Figure 1
AITC selectively induces apoptosis and mitochondrial injury in transformed and primary human leukemia cells. U937 cells were treated without or with various concentrations of AITC for 24 h, or treated with 20 μM AITC for different time intervals as indicated. (A) Apoptosis and reduction in △ψm were determined using flow cytometry as described in Methods. ‘Low’ △ψm values are expressed as the percentage of cells exhibiting a diminished mitochondrial membrane potential. Error bars represent means ± SD for (n=3). (B) Total cellular extracts and cytosolic fractions (C) were prepared and subjected to Western blot analysis using antibodies against PARP, cleaved-caspase 3 (C-Caspase 3), cleaved-caspase 9 (C-Caspase 9) , cytochrome c (Cyto c) and GADPH to ensure equivalent loading. Two additional studies yielded equivalent results. (C-D) U937, Jurkat, and HL-60 cells were treated without or with 20 μM AITC for 24 h, flow cytometry was used to determine apoptosis, and Western blot assay was used to determine the expression of PARP, C-Caspase 3, C-Caspase 9, and Cyto c. Error bars represent means ± SD (n=3). ** P< 0.01 compared with control. (E) Primary leukemia blasts were isolated from the peripheral blood of 17 patients with AML as described in Methods. After exposure to 20 μM AITC for 24 h, apoptosis was determined using flow cytometry. Error bars represent means ± SD (n=3). (F) Whole cell lysates and cytosolic fractions (C) in blasts from 2 AML patients were obtained and subjected to Western blot analysis. (G) Normal CD34+ cells were exposed to 20 μM AITC for 24 h, after which apoptosis was determined using flow cytometry. Error bars represent means ± SD (n=3). P = 0.056.