Early events of EBV mediated B cell transformation

Sammanfattning: Epstein-Barr virus (EBV) efficiently immortalizes B cells in vitro leading to the establishment of lymphoblastoid cell lines (LCLs). Genes encoding RB and p53 are frequently deleted or mutated in human tumors. EBV immortalized LCLs, however, retain both genes in their wild type state. We aimed to examine the RB and p53 behaviour after in vitro EBV infection compare to the mitogen stimulated B cells and their expression in the established LCLs. Both Rb and p53 were up-regulated 24 hours after EBV infection along with EBNA-2, EBNA-5 and CD23. The levels of RB remained high in the LCLs while the number of p53 positive cells decreased and was restricted to a minor fraction of the cells in the LCLs. It is noteworthy that the increased p53 expression in both EBV infected and CD40L+IL-4 stimulated primary B cells caused neither growth arrest nor apoptosis suggesting that wtp53 protein may be inactive in its growth suppression function in these cells. EBNA-5, that showed strong homogeneous staining in the infected blasts 24 hr after EBV infection, appeared in distinct nuclear foci several days later. In the established LCLs the focal pattern dominates. We found that these foci are the PML-containing nuclear bodies. The lineage specific expression of D-type cyclins implies their involvement in differentiation. LCLs express predominantly cyclin D2. BL lines with an original tumor phenotype express only cyclin D3. The EBV-negative BLs converted by EBV in vitro that acquire, though partially, an LCL-like phenotype, did not express cyclin D2. This suggested that cyclin D2 vs. D3 expression in B cell lines may reflect properties of their cells of origin. BL are believed to originate from the germinal centers (GC) of secondary follicles. Indeed, the centroblasts of GC expressed cyclin D3 but not D2 as the BL tumors and lines. The EBV infection of resting B cells in vitro induces an immunoblastic B cell phenotype. EBV-encoded LMP-1 is, at least in part, responsible for these phenotypic changes. We have described novel high molecular weight cytockeleton- and nucleoskeleton-associated proteins (the pNDCFs) that are absent from primary B cells and BL lines but are induced after both EBV infection and CD40-stimulation of B cells. LMP-1 alone is sufficient to induce these proteins in BL lines. The association of pNDCFs with intermediate filaments in the cytoplasm and, at the same time, with the distinct nuclear foci, may suggest their involvement in signal transduction. The cloning of the genes that encode these proteins will allow to elucidate their function.