ASK1 Is Activated by Treatment with Arsenic Trioxide through Accumulation of Reactive Oxygen Species and May Suppress Apoptosis through NF-kappaB in Leukemia Cells.

Abstract

Arsenic trioxide (ATO), a remarkably effective reagent for treatment of relapsed acute promyelocytic leukemia (APL), is reported to induce differentiation of APL cells at low concentrations and apoptosis at high concentrations. Induction of granulocytic differentiation is considered to be a specific effect of ATO on APL cells. By contrast, ATO commonly induces apoptosis of various tumor cells of hematological malignancies, including chronic myeloid leukemia cells expressing the BCR/ABL fusion kinase, as well as those of solid tumors. It was reported that ATO treatment induced intracellular accumulation of reactive oxygen species (ROS) by inhibiting glutathione peroxidase activity. Accumulated ROS induced a decline in cellular mitochondrial membrane potential, followed by cytochrome c release, caspase 3 activation, and apoptosis of cells. On the other hand, it is well known that adequate dose of ROS is indispensable for proliferation and survival of a variety of cells, including hematopoietic cells. Thus, various intracellular signaling pathways are strictly regulated and activated downstream of ROS to promote or suppress apoptosis, and the signaling pathways activated by ROS accumulation induced by ATO need to be defined to understand the mechanisms for ATO-induced apoptosis.Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the JNK and p38 signaling pathways and is involved in regulation of apoptosis. Here we find that ATO treatment of NB4 and K562 leukemia cells induces activation of ASK1. ATO induced accumulation of ROS, and the ASK1 activation was suppressed by cotreatment with an antioxidant, N-acetyl-l-cysteine. Although the Rho family GTPases Rac and cdc42 were activated by ATO, overexpression of their dominant-negative mutants did not suppress ATO-induced ASK1 activation. ASK1 activation was induced most significantly at low concentrations of ATO, where G2/M arrest but not apoptosis was induced. On the other hand, ASK1 activation induced by ATO was barely detectable at high concentrations, where apoptosis was induced significantly. By contrast, JNK and p38 were activated in dose-dependent manners by ATO. Murine embryonic fibroblasts (MEFs) derived from ASK1-deficient mice were more prone to ATO-induced apoptosis than control MEFs. Moreover, ASK1 was activated by ATO in a more sustained manner in ATO-resistant leukemia cell lines than in sensitive cell lines. Finally, a dominant-negative ASK1 mutant reduced ATO-induced NF-kappaB activation in leukemia cells. Together, the results indicate that ASK1 is activated by ATO through ROS generation independent of activation of Rac and cdc42 and may play a negative role in induction of apoptosis, possibly through activation of NF-kappaB.