Despite the fact that EBV-immortalized B cells are capable of continuous growth when cultured under optimal culture conditions, they die when incubated at sufficiently low cell densities. We have examined the process of death in EBV-immortalized cells cultured at critically low cell densities. We asked whether death occurs by necrosis or by apoptosis. Two lines of evidence suggest that death occurs through apoptosis. First, the cellular DNA was found to separate in discrete fragments on agarose gels. Second, the protein synthesis inhibitor cycloheximide inhibited cell death, suggesting that the process involves protein synthesis. A number of gene products have been implicated in apoptic death processes, including BCL-2, p53 and C-myc. So far, we have examined the role of two of these genes in our system, BCL-2 and p53. Levels of expression of BCL-2 did not change significantly in EBV-immortalized cells destined to die by apoptosis. In contrast, levels of p53 in RNA were increased by 2-3 fold 6 to 12 hours before death occurred. Thus, EBV-immortalized cells die by apoptosis when deprived of autocrine growth factors and this process is associated with increased expression of the p53 tumor suppressor gene.
