| Arsenic trioxide (As2O3) is an effective drug for the treatment of acute promyelocytic leukemia (APL), but is less effective against other malignancies. Although the response of APL cells to AS 2O3 has been linked to degradation of the PML/RARalpha fusion oncoprotein characteristic of this disease, we present evidence that PML/RARalpha does not confer sensitivity to As2O3-induced apoptosis. In two arsenic-resistant APL cell lines, AsR2 and AsR3, that we established to study mechanisms of action of arsenic trioxide, PML/RARalpha expression was regulated normally by As2O3. However, both arsenic-resistant clones contained high glutathione (GSH) levels, and we found that GSH depletion dramatically synergized with As2O 3 in the induction of apoptosis in these cells. This apoptosis was dependent upon the generation of reactive oxygen species (ROS) and oxidative stress, which were only detected upon cotreatment with arsenic and the GSH depletor, buthionine sulfoximine (BSO). c-Jun N-terminal kinase (JNK) is known to be induced by oxidative stress and As2O3, and its activation has been implicated in the induction of apoptosis in some cell systems. Therefore, we sought to determine whether activation of JNK could mediate the effects of As2O3 in APL. We found that AsR2 and AsR3 cells were not only resistant to arsenic-induced apoptosis, but to JNK1 activation as well. Only under conditions of GSH depletion, a condition that strongly synergized in inducing apoptosis, could As2O3-induced JNK activity be elicited in either resistant cell line. Furthermore, inhibition of JNK signaling by chemical and genetic strategies significantly increased survival of NB4 and mouse embryo fibroblast cells, respectively, in response to As 2O3, but had no effect on doxorubicin-induced apoptosis. To investigate the effects of As2O3 on events downstream of JNK, we performed transient transfection and electrophoretic mobility shift assays to examine AP-1 activation. We found that As2O3 induced a dose-dependent increase in reporter gene activity driven by both natural and synthetic AP-1-response elements (TRE), as well as an increase in AP-1 DNA-binding to an oligonucleotide bearing a consensus THE sequence. Paradoxically, however, we found that combined As2O3 and BSO treatment repressed arsenic-induced AP-1 activity, yet enhanced DNA binding. Similar results were observed using the related metal, antimony, which, though less toxic alone, shows greater synergy with BSO. (Abstract shortened by UMI.)... |
