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 characteristic feature of apoptosis but not necrosis. We tested whether death in factor deprived EBV-immortalized cells occurs at a particular stage of cell cycle. Much of the data supports the view that cell death under these experimental conditions occurs as all stages of cell cycle, without a clear preference for a given stage of cell cycle. In addition, cell cycle analysis of factor deprived cells indicated that the cells fail to growth arrest in GO/1. A number of gene products have been implicated in apoptic death processes, including BCL-2, p53 and C-myc. Levels of expression of BCL-2 and C-myc did not change significantly in EBV- immortalized cells destined to die by apoptosis. Levels of p53 RNA were increased by 1.8-2 fold 6 to 12 hours before death occurred. The observation that growth-factor deprived EBV-immortalized cells die at all stages of the cell cycle without undergoing growth arrest, together with the observation that C-myc expression remains unchanged in these cells, suggests that dysregulated myc expression is an important element of cell death in these cells. Thus, EBV-immortalized cells die by apoptosis when deprived of autocrine growth factors and this process is associated with insignificant changes in the expression of C-myc, BCL-2 and p53.
