Abstract
Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-kappaB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (gamma-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-kappaB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-kappaB activation (e.g. in I kappaB alpha super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-kappaB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-kappaB activation, blocked TNFalpha- but not HDACI-mediated NF-kappaB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-kappaB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, gamma-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-kappaB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-kappaB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.
Highlights
HDACI-mediated Mn-SOD2 induction and increased reactive oxygen species (ROS) accu- tic proteins (e.g. Bim), down-regulation of anti-apoptotic mulation, DNA damage, and apoptosis
We have examined the roles of the components of the DNA damage response pathway, ATM and NEMO, in p65 activation by HDACIs
The present findings identify the ATM/NEMO DNA damage pathway as a critical mediator of p65 activation in human leukemia cells exposed to HDACIs
Summary
HDACI-mediated Mn-SOD2 induction and increased ROS accu- tic proteins (e.g. Bim), down-regulation of anti-apoptotic mulation, DNA damage, and apoptosis. HDACI-mediated NF-B activation, siNEMO clones exposed to LBH-589 exhibited pronounced attenuation of Mn-SOD2 protein and mRNA expression (Fig. 6A, left panel, inset and bar graph), accompanied by persistent LBH-589-induced ROS accumulation, compared with U937/siC control cells (Fig. 6A, right panel).
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