Figure 1
AZA treatment of Dex-resistant C1-15 restores GC-dependent sensitivity to CEM clones. A. CEM-C1-15 cells - treated with 100 nM AZA or DMSO for 48 hours were cloned by serial dilution to single cells and allowed to regrow. 105 DMSO and 94 AZA-treated outgrown clones were then exposed to 1 μM Dex or vehicle for 96 h and characterized for viable ( exclude trypan blue) and apoptotic (intact, trypan blue positive) cells. Plotted are % growth repression (total reduction in viable cells, x-axis) and % apoptotic cells within the remaining intact cell population (y-axis). B-D, Studies of revertant clone CEM-IV B9. B. Overall methylation of DNA from untreated CEM-C1-15 cells vs - clone CEM-IV B9, by cytosine extension assay- (DPMs of incorporated 3H dCTP per μg of genomic DNA). Error bars = 1 SD, n = 5. p = 0.0003. C. GC (Dex) sensitivity. Triplicate cultures of naturally Dex-sensitive CEM-C7-14 (dashed line, closed diamonds), Dex-resistant CEM-C1-15 (closed circles), and two - revertant clones: CEM-C1-6 (spontaneous revertant from C1-15, closed squares), and CEM IV B9 (demethylated revertant, open circles) were treated with 1 μM Dex and followed for 96 hours for numbers of viable cells. - Shown is the average percentage - of viable cells. Error bars = 1 SD -. D. Flow cytometry. The four - clones -of panel C were treated with vehicle (control) or 1 μM Dex for 96 hours and evaluated by flow cytometry. Data in graphs representative of biological n = 2. For IV B9 cells, extensive cell death and secondary, post apoptotic lysis allowed modelling of <20,000 events. All four panels are at same scale. Cell cycle results (avg. of 2 expts), see Table 1.