Effects of DAC on DNMT1 depletion, DNA damage, and apoptosis in normal hematopoietic precursors. (A) DAC 0.005μM depletes DNMT1 in normal hematopoietic precursors. Normal CD34⁺ cells were isolated from cord blood. DAC 0.005μM was added once daily on days 1-4 and DNMT1 was quantified by Western blot on day 5. (B-C) DAC > 0.5μM was required to induce measurable DNA damage. Twenty-four hours after DAC or AraC exposure, DNA damage was measured by flow-cytometric assessment for phosphorylation of histone H2AX (γH2AX; B) or the Fast Micromethod for DNA scission (C). Equimolar levels of AraC were used as positive controls. Gray histogram is the isotype control. (D) DAC > 0.5μM was required to induce apoptosis. Twenty-four hours after DAC or AraC exposure, apoptosis was measured by flow-cytometric assessment for annexin staining. Double annexin/7-amino-actinomycin D (7AAD)–positive cells represent late apoptosis/necrosis. (E) DAC up to 0.5μM in combination with THU did not cause significant DNA damage, as measured by flow-cytometric assessment of γH2AX levels 24 hours after addition of the drug to normal hematopoietic precursors. Results are expressed as a percentage of vehicle-treated controls.