The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage
Graphical abstract
Highlights
► H2O2 driven ROS activates NF-κB and arrests normal HDF and A549 tumor cell cycling. ► HDFs prematurely senesce while A549 cells become apoptotic in response to ROS. ► Suppressing NF-κB activation exacerbates the ROS-mediated HDF senescence response. ► Inhibiting canonical NF-κB enhances ROS-induced A549 tumor cell apoptosis. ► The p53–p21Cip1/Waf1 axis drives the ROS-mediated senescence response of HDFs.
Introduction
Oxidative DNA damage results from the accumulation of reactive oxygen species (ROS) due to an imbalance between ROS production and removal by antioxidant systems. ROS cause a variety of genetic damage, mostly base lesions and DNA single strand breaks (SSBs) but also double stranded DNA breaks (DSBs). In effect, replication can stall due to DNA damage resulting from ROS in the cell, and failure to stabilize stalled replication forks can result in their collapse and ultimately genetic instability [1], [2]. Oxidative damage can provoke cell cycle arrest and senescence, but it can also induce apoptosis with normal cells being more resistant to apoptosis than tumor cells [1], [2], [3].
Normal human diploid fibroblasts (HDFs) undergo a finite number of cell divisions in culture, a phenomenon termed cellular or replicative senescence. In vitro cell propagation causes telomere shortening, recognized as DSBs that activates a DNA damage checkpoint response (DDR), culminating in replicative senescence. In addition, human cells undergoing senescence and aging mice accumulate DNA lesions with irreparable DSBs outside telomeres, suggesting that their accumulation may have a causal role in mammalian aging [4], [5], [6]. DDR involves activation of the kinases ATM and Chk2 and their downstream effector p53 and its target p21Cip1/Waf1 [7], [8]. Normal HDFs can also undergo senescence in response to oxidative stress, referred to as stress-induced premature senescence (SIPS) [8], [9]. Hydrogen peroxide (H2O2) produces thymidine glycols that can lead to DSBs at replication forks [1] and to the appearance of DNA damage foci in HDFs [4], [9], [10]. ROS accumulation was shown to accelerate HDF senescence by DDR in conjunction with the induction and stabilization of p53 and p21Cip1/Waf1 [11], [12], [13], [14], [15], [16]. However, dependent on cell type and dosage, H2O2-mediated ROS accumulation has also been reported to induce the death of normal and tumor cells [1], [3], [17], [18].
NF-κB transcription factors are critical regulators of most if not all pro-inflammatory/stress-like responses. NF-κBs bind to DNA as dimers of five possible subunits (RelA/p65, c-Rel, RelB, p50, p52). Archetypical p65/p50 heterodimers are cytoplasmically restrained by IκBs (inhibitors of NF-κBs) in most cells. Canonical NF-κB activation generally requires the phosphorylation of serines 32 and 36 in ΙκΒα's signal response domain (SRD), causing its ubiquitination and subsequent proteasomal destruction, allowing p65/50 dimers to translocate to the nucleus and activate their target genes. IκBα SRD phosphorylation requires the IKK signalsome complex (IKKα and IKKβ serine–threonine kinases, and NEMO/IKKγ, a regulatory/adapter protein). IKKβ activation by phosphorylation of its T-activating loop serines 177/181 requires NEMO and rapidly occurs in response to a host of pro-inflammatory/stress-related extracellular signals. In contrast to IKKβ, IKKα activation, by phosphorylation of T-loop serines 176/180, is NEMO-independent, requires de novo protein synthesis and is mainly mediated by stimuli invoked in adaptive immune responses. In vivo, unlike IKKβ, IKKα does not generally target IκBs but instead phosphorylates an IκB-like signal SRD in NF-κB p100, which induces its ubiquitination/proteasome-dependent processing yielding the NF-κB p52 subunit. Thus, NF-κB p100 functions akin to IκBα by sequestering NF-κB RelB in the cytoplasm and also yields NF-κB p52, which translocates to the nucleus together with RelB, to activate other unique NF-κB target genes [19], [20]. Some p100 also sequesters a subset of p65/p50 heterodimers, whose activation can only be initiated by IKKα [21]. IKK catalytic activity also regulates other cellular responses independent of NF-κB activation. For example, IKKβ controls anti-apoptotic, pro-inflammatory and proliferative responses by targeting p53, FoxO and other cell cycle effectors [19], [20], [22].
NF-κB is activated by moderate ROS levels in a cell-type specific manner by different mechanisms, including the classical IKKβ-dependent pathway [23], [24]. In addition, genotoxic stimuli producing DSBs cause ATM/NEMO-dependent IKKβ-mediated canonical NF-κB activation, which facilitates cell survival and cycle arrest thereby giving cells precious time to repair their damaged DNA before p53-dependent apoptotic pathways are invoked [25], [26]. A role for IKKα in DDR invoked by oxidative stress or chemotherapeutics has also been suggested [27], [28]. Collectively, these studies provided evidence that the induction of NF-κB signaling by DNA damage is important for cell survival. However, to our knowledge little if any evidence is available on how canonical NF-κB activity functionally contributes to ROS-induced HDF senescence responses.
To address NF-κB's functional roles in ROS-mediated DDR in the context of normal vs. human tumor cells, we inhibited canonical NF-κB activation in MRC-5 and IMR-90 normal HDFs and suppressed NF-κB signaling in A549 human lung tumor epithelial cells. Inhibition of canonical NF-κB in both cell types exacerbated ROS initiated DDR by up-regulating the p53–p21Cip1 axis, leading to the enhanced accumulation of cells at the G2 or G2-M checkpoint. Importantly, the final physiological outcomes of ROS-induced DDR greatly differed in normal vs. human cancer cells with the former normal human diploid fibroblasts (HDFs) succumbing to premature senescence and the latter human lung cancer epithelial cells undergoing exacerbated apoptosis.
Section snippets
Cell culture and retroviral transduction
MRC-5, human embryonic diploid fibroblasts (HDFs), originally from the National Institute for Biological Standards and Controls, South Mimms, UK [29], were kept at the cell stock of the Genetics Division, National Institute for Medical Research, London UK, and obtained from Dr. Lily Huschtscha-Holliday and used in several previous studies by our group [29], [30], [31]. IMR-90 HDFs and human epithelial lung tumor cells A549 were originally obtained from ATCC and phoenix retroviral packaging
H2O2 activates a ROS-mediated DNA damage checkpoint response in normal and cancer cells
Increasing concentrations of H2O2 resulted in ROS accumulation in both normal HDFs MRC-5 and A549 lung cancer cells (Fig. 1A), reduced A549 colony formation (Fig. 1B) and also induced DDR characteristic changes in specific cellular proteins. After 2 h of H2O2 exposure followed by 24 h of recovery, phosphorylations were detected for p-ATM (S1981), p-Chk2 (T68) and γ-H2AX in both cell types (Fig. 2A and B). Moreover, p-p53 (Ser15), total p53 and p21Cip1 levels were also elevated in an H2O2
Discussion
Oxidative stress induces DNA damage leading to either growth arrest and senescence or apoptosis, depending on the nature and intensity of the stimulus and cell type (i.e., normal vs. tumor cells) [1], [2], [3]. Here we show that H2O2 induced oxidative stress initiated a DDR leading to G2 or G2-M arrest and the premature senescence of MRC-5 normal human lung diploid fibroblasts (HDFs), but instead caused the apoptosis of A549 human lung tumor epithelial cells. NF-κB signaling was induced by
Conflict of interest
The authors have no conflicting financial interests.
Acknowledgments
We thank Dr. R. Agami, The Netherlands Cancer Institute, Amsterdam, for pRetro.Super-Puro and pRS-shp53 retrovectors. This research was co-funded by EU (European Social Fund) and the Hellenic Ministry of Education within the framework of the program ‘Pythagoras IΙ’ Grant No: 1887 (EK); and also in part by PENED'03 (Program for Supporting Research Manpower) program grant No: 61/2055 and ‘THALIS’ grants (EK) of the General Secretariat for Research and Technology, the Empeirikeion Foundation,
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- 1
Present address: Institute for Cell and Molecular Biosciences (ICaMB), University of Newcastle Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
- 2
Contributed equally to this work.