MycER, in the presence of Bcl-XL, is not sufficient to drive the proliferation of PTLD1 cells. (A) The level of MycER expression was analyzed by Western blot in Bcl-XL–protected PTLD1 cells and found to be comparable with the level of Myc in several BL cell lines (Oku-BL and Dante-BL) in which Myc is misregulated. (B) The growth of cell populations was measured over time in cells losing EBV but gaining MycER function. Dashed lines represent cells in which dnEBNA1 is induced; solid lines, cells in which dnEBNA1 is uninduced. Gray lines trace cells in which MycER is inactive, red lines cells in which MycER is activated (beginning 4 days after dnEBNA1 was induced). All cells were previously transduced with a Bcl-XL expression vector (Bcl-XL-protected). The average of 4 independent experiments ± SD is shown. The difference in the total number of doublings by day 16 between cells in which MycER is inactive or active is statistically significant (P = .04, one-sided Wilcoxon rank-sum test). (C) The percentage of cells in each stage of the cell cycle was determined by propidium iodide staining and fluorescence-activated cell sorting analysis. PTLD1 cells were analyzed 16 days after dnEBNA1 induction or mock induction and 12 days after activation or mock activation of MycER. The average of 3 independent experiments ± SD is shown. The reduction in the percentage of cells in G1/G0 in the presence of active MycER (from 87% to 82%) is statistically significant (P = .02, one-sided Wilcoxon rank-sum test). (D) RT-PCR of Myc target genes in cells in which EBV is evicted and MycER is active or inactive (day 16 from panel A). Target gene levels were normalized to β-actin levels and then compared with the control (inactive Myc) levels, which were arbitrarily set to one. The average of 3 independent experiments ± SD is shown.
