in latency I BL cells is silenced due to hypermethylation, but by another mechanim in latency III BL cells. These results show that PGs expression patterns follow the EBV latency programs . It will be highly interesting to further investigate if EBV and its transformation associated genes are directly involved in control of PGs, as well as how PGs may contribute to major phenotypic properties of EBV-carrying cell lines, such as adhesion, migration and growth in soft agarose – a property associated with the malignant phenotype of BLs.