Arsenic trioxide-dependent activation of thousand-and-one amino acid kinase 2 and transforming growth factor-beta-activated kinase 1

Abstract

Arsenic trioxide (As(2)O(3)) has potent antileukemic properties in vitro and in vivo, but the mechanisms by which it generates its effects on target leukemic cells are not well understood. Understanding cellular mechanisms and pathways that are activated in leukemic cells to control the generation of As(2)O(3) responses should have important implications in the development of novel approaches using As(2)O(3) for the treatment of leukemias. In this study, we used immunoblotting and immune complex kinase assays to provide evidence that the kinases thousand-and-one amino acid kinase 2 (TAO2) and transforming growth factor-beta-activated kinase 1 (TAK1) are rapidly activated in response to treatment of acute leukemia cells with As(2)O(3). Such activation occurs after the generation of reactive oxygen species and regulates downstream engagement of the p38 mitogen-activated protein kinase. Our studies demonstrate that siRNA-mediated knockdown of TAO2 or TAK1 or pharmacological inhibition of TAK1 enhances the suppressive effects of As(2)O(3) on KT-1-derived leukemic progenitor colony formation and on primary leukemic progenitors from patients with acute myelogenous leukemia. These results indicate key negative-feedback regulatory roles for these kinases in the generation of the antileukemic effects of As(2)O(3). Thus, molecular or pharmacological targeting of these kinases may provide a novel approach to enhance the generation of arsenic-dependent antileukemic responses.

Fig. 1.

As₂O₃-dependent phosphorylation of TAO2 in leukemic cell lines. A, U937 cells were incubated in the absence or presence of As₂O₃ (2 μM) for the indicated times. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-TAO2 (Ser181) antibody (top). The same blot was reprobed with an anti-GAPDH antibody to control for protein loading (bottom). B, as in A, but using NB4 cells. C, as in A, but using NB4.306 cells. D, U937 cells were incubated with As₂O₃ (2 μM) as indicated. Cell lysates were subjected to in vitro kinase assays using activating transcription factor 2 as an exogenous substrate. Proteins were resolved by SDS-PAGE, and phosphorylated proteins were detected by autoradiography (top). Longer exposure of the same membrane is also shown (bottom).

Fig. 2.

As₂O₃-dependent phosphorylation of TAK1 in leukemic cell lines. A, NB4 cells were incubated in the absence or presence of As₂O₃ (2 μM) for the indicated times. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-TAK1 (Ser412) antibody (top). The same blot was reprobed with an anti-GAPDH antibody to control for protein loading (bottom). B, as in A, but using KT-1 cells. C, as in A, but using NB4.306 cells. D, KT-1 cells were incubated in the absence or presence of As₂O₃ at varying times and concentrations as indicated. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-TAO2 (Ser181) antibody (top) or an anti-phospho-TAK1 (Ser412) antibody (middle). The same blot was reprobed with an anti-GAPDH antibody to control for protein loading (bottom).

Fig. 3.

As₂O₃-induced phosphorylation of TAO2 and TAK1 is diminished by the reducing agents DTT and NAC. A, NB4 cells were preincubated for 1 h with DTT (1 mM) and subsequently incubated with As₂O₃ (2 μM) for 30 min. Cells were then analyzed by flow cytometry for the presence of ROS as described under Materials and Methods. Data are expressed as the fold increase in mean fluorescence over untreated samples and represent the means ± S.E. of two independent experiments. B, NB4 cells were incubated with or without combinations of DTT (1 mM), NAC (10 mM), and As₂O₃ (2 μM) as indicated. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-TAO2 (Ser181) antibody (top). The same blot was reprobed with an anti-GAPDH antibody to control for protein loading (bottom). C, similar experiment as in B, demonstrating immunoblotting with an anti-phospho-TAK1 (Ser412) antibody (top).

Fig. 4.

Knockdown of TAO2 or TAK1 and pharmacological inhibition of TAK1 block As₂O₃-induced phosphorylation of p38 MAPK. A, left, U937 cells were transfected with control siRNA or TAO2-specific siRNA. Expression of mRNA for TAO2 gene was evaluated by quantitative real-time RT-PCR using GAPDH gene for normalization. Data represent means ± S.E. of two experiments. Right, as on the left, but using TAK1-specific siRNA. B, U937 cells were transfected with control siRNA or TAO2-specific siRNA, and cells were incubated in the absence or presence of As₂O₃ (2 μM) for 30 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-p38 (Thr180/Tyr182) antibody (top). Equal amounts of cell lysates from the same experiment were resolved separately by SDS-PAGE and immunoblotted with an anti-p38 antibody (bottom). C, similar experiment as in B, but using TAK1 siRNA instead of TAO2 siRNA. D, KT-1 cells were pretreated for 60 min with 5Z-7-oxozeaenol (500 nM) and were subsequently incubated with As₂O₃ (2 μM) for 30 min in the continuous absence or presence of 5Z-7-oxozeaenol as indicated. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-p38 (Thr180/Tyr182) antibody (top). The same blot was reprobed with an anti-p38 antibody to control for protein loading (bottom).

Fig. 5.

siRNA-mediated knockdown of TAO2 and TAK1 and pharmacological inhibition of TAK1 enhance the suppressive effects of As₂O₃ on leukemic progenitor (CFU-L) growth. A, left, KT-1 cells were transfected with control siRNA or TAO2-specific siRNA. Expression of mRNA for TAO2 gene was evaluated by quantitative real-time RT-PCR using GAPDH gene for normalization. Data are expressed as the percentage of control samples and represent means ± S.E. of three experiments. Right, as on the left, but using TAK1-specific siRNA. B, KT-1 cells transfected with control siRNA or TAO2-specific siRNA were subsequently incubated in methylcellulose in the absence or presence of As₂O₃ (0.5 μM), and leukemic CFU-L colony formation was assessed. Data are expressed as the percentage of control colony formation of samples treated with control siRNA and represent means ± S.E. of four independent experiments as shown. Paired t test analysis comparing the effects of As₂O₃ in the absence or presence of TAO2 siRNA showed a paired p value of 0.0006. C, as in B, but using TAK1-specific siRNA. Paired t test analysis comparing the effects of As₂O₃ in the absence or presence of TAK1 siRNA showed a paired p value of 0.0087. D, KT-1 cells were incubated in methylcellulose with As₂O₃ (0.5 μM), in the absence or presence of 5Z-7-oxozeaenol (100 nM) as indicated, and leukemic CFU-L colony formation was assessed. Data are expressed as the percentage of control colony formation of untreated samples and represent means ± S.E. of five independent experiments as shown. Paired t test analysis comparing the effects of As₂O₃ in the absence or presence of 5Z-7-oxozeaenol showed a paired p value of 0.005935. UT, untreated.

Fig. 6.

siRNA-Mediated knockdown of TAO2 and TAK1 and pharmacological inhibition of TAK1 enhance As₂O₃-induced growth suppression of CFU-L colony formation from AML patients. A, peripheral blood mononuclear cells from two AML patients were transfected with control siRNA or TAO2 siRNA and were subsequently incubated in methylcellulose in the absence or presence of As₂O₃ (0.5 μM). CFU-L colony formation was assessed, and data are expressed as means ± S.E. of the percentage of colony formation of samples treated with control siRNA only. B, as in A, but using TAK1-specific siRNA. C, peripheral blood mononuclear cells from three AML patients were plated in a methylcellulose assay system with As₂O₃ (0.5 μM), in the absence or presence of 5Z-7-oxozeaenol (100 nM), as indicated. CFU-L colony formation was assessed and data are expressed as means ± S.E. of the percentage of colony formation of untreated samples. Paired t test analysis comparing the effects of As₂O₃ in the absence or presence of 5Z-7-oxozeaenol showed a paired p value of 0.01009. UT, untreated.