DATS was purchased from LKT Laboratories (St Paul, MN). 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT), propidium iodide (PI), 5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), 4,6-Diamidino-2-phenyllindile (DAPI), 5-(and 6)-carboxy-2’7’-dichlorodihydrofluorescein diacetate (DCFDA), and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) and caspase activity assay kits were obtained from GIBCO-BRL (Gaithersburg, MD) and R&D Systems (Minneapolis, MN), respectively. DNA staining kit (CycleTEST™ PLUS Kit) and enhanced chemiluminescence (ECL) kit were purchased from Becton Dickinson (San Jose, CA) and Amersham (Arlington Heights, IL), respectively. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

The human leukemia cell lines used in our studies included human monocytic leukemia (U937 and THP-1), human acute myeloblastic leukemia (HL60) and human erythroid chronic myeloid leukemia (K562) were purchased from the American Type Culture Collection (Rockville, MD). They were maintained at 37°C in humidified 95 % air and 5 % CO₂ in RPMI1640 supplemented with 10 % heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. DATS was dissolved in dimethyl sulfoxide (DMSO) as a stock solution at a 100 mM concentration, and the stock solution was then diluted with the medium to the desired concentration prior to use. For the cell viability study, cells were grown to 70 % confluence and treated with DATS. Control cells were supplemented with complete media containing 0.1 % DMSO (vehicle control). Following treatment, cell viability was determined by use of the MTT assay, which is based on the conversion of MTT to MTT-formazan by mitochondrial enzymes 30 . The effect of DATS on inhibition of cell growth was assessed as the percentage of cell viability, where vehicle-treated cells were considered 100 % viable.

For DAPI staining, cells were washed with phosphate-buffered saline (PBS) and fixed with 3.7 % paraformaldehyde (Sigma-Aldrich) in PBS for 10 min at room temperature. Fixed cells were washed with PBS and stained with 2.5 μg/ml DAPI solution for 10 min at room temperature. Cells were then washed twice with PBS and analyzed using a fluorescence microscope (Carl Zeiss, Germany).

Following DATS treatment, cells were lysed in a buffer containing 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.5 % Triton X-100 for 1 h at room temperature. Lysates were vortexed and cleared by centrifugation at 19,000 g for 30 min at 4°C. A 25:24:1 (v/v/v) equal volume of neutral phenol : chloroform : isoamyl alcohol (Sigma-Aldrich) was used for extraction of DNA in the supernatant, followed by electrophoretic analysis on 1.0 % agarose gels containing 0.1 μg/ml ethidium bromide (EtBr, Sigma-Aldrich) 31 .

For analysis of the cell cycle, cells were collected, washed with cold PBS, and fixed in 75 % ethanol at 4°C or 30 min. A DNA staining kit was used according to the manufacturer’s instructions for measurement of the DNA content of the cells 32 . Flow cytometric analyses were carried out using a flow cytometer and the relative DNA content was determined using CellQuest software based on the presence of red fluorescence. MMP (ΔΨm) was determined using the dual-emission potential-sensitive probe, JC-1. Cells were collected and incubated with 10 μM JC-1 for 20 min at 37°C in the dark. Cells were then washed once with PBS and analyzed using a flow cytometer 33 . For measurement of ROS generation, cells were treated with DATS for various periods and the medium was discarded; cells were then incubated with new culture medium containing 10 μM of DCFDA at 37°C in the dark for 20 min. Cell lysates were used for evaluation of ROS generation using a flow cytometer 34 .

Cells were harvested and washed twice in PBS at 4°C. Total cells lysates were lysed in lysis buffer (40 mM Tris (pH 8.0), 120 mM, NaCl, 0.5 % NP-40, 0.1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 100 μg/ml phenymethylsulfonyl fluoride). Supernatants were collected and protein concentrations were then measured using protein assay reagents (Pierce, Rockford, IL). Equal amounts of protein extracts were denatured by boiling at 95°C for 5 min in sample buffer (0.5 M Tris–HCl, pH 6.8, 4 % SDS, 20 % glycerol, 0.1 % bromophenol blue, 10 % β-mercaptoethanol) at a ratio of 1:1, subjected to SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH) by electroblotting. Membranes were blocked with 5 % non-fat dry milk in PBS with Tween 20 buffer (PBS-T) (20 mM Tris, 100 mM NaCl, pH 7.5, and 0.1 % Tween 20) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with the primary antibodies, probed with enzyme-linked secondary antibodies, and visualized using an ECL kit, according to the manufacturer's instructions.

Activities of caspases were determined by use of colorimetric assay kits, which utilize synthetic tetrapeptides (Asp-Glu-Val-Asp (DEAD) for caspase-3; Ile-Glu-Thr-Asp (IETD) for caspase-8; Leu-Glu-His-Asp (LEHD) for caspase-9, respectively) labeled with p-nitroaniline (pNA). Briefly, DATS-treated and untreated cells were lysed in the supplied lysis buffer. Supernatants were collected and incubated with the supplied reaction buffer containing DTT and DEAD-pNA, IETD-pNA, or LEHD-pNA as substrates at 37°C. The reactions were measured by changes in absorbance at 405 nm using the VERSAmax tunable microplate reader.

Unless otherwise indicated, each result is expressed as the mean ± SD of data obtained from triplicate experiments. Statistical analysis was performed using a paired Student t-test. Differences at p < 0.05 were considered statistically significant.

To evaluate the effects of DATS on leukemia cell viability, four leukemia cell lines (U937, THP-1, HL60 and K562) were stimulated with various concentrations of DATS for 48 h or with 20 μM DATS for the indicated times, and an MTT assay was performed. As shown in Figure 1, DATS induced a decrease in cell viability in a dose- and time-dependent manner in all four models of leukemia. For example, treatment with 20 μM DATS for 24 h and 48 h in U937 cells resulted in 53 % and 62 % inhibition, respectively, which was associated with many morphological changes (Figure 2A).

In order to determine whether the decrease in leukemia cell viability by DATS treatment was due to induction of apoptosis, three established criteria were subsequently used for assessment of apoptosis. First, morphological changes of cells were determined using DAPI staining; as shown in Figure 2B and C, treatment with DATS resulted in observation of a significant number of cells with chromatin condensation, loss of nuclear construction, and formation of apoptotic bodies, whereas these features were not observed in control cells. Second, we analyzed DNA fragmentation, which is another hallmark of apoptosis. Following agarose gel electrophoresis of DNAs from cells treated with DATS, a typical ladder pattern of internucleosomal fragmentation was observed. In contrast, DNA fragmentation in control cells was barely detected (Figure 2D). In addition, the degrees of apoptosis in cells treated with DATS were determined using flow-cytometric analysis for detection of hypodiploid cell populations. As shown in Figure 3, addition of DATS to leukemia cells resulted in increased accumulation of cells in the sub-G1 phase in a manner similar to that observed with DATS-induced viability inhibition, formation of apoptotic bodies, and accumulation of extranuclear fragmented DNA. This finding suggests that leukemia cells may undergo apoptosis after exposure to DATS, and there is a good correlation between the extent of apoptosis and inhibition of growth.

To reveal the mechanisms underlying the apoptotic effect of DATS on leukemia cells, we determined the expression levels and activities of caspase-3, -8, and −9 using U937 cells. As shown in Figure 4A, immunoblotting results showed that DATS treatment induced a concentration-dependent decrease in levels of procaspase-3, -8, and −9 proteins. For further quantification of the proteolytic activation of caspases, protein in the lysates of cells treated with DATS was normalized and then assayed for in vitro activities using fluorogenic substrates. As shown in Figure 4B, treatment with DATS resulted in a significant concentration-dependent increase of the activities of caspase-3, -8, and −9, compared with control cells. In addition, DATS treatment led to progressive proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and β-catenin, well-known substrate proteins of activated caspase-3, demonstrating an association of DATS-induced apoptosis with caspase activation.

The role of Bcl-2 and IAP family proteins in DATS-mediated apoptosis was determined by Western blotting for measurement of expression of Bcl-2 and IAP family members. As shown in Figure 5A, the levels of total Bid and Bcl-2 proteins were decreased in response to DATS treatment; however the levels of pro-apoptotic Bax remained unchanged. In addition, the levels of anti-apoptotic XIAP and cIAP-1 were also markedly inhibited by DATS treatment in a concentration-dependent manner.

Mitochondria, which play an essential role in apoptosis, are specialized organelles, which contain an outer membrane separated from an inner membrane by an intermembrane space that contains many proapoptotic proteins, including cytochrome c. Decrease of MMP causes disruption of the outer mitochondrial membrane, which in turn contributes to release of cytochrome c. Because generation and acumination of ROS in cancer cells might be related to mitochondrial dysfunction and cell apoptosis, we attempted to characterize the relationship between ROS production and changes in the MMP. For this study, the effects of DATS on the levels of MMP were monitored via a flow cytometer using the mitochondrial-specific probe, JC-1. As shown in Figure 5B, MMP values showed a concentration-dependent decrease by DATS treatment, indicating that DATS induced mitochondrial membrane hyperpolarization by depolarization. Next, ROS production was measured using a cell-permeant, oxidation-sensitive dye, DCFDA. The results indicated that extending the time of DATS treatment to 0.5 h and 1 h resulted in increased ROS production to greater than 5.5 and 6.2 times that of the control, respectively (Figure 6A).

In order to show that generation of ROS is a key step in the DATS-induced apoptotic pathway, cells were pretreated with 10 mM of NAC, a commonly used reactive oxygen intermediate scavenger, for 1 h, followed by treatment with DATS for 48 h. Blocking of ROS generation by pretreatment of cells with NAC effectively prevented DATS-induced down-regulation of Bcl-2 and XIAP expression, activation of caspases, and cleavage of PARP (Figure 6B). In addition, NAC had no effect on cell viability and apoptosis induction at a concentration of 10 mM; however, the presence of NAC almost completely suppressed DATS-induced apoptosis, as demonstrated by a near-complete reversal of the percentage of sub-G1 cells that were observed (Figure 7A), which was associated with recovered cell viability (Figure 7B). As expected, blocking of generation of ROS by pretreatment of cells with NAC also prevented DATS-induced chromatin condensation (Figure 7C). Collectively, these findings suggest that an increase in ROS generation is required for occurrence of DATS-induced apoptosis in U937.