Analysis on the mechanisms of NF-κB/Reactive Oxigen Species-mediated survival of hematopoietic cells and its clinical application

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To examine the roles for NF-κB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-κB (IκBSR), which can inhibit the function of all NF-κB family proteins, in a factor-dependent cell line, Ba/F3, in an inducible manner. Although the induced IκBSR neither affected thrombopoietin-dependent nor gp130-mediated growth of Ba/F3 cells, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IκBSR enhanced factor-deprived apoptosis, which was associated with the reduction of the mitochondrial membrane potential and accumulation of reactive oxygen species (ROS). Since IκBSR-enhanced apoptosis was cancelled by ROS scavenger enzymes such as MnSOD and thioredoxin X (TRX) almost completely, ROS were supposed be involved in the IκBSR-enhanced apoptosis. When IκBSR was expressed in normal hematopoietic stem/progenitor cells, IκBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS, which was also r … More elieved by ROS scavenger enzymes, MCI-186, N-acetyl-cysteine (NAC), and TRX. As for the mechanism of the IκBSR-induced ROS accumulation and apoptosis, semiquantitative reverse transcriptese-PCR analyses showed that the expression of ROS scavenger enzymes (MnSOD, glutathione peroxidase, and TRX) was suppressed by IκBSR. In addition, the expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL and A1) was also suppressed. Next, we expressed IκBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IκBSR was expressed at the stage of Flk-1^+ cells (putative hemangioblasts), IκBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IκBSR was expressed affer the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-κB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS. Less