Combined treatment with adaphostin and MG-132 markedly induces death in primary AML cells while exhibiting little toxicity toward normal hematopoietic cells. (A) Primary human AML blasts (FAB M2 subtype in 4 samples; M5 in 1 sample) were suspended in medium containing 10% FCS at a cell density of 0.8 × 106/mL in the presence of 750 nM adaphostin plus or minus 300 nM MG-132 for 24 hours. At the end of drug exposure, apoptotic cells were monitored by annexin V/PI staining. Cell death for control blasts was generally less than 15% to 25%. (B) Primary AML blasts (sample 2) were exposed to drugs alone and in combination as in panel A for 18 hours, after which cell lysates were obtained and Western blot analysis performed to monitor PARP and MEK1 cleavage and levels of phospho-JNK and Raf-1. Each lane was loaded with 30 μg protein, and blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. (C) CD34+ cells obtained from the bone marrow of a patient undergoing a routine diagnostic procedure for a nonmyeloid hematologic disorder (eg, thrombocytopenia) were isolated by an immunomagnetic bead separation technique as described in “Materials and methods” and exposed to adaphostin (1.0 μM) ± MG-132 (300 nM) for 24 hours. At the end of this period, the percentage of apoptotic cells was determined by annexin V/PI staining and flow cytometry. A parallel experiment was also performed with normal peripheral-blood mononuclear cells (NPBMNC) from a healthy donor. Values represent the means plus or minus SD for triplicate determinations.
