DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway

Abstract

The DNA damage response (DDR) cascade and ROS (reactive oxygen species) signaling are both involved in the induction of cell death after DNA damage, but a mechanistic link between these two pathways has not been clearly elucidated. This study demonstrates that ROS induction after treatment of cells with neocarzinostatin (NCS), an ionizing radiation mimetic, is at least partly mediated by increasing histone H2AX. Increased levels of ROS and cell death induced by H2AX overexpression alone or DNA damage leading to H2AX accumulation are reduced by treating cells with the antioxidant N-Acetyl-L-Cysteine (NAC), the NADP(H) oxidase (Nox) inhibitor DPI, expression of Rac1N17, and knockdown of Nox1, but not Nox4, indicating that induction of ROS by H2AX is mediated through Nox1 and Rac1 GTPase. H2AX increases Nox1 activity partly by reducing the interaction between a Nox1 activator NOXA1 and its inhibitor 14-3-3zeta. These results point to a novel role of histone H2AX that regulates Nox1-mediated ROS generation after DNA damage.

Figure 1

Exposure to DNA-damaging agents leads to increased levels of ROS that contribute to cell death. (a) Sub-confluent U2OS and HBL100 cells were treated with the DNA-damaging agents neocarzinostatin (NCS, 0.5 μg/ml), hydroxyurea (HU, 2 mM), and doxorubicin (8.5 μM) for 20 min to 5 h. ROS levels were quantified using DCFH-DA and a fluorescent microplate reader. (b) Addition of 6 or 30 mM NAC 2 h after NCS treatment suppressed the latent NCS-induced increase in ROS. (c) Suppression of the latent NCS-induced ROS by NAC treatment reduced NCS-induced apoptosis

Figure 2

Upregulation and phosphorylation of H2AX is an early event in the DDR cascade. U2OS and HBL100 cells were treated with NCS (0.5 μg/ml), for 20 min to 5 h in PBS buffer (to mimic the condition of cells used for ROS assays). Levels and phosphorylation of H2AX and ATM protein were assayed by immunoblot analysis using antibodies indicated

Figure 3

Histone H2AX increases basal and DNA damage-induced ROS. Overexpression of Flag-H2AX increased basal and NCS-induced ROS in U2OS (a) and HBL-100 (b) cells, whereas inducible H2AX knockdown reduces NCS-induced ROS in HCT-116 (c) and HBL100 (d) cells. Immunoblot analysis was performed at 5 h after NCS treatment. ^**Flag-tagged H2AX, ^*endogenous H2AX

Figure 4

H2AX increases ROS through Nox1/Rac1 pathway. Control U2OS cells (a, white bar), Flag-H2AX U2OS cells (a, black bar), control HBL100 cells (b, white bar), and Flag-H2AX HBL100 cells (b, black bar) were treated with NCS (0.5 μg/ml, 5 h) with or without the Nox-inhibitor, DPI (0.5 μM) and fold-induction of ROS was measured. (c) Vector control cells or Flag-H2AX U2OS cells were transiently transfected with an indicated plasmid with a control vector for 12 h, followed by NCS treatment (0.5 μg/ml) for 5 h. Levels of ROS were measured in more than three independent experiments. (d) Levels of proteins were immunoblotted to confirm expression or knockdown of indicated proteins. (e) Expression of H2AX inhibited coimmunoprecipitation of 14-3-3zeta with NOXA1. U2OS cells were transfected with the indicated plasmids, followed by GST-pulldown of 14-3-3zeta. ^**Flag-taged H2AX, ^*endogenous H2AX

Figure 5

Model of ROS induction by H2AX after DNA damage. DNA damage induces levels and phosphorylation of H2AX. Then, H2AX activates Rac1 GTPase and Nox1 by sequestering 14-3-3zeta, leading to increase in intracellular levels of ROS. H2AX can also induce ROS and apoptosis by the alternative pathways that are usually inhibited by Nox1

Figure 6

High levels of H2AX induces apoptosis without DNA damage, which is decreased by Rac1N17. (a) U2OS cells stably expressing Flag-H2AX show constitutive apoptosis without DNA damage, compared with vecetor control cells. 293T (b) or U2OS (c) cells were transfected with expression vectors of Falg-H2AX, HA-Rac1N17 or both for 48 h, and apoptosis was compared with that of vector control cells. A bar graph represents fraction of apoptotic cells