Effect of the timing of etoposide application and of mcl-1 induction on the viability of FDC-P1 transfectants. (A) Effect of short-term exposure to etoposide. Cells were treated as in Fig 2A, except that etoposide (20 μg/mL) was washed out after the indicated times and replaced with drug-free medium. For samples exposed to dexamethasone, dexamethasone was again added to the medium after drug wash-out. Cell viability was assayed at 24 hours; thus, the point indicating 24 hours of exposure to etoposide represents continuous exposure. In separate experiments, cells exposed to etoposide for 0.5 hours were observed for 3 days. Cell viability in clone 8.5-MAMneo (with or without dexamethasone) and clone 7.5-MAMmcl-1 (without dexamethasone) averaged 78% to 83% on day 1 and 57% to 65% on days 2 and 3. These values for clone 7.5-MAMmcl-1 (with dexamethasone) averaged 95% on day 1 and 79% to 81% on days 2 and 3. (B) Effect of expression of mcl-1 after exposure to etoposide. Cells were treated essentially as in Fig 2A, except that dexamethasone was added at various times either before, concomitant with, or after the time of addition of etoposide (20 μg/mL). The inoculum density was 5 × 105 cells/mL. Cell viability was assayed at the 24-hour time point. The time point labeled “0” indicates concomitant addition of dexamethasone and etoposide. The point labeled “−2” indicates addition of dexamethasone 2 hours before the addition of etoposide. For both (A) and (B), the values shown are the mean ± SD of three independent experiments. Where no bars are seen, the SD fell within the symbol.
