Figure 2
Diagnostic AML blasts from patients either at first diagnosis or at relapse are equally sensitive to MK-1775. Panel A: Freshly isolated AML patient samples were purified by standard Ficoll-Hypaque density centrifugation then treated with MK-1775 for 48 h and apoptotic events were determined by annexin V/PI staining and flow cytometry analyses. Panels B and C: Ex vivo MK-1775 (MK) sensitivity was determined using MTT assays. The horizontal lines indicate median MK-1775 IC50s in each group of AML patient samples. Panel D: Cytarabine sensitivity was determined using MTT assays for the HL-60 and HL-60/Ara-C cell lines. Panel E: The HL60 and HL60/Ara-C cells were treated with MK-1775 for 48 h and apoptotic events were determined by annexin V/PI staining and flow cytometry analyses. The data are presented as mean of triplicates ± standard errors from one representative experiment. Panel F: Freshly isolated cells from patient AML#10 were purified by standard Ficoll-Hypaque density centrifugation. AML#10, HL-60, and HL-60/Ara-C cells were treated with MK-1775 for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-CDK1, -CDK1, -p-CDK2, -CDK2, -γH2AX, or -β-actin antibody.