Figure 3
AZA restores GC-dependent sensitivity to clones of multiple myeloma cells derived from the resistant RPMI 8226 parent. A. RPMI 8226 cells were treated with 100 nM AZA or an equivalent volume of DMSO for 48 hours. Cells were then dilution cloned to single cells, and viable cell populations were allowed to regrow. Percentages of growth repression vs. apoptosis after 96 hours of 1 μM Dex treatment for control (50 clones, open diamonds) or AZA (47 clones, closed squares) are plotted as in Figure 1A. Revertant clone RPMI-II E6 is indicated. B. The methylation states of DNA for uncloned RPMI 8226 cells without AZA treatment and of revertant clone RPMI-II E6 were evaluated by cytosine extension assay. Data are presented as DPMs of incorporated 3H dCTP per μg of genomic DNA. Error bars = 1 standard deviation from the mean. n = 5 for RPMI-II E6 and 6 for RPMI 8226. p = 0.08, RPMI 8226 vs. RPMI-II E6. C. RPMI 8226 and II E6 cells were treated in triplicate with ethanol (control, closed diamond, closed triangles) or 1 μM Dex (Dex, open diamonds, open triangles) for 96 hours with final evaluation using Trypan blue exclusion to estimate vialble cells. Shown is the average percentage of 3 independent replicates for cells able to exclude the dye (% culture viability). Error bars = 1 standard deviation from the mean. D. RPMI 8228 and clone II E6 were treated with ethanol vehicle (control) or 1 μM Dex for 96 hours, then stained with PI and evaluated by flow cytometry. Each histogram represents the results from the collection of 20,000 events where possible. n = 3. Note that Y-axis scales are all identical; in the lower two panels, loss of cells due to lysis reduced the number of countable “events” in the cytometer. Also, see Table 1.